J - Coronal section through rostral telencephalon of an E14 . 5 Rx - Dicer mutant illustrating high abundance location of rosettes ( arrows ) , as revealed by distribution of mitoses ( PH3 ) neurons ( Tuj1 ) . K - S - of regional identity of rosettes in E14 . 5 Rx - Dicer mutants . ( K ) Schema of normal transcription factor expression patterns defining telencephalic regional identity . Expression of Ngn2 , Tbr2 Pax6 identifies rosettes in rostro - dorsal telencephalon as having dorsal identity ( L - N , P ) ; Gsx2 , Dlx2 Dlx5 identify rosettes in ventral telencephalon as having ventral identity ( O , Q ) ; Nkx2 . 1 identifies MGE rosettes as being normotopic ( R ) ; absence of Gsx2 in LGE rosette identifies them as ectopic ( S ) . Tuj1 labels neurons . In ( L , M ) , arrowheads indicate border between dorsal ventral territories , dotted indicates outer border of telencephalon . LGE , lateral ganglionic eminence ; MGE , medial ganglionic eminence ; P , pallium ; SP , subpallium .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J - Coronal section through the rostral telencephalon of an E14.5 Rx-Dicer mutant embryo illustrating the high abundance and location of rosettes (arrows), as revealed by the distribution of mitoses (PH3) and neurons (Tuj1). K-S - Analysis of the regional identity of rosettes in E14.5 Rx-Dicer mutants. (K) Schema of normal transcription factor expression patterns defining telencephalic regional identity. Expression of Ngn2, Tbr2 and Pax6 identifies rosettes in the rostro-dorsal telencephalon as having dorsal identity (L-N,P); Gsx2, Dlx2 and Dlx5 identify rosettes in the ventral telencephalon as having ventral identity (O,Q); Nkx2.1 identifies MGE rosettes as being normotopic (R); absence of Gsx2 in LGE rosette cells identifies them as ectopic (S). Tuj1 labels neurons. In (L,M), arrowheads indicate the border between dorsal and ventral territories, and dotted line indicates the outer border of the telencephalon. LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; P, pallium; SP, subpallium.


A - Coronal section through rostral telencephalon of an E14 . 5 Rx - Dicer mutant displaying three types of rosettes .

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A - Coronal section through the rostral telencephalon of an E14.5 Rx-Dicer mutant embryo displaying the three types of rosettes.


B - High magnification of rosettes in Rx - Dicer mutants at E12 . 5 ( Type 1 ) , E14 . 5 ( Type 2 ) E17 . 5 ( Type 3 ) , immunostained for PH3 Tuj1 . Dashed indicates ventricular surface ; arrowheads indicate basal mitoses ; arrows indicate a stream of apical mitoses connecting lumen of rosette with lumen of telencephalic ventricle .

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B - High magnification of rosettes in Rx-Dicer mutants at E12.5 (Type 1), E14.5 (Type 2) and E17.5 (Type 3), immunostained for PH3 and Tuj1. Dashed line indicates ventricular surface; arrowheads indicate basal mitoses; arrows indicate a stream of apical mitoses connecting the lumen of the rosette with the lumen of the telencephalic ventricle.


C , D - Abundance of rosette types at indicated ages ( C ) , rostro - caudal distribution of total rosette abundance per , independent of type ( D ) . n ≥ 2 brains per .

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C,D - Abundance of rosette types at the indicated ages (C), and rostro-caudal distribution of total rosette abundance per age, independent of type (D). n ≥ 2 brains per age.


F , G - Distribution of neurons ( Tuj1 + ) apical adherens junction Par3 in OB primordium of control Rx - Dicer mutant at indicated ages . Dotted lines indicate basal border , dashed lines indicate apical border . arrowheads indicate ectopic neurons / or absence of Par3 . Arrow in ( G ) indicates accumulation of Par3 at lumen of a nascent rosette .

O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O

F,G - Distribution of neurons (Tuj1+ cells) and apical adherens junction protein Par3 in the OB primordium of control and Rx-Dicer mutant embryos at the indicated ages. Dotted lines indicate basal border, dashed lines indicate apical border. White arrowheads indicate ectopic neurons and/or absence of Par3. Arrow in (G) indicates accumulation of Par3 at the lumen of a nascent rosette.


A , B - Changes in expression of miRNAs between control Rx - Dicer mutants at E11 . 5 in rostral telencephalon . Red dots indicate statistically differentially expressed miRNAs according to FDR correction of P values on Wald statistic ( DESeq2 ) ( B ) . Dashes lines indicate stringency limits in evaluation of results .

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A,B - Changes in expression levels of miRNAs between control and Rx-Dicer mutants at E11.5 in the rostral telencephalon. Red dots indicate statistically differentially expressed miRNAs according to FDR correction of P values based on the Wald statistic (DESeq2 analysis) (B). Dashes lines indicate stringency limits in evaluation of results.


D , E - Changes in mRNA expression between control Rx - Dicer mutants at E11 . 5 in rostral telencephalon . Statistically differentially expressed mRNAs according to FDR correction of P values on Wald statistic ( DESeq2 ; Adj . P < 0 . 1 FC > 1 . 25 ) are highlighted in red , three with highest Adj . P value are identified by name . Blue dot indicates Irs2 : FC = 1 . 748 , Adj . P = 5 . 95e - 08 .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

D,E - Changes in mRNA expression levels between control and Rx-Dicer mutants at E11.5 in the rostral telencephalon. Statistically differentially expressed mRNAs according to FDR correction of P values based on the Wald statistic (DESeq2 analysis; Adj. P < 0.1 and FC > 1.25) are highlighted in red, and the three with the highest Adj. P value are identified by name. Blue dot indicates Irs2: FC=1.748, Adj. P = 5.95e-08.


G - Heatmaps of relative expression of DEGs related to apoptosis ( top ) proliferation ( bottom ) . Rows correspond to independent biological replicates . Indicated are some example genes .

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G - Heatmaps of relative expression levels of DEGs related to apoptosis (top) and proliferation (bottom). Rows correspond to independent biological replicates. Indicated are some example genes.


H - Enrichment plots from GSEA for MSigDB Hallmark Neurogenesis ( NES = 2 . 98 ; p = 0 ; Adj . P = 0 . 00045 ) , Regulation of proliferation ( NES = 2 . 40 ; p = 0 ; Adj . P = 0 . 0033 ) , Positive regulation of adhesion ( NES = 2 . 06 ; p = 0 . 0058 ; Adj . P = 0 . 013 ) , Regulation of response to stress ( NES = 2 . 50 ; p = 0 ; Adj . P = 0 . 0023 ) , Positive regulation of ( NES = 1 . 84 ; p = 0 . 0179 ; Adj . P = 0 . 0326 ) , Apoptotic signaling pathway ( NES = 1 . 59 ; p = 0 . 047 ; Adj . P = 0 . 084 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H - Enrichment plots from GSEA for MSigDB Hallmark Neurogenesis (NES = 2.98; p = 0; Adj. P = 0.00045), Regulation of cell proliferation (NES = 2.40; p=0; Adj. P = 0.0033), Positive regulation of cell adhesion (NES = 2.06; p = 0.0058; Adj. P = 0.013), Regulation of response to stress (NES = 2.50; p = 0; Adj. P = 0.0023), Positive regulation of cell death (NES = 1.84; p = 0.0179; Adj. P = 0.0326), Apoptotic signaling pathway (NES = 1.59; p= 0.047; Adj. P = 0.084).


I , J - ISH stains of Irs2 mRNA , qPCR for Irs2 mRNA let - 7 - 5p miRNA , in rostral telencephalon of control Rx - Dicer mutant . Dashed indicates basal border , dotted indicates apical surface . Arrowheads indicate with greatest increase in Irs2 expression . BG , basal ganglia ; H , hippocampus ; NCx , neocortex ; OB , olfactory bulb ; Spt , septum . Histograms represent mean ± ( logarithmic scale ) ; symbols in plots indicate values for ; t - , * P < 0 . 05 , * * P < 0 . 01 . N = 6 - 9 replicates per group . Scale bar , 100 µm .

O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I,J - ISH stains of Irs2 mRNA, and qPCR for Irs2 mRNA and let-7-5p miRNA, in the rostral telencephalon of control and Rx-Dicer mutant embryos. Dashed line indicates basal border, dotted line indicates apical surface. Arrowheads indicate area with the greatest increase in Irs2 expression. BG, basal ganglia; H, hippocampus; NCx, neocortex; OB, olfactory bulb; Spt, septum. Histograms represent mean ± SEM (logarithmic scale); symbols in plots indicate values for individual embryos; t-test, *P <0.05, **P <0.01. N = 6-9 replicates per group. Scale bar, 100 µm.


A - C - Immunostains for activated p53 ( red ) in rostral telencephalon of control Rx - Dicer mutant at E12 . 5 , of positive ( C ) . Dashed lines indicate apical side , dotted lines indicate basal side . N = 4 replicates per genotype . Scale bar , 50µm .

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A-C - Immunostains for activated p53 (red) in the rostral telencephalon of control and Rx-Dicer mutant embryos at E12.5, and quantification of positive cells (C). Dashed lines indicate apical side, dotted lines indicate basal side. N = 4 replicates per genotype. Scale bar, 50µm.


D - L - Rostral telencephalon of E12 . 5 of indicated genotypes immunostained for detection of apoptotic ( Casp3 ) , neurons ( Tuj1 ) apical Par3 , of positive for Casp3 ( H ) . N = 4 - 7 replicates per genotype . Scale bar , 50 µm .

