H , I , Immunostaining for Tbr2 ( red ) Ngn1 ( green ) in Gpr124KO cortices after exposure of pregnant dams to 21 % O2 ( H ) or 80 % O2 ( I ) . J - L of neurogenic ( Ngn1 + ) RGs BPs ( J ) , newborn BPs ( K ) or expanding ( Tbr2 - Ngn1 - ) RGs ( L ) in Gpr124KO cortices after exposure of pregnant dams to 21 % O2 or 80 % O2 ( mean ± ; N = 4 for 21 % O2 N = 5 for 80 % O2 ; * p < 0 . 05 , * * p < 0 . 01 ) . M , N , Immunostaining for Tbr2 ( red ) Ngn1 ( green ) in control cortices after exposure of pregnant dams to 21 % O2 ( M ) or 80 % O2 ( N ) . O - Q of neurogenic ( Ngn1 + ) RGs BPs ( O ) , newborn BPs ( P ) or expanding ( Tbr2 - Ngn1 - ) RGs ( Q ) in control cortices after exposure of pregnant dams to 21 % O2 or 80 % O2 ( mean ± ; N = 5 ; * * p < 0 . 01 ) . Full , dotted dashed lines indicate basal apical boundaries of cortex or boundaries of cortical zones , respectively . CP , cortical plate ; IZ , intermediate zone ; SVZ , subventricular zone ; VZ , ventricular zone . Scale bar : 100

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

H,I, Immunostaining for Tbr2 (red) and Ngn1 (green) in Gpr124KO cortices after exposure of the pregnant dams to 21% O2 (H) or 80% O2 (I). J-L Quantification of neurogenic (Ngn1+) RGs and BPs (J), newborn BPs (K) or expanding (Tbr2- Ngn1-) RGs (L) in Gpr124KO cortices after exposure of the pregnant dams to 21% O2 or 80% O2 (mean±SEM; N=4 for 21% O2 and N=5 for 80% O2; * p<0.05, ** p<0.01). M,N, Immunostaining for Tbr2 (red) and Ngn1 (green) in control cortices after exposure of the pregnant dams to 21% O2 (M) or 80% O2 (N). O-Q Quantification of neurogenic (Ngn1+) RGs and BPs (O), newborn BPs (P) or expanding (Tbr2-Ngn1-) RGs (Q) in control cortices after exposure of the pregnant dams to 21% O2 or 80% O2 (mean±SEM; N=5; ** p<0.01). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm


A , qRT - PCR of glycolytic enzymes in Prom1 + VZ their Prom1 - differentiated progeny ( BPs neurons ) in WT cortices at E14 . 5 ( mean ± ; N = 3 ( Hk2 , Gapdh ) ; N = 6 ( Eno1 ; Ldha ; * * p < 0 . 01 ) .

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

A, qRT-PCR analysis of glycolytic enzymes in Prom1+ VZ cells and their Prom1- differentiated progeny (BPs and neurons) in WT cortices at E14.5 (mean±SEM; N=3 (Hk2, Gapdh); N=6 (Eno1; Ldha; ** p<0.01).


B , Measurement of lactate secretion from proliferating NSCs , of NSCs differentiating for 3 days ( switch to neurogenesis ) or for 7 days ( fully differentiated ) , normalized to content ( mean ± ; N = 6 ; * * * p < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Measurement of lactate secretion from proliferating NSCs, and of NSCs differentiating for 3 days (switch to neurogenesis) or for 7 days (fully differentiated), normalized to cellular protein content (mean±SEM; N=6; *** p<0.001).


C , qRT - PCR of Pfkfb3 expression in proliferating NSCs , transduced with scr or Pfkfb3shRNA # 1 ( mean ± ; N = 4 ; * p < 0 . 05 ) .

O O O O O O B-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

C, qRT-PCR analysis of Pfkfb3 expression in proliferating NSCs, transduced with scr or Pfkfb3shRNA#1 (mean±SEM; N=4; * p<0.05).


D , Measurement of lactate secretion from proliferating NSCs transduced with scr or Pfkfb3shRNA # 1 , normalized to content ( mean ± ; N = 3 ; * p < 0 . 05 ) .

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

D, Measurement of lactate secretion from proliferating NSCs transduced with scr or Pfkfb3shRNA#1, normalized to cellular protein content (mean±SEM; N=3; * p<0.05).


E , Measurement of mitochondrial respiration rate ( oxygen consumption rate , OCRMITO ; see supplemental methods ) in proliferating NSCs , transduced with scr or Pfkfb3shRNA # 1 , normalized to content ( mean ± ; N = 3 ; 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 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

E, Measurement of mitochondrial respiration rate (oxygen consumption rate, OCRMITO; see supplemental methods) in proliferating NSCs, transduced with scr or Pfkfb3shRNA#1, normalized to cellular protein content (mean±SEM; N=3; N.S., not significant).


F - H , Epifluorescent of E15 . 5 cortex , 3 days after in utero electroporation ( IUE ) with EGFP + scr shRNA ( F ) , EGFP + Pfkfb3 shRNA # 1 ( G ) or EGFP + Pfkfb3 shRNA # 2 ( H ) . J , of EGFP + in cortical zones VZ / SVZ , IZ CP shown in panels F - H ( mean ± ; N = 4 for scr N = 5 for Pfkfb3 shRNAs ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) . K - M , Epifluorescent of mouse cortex after in utero electroporation ( IUE ) with HIF - 1α scr shRNA ( K ) or with HIF - 1α two different Pfkfb3 shRNAs ( L , M ) at E16 . 5 , 3 days after transfection . N , of EGFP + in cortical zones VZ / SVZ , IZ CP shown in K - M ( mean ± ; N = 5 ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) . Full , dotted dashed lines indicate basal apical boundaries of cortex or boundaries of cortical zones , respectively . CP , cortical plate ; IZ , intermediate zone ; SVZ , subventricular zone ; VZ , ventricular zone . Scale bar : 100

O O O O O O O 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-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 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 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 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 O O O O O O O O O O O O O O O O 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-H, Epifluorescent images of the E15.5 cortex, 3 days after in utero electroporation (IUE) with EGFP + scr shRNA (F), EGFP + Pfkfb3 shRNA#1 (G) or EGFP + Pfkfb3 shRNA#2 (H). J, Quantification of EGFP+ cells in the cortical zones VZ/SVZ, IZ and CP shown in panels F-H (mean±SEM; N=4 for scr and N=5 for Pfkfb3 shRNAs; * p<0.05, ** p< 0.01, *** p< 0.001). K-M, Epifluorescent images of the mouse cortex after in utero electroporation (IUE) with HIF-1α and scr shRNA (K) or with HIF-1α and two different Pfkfb3 shRNAs (L,M) at E16.5, 3 days after transfection. N, Quantification of EGFP+ cells in the cortical zones VZ/SVZ, IZ and CP shown in K-M (mean±SEM; N=5; * p<0.05, ** p< 0.01, *** p< 0.001). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm


A Affinity measurements by biolayer interferometry ( BLI ) of bipNbs NM1267 NM1268 . NM1267 NM1268 were applied with concentrations ranging from 5 - 0 . 625 nM 20 ‑ 2 . 5 nM , respectively ( illustrated with gradually lighter shades ) on immobilized RBD derived from SARS - CoV - 2 B . 1 ( RBDB . 1 ) . Global 1 : 1 fits are illustrated as dashed lines . Global 1 : 1 fits are illustrated as dashed lines binding affinity ( KD ) , association ( kon ) dissociation constant ( koff ) determined for bipNbs are summarized .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Affinity measurements by biolayer interferometry (BLI) of bipNbs NM1267 and NM1268. NM1267 and NM1268 were applied with concentrations ranging from 5-0.625 nM and 20‑2.5 nM, respectively (illustrated with gradually lighter shades) on immobilized RBD derived from SARS-CoV-2 B.1 (RBDB.1). Global 1:1 fits are illustrated as dashed lines. Global 1:1 fits are illustrated as dashed lines and binding affinity (KD), association (kon) and dissociation constant (koff) determined for the individual bipNbs are summarized.


A - P Affinity measurements by BLI of bipNb NM1267 NM1268 on recently identified RBD variants B . 1 . 1 . 7 ( Alpha ) ( A , I ) , B . 1 . 351 ( Beta ) ( B , J ) , P1 ( Gamma ) ( C , K ) , B . 1 . 617 . 2 ( Delta ) ( D , L ) , B . 1 . 429 ( Epsilon ) ( E , M ) , P3 ( Theta ) ( F , N ) , B . 1 . 617 . 1 ( Kappa ) ( G , O ) A . 23 . 1 ( H , P ) . NM1267 NM1268 were applied with concentrations ranging from 5 - 0 . 625 nM 20 ‑ 2 . 5 nM , respectively ( illustrated with gradually lighter shades ) on immobilized RBD variants . Global 1 : 1 fits are illustrated as dashed lines .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-P Affinity measurements by BLI of bipNb NM1267 and NM1268 on recently identified RBD variants B.1.1.7 (Alpha) (A, I), B.1.351 (Beta) (B, J), P1 (Gamma) (C, K), B.1.617.2 (Delta) (D, L), B.1.429 (Epsilon) (E, M), P3 (Theta) (F, N), B.1.617.1 (Kappa) (G, O) and A.23.1 (H, P). NM1267 and NM1268 were applied with concentrations ranging from 5-0.625 nM and 20‑2.5 nM, respectively (illustrated with gradually lighter shades) on immobilized RBD variants. Global 1:1 fits are illustrated as dashed lines.


A - F Neutralization potency of NM1267 NM1268 were analyzed in Caco ‑ 2 using SARS - CoV - 2 B . 1 ( A , D ) SARS - CoV - 2 B . 1 . 351 ( Beta ) ( B , E ) SARS - CoV - 2 B . 1 . 617 . 2 ( Delta ) ( C , F ) . Infection normalized to - only infection control is illustrated as of infection ( infection [ % ] ) . Data are presented as mean ± of three ( n = 3 ) biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F Neutralization potency of NM1267 and NM1268 were analyzed in Caco‑2 cells using the SARS-CoV-2 B.1 (A, D) SARS-CoV-2 B.1.351 (Beta) (B, E) and SARS-CoV-2 B.1.617.2 (Delta) (C, F). Infection normalized to virus-only infection control is illustrated as percent of infection (infection [%]). Data are presented as mean ± SEM of three (n = 3) biological replicates.


C Hemizygous K18 - hACE2 mice were treated intranasally with 20 µg of NM1251 ( n = 15 ) or NM1267 ( n = 12 ) seven hours prior to infection with 3 * 103 PFU SARS - CoV - 2 B . 1 . survival were monitored for 14 days . , * * * * P < 0 . 0001 , by log - rank

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Hemizygous K18-hACE2 mice were treated intranasally with 20 µg of NM1251 (n = 15) or NM1267 (n = 12) seven hours prior to infection with 3*103 PFU SARS-CoV-2 B.1. survival were monitored for 14 days. , ****P < 0.0001, by log-rank test


D Nasal swabs were collected from six mice per group ( n = 6 ) at indicated time points viral load was determined by plaque - . Bars represent mean ± . * * P < 0 . 01 , by unpaired t

O O O O O O O O O O O O O O O O O O O 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 Nasal swabs were collected from six mice per group (n = 6) at the indicated time points and viral load was determined by plaque-assay. Bars represent mean ± SEM. **P < 0.01, by unpaired t test


Histopathological of mice , which were intranasally treated with bipNb NM1267 or control Nb NM1251 subsequently infected with 3 * 103 PFU SARS - CoV - 2 B . 1 . A - B Serial tissue sections revealed severe inflammation ( H & E ) numerous widespread SARS - CoV - 2 positive alveolar epithelia macrophages ( in situ hybridization ( ISH ) ) in lungs of infected , control - treated mice . In infected NM1267 - treated no inflammation or only small focal areas with inflammation a few SARS - CoV - 2 positive were observed . Scale bars represent 1 mm 200 µm , respectively .

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

Histopathological analysis of mice, which were intranasally treated with bipNb NM1267 or control Nb NM1251 and subsequently infected with 3*103 PFU SARS-CoV-2 B.1. A-B Serial tissue sections revealed severe inflammation (H&E) and numerous widespread SARS-CoV-2 RNA positive alveolar epithelia cells and macrophages (in situ hybridization (ISH)) in lungs of infected, control-treated mice. In infected and NM1267-treated animals no inflammation or only small focal areas with inflammation and a few SARS-CoV-2 RNA positive cells were observed. Scale bars represent 1 mm and 200 µm, respectively.


B , Hemizygous K18 - hACE2 mice were treated intranasally with 20 µg of NM1251 ( n = 8 ) , NM1267 ( n = 9 ) or NM1268 ( n = 9 ) seven hours prior to infection with 3 * 103 PFU SARS - CoV - 2 B . 1 . 617 . 2 ( Delta ) . loss were monitored for 14 days . Dashed indicates humane endpoint symbols represent mean ± * * * * P < 0 . 0001 in orange between NM1251 NM1267 in purple between NM1251 NM1268 , * P < 0 . 1 in black between NM1267 NM1268 , by two - way ANOVA with Sidak ' s multiple comparison

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Hemizygous K18-hACE2 mice were treated intranasally with 20 µg of NM1251 (n = 8), NM1267 (n = 9) or NM1268 (n = 9) seven hours prior to infection with 3*103 PFU SARS-CoV-2 B.1.617.2 (Delta). Weight loss were monitored for 14 days. Dashed line indicates humane endpoint and symbols represent mean ± SEM ****P < 0.0001 in orange between NM1251 and NM1267 and in purple between NM1251 and NM1268, *P < 0.1 in black between NM1267 and NM1268, by two-way ANOVA with Sidak's multiple comparison test


C Hemizygous K18 - hACE2 mice were treated intranasally with 20 µg of NM1251 ( n = 8 ) , NM1267 ( n = 9 ) or NM1268 ( n = 9 ) seven hours prior to infection with 3 * 103 PFU SARS - CoV - 2 B . 1 . 617 . 2 ( Delta ) . survival were monitored for 14 days . * * * P < 0 . 001 in orange between NM1251 NM1267 * * * * P < 0 . 0001 in purple between NM1251 NM1268 , by log - rank

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Hemizygous K18-hACE2 mice were treated intranasally with 20 µg of NM1251 (n = 8), NM1267 (n = 9) or NM1268 (n = 9) seven hours prior to infection with 3*103 PFU SARS-CoV-2 B.1.617.2 (Delta). survival were monitored for 14 days. ***P < 0.001 in orange between NM1251 and NM1267 and ****P < 0.0001 in purple between NM1251 and NM1268, by log-rank test


B , C Relative abundance of sgRNAs in populations with high versus low signal were visualized as log2 fold chances for basal as well as activated autophagy conditions for ( B ) p62 or ( C ) NDP52 . Entire data is reported in Dataset EV1

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

B, C Relative abundance of sgRNAs in cell populations with high versus low signal were visualized as log2 fold chances for basal as well as activated autophagy conditions for (B) p62 or (C) NDP52. Entire data is reported in Dataset EV1


D - F Validation of TMEM41B . H4 Cas9 were infected with sgRNAs targeting TMEM41B or a non - targeting ( NT ) control , treated with 500 nM AZD8055 , 50nM Bafilomycin A1 ( BafA1 ) or DMSO vehicle control for 24 h , analyzed by immunoblotting . ( E , F ) p62 NDP52 band intensities are depicted as total relative to vehicle control in H4 Cas9 NT ( E ) , or as flux by calculating in BafA1 - treated versus vehicle control ( F ) . Data are presented as mean ± SD ( n = 6 independent experiments ) with paired t - values

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 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

D-F Validation of TMEM41B. H4 Cas9 cells were infected with sgRNAs targeting TMEM41B or a non-targeting (NT) control, treated with 500 nM AZD8055, 50nM Bafilomycin A1 (BafA1) or DMSO vehicle control for 24 h, and analyzed by immunoblotting. (E, F) p62 and NDP52 band intensities are depicted as total levels relative to vehicle control in H4 Cas9 NT cells (E), or as flux by calculating the ratio in BafA1-treated cells versus vehicle control (F). Data are presented as mean ± SD (n=6 independent experiments) with paired t-test values


G LAMP1 p62 were co - stained imaged using an LSM700 confocal microscope . Arrowheads point at p62 - filled empty LAMP1 - positive organelles . Scale bar : 5 µm

O B-MEASURED_VAR I-MEASURED_VAR 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

G LAMP1 and p62 were co-stained and imaged using an LSM700 confocal microscope. Arrowheads point at p62-filled and empty LAMP1-positive organelles. Scale bar: 5 µm


H LAMP1 - positive organelles were manually quantified for containing a p62 - filled or empty lumen . A total of 50 - 60 LAMP1 - positive organelles were analyzed in each experiment . Data are presented as mean ± SD ( n = 3 independent experiments ) with paired t - values

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H LAMP1-positive organelles were manually quantified for containing a p62-filled or empty lumen. A total of 50-60 LAMP1-positive organelles were analyzed in each experiment. Data are presented as mean ± SD (n=3 independent experiments) with paired t-test values


I HeLa were transduced with Cas9 TMEM41B or NT sgRNAs followed by infection with luciferase - expressing S . typhimurium . Luciferase readings were taken up to 8 . 5 h post - infection . Results from one representative experiment are presented as mean ± SD ( n = 8 technical replicates )

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

I HeLa cells were transduced with Cas9 and TMEM41B or NT sgRNAs followed by infection with luciferase-expressing S. typhimurium. Luciferase readings were taken up to 8.5 h post-infection. Results from one representative experiment are presented as mean ± SD (n=8 technical replicates)


A H4 Cas9 were infected with sgRNAs targeting ATG7 , TMEM41B or NT control , treated with 500 nM AZD8055 or vehicle control for 24 h , analyzed by immunoblotting . B of LC3 - II to LC3 - I band intensities are depicted as mean ± SD ( n = 6 independent experiments ) with Wilcoxon values

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

A H4 Cas9 cells were infected with sgRNAs targeting ATG7, TMEM41B or NT control, treated with 500 nM AZD8055 or vehicle control for 24 h, and analyzed by immunoblotting. B Ratio of LC3-II to LC3-I band intensities are depicted as mean ± SD (n=6 independent experiments) with Wilcoxon test values


C H4 Cas9 TMEM41B NT control were probed by LC3B immunostaining imaged with an automated CV7000 confocal microscope . Scale bar : 20 μm . D number of LC3 per was quantified using Yokogawa Software ( YAS ) depicted as mean ± SD ( n = 3 technical replicates )

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

C H4 Cas9 TMEM41B and NT control cells were probed by LC3B immunostaining and imaged with an automated CV7000 confocal microscope. Scale bar: 20 μm. D The number of LC3 puncta per cell was quantified using Yokogawa Analysis Software (YAS) and depicted as mean ± SD (n=3 technical replicates)


E LC3 WIPI2 were co - stained imaged using an LSM700 confocal microscope . Arrowheads point at co - localized LC3 WIPI2 . Scale bar : 5 µm

O B-MEASURED_VAR I-MEASURED_VAR 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

E LC3 and WIPI2 were co-stained and imaged using an LSM700 confocal microscope. Arrowheads point at co-localized LC3 and WIPI2 puncta. Scale bar: 5 µm


F number of WIPI2 positive for LC3 was quantified using ImageJ depicted as mean ± SD ( n = 5 technical replicates ) . G total number of WIPI2 per was quantified using Harmony software on acquired with an automated Operetta microscope . Data are depicted as mean ± SD ( n = 3 technical replicates )

O O O B-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 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

F The number of WIPI2 puncta positive for LC3 was quantified using ImageJ and depicted as mean ± SD (n=5 technical replicates). G The total number of WIPI2 puncta per cell was quantified using the Harmony software on images acquired with an automated Operetta microscope. Data are depicted as mean ± SD (n=3 technical replicates)


H H4 Cas9 TMEM41B NT control were infected with a RFP - DFCP1 expression construct for 72h , fixed , stained with LC3 antibodies imaged with an automated CV7000 confocal microscope . Arrowheads point at co - localized LC3 RFP - DFCP1 . Scale bar : 5 µm . I number of RFP - DFCP1 positive for LC3 was quantified using ImageJ depicted as mean ± SD ( n = 10 technical replicates )

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 B-MEASURED_VAR O O B-MEASURED_VAR 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

H H4 Cas9 TMEM41B and NT control cells were infected with a RFP-DFCP1 expression construct for 72h, fixed, stained with LC3 antibodies and imaged with an automated CV7000 confocal microscope. Arrowheads point at co-localized LC3 and RFP-DFCP1 puncta. Scale bar: 5 µm. I The number of RFP-DFCP1 puncta positive for LC3 was quantified using ImageJ and depicted as mean ± SD (n=10 technical replicates)


J mCherry - GFP - LC3 was expressed in H4 Cas9 TMEM41B KO NT control which were treated with 50nM BafA1 or vehicle control for 24 h , fixed imaged . Scale bar : 20 μm . number of mCherry - GFP - positive per was quantified using YAS are depicted as mean ± SD ( n = 3 technical replicates )

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J mCherry-GFP-LC3 was expressed in H4 Cas9 TMEM41B KO and NT control cells which were treated with 50nM BafA1 or vehicle control for 24 h, fixed and imaged. Scale bar: 20 μm. The number of mCherry- and GFP-positive puncta per cell was quantified using YAS and are depicted as mean ± SD (n=3 technical replicates)


A H4 Cas9 stably expressing NT , TMEM41B or ATG7 sgRNAs were stained with BODIPY 493 , NBD cholesterol or BODIPY FL C12 probes for 2 h at 37 ° C imaged live with an automated CV7000 confocal microscope . per was quantified using YAS depicted as mean ± SD ( n = 4 technical replicates ) . Scale bar : 20 μm

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A H4 Cas9 cells stably expressing NT, TMEM41B or ATG7 sgRNAs were stained with BODIPY 493, NBD cholesterol or BODIPY FL C12 probes for 2 h at 37°C and imaged live with an automated CV7000 confocal microscope. Puncta area per cell was quantified using YAS and depicted as mean ± SD (n=4 technical replicates). Scale bar: 20 μm


B H4 Cas9 TMEM41B KO NT control were treated overnight with 400 μM BSA - conjugated oleic acid or 0 . 1 % BSA as vehicle control , stained for 2 h with HCS LipidTox Green Neutral Lipid Stain imaged with an automated Operetta microscope . Scale bar : 20 μm

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B H4 Cas9 TMEM41B KO and NT control cells were treated overnight with 400 μM BSA-conjugated oleic acid or 0.1% BSA as vehicle control, stained for 2 h with HCS LipidTox Green Neutral Lipid Stain and imaged with an automated Operetta microscope. Scale bar: 20 μm


C Lipid droplet , number mean was quantified using Harmony software . Data are presented as mean ± SD ( n = 3 technical replicates ) . An average of 1500 was analyzed per replicate

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C Lipid droplet size, number and mean fluorescence intensity was quantified using Harmony software. Data are presented as mean ± SD (n=3 technical replicates). An average of 1500 cells was analyzed per replicate


D H4 Cas9 TMEM41B KO NT control were probed by ADRP immunostaining imaged with an automated CV7000 confocal microscope . Scale bar : 20 μm . E of ADRP droplets was quantified with YAS depicted as mean ± SD ( n = 3 technical replicates )

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D H4 Cas9 TMEM41B KO and NT control cells were probed by ADRP immunostaining and imaged with an automated CV7000 confocal microscope. Scale bar: 20 μm. E Size of ADRP droplets was quantified with YAS and depicted as mean ± SD (n=3 technical replicates)


F level of ADRP was probed by immunoblotting in H4 Cas9 TMEM41B KO NT control . G ADRP band intensities were quantified depicted as mean ± SD ( n = 4 independent experiments ) with paired t - values

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F Protein level of ADRP was probed by immunoblotting in H4 Cas9 TMEM41B KO and NT control cells. G ADRP band intensities were quantified and depicted as mean ± SD (n=4 independent experiments) with paired t-test values


A H4 Cas9 TMEM41B KO NT control were pulsed with fatty acid analog Red C12 , chased in complete medium or upon serum - deprivation , stained with MitoTracker . were imaged live with an automated CV7000 confocal microscope . Scale bar : 20 μm

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A H4 Cas9 TMEM41B KO and NT control cells were pulsed with the fatty acid analog Red C12, chased in complete medium or upon serum-deprivation, and stained with MitoTracker. Cells were imaged live with an automated CV7000 confocal microscope. Scale bar: 20 μm


C - E H4 Cas9 TMEM41B KO NT control were plated in XF96 plates analyzed using a Seahorse Bioscience XF96 . ( C ) Basal oxygen consumption rates ( OCR ) extra acidification rates ( ECAR ) were measured in grown in complete medium . OCR / ECAR is also reported . Data are presented as mean ± ( n = 30 - 32 technical replicates ) from one representative experiment

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C-E H4 Cas9 TMEM41B KO and NT control cells were plated in XF96 plates and analyzed using a Seahorse Bioscience XF96. (C) Basal oxygen consumption rates (OCR) and extra cellular acidification rates (ECAR) were measured in cells grown in complete medium. OCR/ECAR ratio is also reported. Data are presented as mean ± SEM (n=30-32 technical replicates) from one representative experiment


( D ) OCR was measured in grown in substrate - limited medium treated in absence or presence of Etomoxir ( Eto ) . Oligomycin ( OA ) , FCCP ( FC ) a mixture of rotenone antimycin A ( RA ) was added as indicated . Data are shown as mean ± ( n = 69 technical replicates )

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(D) OCR was measured in cells grown in substrate-limited medium and treated in the absence or presence of Etomoxir (Eto). Oligomycin (OA), FCCP (FC) and a mixture of rotenone and antimycin A (RA) was added as indicated. Data are shown as mean ± SEM (n= 69 technical replicates)


( E ) Endogenous FA utilization was calculating by subtracting basal normalized OCR values of Eto - treated from untreated . Data are shown as mean ± ( n = 69 technical replicates )

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(E) Endogenous FA utilization was calculating by subtracting basal normalized OCR values of Eto-treated cells from untreated cells. Data are shown as mean ± SEM (n= 69 technical replicates)


A H4 Cas9 stably expressing N - terminally ( Myc - TMEM41B ) or C - terminally Myc - tagged TMEM41B ( TMEM41B - Myc ) were probed by calnexin ( CANX ) Myc tag immunostaining imaged with an automated CV7000 confocal microscope . Scale bar : 20 μm

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A H4 Cas9 cells stably expressing N-terminally (Myc-TMEM41B) or C-terminally Myc-tagged TMEM41B (TMEM41B-Myc) were probed by calnexin (CANX) and Myc tag immunostaining and imaged with an automated CV7000 confocal microscope. Scale bar: 20 μm


B - D Endogenous TMEM41B locus was tagged with C - terminal Myc using CRISPR - directed homologous recombination in HeLa . HeLa knock - in ( KI ) were compared to parental HeLa ( - ) HeLa stably transduced with a TMEM41B - Myc over - expression ( OE ) construct . ( B ) Confocal of stained with antibodies against CANX Myc confirming ER localization of both KI OE TMEM41B - Myc . Scale bar represents 20 μm

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B-D Endogenous TMEM41B locus was tagged with C-terminal Myc using CRISPR-directed homologous recombination in HeLa cells. HeLa knock-in (KI) cells were compared to parental HeLa (-) and HeLa cells stably transduced with a TMEM41B-Myc over-expression (OE) construct. (B) Confocal micrographs of cells stained with antibodies against CANX and Myc confirming ER localization of both KI and OE TMEM41B-Myc. Scale bar represents 20 μm


μm . ( C ) Immunoblot confirming expression of both KI OE TMEM41B -

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μm. (C) Immunoblot analysis confirming expression of both KI and OE TMEM41B-Myc


( D ) PCR with primers binding to TMEM41B last intron to Myc tag confirming successful targeting of endogenous TMEM41B locus with Myc ( band labeled with asterisk ) . M =

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(D) PCR analysis with primers binding to TMEM41B last intron and to Myc tag confirming successful targeting of the endogenous TMEM41B locus with Myc (band labeled with asterisk). M=size marker


E H4 Cas9 H4 Cas9 stably expressing Myc - TMEM41B or TMEM41B - Myc were lysed subjected to anti - Myc IP . Eluates were analyzed by mass spectrometry . Enrichment of in IPs from Myc - TMEM41B or TMEM41B - Myc versus H4 Cas9 is depicted as log2 fold changes . Entire data is reported in Dataset EV2

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E H4 Cas9 cells and H4 Cas9 cells stably expressing Myc-TMEM41B or TMEM41B-Myc were lysed and subjected to anti-Myc IP. Eluates were analyzed by mass spectrometry. Enrichment of proteins in IPs from Myc-TMEM41B or TMEM41B-Myc cells versus H4 Cas9 cells is depicted as log2 fold changes. Entire data is reported in Dataset EV2


( a ) Synchronization dynamics of a population of HeLa within almost 3 complete cycles measured via PI followed by FACS . increase in number following each mitosis is shown by an overlaid curve ( in orange ; mean s . d . of n = 3 ) .

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(a) Synchronization dynamics of a population of HeLa cells within almost 3 complete cell cycles measured via PI staining followed by FACS analysis. The increase in cell number following each mitosis is shown by an overlaid curve (in orange; mean and s.d. of n=3).


( b ) Computational modelling of synchronization loss , considering 11 % - variation in doubling time , shows that simulated fraction of in G1 , S , G2 / M in synchronized throughout cycle ( straight lines ) match experimental measurements ( marked with asterisk ) .

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(b) Computational modelling of the synchronization loss, considering 11% cell-cell variation in doubling time, shows that the simulated fraction of the cells in G1, S, and G2/M in the synchronized cells throughout the cell cycle (straight lines) match experimental measurements (marked with asterisk).


( c ) measured average volume in synchronized population ( red ; mean s . d . of n = 3 ; in Equation 6 ) , deconvoluted signal ( in case of no synchronization loss ; green ; in Equation 6 ) , simulated average volume considering loss in synchronization ( black ; matching measured data ; Equation 6 ) .

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(c) The measured average cell volume in the synchronized cell population (red; mean and s.d. of n=3; in Equation 6), the deconvoluted signal (in case of no synchronization loss; green; in Equation 6), and the simulated average cell volume considering the loss in synchronization (black; matching the measured concentration data; Equation 6).


( d ) measured of CTP in synchronized shown in red ( mean s . d . of n = 5 ; ) in Equation 7 ; deconvoluted dynamics , in case of no synchronization loss ( green ; in Equation 7 ) ; expected dynamics on deconvoluted concentrations considering loss in synchronization , matching measured concentrations ( black ; Equation 7 ) . measured concentrations converge towards steady - state concentrations measured in non - synchronized ( horizontal blue ) .

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(d) The measured concentration of CTP in synchronized cells shown in red (mean and s.d. of n=5; ) in Equation 7; the deconvoluted concentration dynamics, in case of no synchronization loss (green; in Equation 7); and the expected concentration dynamics based on the deconvoluted concentrations and considering the loss in synchronization, matching the measured concentrations (black; Equation 7). The measured concentrations converge towards the steady-state concentrations measured in non-synchronized cells (horizontal blue line).


Figure 2 : Oscillation in metabolite concentrations throughout cycle in HeLa . figure shows metabolites found to significantly oscillate throughout cycle ( concentrations normalized per metabolite ; maximal in red ; minimal in blue ) . amplitude of oscillations is shown on right . Metabolites are color - coded according to metabolic pathways : Energy / redox cofactors ( purple ) ; ( blue ) ; glycolytic metabolites ( red ) ; TCA cycle metabolites ( green ) .

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Figure 2: Oscillation in metabolite concentrations throughout the cell cycle in HeLa cells. The figure shows metabolites found to significantly oscillate throughout the cell cycle (concentrations normalized per metabolite; maximal concentration in red; minimal concentration in blue). The amplitude of the oscillations is shown on the right. Metabolites are color-coded according to metabolic pathways: Energy/redox cofactors (purple); amino acids (blue); glycolytic metabolites (red); TCA cycle metabolites (green).


( c - e ) Measured relative fraction of m + 2 labeling of TCA cycle intermediates after feeding [ U - 13C ] - glucose ( red ; mean s . d . of n = 3 ) , deconvoluted signal ( green ) , expected labeling dynamics considering loss in synchronization ( black ; representing TCA cycle oxidation of glucose - derived acetyl - CoA ) .

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(c-e) Measured relative fraction of the m+2 labeling of TCA cycle intermediates after feeding [U-13C]-glucose (red; mean and s.d. of n=3), the deconvoluted signal (green), and the expected labeling dynamics considering the loss in synchronization (black; representing TCA cycle oxidation of glucose-derived acetyl-CoA).


( f ) Oscillations in citrate m + 4 labeling after feeding [ U - 13C ] - glutamine ( mean s . d . of n = 3 ) .

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(f) Oscillations in citrate m+4 labeling after feeding [U-13C]-glutamine (mean and s.d. of n=3).


( g ) Oscillations in total citrate throughout cycle ( mean s . d . of n = 5 ) .

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(g) Oscillations in the total citrate concentration throughout the cell cycle (mean and s.d. of n=5).


( h ) measured lactate secretion flux in synchronized shown in red ( mean s . d . of n = 5 ; in Equation 9 ; ) ; deconvoluted secretion flux dynamics , in case of no synchronization loss ( green ; in Equation 10 ) ; expected secretion flux on deconvoluted fluxes considering loss in synchronization , matching measured fluxes ( black ; Equation 10 ) .

(h) The measured lactate secretion flux in synchronized cells shown in red (mean and s.d. of n=5; in Equation 9;); the deconvoluted secretion flux dynamics, in case of no synchronization loss (green; in Equation 10); and the expected secretion flux based on the deconvoluted fluxes and considering the loss in synchronization, matching the measured fluxes (black; Equation 10).


Oscillations in aspartate ( a ) concentrations throughout cycle when feeding [ U - 13C ] - glutamine ( representing oxidative TCA cycle activity ) .

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Oscillations in aspartate (a)concentrations throughout the cell cycle when feeding [U-13C]-glutamine (representing oxidative TCA cycle activity).


Oscillations in malate ( b ) concentrations throughout cycle when feeding [ U - 13C ] - glutamine ( representing oxidative TCA cycle activity ) .

Oscillations in malate (b) concentrations throughout the cell cycle when feeding [U-13C]-glutamine (representing oxidative TCA cycle activity).


( c ) Uniform malate m + 4 labeling throughout cycle ( combined with increase in malate in S phase representing increased oxidative TCA cycle flux in S phase ) .

(c) Uniform malate m+4 labeling throughout the cell cycle (combined with the increase in malate concentration in S phase representing increased oxidative TCA cycle flux in S phase).


( d ) Oscillations in pyrimidines m + 3 labeling throughout cycle when feeding [ U - 13C ] - glutamine ( representing de novo pyrimidine biosynthesis ) .

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(d) Oscillations in pyrimidines m+3 labeling throughout the cell cycle when feeding [U-13C]-glutamine (representing de novo pyrimidine biosynthesis).


( e ) Oscillations in lactate m + 3 labeling throughout cycle when feeding [ U - 13C ] - glutamine ( representing malic enzyme activity ) .

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(e) Oscillations in lactate m+3 labeling throughout the cell cycle when feeding [U-13C]-glutamine (representing malic enzyme activity).


( f ) Oscillations in malate m + 3 throughout cycle when feeding [ U - 13C ] - glucose ( representing pyruvate carboxylase activity ) .

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(f) Oscillations in malate m+3 throughout the cell cycle when feeding [U-13C]-glucose (representing pyruvate carboxylase activity).


( g ) Oscillations in citrate m + 5 when feeding [ U - 13C ] - glutamine throughout cycle ( representing reductive IDH flux ) .

(g) Oscillations in citrate m+5 when feeding [U-13C]-glutamine throughout the cell cycle (representing reductive IDH flux).


( h ) Oscillations in acetyl - CoA m + 2 when feeding [ U - 13C ] - glucose , representing oxidative glucose metabolism .

(h) Oscillations in acetyl-CoA m+2 when feeding [U-13C]-glucose, representing oxidative glucose metabolism.


Oscillations in acetyl - CoA m + 2 when feeding [ U - 13C ] - glutamine , representing reductive glutamine metabolism ( i ) . For measurements of metabolite concentrations ( a - b ) , red marks represent mean s . d . of n = 5 . For measurements of fractional isotopic labeling ( c - i ) , red marks represent mean s . d . of n = 3 .

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Oscillations in acetyl-CoA m+2 when feeding [U-13C]-glutamine, representing reductive glutamine metabolism (i). For measurements of metabolite concentrations (a-b), red marks represent mean and s.d. of n=5. For measurements of fractional isotopic labeling (c-i), red marks represent mean and s.d. of n=3.


Figure 5 : Complementary oscillations of glucose versus glutamine - derived fluxes in TCA cycle . ( a - i ) Oscillations in metabolic flux throughout cycle ( in mM / h ) , computed on metabolic modelling of measured oscillations in metabolite concentrations isotopic labeling ( red green marks represent optimal estimates of transient flux with 95 % c . i . ) . Blue lines represent average fluxes inferred in a non - synchronized population . As shown , glucose - derived flux into TCA cycle peak in late G1 phase , while oxidative reductive glutamine metabolism dominates S phase .

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Figure 5: Complementary oscillations of glucose versus glutamine-derived fluxes in TCA cycle. (a-i) Oscillations in metabolic flux throughout the cell cycle (in mM/h), computed based on metabolic modelling of measured oscillations in metabolite concentrations and isotopic labeling (red and green marks represent optimal estimates of transient flux with 95% c.i.). Blue lines represent average fluxes inferred in a non-synchronized cell population. As shown, glucose-derived flux into TCA cycle peak in late G1 phase, while oxidative and reductive glutamine metabolism dominates S phase.


( a - b ) One hour treatment of synchronized with DCA inhibits oscillations in citrate m + 2 ( a ) from isotopic glucose ( representing glycolytic flux into TCA cycle ) .

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(a-b) One hour treatment of synchronized cells with DCA inhibits the oscillations in citrate m+2 (a) from isotopic glucose (representing glycolytic flux into TCA cycle).


( a - b ) One hour treatment of synchronized with DCA inhibits oscillations in acetyl - CoA m + 2 ( b ) from isotopic glucose ( representing glycolytic flux into TCA cycle ) .

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(a-b) One hour treatment of synchronized cells with DCA inhibits the oscillations in acetyl-CoA m+2 (b) from isotopic glucose (representing glycolytic flux into TCA cycle).


It further inhibits oscillations in citrate m + 5 / m + 4 ( c ) from isotopic glutamine ( representing oxidative versus reductive TCA cycle flux ) .

It further inhibits oscillations in citrate m+5/m+4 ratio (c) from isotopic glutamine (representing oxidative versus reductive TCA cycle flux).


It further inhibits oscillations in acetyl - CoA m + 2 ( d ) from isotopic glutamine ( representing oxidative versus reductive TCA cycle flux ) .

It further inhibits oscillations in acetyl-CoA m+2 (d) from isotopic glutamine (representing oxidative versus reductive TCA cycle flux).


( e ) fraction of in G1 , S , G2 / M phases in synchronized HeLa after 3 hour treatment with DCA ( normalized by measurements in untreated control ) .

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(e) The fraction of cells in G1, S, and G2/M phases in synchronized HeLa cells after 3 hour treatment with DCA (normalized by measurements in untreated control cells).


( f ) fraction of in G1 , S , G2 / M phases in non - synchronized HeLa HCT - 116 after 24 hour treatment with DCA ( normalized by measurements in untreated control ) . As shown , DCA significantly increases fraction of in S phase , inhibiting progression into G2 phase ; showing mean s . d . of n = 3 for isotopic labeling forms FACS measurements ( a - f ) . Statistical significance of changes in fraction of in each cycle phase following DCA treatment is calculated on two - , unequal variance t - ( e - f ) .

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(f) The fraction of cells in G1, S, and G2/M phases in non-synchronized HeLa and HCT-116 cells after 24 hour treatment with DCA (normalized by measurements in untreated control cells). As shown, DCA significantly increases the fraction of cells in S phase, inhibiting cellular progression into G2 phase; showing mean and s.d. of n=3 for all isotopic labeling forms and FACS measurements (a-f). Statistical significance of changes in the fraction of cells in each cell cycle phase following DCA treatment is calculated based on two-tailed, unequal variance t-test (e-f).


B - D BSP amyloid aggregation in vitro is pH - dependent in 10 mM EDTA ( B ) , - dependent in 10 mM EDTA at pH 4 . 5 ( C ) strongly inhibited by millimolar Ca2 + at various concentrations ( D ) .

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B-D BSP amyloid aggregation in vitro is pH-dependent in 10 mM EDTA (B), protein concentration-dependent in 10 mM EDTA and at pH 4.5 (C) and strongly inhibited by millimolar Ca2+ at various protein concentrations (D).


E - H showing fibril of BSP12 ( E ) , BSP123 ( F ) , BSP3 ( G ) , BSP45 ( H ) .

E-H TEM images showing fibril morphology of BSP12 (E), BSP123 (F), BSP3 (G), BSP45 (H).


I - J Amyloid aggregation by BSP subconstructs . BSP12 exhibits more prominent aggregation propensity than BSP3 BSP45 in 10 mM EDTA at pH 4 . 5 ( I ) . conjunction of module 3 with modules 1 2 promotes aggregation ( J ) .

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I-J Amyloid aggregation by BSP subconstructs. BSP12 exhibits more prominent aggregation propensity than BSP3 and BSP45 in 10 mM EDTA and at pH 4.5 (I). The conjunction of module 3 with modules 1 and 2 promotes aggregation (J).


B Representative microscopy of BSP its subconstructs ( 20 μM ) at varying pHs . PEG 8K is 2 % . Scale bar , 20 μm .

B Representative fluorescence microscopy images of BSP and its subconstructs (20 μM) at varying pHs. PEG 8K concentration is 2 %. Scale bar, 20 μm.


E of droplet is shown for each under various Ca2 + conditions . Each sample is quantified by total ( left ) mean ( right ) . Each experiment was repeated three times , data shown as mean ± SD .

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E Quantification of droplet area is shown for each protein under various Ca2+ concentration conditions. Each sample is quantified by total area (left) and mean area (right). Each experiment was repeated three times, data shown as mean ± SD.


( A ) Identified acetylated histone peptides plotted against their log2 SILAC ( 1h after 16 J / m2 UV / mock ) , ranked by SILAC

(A) Identified acetylated histone peptides plotted against their log2 SILAC ratio (1h after 16 J/m2 UV/mock), ranked by SILAC ratio


( C ) Western blot of acid extracted histones WCE from HeLa , treated with HATi ( CTK7A , 100 μM CPTH2 , 50 μM ) or HDACi ( TSA , 1 μM ) for 4h or mock treated as

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(C) Western blot of acid extracted histones and WCE from HeLa cells, treated with HATi (CTK7A, 100 μM and CPTH2, 50 μM) or HDACi (TSA, 1 μM) for 4h or mock treated as indicated


( D ) A representative western blot of histone acetylation of HeLa lysed at indicated time points after UV - irradiation ( 16 J / m2 ) . Blots were stained with α - acetyl - lysine ( top panels , high low exposure ) , α - histone H4 ( middle panel ) α - tubulin ( bottom panel )

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(D) A representative western blot of histone acetylation levels of HeLa cells lysed at indicated time points after UV-irradiation (16 J/m2). Blots were stained with α-acetyl-lysine (top panels, high and low exposure), α-histone H4 (middle panel) and α-tubulin (bottom panel)


( F ) Representative western blots of HeLa WCE from different time points after UV - C irradiation ( 16J / m2 ) using indicated histone modification specific antibodies . Histone H4 tubulin were used as loading

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(F) Representative western blots of HeLa WCE from different time points after UV-C irradiation (16J/m2) using the indicated histone modification specific antibodies. Histone H4 and tubulin were used as loading controls


( A ) Representative western blots of histone acetylation of HeLa lysed at indicated time points after UV irradiation ( 16 J / m2 , left panel ) , α - amanitin ( second panel , 100 µg / ml ) , THZ1 ( third panel , 1 μM ) or flavopiridol ( right panel , 1 μM ) treatment . Blots were stained with α - acetyl - lysine ( top panel ) , α - histone H4 ( middle panel ) α - tubulin ( bottom panel )

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(A) Representative western blots of histone acetylation levels of HeLa cells lysed at indicated time points after UV irradiation (16 J/m2, left panel), α-amanitin (second panel, 100 µg/ml), THZ1 (third panel, 1 μM) or flavopiridol (right panel, 1 μM) treatment. Blots were stained with α-acetyl-lysine (top panel), α-histone H4 (middle panel) and α-tubulin (bottom panel)


( C ) Representative western blots of histone acetylation of HeLa pre - treated with transcription inhibitors ( mock ( left ) , THZ1 ( middle , 1 μM ) or flavopiridol ( right , 1 μM ) an hour before UV irradiation lysed at indicated time points after UV ( 16J / m2 ) . Blots were stained with indicated

(C) Representative western blots of histone acetylation levels of HeLa cells pre-treated with transcription inhibitors (mock (left), THZ1 (middle, 1 μM) or flavopiridol (right, 1 μM) an hour before UV irradiation and lysed at the indicated time points after UV (16J/m2). Blots were stained with the indicated antibodies


( A ) