C ) Representative of 14DIV WT PTX3 KO neurons stained for surface AMPARs ( GluA , green ) , Bassoon ( blue ) tubulin ( red ) . Arrowheads point to postsynaptic GluA clusters . Scale bar : 5µm . D ) of surface synaptic AMPARs normalized to total number of Bsn shows a reduction in PTX3 KO cultures with respect to WT ( WT = 1 ± 0 . 065 , KO = 0 . 771 ± 0 . 0 . 92 ; number of fields examined : 36 42 respectively ; Mann - Whitney ; number of 5 WT 6 PTX3 KO , data are presented as normalized mean values ± )

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

C) Representative images of 14DIV WT and PTX3 KO neurons stained for surface AMPARs (GluA, green), Bassoon (blue) and tubulin (red). Arrowheads point to postsynaptic GluA clusters. Scale bar: 5µm. D) Quantification of the surface synaptic AMPARs normalized to the total number of Bsn shows a reduction in PTX3 KO cultures with respect to WT (WT=1±0.065, KO=0.771±0.0.92; number of fields examined: 36 and 42 respectively; Mann-Whitney test; number of animals 5 WT and 6 PTX3 KO, data are presented as normalized mean values±SEM)


E ) Examples of mEPSCs recorded in indicated experimental conditions . F ) Quantitation of mEPSC inter - event interval showing a rescue of mEPSC frequency in PTX3 KO cultures treated with N - terminal fragment of PTX3 ( WT = 1158 ± 34 . 67 ; WT + N - term = 979 . 1 ± 28 . 27 ; PTX3 KO = 1263 ± 38 . 99 ; PTX3 KO + Nterm = 1008 ± 30 . 14 . Number of neurons : WT , Ctr = 25 , WT + N - term = 29 ; PTX3 KO , Ctr = 24 ; PTX3 KO + N - term = 22 ; 3 independent experiments . Kruskall - Wallis , p < 0 . 0001 followed by Dunn " s as indicated in figure , data are presented as mean ± ) . G ) Quantitation of mEPSC amplitude showing a rescue in PTX3 KO cultures treated with N - terminal fragment of PTX3 ( pA , WT = 20 . 85 ± 0 . 249 WT + N - term = 22 . 37 ± 0 . 234 ; PTX3 KO = 20 . 31 ± 0 . 264 ; PTX3 KO + Nterm = 23 . 8 ± 0 . 316 . Number of neurons : WT , Ctr = 25 , WT + N - term = 29 ; PTX3 KO , Ctr = 24 ; PTX3 KO + N - term = 22 ; 3 independent experiments . Kruskall - Wallis , p < 0 . 0001 followed by Dunn " s as indicated in figure , data are presented

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

E) Examples of mEPSCs recorded in the indicated experimental conditions. F) Quantitation of mEPSC inter-event interval showing a rescue of mEPSC frequency in PTX3 KO cultures treated with the N-terminal fragment of PTX3 (WT=1158±34.67; WT+N-term=979.1±28.27; PTX3 KO=1263±38.99; PTX3 KO+Nterm=1008±30.14. Number of neurons: WT, Ctr=25, WT+N-term= 29; PTX3 KO, Ctr= 24; PTX3 KO+N-term=22; 3 independent experiments. Kruskall-Wallis test, p<0.0001 followed by Dunn"s test as indicated in figure, data are presented as mean±SEM). G) Quantitation of mEPSC amplitude showing a rescue in PTX3 KO cultures treated with the N-terminal fragment of PTX3 (pA, WT=20.85±0.249 WT+N-term=22.37±0.234; PTX3 KO=20.31±0.264; PTX3 KO+Nterm=23.8±0.316. Number of neurons: WT, Ctr=25, WT+N-term= 29; PTX3 KO, Ctr= 24; PTX3 KO+N-term=22; 3 independent experiments. Kruskall-Wallis test, p<0.0001 followed by Dunn"s test as indicated in figure, data are presented as


H ) Representative of 14DIV PTX3 KO neurons ( Ctr , + N terminal PTX3 , + C terminal PTX3 ) stained for surface AMPARs ( GluA , green ) , Bassoon ( blue ) tubulin ( red ) . Arrowheads point to postsynaptic GluA clusters . Scale bar : 5µm . I ) of surface synaptic AMPARs normalized to total number of Bsn shows an increase in PTX3 KO treated with N terminal fragment of PTX3 but not with C terminal fragment ( PTX3 KO = 1 . 000 ± 0 . 042 , PTX3 KO + C - term = 1 . 103 ± 0 . 070 , PTX3 KO + N - term = 1 . 428 ± 0 . 061 . Number of fields examined : 63 , 38 , 57 respectively ; Kruskall - Wallis , p < 0 . 0001 followed by Dunn " s ; 3 independent experiments , data are presented as normalized mean

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

H) Representative images of 14DIV PTX3 KO neurons (Ctr, +N terminal PTX3, +C terminal PTX3) stained for surface AMPARs (GluA, green), Bassoon (blue) and tubulin (red). Arrowheads point to postsynaptic GluA clusters. Scale bar: 5µm. I) Quantification of the surface synaptic AMPARs normalized to the total number of Bsn shows an increase in PTX3 KO treated with N terminal fragment of PTX3 but not with the C terminal fragment (PTX3 KO=1.000±0.042, PTX3 KO+C-term=1.103±0.070, PTX3 KO+N-term=1.428±0.061. Number of fields examined: 63, 38, 57 respectively; Kruskall-Wallis test, p<0.0001 followed by Dunn"s test; 3 independent experiments, data are presented as normalized mean values


J - L ( J ) Examples of mEPSCs recordings in WT PTX3 KO ( ) hippocampal slices at P8 - 9 showing that frequency ( K ) amplitude ( L ) are significantly decreased in KO neurons with respect to WT ( Hz : WT = 1 . 54 ± 0 . 136 ; PTX3 KO = 0 . 912 ± 0 . 083 ; Mann Whitney . pA : WT = 16 . 57 ± 1 . 334 ; PTX3 KO = 12 . 55 ± 1 . 037 ; unpaired t - . Data are presented as a distribution plus mean ± . WT : 18 , 4 mice ; PTX3 KO : 20 , 5 mice )

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 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-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 O O O O O O O

J-L (J) Examples of mEPSCs recordings in WT and PTX3 KO (littermates) hippocampal slices at P8-9 showing that frequency (K) and amplitude (L) are significantly decreased in KO neurons with respect to WT (Hz: WT=1.54±0.136; PTX3 KO=0.912±0.083; Mann Whitney test. pA: WT=16.57±1.334; PTX3 KO=12.55±1.037; unpaired t-test. Data are presented as a distribution plus mean±SEM. WT: 18 cells, 4 mice; PTX3 KO: 20 cells, 5 mice)


N ) Examples of mEPSCs recordings of P30 WT PTX3 KO hippocampal slices . O ) of mEPSC frequency showing reduced frequency in PTX3 KO mice with respect to WT ( Hz : WT = 2 . 27 ± 0 . 115 ; PTX3 KO = 1 . 74 ± 0 . 136 . WT : 10 , 4 mice ; PTX3 KO : 11 , 5 mice . Unpaired t - p = 0 . 008 . Data are presented as a distribution plus mean ± ) . P , Q ( P ) No difference in average amplitude is evident ( pA : WT = 11 . 95 ± 0 . 589 ; PTX3 KO = 10 . 69 ± 0 . 438 ; unpaired t - p = 0 . 099 . Data are presented as a distribution plus mean ± . WT : 10 , 4 mice ; PTX3 KO : 11 , 5 mice ) ; however cumulative probability plot of amplitudes ( Q ) for mEPSCs in WT PTX3 KO shows that there is a significant shift in distribution by Kolmogorov - Smirnov ( p < 0 . 0001 , D = 0 . 112 ) .

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 B-CONTROLLED_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 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 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 B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O

N) Examples of mEPSCs recordings of P30 WT and PTX3 KO hippocampal slices. O) Quantification of mEPSC frequency showing reduced frequency in PTX3 KO mice with respect to WT (Hz: WT=2.27±0.115; PTX3 KO=1.74±0.136. WT: 10 cells, 4 mice; PTX3 KO: 11 cells, 5 mice. Unpaired t-test p=0.008. Data are presented as a distribution plus mean±SEM). P, Q (P) No difference in the average amplitude is evident (pA: WT=11.95±0.589; PTX3 KO=10.69±0.438; unpaired t-test p=0.099. Data are presented as a distribution plus mean±SEM. WT: 10 cells, 4 mice; PTX3 KO: 11 cells, 5 mice); however the cumulative probability plot of amplitudes (Q) for mEPSCs in WT and PTX3 KO shows that there is a significant shift in the distribution by the Kolmogorov-Smirnov test (p <0.0001, D=0.112).


( A ) intracellular distribution of DOR is modified by starvation . HeLa were transiently transfected with DOR incubated for 1 h with DMEM ( basal ) , HBSS ( starvation ) , 50 μM chloroquine or 10 mM 3MA . intracellular localization of DOR was analysed by immunofluorescence is shown in red . nuclei are shown in blue ( DAPI ) . Scale bars , 10 μm .

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 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

(A) The intracellular distribution of DOR is modified by starvation. HeLa cells were transiently transfected with DOR and incubated for 1 h with DMEM (basal), HBSS (starvation), 50 μM chloroquine or 10 mM 3MA. The intracellular localization of DOR was analysed by immunofluorescence and is shown in red. The nuclei are shown in blue (DAPI staining). Scale bars, 10 μm.


( B ) Endogenous DOR leaves nucleus under starvation . C2C12 myoblasts were incubated for 30 min at 37 ° C with DMEM ( basal ) or HBSS ( starvation ) . DOR is shown in red nuclei are in blue ( DAPI ) . Scale bars , 5 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

(B) Endogenous DOR leaves the nucleus under starvation. C2C12 myoblasts were incubated for 30 min at 37°C with DMEM (basal) or HBSS (starvation). DOR is shown in red and nuclei are in blue (DAPI staining). Scale bars, 5 μm.


( C , D ) De ‐ activation of autophagy returns DOR to nucleus . HeLa were transiently transfected with DOR incubated for 1 h with DMEM ( basal ) or HBSS ( starvation ) . were then incubated with normal DMEM for an additional hour ( starvation + GM ) . intracellular distribution of DOR was determined by immunofluorescence . DOR is shown in red nuclei are in blue . Scale bars , 5 μm . lysates were also obtained western blot assays were performed with specific antibodies . 3MA , 3 ‐ methyladenine ; DAPI , 4 ′ , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; DMEM , Dulbecco ' s modified Eagle medium ; DOR , diabetes ‐ obesity ‐ regulated gene ; HBSS , Hank ' s balanced salt solution ; LC3 , microtubule ‐ associated 1A / 1B ‐ chain 3 .

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

(C,D) De‐activation of autophagy returns DOR to the nucleus. HeLa cells were transiently transfected with DOR and incubated for 1 h with DMEM (basal) or HBSS (starvation). Cells were then incubated with normal DMEM for an additional hour (starvation+GM). The intracellular distribution of DOR was determined by immunofluorescence. DOR is shown in red and the nuclei are in blue. Scale bars, 5 μm. Cell lysates were also obtained and western blot assays were performed with specific antibodies. 3MA, 3‐methyladenine; DAPI, 4′,6‐diamidino‐2‐phenylindole; DMEM, Dulbecco's modified Eagle medium; DOR, diabetes‐ and obesity‐regulated gene; HBSS, Hank's balanced salt solution; LC3, microtubule‐associated protein 1A/1B‐light chain 3.


DOR localizes to autophagosomes when autophagy is activated . ( A ) Confocal of HeLa transiently transfected with DOR GFP ‐ LC3 , incubated for 1h with DMEM , HBSS ( starvation ) , 2 μM rapamycin or HBSS containing 50 μM chloroquine . Nuclei were labelled with DAPI . Scale bars , 10 μm .

B-MEASURED_VAR 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

DOR localizes to autophagosomes when autophagy is activated. (A) Confocal images of HeLa cells transiently transfected with DOR and GFP‐LC3, and incubated for 1h with DMEM, HBSS (starvation), 2 μM rapamycin or HBSS containing 50 μM chloroquine. Nuclei were labelled with DAPI. Scale bars, 10 μm.


( B , C ) Confocal of HeLa transiently transfected with DOR or GFP ‐ LC3 incubated with 50 nM Lysotracker in DMEM or HBSS . Scale bars , 10 μm .

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

(B,C) Confocal images of HeLa cells transiently transfected with DOR or GFP‐LC3 and incubated with 50 nM Lysotracker in DMEM or HBSS. Scale bars, 10 μm.


( D ) Confocal of HeLa transiently transfected with DOR LAMP1 ‐ GFP , or with YFP ‐ LC3 LAMP1 ‐ GFP . Scale bars , 10 μm . Contrast ‐ corrected merge RGB pictures Z ‐ projection of Product of differences from mean ( PDM ) are shown in panels . Colour scales of PDM have different maximal values , so different conditions are not comparable from these projections . Product of differences from mean ( PDM ) values closer to 1 show reliable colocalized pixels . DAPI , 4 ′ , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; DMEM , Dulbecco ' s modified Eagle medium ; DOR , diabetes ‐ obesity ‐ regulated gene ; GFP , green ; HBSS , Hank ' s balanced salt solution ; LAMP1 , lysosomal ‐ associated 1 ; LC3 , microtubule ‐ associated 1A / 1B ‐ chain 3 .

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

(D) Confocal images of HeLa cells transiently transfected with DOR and LAMP1‐GFP, or with YFP‐LC3 and LAMP1‐GFP. Scale bars, 10 μm. Contrast‐corrected merge RGB pictures and the Z‐projection of Product of the differences from the mean (PDM) images are shown in all panels. Colour scales of PDM images have different maximal values, so the different conditions are not comparable from these projections. Product of the differences from the mean (PDM) values closer to 1 show reliable colocalized pixels. DAPI, 4′,6‐diamidino‐2‐phenylindole; DMEM, Dulbecco's modified Eagle medium; DOR, diabetes‐ and obesity‐regulated gene; GFP, green fluorescent protein; HBSS, Hank's balanced salt solution; LAMP1, lysosomal‐associated membrane protein 1; LC3, microtubule‐associated protein 1A/1B‐light chain 3.


( A ) DOR gain ‐ of ‐ function accelerates degradation of of middle - long half ‐ life . Untreated HeLa those transiently transfected with DOR were incubated in DMEM or HBSS . * Significant effects of starvation , P0 . 01 ; τsignificant effects caused by DOR overexpression , P0 . 01 .

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 B-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 O O O O O O

(A) DOR gain‐of‐function accelerates the degradation of proteins of middle-long half‐life. Untreated HeLa cells and those transiently transfected with DOR were incubated in DMEM or HBSS. *Significant effects of starvation, P0.01; τsignificant effects caused by DOR overexpression, P0.01.


( B ) DOR overexpression enhances autophagosomal formation . LC3 ‐ GFP ‐ positive vacuoles were counted in 100 transfected HeLa ( with GFP ‐ LC3 TRα1 , or GFP ‐ LC3 DOR ) . * Significant effects of starvation , P0 . 001 ; τsignificant effects of DOR overexpression , P0 . 001 .

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

(B) DOR overexpression enhances autophagosomal formation. LC3‐GFP‐positive vacuoles were counted in 100 transfected HeLa cells (with GFP‐LC3 and TRα1, or GFP‐LC3 and DOR). *Significant effects of starvation, P0.001; τsignificant effects of DOR overexpression, P0.001.


( C , D ) HeLa transiently transfected with empty pCDNA3 or DOR were incubated in DMEM ( basal ) or in HBSS ( starvation ) . were processed by transmission electron microscopy . Arrows indicate autophagosomes . Insets are autophagosomes ( diameter , 407 ± 63 nm ) . Scale bars , 1 μm . Autophagosomes were counted in 50 randomly chosen transfected . * Significant effects of starvation , P0 . 05 ; τsignificant effects of DOR overexpression , P0 . 05 . DMEM ; Dulbecco ' s modified Eagle medium ; DOR , diabetes ‐ obesity ‐ regulated gene ; GFP , green ; HBSS ; Hank ' s balanced salt solution ; LC3 , microtubule ‐ associated 1A / 1B ‐ chain 3 .

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 O O O O O O B-CONTROLLED_VAR 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

(C,D) HeLa cells transiently transfected with empty pCDNA3 or DOR were incubated in DMEM (basal) or in HBSS (starvation). Cells were processed by transmission electron microscopy. Arrows indicate autophagosomes. Insets are autophagosomes (diameter, 407±63 nm). Scale bars, 1 μm. Autophagosomes were counted in 50 randomly chosen transfected cells. *Significant effects of starvation, P0.05; τsignificant effects of DOR overexpression, P0.05. DMEM; Dulbecco's modified Eagle medium; DOR, diabetes‐ and obesity‐regulated gene; GFP, green fluorescent protein; HBSS; Hank's balanced salt solution; LC3, microtubule‐associated protein 1A/1B‐light chain 3.


( A , B ) C2C12 myoblasts were previously infected with lentiviruses encoding scrambled , DOR siRNA1 or DOR siRNA2 . Endogenous LC3 was detected by immunofluorescence . LC3 ‐ positive were counted in 100 per group under basal conditions or starved for 20 min . Scale bars , 10 μm . * Significant effects of starvation , P0 . 001 ; τsignificant effects caused by DOR knockdown , P0 . 001 .

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

(A,B) C2C12 myoblasts were previously infected with lentiviruses encoding scrambled RNA, DOR siRNA1 or DOR siRNA2. Endogenous LC3 was detected by immunofluorescence. LC3‐positive puncta were counted in 100 cells per group under basal conditions or starved for 20 min. Scale bars, 10 μm. *Significant effects of starvation, P0.001; τsignificant effects caused by DOR knockdown, P0.001.


( C , D ) Scramble or DOR siRNA1 C2C12 were processed by transmission electron microscopy . Arrows indicate autophagosomes . Insets are autophagosomes . Scale bars , 1 μm . Autophagosomes were counted in 50 randomly chosen . * Significant effects of DOR repression , P0 . 001 .

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 B-CONTROLLED_VAR O O O O O O

(C,D) Scramble or DOR siRNA1 C2C12 cells were processed by transmission electron microscopy. Arrows indicate autophagosomes. Insets are autophagosomes. Scale bars, 1 μm. Autophagosomes were counted in 50 randomly chosen cells. *Significant effects of DOR repression, P0.001.


( E ) Scramble or DOR siRNA1 C2C12 were incubated in DMEM with without 10 mM 3MA . * Significant effects of DOR knockdown , P0 . 01 ; τsignificant effects caused by 3MA , P0 . 01 . 3MA , 3 ‐ methyladenine ; DMEM , Dulbecco ' s modified Eagle medium ; DOR , diabetes ‐ obesity ‐ regulated gene ; LC3 , microtubule ‐ associated 1A / 1B ‐ chain 3 .

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

(E) Scramble or DOR siRNA1 C2C12 cells were incubated in DMEM with and without 10 mM 3MA. *Significant effects of DOR knockdown, P0.01; τsignificant effects caused by 3MA, P0.01. 3MA, 3‐methyladenine; DMEM, Dulbecco's modified Eagle medium; DOR, diabetes‐ and obesity‐regulated gene; LC3, microtubule‐associated protein 1A/1B‐light chain 3.


( A ) Decrease of mRNA in TubG4 , UAS ‐ CG11347 ‐ RNAi compared with TubG4 controls . measurements were normalized to rp49 . * Significant change , P0 . 05 .

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

(A) Decrease of mRNA levels in TubG4, UAS‐CG11347‐RNAi animals compared with TubG4 controls. All measurements were normalized to rp49. *Significant change, P0.05.


( B ) Lysotracker of fat bodies . No Lysotracker is observed in feeding early third ‐ instar larvae , whereas Lysotracker ‐ positive granules ( red ) accumulate in fat of wandering late third ‐ instar controls ( upper panels , TubG4 / + ) . dDOR knockdown ( TubG4 , UAS ‐ CG11347 ‐ RNAi T4 , lower panels ) show decreased of Lysotracker ‐ positive granules in wandering late third ‐ instar larvae . Nuclei are shown in blue . Scale bars , 50 μm . ( C ) number of Lysotracker ‐ positive per unit are shown for each genotype ( control RNAi ) , normalized to value for control processed in parallel . * Significant change , P0 . 05 .

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

(B) Lysotracker staining of fat bodies. No Lysotracker staining is observed in feeding early third‐instar larvae, whereas Lysotracker‐positive granules (red) accumulate in fat body cells of wandering late third‐instar controls (upper panels, TubG4/+). dDOR knockdown animals (TubG4, UAS‐CG11347‐RNAi T4, lower panels) show decreased staining of Lysotracker‐positive granules in wandering late third‐instar larvae. Nuclei are shown in blue. Scale bars, 50 μm. (C) The number of Lysotracker‐positive puncta per unit area are shown for each genotype (control and RNAi), normalized to the value for control cells processed in parallel. *Significant change, P0.05.


( D ) Transmission electron microscopy of fat from wandering late third ‐ instar larvae . Developmental autophagy results in accumulation of large autolysosomes ( AL ) in control ( TubG4 / + ) larvae , whereas only a few autolysosomes are seen in CG11347 RNAi fat of same . Control RNAi fat bodies present lipid droplets ( LP ) with a ' striped ' appearance , which is an electron microscopy artefact . Scale bars , 5 nm . dDor , CG11347 , a homologue of DOR ; DOR , diabetes ‐ obesity ‐ regulated gene ; mRNA , messenger ; wt , wild type .

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

(D) Transmission electron microscopy images of fat body cells from wandering late third‐instar larvae. Developmental autophagy results in the accumulation of large autolysosomes (AL) in control (TubG4/+) larvae, whereas only a few autolysosomes are seen in CG11347 RNAi fat body cells of the same age. Control and RNAi fat bodies present lipid droplets (LP) with a 'striped' appearance, which is an electron microscopy artefact. Scale bars, 5 nm. dDor, CG11347, a homologue of DOR; DOR, diabetes‐ and obesity‐regulated gene; mRNA, messenger RNA; wt, wild type.


C ) . Western blot of LMF1 - partners after DSP crosslinking affinity tag purification using LMF1 ' s C - terminal His tag . Tetracycline induces LMF1 expression . Loading dye with 50mM DTT was used to break disulfide bonds between LMF1 its interacting partners . LMF1 complexes are labeled with asterisks an arrow points to LMF1

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C). Western blot of LMF1-partners after DSP crosslinking and affinity tag purification using LMF1's C-terminal His tag. Tetracycline induces LMF1 expression. Loading dye with 50mM DTT was used to break the disulfide bonds between LMF1 and its interacting partners. LMF1 complexes are labeled with asterisks and an arrow points to LMF1


D ) . Western blot of LMF1 - partners after pBPA photo - crosslinking affinity tag purification . DMSO panel does not include pBPA serves as a negative control . Fractions were eluted with increasing concentrations of imidazole . An arrow points to LMF1

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D). Western blot of LMF1-partners after pBPA photo-crosslinking and affinity tag purification. The DMSO panel does not include pBPA and serves as a negative control. Fractions were eluted with increasing concentrations of imidazole. An arrow points to LMF1


E ) . SYPRO orange stain of DSP crosslinked samples . Samples without tetracycline but with DSP DTT ( negative control ) , with tetracycline , DSP DTT were sent for LC MS / MS . LMF1 complexes are labeled with asterisks an arrow points to LMF1

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E). SYPRO orange protein stain of DSP crosslinked samples. Samples without tetracycline but with DSP and DTT (negative control), and with tetracycline, DSP and DTT were sent for LC MS/MS. LMF1 complexes are labeled with asterisks and an arrow points to LMF1


F ) . SYPRO orange stained gel of pBPA samples . An arrow points to LMF1

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F). SYPRO orange stained gel of pBPA samples. An arrow points to LMF1


A - B ) . HEK293 stably expressing LPL - V5 or PL - V5 were transfected with scrambled negative control siRNA ( NC ) or siRNA against LMF1 - interacting partners . Media fractions were probed for lipase V5 tags , lysate fractions were probed for V5 LMF1 interacting - partners . GAPDH is a loading

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A-B). HEK293 cells stably expressing LPL-V5 or PL-V5 were transfected with scrambled negative control siRNA (NC) or siRNA against LMF1-interacting partners. Media fractions were probed for the lipase V5 tags, and lysate fractions were probed for V5 and the LMF1 interacting-partners. GAPDH is a loading control


siRNA knockdowns were performed in triplicate , lipase secretion was quantified relative to NC siRNA . Error bars indicate standard error . of of each remaining from three independent experiments . Error bars indicate standard error .

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siRNA knockdowns were performed in triplicate, and lipase secretion was quantified relative to NC siRNA. Error bars indicate the standard error. Quantification of the percent of each protein remaining from three independent experiments. Error bars indicate the standard error.


E ) HEK293 stably expressing LPL - V5 or PL - V5 were transfected with NC siRNA or siRNA against TRX . Lipase TRX were tested as for Fig 1A

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E) HEK293 cells stably expressing LPL-V5 or PL-V5 were transfected with NC siRNA or siRNA against TRX. Lipase and TRX levels were tested as for Fig 1A


F ) Txnip overexpression in HEK293 stably expressing LPL - V5 or PL - V5 hinders LPL , but not PL , secretion

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F) Txnip overexpression in HEK293 cells stably expressing LPL-V5 or PL-V5 hinders LPL, but not PL, secretion


A ) . A Western blot against C - terminal V5 tags of both lipases shows that LPL , but not PL , forms intermolecular disulfide bonds in . Lipase monomers are marked with a single arrow , are marked with double triple arrows . LPL are not present when samples are treated with DTT . There is no PL in pellet fraction because PL does not aggregate

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A). A Western blot against the C-terminal V5 tags of both lipases shows that LPL, but not PL, forms intermolecular disulfide bonds in cells. Lipase monomers are marked with a single arrow, and aggregates are marked with double and triple arrows. The LPL aggregates are not present when samples are treated with DTT. There is no PL in the pellet fraction because PL does not aggregate


B ) . A Western blot of a non - reducing gel of lysate pellet fraction of LPL grown in lacking LMF1 ( HEK293 ∆ LMF1 ) , normal HEK293 or with additional LMF1 ( + LMF1 ) . LPL monomers are marked as above

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B). A Western blot of a non-reducing gel of the lysate and pellet fraction of LPL grown in cells lacking LMF1 (HEK293∆LMF1), normal HEK293 cells or cells with additional LMF1 (+LMF1). LPL monomers and aggregates are marked as above


C ) . Co - translational LPL folding was carried out in presence of SP HEK293 or HEK293 ∆ LMF1 . At indicated time points , AMS was added to reactions to differentiate reduced ( R ) oxidized ( O ) LPL . position of stacker layer is indicated

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C). Co-translational LPL folding was carried out in the presence of SP HEK293 or HEK293∆LMF1 cells. At the indicated time points, AMS was added to reactions to differentiate reduced (R) and oxidized (O) LPL. The position of the stacker layer is indicated


amount of reduced / total LPL was quantified . Each point is average of three independent experiments , error bars represent standard error . Significance was determined by a 2 - student ' s t - .

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The amount of reduced/total LPL intensity was quantified. Each point is the average of three independent experiments, and error bars represent the standard error. Significance was determined by a 2-tailed student's t-test.


amount of oxidized / total LPL ( E ) of stacker / total LPL ( F ) was quantified . Each point is average of three independent experiments , error bars represent standard error . Significance was determined by a 2 - student ' s t -

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the amount of oxidized/total LPL (E) and the intensity of the stacker/total LPL (F) was quantified. Each point is the average of three independent experiments, and error bars represent the standard error. Significance was determined by a 2-tailed student's t-test


G H ) . Data from ( D - F ) was combined to show trends in LPL folding over time for reactions using HEK293 or HEK293 ∆ LMF1 . Each point is average of three independent experiments , error bars represent standard deviation

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G and H). Data from (D-F) was combined to show the trends in LPL folding over time for reactions using HEK293 or HEK293∆LMF1 cells. Each point is the average of three independent experiments, and error bars represent the standard deviation


Differential sensitivity of HEK293 containing lacking LMF1 ( A ) to drugs that perturb redox homeostasis . were treated with BSO , DTT , or tunicamycin as described in materials methods . Treated untreated were counted , fraction of surviving was calculated . Three wells were counted averaged for each data point , three independent trials were carried out per condition . Significance was determined by a 2 - student ' s t - .

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Differential sensitivity of HEK293 cells containing and lacking LMF1 (A) to drugs that perturb cellular redox homeostasis. Cells were treated with BSO, DTT, or tunicamycin as described in materials and methods. Treated cell and untreated cells were counted, and the fraction of surviving cells was calculated. Three wells were counted and averaged for each data point, and three independent trials were carried out per condition. Significance was determined by a 2-tailed student's t-test.


Differential sensitivity of cld / cld wt / cld MEFs ( B ) to drugs that perturb redox homeostasis . were treated with BSO , DTT , or tunicamycin as described in materials methods . Treated untreated were counted , fraction of surviving was calculated . Three wells were counted averaged for each data point , three independent trials were carried out per condition . Significance was determined by a 2 - student ' s t - .

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Differential sensitivity of cld/cld and wt/cld MEFs (B) to drugs that perturb cellular redox homeostasis. Cells were treated with BSO, DTT, or tunicamycin as described in materials and methods. Treated cell and untreated cells were counted, and the fraction of surviving cells was calculated. Three wells were counted and averaged for each data point, and three independent trials were carried out per condition. Significance was determined by a 2-tailed student's t-test.


C ) . redox sensor ERroGFP - S4 shows that HEK293 ∆ LMF1 have a more oxidized ER than HEK293 HEK293 + LMF1 both untreated when stressed by expression of LPL - mCherry . A two - student ' s t - was used to calculate significance of difference in of oxidized / reduced GFP between types . Each data point is one of three spots measured per , 17 were measured per condition . Significance was determined by a 2 - student ' s t -

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C). The redox sensor ERroGFP-S4 shows that HEK293∆LMF1 cells have a more oxidized ER than HEK293 and HEK293+LMF1 cells both untreated and when stressed by expression of LPL-mCherry. A two-tailed student's t-test was used to calculate the significance of the difference in the ratio of oxidized/reduced GFP fluorescence intensity between cell types. Each data point is one of three spots measured per cell, 17 cells were measured per condition. Significance was determined by a 2-tailed student's t-test


) . Secretion of LPL - V5 from HEK293 ∆ LMF1 transiently transfected with LMF1 bearing indicated cysteine to alanine mutations . Lipase in lysate pellet , LMF1 in pellet ( α - HIS ) , are also probed . GAPDH serves as a loading control .

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). Secretion of LPL-V5 from HEK293∆LMF1 cells transiently transfected with LMF1 bearing the indicated cysteine to alanine mutations. Lipase levels in the lysate and pellet, and LMF1 levels in the pellet (α-HIS), are also probed. GAPDH serves as a loading control.


α - His Western blot to detect redox state of LMF1 . In first two lanes samples were untreated resolved in reducing or non - reducing loading dye . next 4 lanes were subject to indicated treatments prior to lysis then treated with AMS prior to non - reducing SDS - PAGE .

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α-His Western blot to detect the redox state of LMF1. In the first two lanes samples were untreated and resolved in reducing or non-reducing loading dye. The next 4 lanes were subject to the indicated treatments prior to cell lysis then treated with AMS prior to non-reducing SDS-PAGE.


α - His Western blot to detect acid trapped partners of LMF1 . Samples were TCA precipitated , treated with NEM , LMF1 was purified via its C - terminal His tag . Samples were resolved by reducing or nonreducing SDS - PAGE . lanes marked - LMF1 are HEK293 ∆ LMF1 included as a negative control .

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α-His Western blot to detect acid trapped partners of LMF1. Samples were TCA precipitated, treated with NEM, and LMF1 was purified via its C-terminal His tag. Samples were resolved by reducing or nonreducing SDS-PAGE. The lanes marked -LMF1 are HEK293∆LMF1 cells included as a negative control.


Samples were TCA precipitated , treated with NEM , LMF1 was purified via its C - terminal His tag . Samples were resolved by reducing or nonreducing SDS - PAGE . lanes marked - LMF1 are HEK293 ∆ LMF1 included as a negative control . Samples were probed with an antibody for TRX .

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Samples were TCA precipitated, treated with NEM, and LMF1 was purified via its C-terminal His tag. Samples were resolved by reducing or nonreducing SDS-PAGE. The lanes marked -LMF1 are HEK293∆LMF1 cells included as a negative control. Samples were probed with an antibody for TRX.


Bottom , center top slices from a z - stack of a cld / wt or cld / cld MEF . YPet - tagged fibronectin is in green mCherry - Sec61β , an ER marker , is in red . Note fibronectin assembly into extracellular matrix in cld / wt . cld / wt had significantly higher amounts of fibronectin outside of ER than cld / cld by a two - student ' s t - .

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Bottom, center and top slices from a z-stack of a cld/wt or cld/cld MEF cell. YPet-tagged fibronectin is in green and mCherry-Sec61β, an ER marker, is in red. Note fibronectin assembly into the extracellular matrix in cld/wt cells. cld/wt cells had significantly higher amounts of fibronectin outside of the ER than cld/cld cells by a two-tailed student's t-test.


Bottom , center top slices show that LDLR is retained in ER in HEK293 ∆ LMF1 . LDLR - GFP is in green mCherry - Sec61β is in red . Note clumping of LDLR in ER in HEK293 ∆ LMF1 . of LDLR within ER / total LDLR was significantly higher for HEK293 ∆ LMF1 than HEK293 or HEK293 + LMF1 by a 2 - student ' s t - .

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Bottom, center and top slices show that LDLR is retained in the ER in cells HEK293∆LMF1 cells. LDLR-GFP is in green and mCherry-Sec61β is in red. Note the clumping of LDLR in the ER in HEK293∆LMF1 cells. The ratio of LDLR within the ER/total LDLR was significantly higher for HEK293∆LMF1 cells than HEK293 or HEK293+LMF1 cells by a 2-tailed student's t-test.


A . Lysates of HK2 cultured in FM or STV ( 2h ) were immunoprecipitated with anti - OFD1 ( left ) anti - ATG13 ( right ) antibodies or IgG analyzed by WB ; ACTIN was used as loading control . n = 3 independent experiments . IP = immunoprecipitation , FM = full medium , STV = HBSS starvation .

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A. Lysates of HK2 cells cultured in FM or STV(2h) were immunoprecipitated with anti-OFD1 (left) and anti-ATG13 (right) antibodies or IgG and analyzed by WB; ACTIN was used as loading control. n=3 independent experiments. IP=immunoprecipitation, FM=full medium, STV=HBSS starvation.


B . Airyscan confocal of endogenous FIP200 / γTUBULIN ATG13 / γTUBULIN colocalization with lentiviral delivered HA - OFD1 in HK2 . were cultured in HBSS ( 90min ) . Green , FIP200 ATG13 ; red , HA - OFD1 ; Grey , γTUBULIN ; blue , Hoechst for nuclei . Insets show magnifications single - color channels of selected areas . Scale bar = 3 , 5μm . C . of HA - OFD1 / ATG13 HA - OFD1 / FIP200 colocalization , expressed as % of total OFD1 ( mean ± ) . n = 3 independent experiments ; n = 53 for ATG13 n = 39 for FIP200 were analyzed .

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B. Airyscan confocal analysis of endogenous FIP200/γTUBULIN and ATG13/γTUBULIN colocalization with lentiviral delivered HA-OFD1 in HK2 cells. Cells were cultured in HBSS (90min). Green, FIP200 and ATG13; red, HA-OFD1; Grey, γTUBULIN; blue, Hoechst for nuclei. Insets show magnifications and single-color channels of selected areas. Scale bar=3,5μm. C. Quantification of HA-OFD1/ATG13 and HA-OFD1/FIP200 colocalization, expressed as % of total OFD1 fluorescence (mean ± SEM). n=3 independent experiments; n=53 cells for ATG13 and n=39 for FIP200 were analyzed.


A . Top , WB of ULK1 - components in wt KO - OFD1 assessed in FM ( - ) or STV ( 2 , 4h ) . Bands at same exposure , noncontiguous on same gel . Bottom , ACTIN - normalized are expressed as fold increase compared with wt , represented by dashed ( mean ± ) ; n = 5 independent experiments . FM = full medium , STV = HBSS starvation .

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A. Top, WB of ULK1-complex components in wt and KO-OFD1 cells assessed in FM (-) or STV (2,4h). Bands at same exposure, noncontiguous on the same gel. Bottom, ACTIN-normalized protein levels are expressed as fold increase compared with wt cells, represented by the dashed line (mean ± SEM); n=5 independent experiments. FM=full medium, STV=HBSS starvation.


B . Top , wild - type KO - OFD1 were incubated with 50µg / ml CHX . Immunoblots were probed with indicated antibodies . Bottom , of βTUBULIN ACTIN - normalized versus untreated conditions ( - ) , data are expressed as mean ± ; n = 4 independent experiments . CHX = Cycloheximide , ns = not significative .

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B. Top, wild-type and KO-OFD1 cells were incubated with 50µg/ml CHX. Immunoblots were probed with indicated antibodies. Bottom, quantification of βTUBULIN and ACTIN-normalized protein levels versus untreated conditions (-), data are expressed as mean ± SEM; n=4 independent experiments. CHX=Cycloheximide, ns=not significative.


C . Immunofluorescence of ULK1 ATG13 in wt KO - OFD1 cultured in HBSS ( 90min ) . Green , ATG13 ULK1 ; blue , Hoechst for nuclei . Scale bar = 10μm . ( Right ) Graphs display of / , data are expressed as mean ± ; n = 5 independent experiments , n ≥ 200 / antibody .

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C. Immunofluorescence of ULK1 and ATG13 puncta in wt and KO-OFD1 cells cultured in HBSS (90min). Green, ATG13 and ULK1; blue, Hoechst for nuclei. Scale bar=10μm. (Right) Graphs display quantification of puncta/cell, data are expressed as mean ± SEM; n=5 independent experiments, n≥200 cells/antibody.


D . Western Blot of ULK1 components in KO - OFD1 transfected with empty vector ( Empty ) or 3xFLAG - OFD1 ( OFD1 ) . Histograms show of band intensities normalized versus ACTIN expressed as mean ± . n = 5 independent experiments .

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D. Western Blot of ULK1 complex components in KO-OFD1 cells transfected with empty vector (Empty) or 3xFLAG-OFD1 (OFD1). Histograms show quantification of band intensities normalized versus ACTIN expressed as mean ± SEM. n=5 independent experiments.


E . Western Blot of ULK1 - components in KO - OFD1 transfected with 3xFLAG - OFD1 ( OFD1 ) or empty vector ( Empty ) treated ( + ) or not ( - ) with SAR405 ( 10μM , 6h ) . ACTIN is loading control . On right , ATG13 are expressed as fold increase compared with empty vector ( mean ± ) ; n = 3 independent experiments .

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E. Western Blot of ULK1-complex components in KO-OFD1 cells transfected with 3xFLAG-OFD1 (OFD1) or empty vector (Empty) and treated (+) or not (-) with SAR405 (10μM, 6h). ACTIN is the loading control. On the right, ATG13 levels are expressed as fold increase compared with empty vector (mean ± SEM); n=3 independent experiments.


F . Representative confocal of co - of 3xFLAG - OFD1 with ATG13 in KO - OFD1 incubated in HBSS , treated ( + ) or not ( - ) with Baf - A1 ( 100nM , 90min ) . Green , ATG13 ; red , 3xFLAG - OFD1 ; blue , Hoechst for nuclei . Scale bar = 10μm . ( Right ) ATG13 / are quantified expressed as mean ± ; n = 3 independent experiments , n ≥ 100 .

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F. Representative confocal images of co-staining of 3xFLAG-OFD1 with ATG13 in KO-OFD1 cells incubated in HBSS, treated (+) or not (-) with Baf-A1(100nM, 90min). Green, ATG13; red, 3xFLAG-OFD1; blue, Hoechst for nuclei. Scale bar=10μm. (Right) ATG13 puncta/cell are quantified and expressed as mean ± SEM; n=3 independent experiments, n≥100 cells.


A . Middle . Lysates from 3xFLAG - OFD1 overexpressing HEK293 were added to beads with immobilized GST - fused LC3B , LC3BF52A - V53AdG , dNLC3BdG GST alone . Right . Lysates from 3xFLAG - OFD1 ( OFD1 ) empty vector ( Empty ) overexpressing HEK293 were added to GST - GABARAP - L1 GST . Left . WB of total lysates ( input ) . panels display WB for FLAG , ACTIN is loading control .

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A. Middle. Lysates from 3xFLAG-OFD1 overexpressing HEK293 cells were added to beads with immobilized GST-fused LC3B, LC3BF52A-V53AdG, dNLC3BdG and GST alone. Right. Lysates from 3xFLAG-OFD1(OFD1) and empty vector (Empty) overexpressing HEK293 cells were added to GST-GABARAP-L1 and GST. Left. WB of total lysates (input). All panels display WB for FLAG, ACTIN is the loading control.


B . Representative confocal of endogenous OFD1 ( green ) co - with LC3B ( red ) , of 3xFLAG - OFD1 ( red ) with GABARAP ( green ) in HK2 ( HBSS , 90min ) . Hoechst labels nuclei ( blue ) . Insets show magnifications single - color channels of selected areas . Scale bar = 10μm ; n ≥ 50 / sample .

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B. Representative confocal images of endogenous OFD1 (green) co-staining with LC3B (red), and of 3xFLAG-OFD1 (red) with GABARAP (green) in HK2 cells (HBSS,90min). Hoechst labels nuclei (blue). Insets show magnifications and single-color channels of selected areas. Scale bar=10μm; n≥50 cells/sample.


D . Lysates from 3xFLAG - OFD1 ( OFD1 ) , 3xFLAG - OFD1 ∆ LIR ( OFD1 ∆ LIR ) empty vector ( Empty ) overexpressing were added to GST - GABARAP - L1 , GST - LC3B GST .

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D. Lysates from 3xFLAG-OFD1(OFD1), 3xFLAG-OFD1∆LIR(OFD1∆LIR) and empty vector (Empty) overexpressing cells were added to GST-GABARAP-L1, GST-LC3B and GST.


E . Representative confocal of OFD1 ∆ LIR co - with LC3B ( left ) GABARAP ( right ) in HK2 ( HBSS , 90min ) . Green , LC3B GABARAP ; red , 3xFLAG - OFD1 ∆ LIR ; blue , Hoechst for nuclei . Scale bar = 5μm . n ≥ 50 / sample .

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E. Representative confocal images of OFD1∆LIR co-staining with LC3B (left) and GABARAP (right) in HK2 cells (HBSS,90min). Green, LC3B and GABARAP; red, 3xFLAG-OFD1∆LIR; blue, Hoechst for nuclei. Scale bar=5μm. n≥50 cells/sample.


A . Representative confocal of 3xFLAG - OFD1 ( OFD1 ) 3xFLAG - OFD1 ∆ LIR ( OFD1 ∆ LIR ) co - with ATG13 in KO - OFD1 ( HBSS , 90min ) . Green , ATG13 ; red , FLAG ; blue , Hoechst for nuclei . Scale bar = 10μm . ( Right ) ATG13 / are quantified expressed as mean ± ; n = 4 independent experiments , n ≥ 100 .

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A. Representative confocal images of 3xFLAG-OFD1 (OFD1) and 3xFLAG-OFD1∆LIR (OFD1∆LIR) co-staining with ATG13 in KO-OFD1 cells (HBSS,90min). Green, ATG13; red, FLAG; blue, Hoechst for nuclei. Scale bar=10μm. (Right) ATG13 puncta/cell are quantified and expressed as mean ± SEM; n=4 independent experiments, n≥100 cells.


B . Western blot of ATG13 in KO - OFD1 transfected with 3xFLAG - OFD1 ( OFD1 ) or 3xFLAG - OFD1 ∆ LIR ( OFD1 ∆ LIR ) or empty vector ( Empty ) . ( Right ) ACTIN - normalized ATG13 are expressed as fold increase compared with empty vector ( mean ± ) ; n = 3 independent experiments .

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B. Western blot of ATG13 in KO-OFD1 cells transfected with 3xFLAG-OFD1 (OFD1) or 3xFLAG-OFD1∆LIR (OFD1∆LIR) or empty vector (Empty). (Right) ACTIN-normalized ATG13 protein levels are expressed as fold increase compared with empty vector (mean ± SEM); n=3 independent experiments.


C . Top . OFD1 fragments ( a , b c ) . Bottom left . Lysates from empty vector ( Empty ) , 3xFLAG - OFD1 ( OFD1 ) , 3xFLAG - OFD1a ( OFD1a ) , 3xFLAG - OFD1b ( OFD1b ) , 3xFLAG - OFD1c ( OFD1c ) overexpressing HEK293 were added to GST - ATG13 ; lysates from 3xFLAG - OFD1 ( OFD1 ) overexpressing HEK293 were added to GST as control . Right . WB of total lysates ( input ) . panels display WB for FLAG , HSP90 is loading control . LIR = LC3 interacting region .

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C. Top. OFD1 fragments (a,b and c). Bottom left. Lysates from empty vector (Empty), 3xFLAG-OFD1(OFD1), 3xFLAG-OFD1a (OFD1a), 3xFLAG-OFD1b (OFD1b), 3xFLAG-OFD1c (OFD1c) overexpressing HEK293 cells were added to GST-ATG13; lysates from 3xFLAG-OFD1(OFD1) overexpressing HEK293 cells were added to GST as control. Right. WB of total lysates (input). All panels display WB for FLAG, HSP90 is the loading control. LIR=LC3 interacting region.


D . Scanning confocal microscopy of wt KO - OFD1 incubated in HBSS ( 8h ) , treated ( + ) or not ( - ) with Baf - A1 ( 50nM , 8h ) . Green , LAMP1 ; red , ATG13 ; blue , Hoechst for nuclei . Scale bar = 6μm . Insets show higher magnification single colour channels of boxed . ( Bottom ) Bar graphs show of lysosomes containing ATG13 expressed as % of total amount of LAMP1 / ( mean ± ) . n = 30 WT , n = 28 KO - OFD1 counted ; three independent experiments .

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D. Scanning confocal microscopy analysis of wt and KO-OFD1 cells incubated in HBSS (8h), treated (+) or not (-) with Baf-A1(50nM, 8h). Green, LAMP1; red, ATG13; blue, Hoechst for nuclei. Scale bar=6μm. Insets show higher magnification and single colour channels of the boxed area. (Bottom) Bar graphs show quantification of lysosomes containing ATG13 expressed as % of total amount of LAMP1/cell (mean ± SEM). n=30 WT cells, n=28 KO-OFD1 cells counted; three independent experiments.


E . Time course of OFD1 in HBSS - starved wt HK2 treated with Baf - A1 ( 100nM ) collected at different time points . LC3B is control for Baf - A1 treatment . ( Right ) Graphs show ACTIN - normalized OFD1 versus FM condition ( - ) expressed as mean ± ; n = 4 independent experiments . STV = starvation , Baf - A1 = bafilomycin , Ctrl = control .

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E. Time course of OFD1 levels in HBSS-starved wt HK2 cells treated with Baf-A1 (100nM) and collected at different time points. LC3B is the control for Baf-A1 treatment. (Right) Graphs show ACTIN-normalized OFD1 levels versus FM condition (-) expressed as mean ± SEM; n=4 independent experiments. STV=starvation, Baf-A1=bafilomycin, Ctrl=control.


F . Western blot of FLAG in KO - OFD1 transfected with 3xFLAG - OFD1 ( OFD1 ) or 3xFLAG - OFD1 ∆ LIR ( OFD1 ∆ LIR ) treated or not ( - ) with Baf - A1 ( 100nM , 2h ) . ( Right ) of GAPDH - normalized FLAG versus untreated ( - ) is expressed as mean ± ; n = 3 independent experiments . Baf - A1 = bafilomycin .

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F. Western blot of FLAG in KO-OFD1 cells transfected with 3xFLAG-OFD1 (OFD1) or 3xFLAG-OFD1∆LIR (OFD1∆LIR) and treated or not (-) with Baf-A1 (100nM,2h). (Right) Quantification of GAPDH-normalized FLAG levels versus untreated (-) is expressed as mean ± SEM; n=3 independent experiments. Baf-A1=bafilomycin.


A . Representative blots of ATG13 ( left ) VPS34 ( right ) phosphorylation in wt KO - OFD1 transiently expressing GFP - ATG13 FLAG - VPS34 in FM .

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A. Representative blots of ATG13 (left) and VPS34 (right) phosphorylation levels in wt and KO-OFD1 cells transiently expressing GFP-ATG13 and FLAG-VPS34 in FM.


B . Wild - type KO - OFD1 were starved in HBSS with / without Baf - A1 ( 100nM , 2h ) chloroquine ( CQ ) ( 50μM , 2h ) total lysates were analyzed by immunoblotting using anti - OFD1 , - LC3B ( LC3B - I 18 kDa LC3B - II 16 kDa ) - ACTIN antibodies . ( Right ) ACTIN - normalized LC3B - II are expressed as fold change versus untreated conditions ( - ) of wt . n = 5 independent experiments .

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B. Wild-type and KO-OFD1 cells were starved in HBSS with/without Baf-A1 (100nM, 2h) and chloroquine (CQ) (50μM, 2h) and total lysates were analyzed by immunoblotting using anti-OFD1, -LC3B (LC3B-I 18 kDa and LC3B-II 16 kDa) and -ACTIN antibodies. (Right) ACTIN-normalized LC3B-II levels are expressed as fold change versus untreated conditions (-) of wt cells. n=5 independent experiments.


C . Representative of LC3B WIPI2 in wt KO - OFD1 ( HBSS , 90min ) . Green , LC3B ; red , WIPI2 ; blue , Hoechst , nuclei . Scale bar = 10μm . ( Right ) of LC3B WIPI2 is shown . ≥ 100 analyzed / sample , n = 5 independent experiments .

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C. Representative images of LC3B and WIPI2 puncta in wt and KO-OFD1 cells (HBSS, 90min). Green, LC3B; red, WIPI2; blue, Hoechst, nuclei. Scale bar=10μm. (Right) Quantification of LC3B and WIPI2 puncta is shown. ≥100 cells analyzed/sample, n=5 independent experiments.


D . Transmission electron microscopy ( left ) ( right ) of autophagic vacuoles ( arrows ) in wt KO - OFD1 ( HBSS , 90min ) .

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D. Transmission electron microscopy (left) and quantification (right) of autophagic vacuoles (arrows) in wt and KO-OFD1 cells (HBSS, 90min).


E . Representative of siOFD1 depleted control ( Ctrl ) HeLa transiently expressing mRFP - eGFP - LC3B ( HBSS , 90min ) . Insets show higher magnification of colocalization in selected areas . Scale bar = 10μm . Bottom , tandem structure of mRFP - GFP - LC3B construct of RFP + GFP + RFP + GFP - in siOFD1 - depleted controls , cultured in full medium ( FM ) or kept in HBSS for 2h ( STV ) are depicted . n ≥ 90 / sample analyzed , n = 5 independent experiments .

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E. Representative images of siOFD1 depleted and control (Ctrl) HeLa cells transiently expressing mRFP-eGFP-LC3B (HBSS, 90min). Insets show higher magnification of colocalization in selected areas. Scale bar=10μm. Bottom, tandem structure of the mRFP-GFP-LC3B construct and quantification of RFP+GFP+ and RFP+GFP- puncta in siOFD1-depleted cells and controls, cultured in full medium (FM) or kept in HBSS for 2h (STV) are depicted. n≥90 cells/sample analyzed, n=5 independent experiments.


F . Representative of WIPI2 in wt HK2 transiently expressing eGFP - OFD1 ( HBSS , 90min ) . Green , eGFP - OFD1 , red , WIPI2 ; blue , Hoechst labels nuclei . On right , of WIPI2 in eGFP - OFD1 transfected compared with non - transfected ( Ctrl ) is shown . Scale bar = 10μm . n ≥ 90 analyzed / sample , n = 3 independent experiments .

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F. Representative images of WIPI2 staining in wt HK2 cells transiently expressing eGFP-OFD1 (HBSS,90min). Green, eGFP-OFD1, red, WIPI2; blue, Hoechst labels nuclei. On the right, quantification of WIPI2 puncta in eGFP-OFD1 transfected cells compared with non-transfected cells (Ctrl) is shown. Scale bar=10μm. n≥90 cells analyzed/sample, n=3 independent experiments.


G . Representative of WIPI2 OFD1 co - in KO - OFD1 transfected with empty vector ( Empty ) , 3xFLAG - OFD1 ( OFD1 ) or 3xFLAG - OFD1 ∆ LIR ( OFD1 ∆ LIR ) ( HBSS , 90min ) . Red , WIPI2 ; green , OFD1 ; blue , Hoechst labels nuclei . Scale bars : 10μm . On right , of WIPI2 . ≥ 100 analyzed / sample , n = 4 independent experiments .

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

G. Representative images of WIPI2 and OFD1 co-staining in KO-OFD1 cells transfected with empty vector (Empty), 3xFLAG-OFD1 (OFD1) or 3xFLAG-OFD1∆LIR (OFD1∆LIR) (HBSS,90min). Red, WIPI2; green, OFD1; blue, Hoechst labels nuclei. Scale bars: 10μm. On the right, quantification of WIPI2 puncta. ≥100 cells analyzed/sample, n=4 independent experiments.


A . Western blot of LC3B in lymphoblasts of OFD type I controls , either untreated ( - ) or exposed to Baf - A1 ( 10nM , 2h ) ( + ) . were cultured in complete medium ( FM , top ) or under serum starvation for 4h ( STV , bottom ) . ACTIN GAPDH were used as loading controls .

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A. Western blot of LC3B in lymphoblasts of OFD type I patients and controls, either untreated (-) or exposed to Baf-A1 (10nM, 2h)(+). Cells were cultured in complete medium (FM, top) or under serum starvation for 4h (STV, bottom). ACTIN and GAPDH were used as loading controls.


B . ( Left ) Graphs show LC3B - II normalized on ACTIN in lymphoblasts controls cultured in complete medium ( FM ) . ( Right ) Graphs show LC3B - II normalized on GAPDH in lymphoblasts controls in serum starvation for 4h ( STV ) . Normalized LC3B - II are expressed as fold change versus untreated conditions ( - ) of control , n = 4 3 controls .

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B. (Left) Graphs show LC3B-II levels normalized on ACTIN in patients lymphoblasts and controls cultured in complete medium (FM). (Right) Graphs show LC3B-II levels normalized on GAPDH in patients lymphoblasts and controls in serum starvation for 4h (STV). Normalized LC3B-II levels are expressed as fold change versus untreated conditions (-) of control cells, n=4 patients and 3 controls.


C . Western blot of ATG13 ATG101 in serum starved lymphoblasts from controls OFD type I . ( Right ) relative to ACTIN ( loading control ) are expressed as fold change versus controls , represented by dashed , n = 4 3 controls .

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C. Western blot of ATG13 and ATG101 proteins in serum starved lymphoblasts from controls and OFD type I patients. (Right) Protein levels relative to ACTIN (loading control) are expressed as fold change versus controls, represented by the dashed line, n=4 patients and 3 controls.


A . Western blot of LC3B in kidney homogenates from leupeptin - treated ( 40mg / kg , 6h ) fasted Ofd1fl / y ; creKsp control mice . ( Right ) LC3B - II normalized to Actin ( loading control ) are expressed as fold increase versus control mice , n = 4 mice / group .

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A. Western blot of LC3B in kidney homogenates from leupeptin-treated (40mg/kg, 6h) fasted Ofd1fl/y;creKsp and control mice. (Right) LC3B-II protein levels normalized to Actin (loading control) are expressed as fold increase versus control mice, n=4 mice/group.


B . Representative of GFP - LC3 ( autophagosomes , green ) in aquaporin2 - positive ( AQP2 , red ) renal distal tubules from P8 Ofd1fl / y ; creKsp ; GFP - LC3 Ofd1fl / y ; GFP - LC3 mice . Scale bar = 10μm . Hoechst labels nuclei . Bar graphs on left show quantitative of GFP - LC3 ; n = 4 mice / group for a total of 300 AQP2 positive nuclei / group analyzed .

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B. Representative images of GFP-LC3 puncta (autophagosomes, green) in aquaporin2-positive (AQP2, red) renal distal tubules from P8 Ofd1fl/y;creKsp;GFP-LC3 and Ofd1fl/y;GFP-LC3 mice. Scale bar=10μm. Hoechst labels nuclei. Bar graphs on the left show quantitative analysis of GFP-LC3 puncta; n=4 mice/group for a total of 300 AQP2 positive nuclei/group analyzed.


C . Western blot of LC3B in kidney homogenates from leupeptin - treated ( 40mg / kg , 6h ) fasted Ofd1 - IND control mice . ( Right ) LC3B - II relative to βTubulin ( loading control ) are expressed as fold increase compared with control mice , n = 5 mutant / 8 control mice .

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C. Western blot of LC3B in kidney homogenates from leupeptin-treated (40mg/kg, 6h) fasted Ofd1-IND and control mice. (Right) LC3B-II protein levels relative to βTubulin (loading control) are expressed as fold increase compared with control mice, n=5 mutant/8 control mice .


D . Western blot of Atg13 in Ofd1fl / y ; creKsp control kidneys at pre - cystic ( P8 , left ) cystic ( P22 , right ) stages . Gapdh was used as loading control . Atg13 are expressed as fold change compared with control mice ; n = 7 mutant / 5 control mice at P8 , n = 4mice / group at P22 .

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D. Western blot of Atg13 in Ofd1fl/y;creKsp and control kidneys at pre-cystic (P8, left) and cystic (P22, right) stages. Gapdh was used as loading control. Atg13 protein levels are expressed as fold change compared with control mice; n=7 mutant/5 control mice at P8, n= 4mice/group at P22.


E . Haematoxylin & eosin of kidney sections from Ofd1fl / y ; Atg7 + / + ; creKsp Ofd1fl / y ; Atg7fl / fl ; creKsp mice . ( Right ) Graph shows fold change of cysts number observed in Ofd1fl / y ; Atg7fl / fl ; creKsp compared with Ofd1fl / y ; Atg7 + / + ; creKsp ; n = 12 sections / were analyzed ; n = 6 mice / group . Scale bars = 1mm .

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E. Haematoxylin & eosin staining of kidney sections from Ofd1fl/y;Atg7+/+;creKsp and Ofd1fl/y;Atg7fl/fl;creKsp mice. (Right) Graph shows fold change of the cysts number observed in Ofd1fl/y;Atg7fl/fl;creKsp animals compared with Ofd1fl/y;Atg7+/+;creKsp; n=12 sections/animal were analyzed; n=6 mice/group. Scale bars=1mm.


F . Blood Urea Nitrogen was quantified on blood withdrawn from Ofd1fl / y ; Atg7fl / fl ; creKsp , Ofd1fl / y ; Atg7 + / + ; creKsp control mice at P45 . n = 8 mice per group .

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F. Blood Urea Nitrogen was quantified on blood withdrawn from Ofd1fl/y;Atg7fl/fl;creKsp, Ofd1fl/y;Atg7+/+;creKsp and control mice at P45. n=8 mice per group.


Agar plugs containing actively growing cultures of wild type S . sclerotiorum ( 1980 ) OA deficient A2 mutant were inoculated onto Col - 0 ced - 9 expressing Arabidopsis leaves . ( A ) Wild type inoculations onto Col - 0 resulted in typical lesions for this pathogen including a rapid , spreading ; however , infection was completely suppressed in ced - 9 expressing . expression of this gene had no effect on A2 phenotype .

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Agar plugs containing actively growing cultures of wild type S. sclerotiorum (strain 1980) and the OA deficient A2 mutant were inoculated onto Col-0 and ced-9 expressing Arabidopsis leaves. (A) Wild type inoculations onto Col-0 plants resulted in typical lesions for this pathogen including a rapid, spreading cell death; however, infection was completely suppressed in ced-9 expressing plants. The expression of this gene had no effect on the A2 phenotype.


( B ) Trypan blue indicates extent of for each genotype / combination , including an agar plug control . were recorded 48 hours post inoculation .

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(B) Trypan blue staining indicates the extent of cell death for each genotype/strain combination, including an agar plug control. All images were recorded 48 hours post inoculation.


Microscopic examination of cross sections of tomato leaves at leading edge of lesion following fungal inoculation . S . sclerotiorum A2 ( A ) wild type ( B ) strains were inoculated onto tomato leaves using colonized agar plugs . 24 hours post inoculation ; leaves were post - fixed in osmium tetroxide , embedded in Spurr ' s epoxy resin . A microtome was used to cut 400 nm sections . Toluidine blue stain was used to reveal fungal hyphae . H = hyphae . dotted represents leading edge of visible lesion . were collected using an Olympus DP 70 camera processed with Olympus DP Controller software , version 2 . 2 . 1 . 227 .

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Microscopic examination of cross sections of tomato leaves at the leading edge of the lesion following fungal inoculation.S. sclerotiorum A2 (A) and wild type (B) strains were inoculated onto tomato leaves using colonized agar plugs. 24 hours post inoculation; leaves were post-fixed in osmium tetroxide, and embedded in Spurr's epoxy resin. A microtome was used to cut 400 nm sections. Toluidine blue stain was used to reveal fungal hyphae. H = hyphae. The dotted line represents the leading edge of the visible lesion. Images were collected using an Olympus DP 70 camera and processed with Olympus DP Controller software, version 2.2.1.227.


S . sclerotiorum A2 induces autophagic structures in . S . sclerotiorum wild type A2 strains were inoculated onto tomato leaves using colonized agar plugs . 24 hours post inoculation ; leaves were stained with 100 µM final of MDC ( Sigma ) in PBS for 30 min . was visualized using an Olympus IX81 inverted confocal microscope ( Olympus systems , Germany ) , with an excitation wavelength of 335 nm an emission wavelength of 508 nm . were collected using an Olympus DP 70 camera processed with Olympus DP Controller software , version 2 . 2 . 1 . 227 . Scale bar = 10 µm .

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S. sclerotiorum A2 strain induces autophagic structures in plants.S. sclerotiorum wild type and A2 strains were inoculated onto tomato leaves using colonized agar plugs. 24 hours post inoculation; leaves were stained with 100 µM final concentration of MDC (Sigma) in PBS for 30 min. Fluorescence was visualized using an Olympus IX81 inverted fluorescence confocal microscope (Olympus systems, Germany), with an excitation wavelength of 335 nm and an emission wavelength of 508 nm. Images were collected using an Olympus DP 70 camera and processed with Olympus DP Controller software, version 2.2.1.227. Scale bar = 10 µm.


Transmission Electron Microscopy ( ) fungal inoculated tomato leaves . Representative from four independent experiments . ( A , G ) ; Healthy non - inoculated leaf tissue . ( B - F ) Tomato leaves inoculated with OA deficient A2 . ( H , I ) Tomato leaves inoculated with wild type S . sclerotiorum . Arrows , autolysosomal / autophagosomal - like structures ; C , chloroplast ; V , vacuole ; N , nucleus ; Circle , active dismantlement of chloroplast ; Rectangle , condensation within nucleus . Black scale bars = 2 µm , scale bars = 1 µm . Sections were examined with a Phillips Morgagni 268 transmission electron microscope at an accelerating voltage of 80 kV . Digital were recorded with a MegaViewIII digital camera operated with iTEM software .

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Transmission Electron Microscopy (TEM) fungal inoculated tomato leaves.Representative TEM images from four independent experiments. (A, G); Healthy non-inoculated leaf tissue. (B-F) Tomato leaves inoculated with the OA deficient A2 strain. (H,I) Tomato leaves inoculated with wild type S. sclerotiorum. Arrows, autolysosomal/autophagosomal-like structures; C, chloroplast; V, vacuole; N, nucleus; Circle, active dismantlement of chloroplast; Rectangle, chromatin condensation within the nucleus. Black scale bars = 2 µm, white scale bars = 1 µm. Sections were examined with a Phillips Morgagni 268 transmission electron microscope at an accelerating voltage of 80 kV. Digital images were recorded with a MegaViewIII digital camera operated with iTEM software.


( A , B ) Agar plugs containing actively growing cultures of OA deficient A2 mutant were inoculated onto leaves of Arabidopsis Col - 0 select Arabidopsis autophagy mutant . These mutants showed enhanced susceptibility to normally non - pathogenic A2 . Lesion diameter was monitored over time were recorded 48 hours post inoculation .

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(A,B) Agar plugs containing actively growing cultures of the OA deficient A2 mutant were inoculated onto leaves of Arabidopsis Col-0 and select Arabidopsis autophagy mutant plants. These mutants showed enhanced susceptibility to the normally non-pathogenic A2 strain. Lesion diameter was monitored over time and all images were recorded 48 hours post inoculation.


( C ) Tomato leaves were either pre - infiltrated with water ( control ) or autophagy inhibitors Wortmannin , LY294002 , Chloroquine ( CQ ) , 3 - methyladenine ( 3 - MA ) . Agar plugs containing actively growing A2 were placed on infiltrated leaves to initiate infection . ( B ) 48 hours post inoculation ; Trypan blue was used to determine extent of fungal colonization cleared with acetic acid ethanol ( 1 : 3 , v / v ) . were taken 48 hours post inoculation .

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(C) Tomato leaves were either pre-infiltrated with water (control) or autophagy inhibitors Wortmannin, LY294002, Chloroquine (CQ), and 3-methyladenine (3-MA). Agar plugs containing actively growing A2 were placed on the infiltrated leaves to initiate infection. (B) 48 hours post inoculation; Trypan blue was used to determine the extent of fungal colonization and cleared with acetic acid and ethanol (1: 3, v/v). Images were taken 48 hours post inoculation.


( A ) NBT treated Arabidopsis ( Col - 0 two independent atg8a mutant lines ) following agar plug inoculation with A2 mutant . were collected 48 hours post inoculation . Dotted lines represent edge of observable legion .

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(A) NBT treated Arabidopsis (Col-0 and two independent atg8a mutant lines) following agar plug inoculation with the A2 mutant. Images were collected 48 hours post inoculation. Dotted lines represent the edge of the observable legion.


( B ) RT - PCR was used to evaluate transcript of three catalases ( CAT1 , 2 , 3 ) three superoxide dismutases in Col - 0 following inoculation with wild type S . sclerotiorum ( black bars ) A2 mutant ( grey bars ) . * > 2 fold change , * * > 5 fold change .

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(B) RT-PCR was used to evaluate the transcript levels of three catalases (CAT1, 2, and 3) and three superoxide dismutases in Col-0 plants following inoculation with wild type S. sclerotiorum (black bars) and the A2 mutant strain (grey bars). *>2 fold change, **>5 fold change.


( A , B ) Representative of expression of NEDD4L in skins from IMQ - treated mice ( A ) or psoriasis ( B ) as detected by on left . NEDD4L expression level was calculated by multiplying score extent score is shown on right . ( A , n = 5 , B , normal , n = 15 , psoriasis , n = 36 ) . Data information : Data are shown as mean ± s . e . m . , are representative of three independent experiments Scale bar , 200 μm ( 100 × ) or 50 μm ( 400 × ) . Significant differences were tested using a two - Student ' s t * * P ＜ 0 . 01 , * * * P ＜ 0 . 001 . NS , no significance .

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(A, B) Representative images of the expression of NEDD4L in the skins from IMQ-treated mice (A) or psoriasis patients (B) as detected by IHC on the left. The NEDD4L expression level was calculated by multiplying the staining intensity score and the extent score and is shown on the right. (A, n=5, B, normal, n=15, psoriasis, n=36). Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments Scale bar, 200 μm (100×) or 50 μm (400×). Significant differences were tested using a two-tailed Student's t test ** P＜0.01, *** P＜0.001. NS, no significance.


( C , D ) Representative of expression of EZH2 in skin epidermis from IMQ - treated mice ( C ) psoriasis ( D ) . number of EZH2 - positive per HPF ( high power field ) in each mouse is shown on right . ( C , control , n = 3 , IMQ , n = 6 . D , normal , n = 13 , psoriasis , n = 27 ) . Data information : Data are shown as mean ± s . e . m . , are representative of three independent experiments Scale bar , 200 μm ( 100 × ) or 50 μm ( 400 × ) . Significant differences were tested using a two - Student ' s t * * P ＜ 0 . 01 , * * * P ＜ 0 . 001 . NS , no significance .

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(C, D) Representative images of the expression of EZH2 in the skin epidermis from the IMQ-treated mice (C) and the psoriasis patients (D). The number of EZH2-positive cells per HPF (high power field) in each mouse and patient is shown on the right. (C, control, n=3, IMQ, n=6. D, normal, n=13, psoriasis, n=27). Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments Scale bar, 200 μm (100×) or 50 μm (400×). Significant differences were tested using a two-tailed Student's t test ** P＜0.01, *** P＜0.001. NS, no significance.


( E ) WB of NEDD4L , EZH2 EZH1 expression in EZH2 - specific siRNA ( siEZH2 ) - or negative control siRNA ( siNC ) - transfected NHEKs with IMQ stimulation . Data information : Data are shown as mean ± s . e . m . , are representative of three independent experiments Scale bar , 200 μm ( 100 × ) or 50 μm ( 400 × ) .

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(E) WB analysis of NEDD4L, EZH2 and EZH1 expression in the EZH2-specific siRNA (siEZH2)- or negative control siRNA (siNC)-transfected NHEKs with IMQ stimulation. Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments Scale bar, 200 μm (100×) or 50 μm (400×).


( D ) Representative of HE of back tissues are shown on left . epidermal thickness measured using scale in microscope is shown on right . ( E ) Representative of Ki67 in back tissues are shown on left . number of Ki67 - positive per HPF in each mouse is shown on right . ( F ) Representative of HE of ear tissues are shown on left . epidermis thickness measured using scale in microscope is shown on right . ( G ) Representative of Ki67 in ear tissues ( same specimen as in F ) are shown on left . number of Ki67 positive per HPF in each mouse is shown on right . Data information : Data are shown as mean ± s . e . m . , are representative of three independent experiments . For back tissues , scale bar , 200 μm ( 100 × ) or 50 μm ( 400 × ) . For ear tissues , scale bar , 200 μm ( 100 × ) or 200 μm ( 200 × ) . Significant differences were tested using a two - Student ' s t Control , n = 3 , IMQ , n = 6 . * * P ＜ 0 . 01 , * * * P ＜ 0 . 001 . NS , no significance .

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(D) Representative images of HE staining of the back tissues are shown on the left. The epidermal thickness measured using the scale in the microscope is shown on the right. (E) Representative images of Ki67 staining in back tissues are shown on the left. The number of Ki67-positive cells per HPF in each mouse is shown on the right. (F) Representative images of HE staining of the ear tissues are shown on the left. The epidermis thickness measured using the scale in the microscope is shown on the right. (G) Representative images of Ki67 staining in ear tissues (same specimen as in F) are shown on the left. The number of Ki67 positive cells per HPF in each mouse is shown on the right. Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments. For back tissues, scale bar, 200 μm (100×) or 50 μm (400×). For ear tissues, scale bar, 200 μm (100×) or 200 μm (200×). Significant differences were tested using a two-tailed Student's t test Control, n=3, IMQ, n=6. ** P＜0.01, *** P＜0.001. NS, no significance.


NEDD4L was knocked out by CRISPR Cas9 method in NHEKs . WT NEDD4L KO NHEKs were infected with lentivirus overexpressing NEDD4L - WT or NEDD4L - C943S mutants . signaling pathway activation induced by IL - 6 were detected by WB analyses Data information : Data are shown as mean ± s . e . m . , are representative of three independent

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NEDD4L was knocked out by the CRISPR Cas9 method in NHEKs. WT and NEDD4L KO NHEKs were infected with lentivirus overexpressing NEDD4L-WT or NEDD4L-C943S mutants. cell signaling pathway activation induced by IL-6 were detected by WB analyses Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments


( G , H ) keratinocytes were separated from WT Nedd4l KO mice . ( G ) WB of expression in IL - 6 - induced signaling pathway activation . ( H ) WB of in IL - 6 signaling pathway . Data information : Data are shown as mean ± s . e . m . , are representative of three independent

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(G, H) Primary keratinocytes were separated from the WT and Nedd4l KO mice. (G) WB analysis of the protein expression in IL-6-induced cell signaling pathway activation. (H) WB analysis of the proteins in the IL-6 signaling pathway. Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments


Six - to eight - old WT Nedd4l cKO mice were treated with IMQ for 6 consecutive days at beginning of treatment , mice were injected intra - epidermally with 10 μg anti - GP130 antibody or with same dosage of isotype on every other day . Representative of Ki67 in back tissues are on left . number of Ki67 - positive per HPF is shown on right . Data information : Data are shown as mean ± s . e . m . , are representative of two independent experiments . Scale bar , 200 μm ( 100 × ) or 50 μm ( 400 × ) . Significant differences were tested using a two - Student ' s t C , control , n = 5 , IMQ , n = 6 . * P ＜ 0 . 05 , * * P ＜ 0 . 01 , * * * P ＜ 0 . 001 . NS , no significance .

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Six- to eight-weeks old WT and Nedd4l cKO mice were treated with IMQ for 6 consecutive days and at the beginning of the treatment, the mice were injected intra-epidermally with 10 μg anti-GP130 antibody or with same dosage of isotype on every other day. Representative images of Ki67 staining in back tissues are on the left. The number of Ki67-positive cells per HPF is shown on the right. Data information: Data are shown as mean ± s.e.m., and are representative of two independent experiments. Scale bar, 200 μm (100×) or 50 μm (400×). Significant differences were tested using a two-tailed Student's t test C, control, n=5, IMQ, n=6. * P＜0.05, ** P＜0.01, *** P＜0.001. NS, no significance.


Six - to eight - old WT Nedd4l cKO mice were treated with IMQ for 6 consecutive days at beginning of treatment , mice were injected intra - epidermally with 10 μg anti - GP130 antibody or with same dosage of isotype on every other day . Keratinocyte proliferation associated genes in skin tissues are detected using real - time PCR . Data information : Data are shown as mean ± s . e . m . , are representative of two independent experiments . Scale bar , 200 μm ( 100 × ) or 50 μm ( 400 × ) . Significant differences were tested using a two - Student ' s t D , control , n = 3 , IMQ , n = 6 . * P ＜ 0 . 05 , * * P ＜ 0 . 01 , * * * P ＜ 0 . 001 . NS , no significance .

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Six- to eight-weeks old WT and Nedd4l cKO mice were treated with IMQ for 6 consecutive days and at the beginning of the treatment, the mice were injected intra-epidermally with 10 μg anti-GP130 antibody or with same dosage of isotype on every other day. Keratinocyte proliferation associated genes in the skin tissues are detected using real-time PCR analysis. Data information: Data are shown as mean ± s.e.m., and are representative of two independent experiments. Scale bar, 200 μm (100×) or 50 μm (400×). Significant differences were tested using a two-tailed Student's t test D, control, n=3, IMQ, n=6. * P＜0.05, ** P＜0.01, *** P＜0.001. NS, no significance.


( A ) Representative pictures of expression of NEDD4L , GP130 p - STAT3 in epidermis of a normal skin section ( same specimen ) sections from psoriasis . Isotype are IgG antibodies from same species served as negative control . ( B ) Semiquantitative scoring of NEDD4L , GP130 p - STAT3 in epidermis detected by . Data information : Each symbol represents one Scale bar , 200 μm ( 100 × ) or 50 μm ( 400 × ) . data are shown as mean ± s . e . m . , significant differences were analyzed using two - Student ' s t ( normal , n = 15 , psoriasis , n = 36 ) . * P ＜ 0 . 05 , * * P ＜ 0 . 01 , * * * P ＜ 0 . 001 .

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(A) Representative pictures of the expression of NEDD4L, GP130 and p-STAT3 in the epidermis of a normal skin section (same specimen) and sections from psoriasis patients. Isotype are IgG antibodies from same species served as negative control. (B) Semiquantitative scoring of NEDD4L, GP130 and p-STAT3 in the epidermis detected by IHC. Data information: Each symbol represents one patient Scale bar, 200 μm (100×) or 50 μm (400×). All data are shown as the mean ± s.e.m., and significant differences were analyzed using two-tailed Student's t test (normal, n=15, psoriasis, n=36). * P＜0.05, ** P＜0.01, *** P＜0.001.


( F ) Full Flag - GP130 , GP130 - aa 616 - 918 , GP130 - aa 616 - 889 , GP130 - aa 616 - 764 , GP130 - aa 616 - 734 were transfected with Myc - NEDD4L into HEK293T , whole lysates were co - IP with ANTI - FLAG M2 magnetic beads , followed by WB with anti - Flag or anti - Myc antibodies .

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(F) Full length Flag-GP130, GP130-aa 616-918, GP130-aa 616-889, GP130-aa 616-764, GP130-aa 616-734 were transfected with Myc-NEDD4L into HEK293T cells, and whole cell lysates were co-IP with ANTI-FLAG M2 magnetic beads, followed by WB with anti-Flag or anti-Myc antibodies.


( C ) WT or NEDD4L KO NHEK was infected with lentivirus that overexpress NEDD4L - WT , NEDD4L WW domain truncated mutant ( NEDD4L - ΔWW ) or its enzymatic activity mutant ( NEDD4L - C943S ) , were followed by denaturation - IP with anti - GP130 antibody WB with anti - Ub antibodies .

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(C) WT or NEDD4L KO NHEK cells was infected with lentivirus that overexpress NEDD4L-WT, NEDD4L WW domain truncated mutant (NEDD4L-ΔWW) or its enzymatic activity mutant (NEDD4L-C943S), the cells were followed by denaturation-IP with anti-GP130 antibody and WB analysis with anti-Ub antibodies.


( E ) WB of polyubiquitination of GP130 in HEK293T co - transfected with full Myc - NEDD4L , HA - Ub Flag - GP130 or its lysine mutants ( K - R ) in intracellular domain , followed by denaturation - IP with ANTI - FLAG M2 magnetic beads WB with anti - HA antibodies .

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(E) WB analysis of the polyubiquitination of GP130 in HEK293T cells co-transfected with full length Myc-NEDD4L, HA-Ub and Flag-GP130 or its lysine mutants (K-R) in the intracellular domain, followed by denaturation-IP with ANTI-FLAG M2 magnetic beads and WB analysis with anti-HA antibodies.


( A - D ) serial dilutions of post - second immunization serum samples were analyzed by ELISA using plates coated with purified RSV rA2 - Line19F - FFL virions . serum samples were serially diluted , detection of antibodies were measured by optical density ( OD ) at 490 nm . End - point titers of serum samples were determined as reciprocal of highest dilution providing an absorbance twice that of negative control ( PBS immunized ) .

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(A-D) The serial dilutions of post-second immunization serum samples were analyzed by ELISA using plates coated with purified RSV rA2-Line19F-FFL virions. The serum samples were serially diluted, and the detection of antibodies were measured by optical density (OD) at 490 nm. End-point titers of the serum samples were determined as the reciprocal of the highest dilution providing an absorbance twice that of the negative control (PBS immunized animal).


( A ) Lung RSV titers ( PFU / gram of lung tissue ) were determined in lungs ( n = 5 mice per group ) collected at 5 days post - RSV infection . Results were determined presented as box whisker plots , where boxes extends from 25th to 75th percentile , whiskers show minimum to maximum value central band represents median value for group .

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(A) Lung RSV titers (PFU/gram of lung tissue) were determined in individual lungs (n = 5 mice per group) collected at 5 days post-RSV infection. Results were determined and presented as box and whisker plots, where boxes extends from 25th to 75th percentile, whiskers show minimum to maximum value and central band represents the median value for the group.


( A ) Serum samples collected from mouse ( M 1 - 5 ) immunized with F 110 - 136 peptide were tested for antibody binding against F - p27 ( 110 - 136 ) peptide or F 1 - 34 peptide ( control ) in ELISA .

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(A) Serum samples collected from individual mouse (M 1-5) immunized with F 110-136 peptide were tested for antibody binding against F-p27 (110-136) peptide or F 1-34 peptide (control) in ELISA.


( B ) A549 were infected with RSV ( MOI = 0 . 1 ) for 16 h , fixed . were treated with mock control rabbit sera ( left panels ) , or rabbit anti - sera against F ( center panels ) , or against F - p27 ( 110 - 136 ) peptide ( right panels ) , followed by Alexa 594 conjugated anti - Rabbit IgG ( red ) . Nuclei are stained with DAPI ( blue ) . Scale bar = 10 µm . number of positive for RSV - F ( middle panel ) RSV - p27 ( right panel ) upon counting of 200 were used to calculate of stained for each antibody are shown in ' merge ' panel . experiments were performed twice variation between two independent expeiremnts was < 6 % .

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(B) A549 cells were infected with RSV (MOI = 0.1) for 16 h, and fixed. Cells were treated with mock control rabbit sera (left panels), or rabbit anti-sera against F (center panels), or against F-p27 (110-136) peptide (right panels), followed by Alexa 594 conjugated anti-Rabbit IgG (red). Nuclei are stained with DAPI (blue). Scale bar = 10 µm. The number of cells positive for RSV-F (middle panel) and RSV-p27 (right panel) upon counting of 200 cells were used to calculate percentage of cells stained for each antibody staining are shown in the 'merge' panel. The experiments were performed twice and variation between the two independent expeiremnts was <6%.