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D-L - Rostral telencephalon of E12.5 embryos of the indicated genotypes immunostained for the detection of apoptotic cells (Casp3), neurons (Tuj1) and the apical complex protein Par3, and quantification of cells positive for Casp3 (H). N = 4-7 replicates per genotype. Scale bar, 50 µm.


M - O - Sagittal sections through rostral telencephalon of E17 . 5 of indicated genotypes . Note rosettes general disorganization in Rx - Dicer single mutants , complete rescue of this phenotype in Rx - Dicer - p53 double mutants . NCx , neocortex ; OB , olfactory bulb . Scale bar , 1 mm .

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M-O - Sagittal sections through the rostral telencephalon of E17.5 embryos of the indicated genotypes. Note the rosettes and general disorganization in Rx-Dicer single mutants, and complete rescue of this phenotype in Rx-Dicer-p53 double mutants. NCx, neocortex; OB, olfactory bulb. Scale bar, 1 mm.


A - Relative Irs2 mRNA in HEK upon transfection with Gfp - or Irs2 - encoding plasmids . Mean ± ; t - , * * * * P < 0 . 0001 . N = 6 replicates per group .

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A - Relative Irs2 mRNA levels in HEK cells upon transfection with Gfp- or Irs2-encoding plasmids. Mean ± SEM; t-test, ****P < 0.0001. N = 6 replicates per group.


B - D - Distribution of neurons ( Tuj1 , red ) , GFP ( green ) mitoses ( PH3 , ) in rostral telencephalon of wild - type E14 . 5 or E17 . 5 mouse , as indicated , electroporated at E12 . 5 with indicated plasmid combinations . Dashed indicates apical surface . Insets show same field of view , with location extent of electroporations . Note overlap of rosettes ( arrows ) with ectopic neurons at apical surface ( arrowheads ) increased basal mitoses , upon overexpression of Irs2 ( C ) . Asterisk indicates absence of malformation in nearby cortex . At E17 . 5 ( D ) GFP + rosettes remain in deep cortical layers ( arrowheads ) .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B-D - Distribution of neurons (Tuj1, red), GFP (green) and mitoses (PH3, white) in the rostral telencephalon of wild-type E14.5 or E17.5 mouse embryos, as indicated, electroporated at E12.5 with the indicated plasmid combinations. Dashed line indicates the apical surface. Insets show the same field of view, with location and extent of electroporations. Note the overlap of rosettes (arrows) with ectopic neurons at the apical surface (arrowheads) and increased basal mitoses, upon overexpression of Irs2 (C). Asterisk indicates absence of malformation in nearby cortex. At E17.5 (D) GFP+ rosettes remain in deep cortical layers (arrowheads).


E - H - Details of rosettes ( large arrows in E ; dashed lines in F - H ) from Irs2 + Gfp - overexpressing E14 . 5 immunostained as indicated . Small arrows indicate apical mitoses , arrowheads indicate basal mitoses ( F , G ) Par3 + apical surface at centre of rosettes ( H ) , dotted lines indicate apical surface , dashed lines indicate basal side of rosettes . Inset in ( H ) is at same scale .

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E-H - Details of rosettes (large arrows in E; dashed lines in F-H) from Irs2+Gfp-overexpressing E14.5 embryos immunostained as indicated. Small arrows indicate apical mitoses, arrowheads indicate basal mitoses (F,G) and Par3+ apical surface at the centre of rosettes (H), dotted lines indicate apical surface, dashed lines indicate basal side of rosettes. Inset in (H) is at the same scale.


I - M - Distribution of neurons ( Tuj1 , red ) , GFP ( green ) mitotic ( PH3 , ) or apoptotic ( Casp3 , ) in rostral telencephalon of wild - type E13 . 5 electroporated at E12 . 5 with indicated plasmids , of Casp3 + ( M ) . Dashed indicates apical surface , dotted lines indicate borders of cortical plate ( CP ) . Nascent rosettes ( arrows ) are next to ectopic ventricular neurons ( arrowheads in K ' ) basal mitoses , without significant apoptosis in VZ ( L ' , M ) . Mean ± ; t - ; ns , not significant , * P < 0 . 05 . N = 3 - 6 replicates per group . N - Distribution of neurons ( Tuj1 , red ) in rostral telencephalon of wild - type E14 . 5 electroporated at E12 . 5 with a combination of plasmids encoding Irs2 , let - 7a , let - 7b let - 7c , Gfp . Dashed indicates apical surface . Formation of rosettes in Irs2 - expressing is rescued by overexpression of let - 7 . Inset shows same field of view , with location extent of electroporation .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O

I-M - Distribution of neurons (Tuj1, red), GFP (green) and mitotic (PH3, white) or apoptotic (Casp3, white) cells in the rostral telencephalon of wild-type E13.5 embryos electroporated at E12.5 with the indicated plasmids, and quantification of Casp3+ cells (M). Dashed line indicates apical surface, dotted lines indicate borders of the cortical plate (CP). Nascent rosettes (arrows) are next to ectopic ventricular neurons (arrowheads in K') and basal mitoses, without significant apoptosis in VZ (L',M). Mean ± SEM; t-test; ns, not significant, *P < 0.05. N = 3-6 replicates per group. N - Distribution of neurons (Tuj1, red) in the rostral telencephalon of wild-type E14.5 embryos electroporated at E12.5 with a combination of plasmids encoding Irs2, let-7a, let-7b and let-7c, and Gfp. Dashed line indicates apical surface. Formation of rosettes in Irs2-expressing embryos is rescued by overexpression of let-7. Inset shows the same field of view, with location and extent of electroporation.


A - Frequency of rosette formation in experimental conditions indicated . n , number of per condition .

A - Frequency of rosette formation in the experimental conditions indicated. n, number of embryos per condition.


B - G - Distribution of neurons ( Tuj1 , red ) , GFP ( green ) mitoses ( PH3 , ) in rostral telencephalon of wild - type E14 . 5 mouse electroporated at E12 . 5 with indicated plasmid combinations . Inset shows same field of view , with location extent of electroporations . In ( C ) ( E ) , arrows indicate rosettes arrowheads indicate neuronal ventricular ectopias . ( D ) ( F ) are high magnification details of rosettes ( dashed lines ) , where arrowheads indicate apical basal mitoses . Loss of endogenous let - 7 drives formation of rosettes ventricular neuronal ectopias ( C ) , which is rescued with additional loss of Irs2 ( G ) .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O

B-G - Distribution of neurons (Tuj1, red), GFP (green) and mitoses (PH3, white) in the rostral telencephalon of wild-type E14.5 mouse embryos electroporated at E12.5 with the indicated plasmid combinations. Inset shows the same field of view, with location and extent of electroporations. In (C) and (E), arrows indicate rosettes and arrowheads indicate neuronal ventricular ectopias. (D) and (F) are high magnification details of individual rosettes (dashed lines), where arrowheads indicate apical and basal mitoses. Loss of endogenous let-7 drives the formation of rosettes and ventricular neuronal ectopias (C), which is rescued with the additional loss of Irs2 (G).


B - G - Stains quantifications of human cerebral organoids electroporated with indicated construct combinations , to reveal frequency of electroporated remaining as progenitors ( % GFP + Ki67 + / GFP + ) . Histograms represent mean ± ; symbols in plots indicate values for organoids ; X2 - , * P < 0 . 05 . N = 8 - 11 replicates per group . Dashed lines indicate ventricular surface , solid arrowheads indicate Ki67 + / GFP + , open arrowheads indicate Ki67 - / GFP + . Scale bars , 50 µm .

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B-G - Stains and quantifications of human cerebral organoids electroporated with the indicated construct combinations, to reveal the frequency of electroporated cells remaining as progenitors (%GFP+Ki67+/GFP+). Histograms represent mean ± SEM; symbols in plots indicate values for individual organoids; X2-test, *P < 0.05. N = 8-11 replicates per group. Dashed lines indicate ventricular surface, solid arrowheads indicate Ki67+/GFP+ cells, open arrowheads indicate Ki67-/GFP+ cells. Scale bars, 50 µm.


q - RT - PCR reveals that miR - 181a / b silencing leads to upregulation of miR - 181a / b predicted targets in SH - SY5Y . N = 3 independent experiments .

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q-RT-PCR reveals that miR-181a/b silencing leads to upregulation of miR-181a/b predicted targets in SH-SY5Y cells. N=3 independent experiments.


miR - 181 - mimic transfection specifically inhibits Luciferase activity of constructs containing WT 3 ' - UTR predicted target sequences . Point mutations ( mut ) in miR - 181a / b binding sites abolish Luciferase repression in cases apart from PPARGC1A . Data are normalized to negative mimic transfection ( dashed ) . N = 6 independent experiments .

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miR-181-mimic transfection specifically inhibits Luciferase activity of constructs containing WT 3'-UTR predicted target sequences. Point mutations (mut) in miR-181a/b binding sites abolish Luciferase repression in all cases apart from PPARGC1A. Data are normalized to negative mimic transfection (dashed line). N=6 independent experiments.


q - RT - PCR reveals upregulation of miR - 181a / b targets in eyes of miR - 181a / b - 1 - / - vs . miR - 181a / b - 1 + / + . n ≥ 5 / genotype .

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q-RT-PCR reveals upregulation of miR-181a/b targets in the eyes of miR-181a/b-1-/- vs. miR-181a/b-1+/+ animals. n≥5 animals/genotype.


miR - 181a / b - 1 - / - mice show increased mtDNA content vs . miR - 181a / b - 1 + / + mice as measured by q - PCR . N = 4 / genotype .

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miR-181a/b-1-/- mice show increased mtDNA content vs. miR-181a/b-1+/+ mice as measured by q-PCR. N=4 animals/genotype.


WB ( left panel ) reveals increased of mitochondrial in eyes of miR - 181a / b - 1 - / - ( - / - ) vs . miR - 181a / b - 1 + / + ( + / + ) mice ( quantified in right panel ) . Data are normalized to either p115 or Gapdh . N ≥ 3 / genotype . Please note that compared bands from + / + - / - samples are from same blots which were cropped shown split for sake of data presentation clarity .

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WB analysis (left panel) reveals increased levels of mitochondrial proteins in the eyes of miR-181a/b-1-/- (-/-) vs. miR-181a/b-1+/+ (+/+) mice (quantified in the right panel). Data are normalized to either p115 or Gapdh. N≥3 animals/genotype. Please note that all compared bands from +/+ and -/- samples are from the same blots which were cropped and shown split for the sake of data presentation clarity.


WB on mitochondrial fractions ( left panel ) show increased of p62 Parkin in mitochondrial fraction from eye of - / - vs . + / + mice ( quantified in right panel ) . Data are normalized to Ndufb8 or Gapdh for mitochondrial fractions , respectively . N = 3 / genotype .

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WB analysis on mitochondrial and cytosolic fractions (left panel) show increased levels of p62 and Parkin in mitochondrial fraction from the eye of -/- vs. +/+ mice (quantified in the right panel). Data are normalized to Ndufb8 or Gapdh for mitochondrial and cytosolic fractions, respectively. N=3 animals/genotype.


shows that miR - 181a / b silencing protects SH - SY5Y from FCCP treatment . N ≥ 7 independent experiments .

O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

Cell death analysis shows that miR-181a/b silencing protects SH-SY5Y cells from FCCP treatment. N≥7 independent experiments.


TUNEL assays on eye brain sections of stage ( st ) 30 medakafish control MO - injected reveal a decrease in number of apoptotic in both hccs - MO / miR - 181a / b - MO - cox7b - MO / miR - 181a / b - MO - injected compared to hccs - MO - cox7b - MO - injected . Scale bars are 20μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O

TUNEL assays on eye and brain sections of stage (st)30 medakafish control and MO-injected embryos reveal a decrease in the number of apoptotic cells in both hccs-MO/miR-181a/b-MO- and cox7b-MO/miR-181a/b-MO-injected compared to hccs-MO- and cox7b-MO-injected embryos. Scale bars are 20μm.


TUNEL - positive / eye . n ≥ 5 eyes for each model .

TUNEL-positive cells/eye. n≥5 eyes for each model.


Caspase assays show restored of caspase - 3 caspase - 9 activities in hccs - MO / miR - 181a / b - MO - cox7b - MO / miR - 181a / b - MO - injected with respect to hccs - MO - cox7b - MO - injected . n ≥ 5 for each model .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O

Caspase assays show restored levels of caspase-3 and caspase-9 activities in hccs-MO/miR-181a/b-MO- and cox7b-MO/miR-181a/b-MO-injected embryos with respect to hccs-MO- and cox7b-MO-injected embryos. n≥5 embryos for each model.


Representative of st30 medaka injected with hccs - MO ( B ) alone or co - injected with miR - 181a / b - MOs Co - injection of miR - 181a / b - MOs rescues microphthalmia microcephaly in both hccs - MO . hccs - MO / miR - 181a / b - MO - b - MO - injected were treated with Baf - A1 , PD98059 or HA14 - 1 . Baf - A1 treatment counteracts protective effect of miR - 181a / b downregulation in hccs - MO / miR - 181a / b - MO PD98059 treatment does not interfere with modulation of MLS phenotype mediated by miR - 181a / b downregulation HA14 - 1 treatment counteracts effect of miR - 181a / b downregulation in hccs - MO / miR - 181a / b - MO model Scale bars are 100μm .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O

Representative images of st30 medaka embryos injected with hccs-MO (B) alone or co-injected with miR-181a/b-MOs Co-injection of miR-181a/b-MOs rescues microphthalmia and microcephaly in both hccs-MO embryos. hccs-MO/miR-181a/b-MO- b-MO-injected embryos were treated with Baf-A1, PD98059 or HA14-1. Baf-A1 treatment counteracts the protective effect of miR-181a/b downregulation in hccs-MO/miR-181a/b-MO embryos PD98059 treatment does not interfere with the modulation of the MLS phenotype mediated by miR-181a/b downregulation HA14-1 treatment counteracts the effect of miR-181a/b downregulation in the hccs-MO/miR-181a/b-MO model Scale bars are 100μm.


Representative of st30 medaka injected with cox7b - MO ( H ) alone or co - injected with miR - 181a / b - MOs Co - injection of miR - 181a / b - MOs rescues microphthalmia microcephaly in cox7b - MO . cox7b - MO / miR - 181a / b - MO - injected were treated with Baf - A1 , PD98059 or HA14 - 1 . Baf - A1 treatment counteracts protective effect of miR - 181a / b downregulation in cox7b - MO / miR - 181a / b - MO PD98059 treatment does not interfere with modulation of MLS phenotype mediated by miR - 181a / b downregulation Scale bars are 100μm .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O

Representative images of st30 medaka embryos injected with cox7b-MO (H) alone or co-injected with miR-181a/b-MOs Co-injection of miR-181a/b-MOs rescues microphthalmia and microcephaly in cox7b-MO embryos. cox7b-MO/miR-181a/b-MO-injected embryos were treated with Baf-A1, PD98059 or HA14-1. Baf-A1 treatment counteracts the protective effect of miR-181a/b downregulation in cox7b-MO/miR-181a/b-MO embryos PD98059 treatment does not interfere with the modulation of the MLS phenotype mediated by miR-181a/b downregulation Scale bars are 100μm.


of with or without MLS phenotype in different conditions in hccs - MO . N ≥ 300 / conditions .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O

Percentage of embryos with or without MLS phenotype in the different conditions in hccs-MO embryos. N≥300 embryos/conditions.


of with or without MLS phenotype in different conditions in cox7b - MO ( N ) . N ≥ 300 / conditions .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

Percentage of embryos with or without MLS phenotype in the different conditions in cox7b-MO (N) embryos. N≥300 embryos/conditions.


Immunofluorescence with anti - NeuN antibody in retina of miR - 181a / b - 1 + / + miR - 181a / b - 1 - / - mice intravitreally injected with Rotenone or DMSO . RGCs are preserved in miR - 181a / b - 1 - / - rotenone - injected mice with respect to controls at both one two after injection . Scale bars are 50μm .

O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescence analysis with anti-NeuN antibody in the retina of miR-181a/b-1+/+ and miR-181a/b-1-/- mice intravitreally injected with Rotenone or DMSO. RGCs are preserved in miR-181a/b-1-/- rotenone-injected mice with respect to controls at both one and two weeks after injection. Scale bars are 50μm.


Number of RGCs / mm ( indicated as % ) at one - two - post - injection . N = 10 .

O O O O O O O O O O O O O O O O O O O O O O O

Number of RGCs/mm (indicated as %) at one- and two-weeks post-injection. N=10.


NADH dehydrogenase histochemical reaction on retinal sections of miR - 181a / b - 1 + / + miR - 181a / b - 1 - / - mice intravitreally injected with Rotenone or DMSO . At one week post - injection , NADH dehydrogenase activity is lost in RGCs ( GCL , areas within dashed lines ) of miR - 181a / b - 1 + / + Rotenone - injected eyes , while it is preserved in those of miR - 181a / b - 1 - / - Rotenone - injected eyes . GCL , Ganglion Layer ONL ; INL , Inner Layer ; Outer Layer . Scale bars are 50μm .

B-MEASURED_VAR I-MEASURED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

NADH dehydrogenase histochemical reaction on retinal sections of miR-181a/b-1+/+ and miR-181a/b-1-/- mice intravitreally injected with Rotenone or DMSO. At one week post-injection, NADH dehydrogenase activity is lost in RGCs (GCL, areas within dashed lines) of miR-181a/b-1+/+ Rotenone-injected eyes, while it is preserved in those of miR-181a/b-1-/- Rotenone-injected eyes. GCL, Ganglion Cells Layer ONL; INL, Inner Nuclear Layer; Outer Nuclear Layer. Scale bars are 50μm.


Immunofluorescence , with an anti - CoxIV antibody , in retina of miR - 181a / b - 1 + / + miR - 181a / b - 1 - / - mice , injected with either Rotenone or DMSO , showed preserved mitochondria in miR - 181a / b - 1 - / - rotenone - injected eyes at one - week post - injection . Dashed boxes indicate of magnifications shown in right panels . Scale bars are 10μm .

O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescence analysis, with an anti-CoxIV antibody, in the retina of miR-181a/b-1+/+ and miR-181a/b-1-/- mice, injected with either Rotenone or DMSO, showed preserved mitochondria in miR-181a/b-1-/- rotenone-injected eyes at one-week post-injection. Dashed boxes indicate the area of magnifications shown in right panels. Scale bars are 10μm.


Graphical representation of results of optokinetic tracking assays reported as fold change of cycles / degree . Visual acuity is preserved in miR - 181a / b - 1 - / - rotenone - injected mice with respect to controls at both one two after injection . N = 10 .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

Graphical representation of the results of the optokinetic tracking assays reported as fold change of cycles/degree. Visual acuity is preserved in miR-181a/b-1-/- rotenone-injected mice with respect to controls at both one and two weeks after injection. N=10.


Immunofluorescence with anti - NeuN antibody in retina of WT , Ndufs4 - / - , Ndufs4 - / - / miR - 181a / b - 1 - / - miR - 181a / b - 1 - / - mice . RGCs are preserved in Ndufs4 - / - / miR - 181a / b - 1 - / - with respect to Ndufs4 - / - mice . Scale bars are 50μm .

O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

Immunofluorescence analysis with anti-NeuN antibody in the retina of WT, Ndufs4-/-, Ndufs4-/-/miR-181a/b-1-/- and miR-181a/b-1-/- mice. RGCs are preserved in Ndufs4-/-/miR-181a/b-1-/- with respect to Ndufs4-/- mice. Scale bars are 50μm.


Graphical representation of results of optokinetic tracking assays reported as fold change of cycles / degree . Visual acuity is preserved in Ndufs4 - / - / miR - 181a / b - 1 - / - with respect to Ndufs4 - / - mice . N ≥ 8 .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O

Graphical representation of the results of the optokinetic tracking assays reported as fold change of cycles/degree. Visual acuity is preserved in Ndufs4-/-/miR-181a/b-1-/- with respect to Ndufs4-/- mice. N≥8.


Electroretinographic reveals amelioration of b - wave patterns in Ndufs4 - / - / miR - 181a / b - 1 - / - with respect to Ndufs4 - / - mice . N ≥ 5 .

O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O

Electroretinographic analysis reveals amelioration of b-wave patterns in Ndufs4-/-/miR-181a/b-1-/- with respect to Ndufs4-/-mice. N≥5.


Electron microscopy shows amelioration of mitochondria increase of mitochondrial number in Ndufs4 - / - / miR - 181a / b - 1 - / - vs . Ndufs4 - / - mice . Scale bars are 1μm . quantitative increase of mitochondria is reported in C as number of mitochondria / RGC , at p30 p55 . N ≥ 2 / genotype .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Electron microscopy analysis shows amelioration of mitochondria morphology and increase of mitochondrial number in Ndufs4-/-/miR-181a/b-1-/- vs. Ndufs4-/- mice. Scale bars are 1μm. The quantitative increase of mitochondria is reported in C as number of mitochondria/RGC, at p30 and p55. N≥2 animals/genotype.


Ndufs4 - / - / miR - 181a / b - 1 - / - mice show increased mtDNA content vs . Ndufs4 - / - mice as measured by q - PCR . N ≥ 4 / genotype .

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Ndufs4-/-/miR-181a/b-1-/- mice show increased mtDNA content vs. Ndufs4-/- mice as measured by q-PCR. N≥4 animals/genotype.


Biochemical activity of MRC complexes I , II IV normalized by of citrate synthase ( CS ) activity in Ndufs4 - / - / miR - 181a / b - 1 - / - vs . Ndufs4 - / - mice . N ≥ 3 / genotype . p ‐ values were calculated by two Student ' s t - ; Error bars are .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Biochemical activity of MRC complexes I, II and IV normalized by the percentage of citrate synthase (CS) activity in Ndufs4-/-/miR-181a/b-1-/- vs. Ndufs4-/- mice. N≥3 animals/genotype. p‐values were calculated by two tailed Student's t-test; Error bars are SEM.


Tap - 1 is required for secretion of TseL . Pellet ( P ) supernatant ( S ) of cultures were analyzed by SDS - PAGE . result of immunoblotting with antisera against TseL Hcp purified antibodies against DnaK is shown . asterisk marks an unspecific band , detected by antiserum against TseL , present in pre - immunized serum . One representative experiment of three independent experiments is shown .

B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

Tap-1 is required for the secretion of TseL. Pellet (P) and supernatant (S) of bacterial cultures were analyzed by SDS-PAGE. The result of immunoblotting with antisera against TseL and Hcp and purified antibodies against DnaK is shown. The asterisk marks an unspecific band, detected by the antiserum against TseL, present in the pre-immunized serum. One representative experiment of three independent experiments is shown.


Tap - 1 is required for TseL - mediated killing . Wild - type C6706 or C6706 lacking tsiV1 were exposed to indicated V52 mutants in a killing . y - axis indicates number of surviving C6706 .

B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

Tap-1 is required for TseL-mediated killing. Wild-type C6706 or C6706 lacking tsiV1 were exposed to the indicated V52 mutants in a killing assay. The y-axis indicates the number of surviving C6706.


Tap - 1 is required for TseL function . Killing assays in which V52Δtap - 1 was mixed with wild - type C6706 or mutants lacking tsiV1 , tsiV2 , or tsiV3 . y - axis indicates surviving C6706 .

B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O

Tap-1 is required for TseL function. Killing assays in which V52Δtap-1 was mixed with wild-type C6706 or mutants lacking tsiV1, tsiV2, or tsiV3. The y-axis indicates surviving C6706.


TseL secretion depends on VgrG - 1 . Western blot of pellet ( P ) supernatant ( S ) samples of indicated strains . Samples were immunoblotted with antisera against TseL Hcp purified antibodies against DnaK . unspecific band detected by rabbit serum is marked with an asterisk . One representative of three experiments is shown .

B-MEASURED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

TseL secretion depends on VgrG-1. Western blot analysis of pellet (P) and supernatant (S) samples of the indicated strains. Samples were immunoblotted with antisera against TseL and Hcp and purified antibodies against DnaK. The unspecific band detected by rabbit serum is marked with an asterisk. One representative of three experiments is shown.


VgrG - 1 is required for TseL - mediated killing . Killing with wild - type V52 or indicated mutants wild - type C6706 or a mutant lacking immunity gene tsiV1 . y - axis indicates surviving C6706 .

B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

VgrG-1 is required for TseL-mediated killing. Killing assay with wild-type V52 or indicated mutants and wild-type C6706 or a mutant lacking the immunity gene tsiV1. The y-axis indicates surviving C6706.


VasX - VgrG - 3 - mediated killing but not TseL - mediated killing occurs in absence of VgrG - 1 . V52ΔvgrG - 1 was mixed in a killing with wild - type C6706 or mutants lacking immunity genes tsiV1 , tsiV2 , or tsiV3 ( specific for TseL , VasX , VgrG - 3 , respectively ) . y - axis indicates surviving C6706 .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

VasX- and VgrG-3-mediated killing but not TseL-mediated killing occurs in the absence of VgrG-1. V52ΔvgrG-1 was mixed in a killing assay with wild-type C6706 or mutants lacking immunity genes tsiV1, tsiV2, or tsiV3 (specific for TseL, VasX, and VgrG-3, respectively). The y-axis indicates surviving C6706.


A , B Interactions between Tap - 1 TseL ( A ) or VgrG - 1 ( B ) . Shown are immunoblots of lysates ( total ) immunoprecipitates with anti - FLAG affinity beads ( IP : FLAG ) of V52Δtap - 1 ( A ) or V52 vgrG - 1 : : myc ( B ) transformed with empty vector or a plasmid encoding His - tagged or FLAG - tagged Tap - 1 .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O

A, B Interactions between Tap-1 and TseL (A) or VgrG-1 (B). Shown are immunoblots of lysates (total) and immunoprecipitates with anti-FLAG affinity beads (IP:FLAG) of V52Δtap-1 (A) or V52 vgrG-1::myc (B) transformed with empty vector or a plasmid encoding His-tagged or FLAG-tagged Tap-1.


C Interaction between TseL VgrG - 1 . Shown are immunoblots of lysates ( total ) immunoprecipitates with an anti - FLAG affinity beads ( IP : FLAG ) of V52 or V52Δtap - 1 transformed with empty vector or a plasmid encoding HIS - tagged or FLAG - tagged VgrG - 1 .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O

C Interaction between TseL and VgrG-1. Shown are immunoblots of lysates (total) and immunoprecipitates with an anti-FLAG affinity beads (IP:FLAG) of V52 or V52Δtap-1 transformed with empty vector or a plasmid encoding HIS-tagged or FLAG-tagged VgrG-1.


Specificity of C - terminal Tap - 1 segment . Killing in which lack of tap - 1 in V52 is complemented by empty vector ( control ) or one of two different alleles of tap - 1 . ability to kill in a TseL - dependent manner is indicated by logarithm of surviving C6706ΔtsiV1 .

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

Specificity of the C-terminal Tap-1 segment. Killing assay in which the lack of tap-1 in V52 is complemented by empty vector (control) or one of two different alleles of tap-1. The ability to kill in a TseL-dependent manner is indicated by the logarithm of surviving C6706ΔtsiV1.


Specificity of N - terminal segment of Tap - 1 . Killing in which lack of tap - 1 in 1587 is complemented by empty vector ( control ) or one of two different alleles of tap - 1 . ability to facilitate killing by effector A55 _ 1502 is indicated by logarithm of surviving 1587ΔA55 _ 1501 - 03 .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

Specificity of N-terminal segment of Tap-1. Killing assay in which the lack of tap-1 in 1587 is complemented by empty vector (control) or one of two different alleles of tap-1. The ability to facilitate killing by the effector A55_1502 is indicated by the logarithm of surviving 1587ΔA55_1501-03.


( A ) time courses of normalized FRET ( Mean ± ) of calcium biosensor before after shockwave stimulation in HBSS Ca2 + medium with ( yellow , n = 6 , N = 3 ) or without Piezo1 expression ( black , n = 6 , N = 3 ) . ( B ) representative FRET / ECFP of calcium biosensor in Piezo1 - expressed HEK before ( top left ) after ( top right ) LIS stimulation . Phase image with initiating point ( left ) image indicating Piezo1 expression ( right ) were also shown on bottom panels ( Scale bar , 40 μm ) . Color scale bars in figures are to show FRET / ECFP ratios , with cold hot colors representing low high ratios , respectively . ( C ) time course of normalized FRET ( Mean ± ) of calcium biosensor in Piezo1 - expressing HEK before after multiple shockwave stimulations in HBSS Ca2 + medium ( n = 6 , N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(A) The time courses of normalized FRET ratio (Mean ± SEM) of calcium biosensor before and after shockwave stimulation in HBSS Ca2+ medium with (yellow line, n = 6, N = 3) or without the Piezo1 expression (black line, n = 6, N = 3). (B) The representative FRET/ECFP ratio images of calcium biosensor in Piezo1-expressed HEK cells before (top left) and after (top right) LIS stimulation. Phase image with laser initiating point (left) and fluorescent image indicating Piezo1 expression (right) were also shown on the bottom panels (Scale bar, 40 μm). Color scale bars in the figures are to show the FRET/ECFP ratios, with cold and hot colors representing low and high ratios, respectively. (C) The time course of normalized FRET ratio (Mean ± SEM) of calcium biosensor in the Piezo1-expressing HEK cells before and after multiple shockwave stimulations in HBSS Ca2+ medium (n = 6, N = 3).


( A ) representative ECFP / FRET of FAK biosensor in Piezo1 overexpressed HEK before ( left ) after ( right ) shockwave stimulation ( Scale bar , 40 μm ) . Color scale bars in figures are to show ECFP / FRET ratios , with cold hot colors representing low high ratios , respectively . ( B ) time courses of normalized ECFP / FRET ( Mean ± ) of FAK FRET biosensor before after shockwave stimulation in HBSS Ca2 + media with ( green , n = 16 , N = 3 ) or without Piezo1 ( black , n = 10 , N = 3 ) . ( C ) change of FRET of FAK FRET biosensor in HEK with or without Piezo1 after shockwave stimulation in HBSS Ca2 + medium . * P < 0 . 05 from student two - t - .

O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

(A) The representative ECFP/FRET ratio images of FAK biosensor in Piezo1 overexpressed HEK cells before (left) and after (right) shockwave stimulation (Scale bar, 40 μm). Color scale bars in the figures are to show the ECFP/FRET ratios, with cold and hot colors representing low and high ratios, respectively. (B) The time courses of normalized ECFP/FRET ratio (Mean ± SEM) of FAK FRET biosensor before and after shockwave stimulation in HBSS Ca2+ media with (green line, n = 16, N = 3) or without Piezo1 (black line, n = 10, N = 3). (C) The percentage change of FRET ratio of the FAK FRET biosensor in HEK cells with or without Piezo1 after shockwave stimulation in HBSS Ca2+ medium. *P < 0.05 from student two-tailed t-test.


( A ) representative ECFP / FRET of FAK biosensor in Piezo1 expressing HEK before after 25 μM Yoda1 stimulation ( Scale bar , 10 μm ) . Color scale bars in figures indicate ECFP / FRET ratios , with cold hot colors representing low high ratios , respectively . ( B ) time courses of normalized FRET ( Mean ± ) of FAK FRET biosensor before after Yoda1 stimulation with ( pink , n = 10 , N = 3 ) or without Piezo1 ( black , n = 11 , N = 3 ) . grey represents DMSO control group ( n = 8 , N = 3 ) .

O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) The representative ECFP/FRET ratio images of FAK biosensor in Piezo1 expressing HEK cells before and after 25 μM Yoda1 stimulation (Scale bar, 10 μm). Color scale bars in the figures indicate ECFP/FRET ratios, with cold and hot colors representing low and high ratios, respectively. (B) The time courses of normalized FRET ratio (Mean ± SEM) of FAK FRET biosensor before and after Yoda1 stimulation with (pink line, n = 10, N = 3) or without Piezo1 (black line, n = 11, N = 3). The grey line represents the DMSO control group (n = 8, N = 3).


( C ) representative FRET / ECFP of calcium biosensor in Piezo1 - expressing HEK before after 25 μM Yoda1 stimulation ( Scale bar , 10 μm ) . Color scale bars in figures indicate FRET / ECFP ratios , with cold hot colors representing low high ratios , respectively . ( D ) time courses of normalized FRET ( Mean ± ) of calcium biosensor in Piezo1 - expressing HEK ( purple , n = 9 , N = 3 ) before after 25 μM Yoda1 stimulation in culture medium .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

(C) The representative FRET/ECFP ratio images of the calcium biosensor in the Piezo1-expressing HEK cells before and after 25 μM Yoda1 stimulation (Scale bar, 10 μm). Color scale bars in the figures indicate FRET/ECFP ratios, with cold and hot colors representing low and high ratios, respectively. (D) The time courses of normalized FRET ratio (Mean ± SEM) of calcium biosensor in the Piezo1-expressing HEK cells (purple line, n = 9, N = 3) before and after 25 μM Yoda1 stimulation in culture medium.


( A ) & ( B ) , time courses ( Mean ± ) ( A ) representative ( Scale bar , 10 μm ) of FRET of SHP2 biosensor ( Y542F ) before after 25 μM or 0 . 5 μM Yoda1 stimulation in with or without Piezo1 overexpression ( red bar , n = 5 , N = 3 ; blue bar , n = 5 , N = 3 ; purple bar , n = 11 , N = 3 ; green bar , n = 5 , N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A)&(B), The time courses (Mean ± SEM) (A) and representative images (Scale bar, 10 μm) of FRET ratio of SHP2 biosensor (Y542F) before and after 25 μM or 0.5 μM Yoda1 stimulation in cells with or without Piezo1 overexpression (red bar, n = 5, N=3; blue bar, n = 5, N=3; purple bar, n = 11, N=3; green bar, n = 5, N=3).


B ) Expression of 21U sensor ( left ) DAPI ( right ) of gonad arms in indicated genetic backgrounds . Gonads are outlined by a dashed . mCherry signal is represented in pseudo - colours [ LUT fire ( ImageJ ) ] to reflect differences in of signal . Number of analysed with indicated phenotype are given in panel . not showing activation of 21U sensor ( + ) were still silenced , that did not show silenced 21U sensor ( RNAe ) state were expressing weakly , comparable to 21U sensor ( + ) . Scale bar : 25 μm .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O

B) Expression of the 21U sensor (left) and DAPI staining (right) of gonad arms in the indicated genetic backgrounds. Gonads are outlined by a dashed line. The mCherry signal is represented in pseudo-colours [LUT fire (ImageJ)] to reflect differences in the intensity of the signal. Number of animals analysed and with indicated phenotype are given in the panel. Animals not showing the activation of the 21U sensor(+) were still silenced, and animals that did not show the silenced 21U sensor(RNAe) state were expressing weakly, comparable to the 21U sensor(+). Scale bar: 25 μm.


C ) Tc1 reversion in different genetic backgrounds . strains tested carried unc - 22 : : Tc1 ( st136 ) allele . Tc1 excision can result in restoration of unc - 22 function , which can be scored visually . Negative control = unc - 22 : : Tc1 ( st136 ) in a wild type background ; positive control = wago - 1 / - 2 / - 3 . Two independent experiments per are represented .

O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O

C) Tc1 reversion assay in different genetic backgrounds. All the strains tested carried the unc-22::Tc1(st136) allele. Tc1 excision can result in restoration of unc-22 function, which can be scored visually. Negative control = unc-22::Tc1(st136) in a wild type background; positive control = wago-1/-2/-3. Two independent experiments per strain are represented.


D ) Crossing scheme to address re - initiation of silencing of 21U sensor . A mut - 7 mutant expressing 21U sensor is crossed with either a wild type ( left ) , prg - 1 ( middle ) or pid - 2 ( right ) mutant hermaphrodite . Their F1 offspring was scored for expression of 21U sensor by microscopy . Three states of expression were scored , represented in a pie chart . three expression states are exemplified by representative at bottom : OFF ( left ) , FAINT ( right ) or ON ( middle ) . DIC are shown above panels . Gonads are outlined by a dashed . Scale bar : 25 µm .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) Crossing scheme to address the re-initiation of silencing of the 21U sensor. A mut-7 mutant male expressing the 21U sensor is crossed with either a wild type (left), prg-1 (middle) or pid-2 (right) mutant hermaphrodite. Their F1 offspring was scored for expression of the 21U sensor by microscopy. Three states of expression were scored, and represented in a pie chart. The three expression states are exemplified by representative images at the bottom: OFF (left), FAINT (right) or ON (middle). DIC images are shown above the fluorescence panels. Gonads are outlined by a dashed line. Scale bar: 25 µm.


E ) Crossing scheme to address if maternal 21U RNAs are sufficient to re - initiate silencing of 21U sensor . A pid - 1 mutant expressing 21U sensor is crossed with a hermaphrodite , heterozygous for same mutation . their F1 offspring inherit a pool of 21U RNAs from hermaphrodite , but in 50 % of F1 , which is pid - 1 homozygous mutant , no zygotic PID - 1 is present , hence no zygotic 21U RNAs can be made . silencing or expression of 21U sensor in pid - 1 homozygous mutant F1 has been scored by microscopy , depicted in a pie chart . At bottom , a representative image of an carrying a silenced 21U sensor ( lower : mCherry signal ; upper : DAPI ) in pid - 1 mutant offspring . Gonads are outlined by a dashed . Scale bar : 25 μm .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O

E) Crossing scheme to address if maternal 21U RNAs are sufficient to re-initiate the silencing of the 21U sensor. A pid-1 mutant male expressing the 21U sensor is crossed with a hermaphrodite, heterozygous for the same mutation. All their F1 offspring inherit a pool of 21U RNAs from the hermaphrodite, but in 50% of the F1, which is pid-1 homozygous mutant, no zygotic PID-1 is present, hence no zygotic 21U RNAs can be made. The silencing or expression of the 21U sensor in the pid-1 homozygous mutant F1 has been scored by microscopy, and depicted in a pie chart. At the bottom, a representative image of an animal carrying a silenced 21U sensor (lower: mCherry signal; upper: DAPI staining) in pid-1 mutant offspring. Gonads are outlined by a dashed line. Scale bar: 25 μm.


F ) Crossing scheme to role of PID - 2 in re - initiating silencing of 21U sensor , mediated by maternally provided 21U RNAs only . expression of 21U sensor in F1 has been scored by microscopy , depicted in a pie chart . arrow heads indicate many arrayed oocytes , typical of a feminized germline . DIC image of a representative are shown at bottom . Gonads are outlined by a dashed . Scale bar : 25 μm .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F) Crossing scheme to test the role of PID-2 in re-initiating the silencing of the 21U sensor, mediated by maternally provided 21U RNAs only. The expression of the 21U sensor in the F1 has been scored by microscopy, and depicted in a pie chart. White arrow heads indicate the many arrayed oocytes, typical of a feminized germline. DIC and fluorescence image of a representative animal are shown at the bottom. Gonads are outlined by a dashed line. Scale bar: 25 μm.


22G RNAs mapping to 21U sensor transgene were identified from small sequencing data , read density was plotted over transgene , which is schematically depicted at bottom . aggregated results of three replicates is shown for each indicated genotype in different panels . shading , in grey , represents standard deviation of read density from three replicates . ( + ) means that sensor was detectably expressed . ( RNAe ) means that sensor was crossed into respective mutant background in an RNAe state ( i . e . its silencing was PRG - 1 - independent ) , its expression remained undetectable by microscopy .

B-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The 22G RNAs mapping to the 21U sensor transgene were identified from small RNA sequencing data, and read density was plotted over the transgene, which is schematically depicted at the bottom. The aggregated results of three replicates is shown for each indicated genotype in the different panels. The shading, in grey, represents the standard deviation of the read density from the three replicates. (+) means that the sensor was detectably expressed. (RNAe) means that the sensor was crossed into the respective mutant background in an RNAe state (i.e. its silencing was PRG-1-independent), and its expression remained undetectable by microscopy.


A ) MA - plot of log2 fold changes ( Y - axis ) versus mean of normalized of 22G RNAs ( X - axis ) for pid - 2 ( xf23 ) mutants , compared to wild type . Red dots : genes with adjusted p - value < 0 . 05 fold - change > 2 . Blue dots : genes with adjusted p - value < 0 . 05 fold - change < - 2 .

O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A) MA-plot of log2 fold changes (Y-axis) versus the mean of normalized counts of 22G RNAs (X-axis) for pid-2(xf23) mutants, compared to wild type. Red dots: genes with adjusted p-value < 0.05 and fold-change > 2. Blue dots: genes with adjusted p-value < 0.05 and fold-change < -2.


B ) displaying overlap significance between different gene sets genes that either up - or down - regulated in pid - 2 ( xf23 ) mutants . Significance was tested with Fisher ' s exact p - values adjusted with Benjamini - Hochberg Procedure . Colour scheme represents odds of overlaps representing strength of association . N . S . : not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B) Heatmap displaying overlap significance between different gene sets and genes that either up- or down-regulated in pid-2(xf23) mutants. Significance was tested with Fisher's exact test and p-values adjusted with the Benjamini-Hochberg Procedure. Colour scheme represents the odds ratio of overlaps representing the strength of association. N.S.: not significant.


C - D ) Cumulative 22G coverage along gene of Mutator CSR - 1 targets . Values represent 22G coverage normalized to total coverage of each gene , for wild type ( N2 ) pid - 2 ( xf23 ) mutants . lines represent average of biological replicates , whereas shading represents standard deviation of biological replicates . a . u . : arbitrary units .

O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-D) Cumulative 22G coverage along the gene body of Mutator and CSR-1 targets. Values represent 22G coverage normalized to the total coverage of each gene, for wild type (N2) and pid-2(xf23) mutants. The lines represent the average of biological replicates, whereas the shading represents the standard deviation of biological replicates. a.u.: arbitrary units.


A ) Volcano plot representing enrichment of interacting with PID - 2 , isolated by immunoprecipitation of eGFP : : PID - 2 , followed by quantitative label - free mass spectrometry . As control , eGFP IPs were performed on extracts from wild - type , non - transgenic . For each IP - MS experiment , quadruplicates were measured analysed . dashed reflects a significance threshold of p - value < 0 . 05 at two - fold enrichment .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A) Volcano plot representing the enrichment of proteins interacting with PID-2, isolated by immunoprecipitation of eGFP::PID-2 protein, followed by quantitative label-free mass spectrometry. As control, eGFP IPs were performed on protein extracts from wild-type, non-transgenic animals. For each IP-MS experiment, quadruplicates were measured and analysed. The dashed line reflects a significance threshold of p-value < 0.05 at two-fold enrichment.


Volcano plot representing enrichment of interacting with PID - 2 , isolated by immunoprecipitation of eGFP : : PID - 2 , followed by quantitative label - free mass spectrometry . In this experiment , endogenous PID - 2 was immunoprecipitated , pid - 2 ( xf23 ) mutant extracts were used as control .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O

Volcano plot representing the enrichment of proteins interacting with PID-2, isolated by immunoprecipitation of eGFP::PID-2 protein, followed by quantitative label-free mass spectrometry. In this experiment, the endogenous PID-2 protein was immunoprecipitated, and pid-2(xf23) mutant protein extracts were used as control.


Volcano plot , representing enrichment of interacting with PID - 4 , by immunoprecipitating endogenously tagged PID - 4 . IPs from extracts from wild - type , non - tagged were used as control .

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O

Volcano plot , representing the enrichment of proteins interacting with PID-4, by immunoprecipitating endogenously tagged PID-4 protein. IPs from protein extracts from wild-type, non-tagged animals were used as control.


Volcano plot , representing enrichment of interacting with PID - 5 , by immunoprecipitating endogenously tagged PID - 5 . IPs from extracts from wild - type , non - tagged were used as control .

Volcano plot, representing the enrichment of proteins interacting with PID-5, by immunoprecipitating endogenously tagged PID-5 protein. IPs from protein extracts from wild-type, non-tagged animals were used as control.


A ) Expression of 21U sensor in indicated genetic backgrounds . Gonads are outlined by a dashed . mCherry signal is represented in pseudo - colours [ LUT fire ( ImageJ ) ] to reflect differences in of signal . panels on right are DIC of respective . Scale bar : 25 μm .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A) Expression of the 21U sensor in the indicated genetic backgrounds. Gonads are outlined by a dashed line. The mCherry signal is represented in pseudo-colours [LUT fire (ImageJ)] to reflect differences in the intensity of the signal. The panels on the right are DIC images of the respective animals. Scale bar: 25 μm.


B ) 22G coverage of 21U sensor ( RNAe ) in indicated genetic backgrounds .

O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O

B) 22G RNA coverage of the 21U sensor(RNAe) in the indicated genetic backgrounds.


C ) MA - plot of log2 fold changes ( on Y - axis ) versus mean of normalized of 22G RNAs ( on X - axis ) for pid - 4 mutants , compared to wild type . Red dots : genes with adjusted p - value < 0 . 05 fold - change > 2 . Blue dots : genes with adjusted p - value < 0 . 05 fold - change < - 2 .

O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C) MA-plot of log2 fold changes (on the Y-axis) versus the mean of normalized counts of 22G RNAs (on the X-axis) for pid-4 mutants, compared to wild type. Red dots: genes with adjusted p-value < 0.05 and fold-change > 2. Blue dots: genes with adjusted p-value < 0.05 and fold-change < -2.


D ) displaying overlap significance between different gene sets genes that either up - or down - regulated in pid - 4 mutants . Colour scheme represents odds of overlaps representing strength of association .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) Heatmap displaying overlap significance between different gene sets and genes that either up- or down-regulated in pid-4 mutants. Colour scheme represents the odds ratio of overlaps representing the strength of association.


MA - plot of log2 fold changes ( on Y - axis ) versus mean of normalized of 22G RNAs ( on X - axis ) , compared to wild type . Red dots : genes with adjusted p - value < 0 . 05 fold - change > 2 . Blue dots : genes with adjusted p - value < 0 . 05 fold - change < - 2 . E ) for pid - 5

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR

MA-plot of log2 fold changes (on the Y-axis) versus the mean of normalized counts of 22G RNAs (on the X-axis) , compared to wild type. Red dots: genes with adjusted p-value < 0.05 and fold-change > 2. Blue dots: genes with adjusted p-value < 0.05 and fold-change < -2. E) for pid-5 mutant


displaying overlap significance between different gene sets genes that either up - or down - regulated in F ) pid - 5

O O O O O O O O O O O O O O O O O O O O O O

Heatmap displaying overlap significance between different gene sets and genes that either up- or down-regulated in F) pid-5 mutant


MA - plot of log2 fold changes ( on Y - axis ) versus mean of normalized of 22G RNAs ( on X - axis ) compared to wild type . Red dots : genes with adjusted p - value < 0 . 05 fold - change > 2 . Blue dots : genes with adjusted p - value < 0 . 05 fold - change < - 2 . G ) for pid - 4 ; pid - 5 double

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O

MA-plot of log2 fold changes (on the Y-axis) versus the mean of normalized counts of 22G RNAs (on the X-axis) compared to wild type. Red dots: genes with adjusted p-value < 0.05 and fold-change > 2. Blue dots: genes with adjusted p-value < 0.05 and fold-change < -2. G) for pid-4;pid-5 double mutant


displaying overlap significance between different gene sets genes that either up - or down - regulated in H ) pid - 4 ; pid - 5 double

O O O O O O O O O O O O O O O O O O O O O O O O O O O

Heatmap displaying overlap significance between different gene sets and genes that either up- or down-regulated in H) pid-4;pid-5 double mutant


I , J ) plots representing fertility over generations for indicated genetic backgrounds at 25oC . Six L2 - L3 larvae for each of indicated backgrounds were - picked to a fresh plate every four or six days , counting as two or three generations , respectively , until no larvae were present on plate to be picked . * : two plates of N2 were contaminated were excluded from .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I, J) Line plots representing fertility over generations for the indicated genetic backgrounds at 25oC. Six L2-L3 larvae for each of the indicated backgrounds were hand-picked to a fresh plate every four or six days, counting as two or three generations, respectively, until no larvae were present on the plate to be picked. *: two plates of N2 were contaminated and were excluded from the assay.


Expression of PID - 2 : : eGFP ( xfSi145 ) ( A ) in perinuclear granules in germline . PGL - 1 : : mTagRFP - T ( A ) mark P granules . indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize granules , their overlaps . One L4 gonad is shown for each . Note that most of L4 gonad is in pachytene stage . Arrow heads indicate condensates . Scale bar : 25 µm .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Expression of PID-2::eGFP(xfSi145) (A) in perinuclear granules in the germline. PGL-1::mTagRFP-T (A) mark P granules. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar: 25 µm.


Expression of , PID - 4 : : mTagRFP - T ( B ) in perinuclear granules in germline . DEPS - 1 : : GFP ( B mark P granules . indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize granules , their overlaps . One L4 gonad is shown for each . Note that most of L4 gonad is in pachytene stage . Arrow heads indicate condensates . Scale bar : 25 µm .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Expression of , PID-4::mTagRFP-T (B) in perinuclear granules in the germline. DEPS-1::GFP (B mark P granules. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar: 25 µm.


Expression of PID - 5 : : mTagRFP - T ( C ) in perinuclear granules in germline . DEPS - 1 : : GFP mark P granules . indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize granules , their overlaps . One L4 gonad is shown for each . Note that most of L4 gonad is in pachytene stage . Arrow heads indicate condensates . Scale bar : 25 µm .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Expression of PID-5::mTagRFP-T (C) in perinuclear granules in the germline. DEPS-1::GFP mark P granules. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar: 25 µm.


Expression of PID - 4 : : mTagRFP - T ( D ) together with 3xFLAG : : GFP : : ZNFX - 1 , a Z granule marker . indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize granules , their overlaps . One L4 gonad is shown for each . Note that most of L4 gonad is in pachytene stage . Arrow heads indicate condensates . Scale bar : 25 µm .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Expression of PID-4::mTagRFP-T (D) together with 3xFLAG::GFP::ZNFX-1, a Z granule marker. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar: 25 µm.


Expression of PID - 5 : : mTagRFP - T ( E ) together with 3xFLAG : : GFP : : ZNFX - 1 , a Z granule marker . indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize granules , their overlaps . One L4 gonad is shown for each . Note that most of L4 gonad is in pachytene stage . Arrow heads indicate condensates . Scale bar : 25 µm .

Expression of PID-5::mTagRFP-T (E) together with 3xFLAG::GFP::ZNFX-1, a Z granule marker. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar: 25 µm.


F ) Box - plots representing ( µm ) between centres of two signals from indicated fusion between each pair of is represented by a dot . Between 4 - 10 different gonads were analyzed for each condition .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F) Box-plots representing the distance (µm) between the centres of two fluorescent signals from the indicated fusion proteins The distance between each pair of fluorescent proteins is represented by a dot. Between 4-10 different gonads were analyzed for each condition.


Expression of PID - 2 : : eGFP ( xfSi145 ) together with PID - 4 : : mTagRFP - T ( G ) indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize granules , their overlaps . One L4 gonad is shown for each . Note that most of L4 gonad is in pachytene stage . Arrow heads indicate condensates . Scale bar = 25

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Expression of PID-2::eGFP (xfSi145) together with PID-4::mTagRFP-T (G) The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar = 25 µ


Expression of PID - 2 : : eGFP ( xfSi145 ) together with PID - 5 : : mTagRFP - T ( H ) . indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize granules , their overlaps . One L4 gonad is shown for each . Note that most of L4 gonad is in pachytene stage . Arrow heads indicate condensates . Scale bar = 25 µm .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Expression of PID-2::eGFP (xfSi145) together with PID-5::mTagRFP-T (H). The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar = 25 µm.


A - E ) Expression of 3xFLAG : : GFP : : ZNFX - 1 PGL - 1 : : mTagRFP - T in a wild type ( A ) , pid - 2 ( B ) , pid - 4 ; pid - 5 ( C ) , pid - 4 ( D ) , pid - 5 ( E ) mutant backgrounds . indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize granules , their overlaps . One L4 gonad is shown . Arrow heads indicate condensates . Scale bar : 25 µm . F ) of Z / P granules in wild type , pid - 2 , pid - 4 / - 5 , pid - 4 pid - 5 mutant backgrounds . number of granules is indicated in brackets , next to genotype .

O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O

A-E) Expression of 3xFLAG::GFP::ZNFX-1 and PGL-1::mTagRFP-T in a wild type (A), pid-2 (B), pid-4;pid-5 (C), pid-4 (D), and pid-5 (E) mutant backgrounds. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown. Arrow heads indicate individual condensates. Scale bar: 25 µm. F) Quantification of the ratio Z / P granules in wild type, pid-2, pid-4/-5, pid-4 and pid-5 mutant backgrounds. The number of granules is indicated in brackets, next to the genotype.


Expression of PID - 4 : : mTagRFP - T ( G ) together with 3xFLAG : : GFP : : ZNFX - 1 , a Z granule marker , in a pid - 2 mutant background . indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize granules , their overlaps . One L4 gonad is shown for each . Scale bar : 25 µm .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Expression of PID-4::mTagRFP-T (G) together with 3xFLAG::GFP::ZNFX-1, a Z granule marker, in a pid-2 mutant background. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Scale bar: 25 µm.


Expression of PID - 4 : : mTagRFP - T H ) together with 3xFLAG : : GFP : : ZNFX - 1 , a Z granule marker , in a pid - 2 mutant background . indicated dashed boxes reflect zoom - ins on specific nuclei to better visualize granules , their overlaps . One L4 gonad is shown for each . Scale bar : 25 µm .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Expression of PID-4::mTagRFP-T H) together with 3xFLAG::GFP::ZNFX-1, a Z granule marker, in a pid-2 mutant background. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Scale bar: 25 µm.


I ) Box - plots representing ( µm ) between centres of two signals from indicated fusion between each pair of is represented by a dot . Between 4 - 10 different gonads were analyzed for each condition . median is represented by a . interquartile range ( IQR ) , 25th to 75th percentile , is represented by upper lower lines , respectively , whiskers represent first quartile ( down to - 1 . 5 * IQR ) or third quartile ( up to + 1 . 5 * IQR ) . P - values were calculated using a t - ( two - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I) Box-plots representing the distance (µm) between the centres of two fluorescent signals from the indicated fusion proteins The distance between each pair of fluorescent proteins is represented by a dot. Between 4-10 different gonads were analyzed for each condition. The median is represented by a line. The interquartile range (IQR), 25th to 75th percentile, is represented by the upper and lower lines, respectively, and whiskers represent the first quartile (down to -1.5*IQR) or the third quartile (up to +1.5*IQR). P-values were calculated using a t-test (two-tailed).


( a ) HEK293 were starved of glucose ( 4 h ) as indicated , endogenous Ulk1 was immunoprecipitated an autophosphorylation was performed . were resolved by SDS - PAGE visualized with autoradiography ( top ) or western blotting ( WB ; bottom ) .

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(a) HEK293 cells were starved of glucose (4 h) as indicated, endogenous Ulk1 was immunoprecipitated and an autophosphorylation assay was performed. Proteins were resolved by SDS-PAGE and visualized with autoradiography (top) or western blotting (WB; bottom).


( b ) were incubated in glucose - free medium ( 4 h ) as indicated lysed . Lysates were incubated with lambda phosphatase ( λ PPase ) as indicated . Endogenous Ulk1 mobility was examined by western blotting .

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(b) Cells were incubated in glucose-free medium (4 h) as indicated and lysed. Lysates were incubated with lambda phosphatase (λ PPase) as indicated. Endogenous Ulk1 mobility was examined by western blotting.


( c ) HA - Ulk1 was transfected into HEK293 together with wild - type ( WT ) AMPKα1 or a kinase - dead ( DN ) mutant . were starved of glucose ( 4 h ; Glu ) or ( - A . A ) treated with compound C ( 20 μM , C . C ) as indicated . Ulk1 mobility as well as phosphorylation of ACC S6K were determined by western blotting .

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(c) HA-Ulk1 was transfected into HEK293 cells together with wild-type (WT) AMPKα1 or a kinase-dead (DN) mutant. Cells were starved of glucose (4 h; Glu) or amino acids (-A.A) and treated with compound C (20 μM, C.C) as indicated. Ulk1 mobility as well as phosphorylation levels of ACC and S6K were determined by western blotting.


( d ) HA - Ulk1 were immunopurified from transfected HEK293 , which had undergone glucose starvation ( 4 h ) as indicated . HA - Ulk1 were treated with λ PPase , in vitro kinase assays were performed in presence of GST - ATG13 . were resolved by SDS - PAGE ; phosphorylated were visualized with autoradiography , HA - Ulk1 by western blotting GST - Atg13 by Coomassie .

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(d) HA-Ulk1 proteins were immunopurified from transfected HEK293 cells, which had undergone glucose starvation (4 h) as indicated. The HA-Ulk1 proteins were treated with λ PPase, and in vitro kinase assays were performed in the presence of GST-ATG13. Proteins were resolved by SDS-PAGE; phosphorylated proteins were visualized with autoradiography , HA-Ulk1 by western blotting and GST-Atg13 by Coomassie staining.


( e ) HA - Ulk1 was immunopurified from transfected HEK293 under glucose - rich media treated with AMPK in presence of cold ATP for 15 min , followed by kinase assays as described in d . In vitro kinase assays were performed in presence of GST - ATG13 . were resolved by SDS - PAGE ; phosphorylated were visualized with autoradiography , HA - Ulk1 by western blotting GST - Atg13 by Coomassie .

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(e) HA-Ulk1 was immunopurified from transfected HEK293 cells under glucose-rich media and treated with AMPK in the presence of cold ATP for 15 min, followed by kinase assays as described in d. In vitro kinase assays were performed in the presence of GST-ATG13. Proteins were resolved by SDS-PAGE; phosphorylated proteins were visualized with autoradiography, HA-Ulk1 by western blotting and GST-Atg13 by Coomassie staining.


( f ) AMPK wild - type ( WT ) α1 / α2 double knockout ( DKO ) MEFs were incubated with or without glucose ( 4 h ) . Endogenous Ulk1 was immunoprecipitated autophosphorylation was measured ( mean ± s . d . , n = 3 ) . Autophosphorylation activity was normalized to Ulk1 level ; relative activity is calculated by normalization to Ulk1 activity from AMPK wild - type MEFs in glucose - rich conditions .

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(f) AMPK wild-type (WT) and α1/α2 double knockout (DKO) MEFs were incubated with or without glucose (4 h). Endogenous Ulk1 was immunoprecipitated and autophosphorylation was measured (mean ± s.d., n = 3). Autophosphorylation activity was normalized to Ulk1 protein level; relative activity is calculated by normalization to Ulk1 activity from AMPK wild-type MEFs in glucose-rich conditions.


( g ) HA - Ulk1 was transfected into HEK293 together with vector ( Vec ) or an AMPKα1 kinase - dead mutant ( DN ) . were starved of glucose ( - Glu ) or ( - A . A ) , or treated with 50 nM rapamycin ( Rapa ) for 3 h before lysis . Left : autophosphorylation activity was assessed normalized as in f ( mean ± s . d . , n = 3 ) . Right : fold induction in Ulk1 autophosphorylation , compared with Ulk1 autophosphorylation from under nutrient - rich conditions . Uncropped of blots are shown in Supplementary Fig . S5 .

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(g) HA-Ulk1 was transfected into HEK293 cells together with vector (Vec) or an AMPKα1 kinase-dead mutant (DN). The cells were starved of glucose (-Glu) or amino acids (-A.A), or treated with 50 nM rapamycin (Rapa) for 3 h before lysis. Left: autophosphorylation activity was assessed and normalized as in f (mean ± s.d., n = 3). Right: fold induction in Ulk1 autophosphorylation, compared with Ulk1 autophosphorylation from cells under nutrient-rich conditions. Uncropped images of blots are shown in Supplementary Fig. S5.


( a ) AMPK phosphorylates Ulk1 S / T domain in vitro . Top : schematic representation of Ulk1 domain structure deletion constructs used to map phosphorylation sites . mouse Ulk1 consists of an N - terminal kinase domain ( KD ; 1 - 278 ) , serine / threonine - rich domain ( S / T domain , 279 - 828 ) , C - terminal domain ( CTD , 829 - 1051 ) . Bottom : indicated Flag - Ulk1 deletion mutants were immunopurified from transfected HEK293 were used for in vitro AMPK as a substrate . Phosphorylation was examined by 32P - autoradiogram level was determined by western blot .

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(a) AMPK phosphorylates the Ulk1 S/T domain in vitro. Top: schematic representation of Ulk1 domain structure and deletion constructs used to map phosphorylation sites. The mouse Ulk1 protein consists of an N-terminal kinase domain (KD; 1-278), serine/threonine-rich domain (S/T domain, 279-828), and C-terminal domain (CTD, 829-1051). Bottom: the indicated Flag-Ulk1 deletion mutants were immunopurified from transfected HEK293 cells and were used for in vitro AMPK assay as a substrate. Phosphorylation was examined by 32P-autoradiogram and protein level was determined by western blot.


( b ) Determination of AMPK phosphorylation sites in Ulk1 . indicated recombinant GST - Ulk1 mutants were expressed purified from Escherichia coli , used as substrates for in vitro phosphorylation by AMPK . Deletion analyses indicated that two Ulk1 fragments in S / T domain , 279 - 425 769 - 782 , were highly phosphorylated by AMPK in vitro . Mutation of Ser 317 abolished majority of phosphorylation in Ulk1 fragment 279 - 425 . Within fragment 769 - 782 , mutations of five serine residues ( Ser 774 , Ser 777 , Ser 778 , Ser 779 Ser 780 ) to alanine , denoted as ( 769 - 782 ) 5SA , completely abolished phosphorylation by AMPK . Reconstitution of Ser 777 in this mutation background , ( 769 - 782 ) 4SA - S777 , but not any of other four residues , restored phosphorylation by AMPK . GST GST - TSC2F ( TSC2 fragment 1300 - 1367 containing AMPK phosphorylation site at Ser 1345 ) were used as negative positive controls for AMPK reaction , respectively . Phosphorylation was determined by 32P - autoradiograph were detected by Coomassie .

O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

(b) Determination of AMPK phosphorylation sites in Ulk1. The indicated recombinant GST-Ulk1 mutants were expressed and purified from Escherichia coli, and used as substrates for in vitro phosphorylation by AMPK. Deletion analyses indicated that two Ulk1 fragments in the S/T domain, 279-425 and 769-782, were highly phosphorylated by AMPK in vitro. Mutation of Ser 317 abolished the majority of phosphorylation in the Ulk1 fragment 279-425. Within the fragment 769-782, mutations of five serine residues (Ser 774, Ser 777, Ser 778, Ser 779 and Ser 780) to alanine, denoted as (769-782) 5SA, completely abolished the phosphorylation by AMPK. Reconstitution of Ser 777 in this mutation background, (769-782) 4SA-S777, but not any of the other four residues, restored the phosphorylation by AMPK. GST and GST-TSC2F (TSC2 fragment 1300-1367 containing AMPK phosphorylation site at Ser 1345) were used as negative and positive controls for AMPK reaction, respectively. Phosphorylation was determined by 32P-autoradiograph and the protein levels were detected by Coomassie staining.


( c ) Ser 317 / Ser 777 are required for glucose - starvation induced Ulk1 phosphorylation in vivo . HA - Ulk1 mutants were transfected into HEK293 . were starved for glucose for 4 h as indicated . HA - Ulk1 was immunoprecipitated examined by western blot for mobility .

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(c) Ser 317/Ser 777 are required for glucose-starvation induced Ulk1 phosphorylation in vivo. HA-Ulk1 and mutants were transfected into HEK293 cells. Cells were starved for glucose for 4 h as indicated. HA-Ulk1 was immunoprecipitated and examined by western blot for mobility.


( d ) Phosphorylation of Ulk1 Ser 317 Ser 777 are induced by AMPK . Wild - type HA - Ulk1 or S317 / 777A mutant were co - transfected with AMPK into HEK293 as indicated . HA - Ulk1 was immunoprecipitated ( IP ) phosphorylation of Ser 317 Ser 777 were determined by western blotting . Uncropped of blots are shown in Supplementary Fig . S5 .

O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) Phosphorylation of Ulk1 Ser 317 and Ser 777 are induced by AMPK. Wild-type HA-Ulk1 or S317/777A mutant were co-transfected with AMPK into HEK293 cells as indicated. HA-Ulk1 was immunoprecipitated (IP) and phosphorylation of Ser 317 and Ser 777 were determined by western blotting. Uncropped images of blots are shown in Supplementary Fig. S5.


( a ) AMPK wild - type or DKO MEFs were starved of glucose ( 4 h ) as indicated . Total lysates were probed for Ulk1 phosphorylation .

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(a) AMPK wild-type or DKO MEFs were starved of glucose (4 h) as indicated. Total cell lysates were probed for Ulk1 protein and phosphorylation.


( b ) Time course of Ulk1 Ser 317 Ser 777 phosphorylation in response to glucose starvation / re - addition . MEFs were starved of glucose ( - Glu ) for indicated times . After 3 h starvation , culture was switched to glucose - containing ( 25 mM ) medium samples were harvested ( Re - Glu ) . In parallel , were treated with - acid - free ( - A . A ) medium or 50 nM rapamycin ( Rapa ) for 3 h .

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(b) Time course of Ulk1 Ser 317 and Ser 777 phosphorylation in response to glucose starvation/re-addition. MEFs were starved of glucose (-Glu) for the indicated times. After 3 h starvation, the culture was switched to glucose-containing (25 mM) medium and samples were harvested (Re-Glu). In parallel, cells were treated with amino-acid-free (-A.A) medium or 50 nM rapamycin (Rapa) for 3 h.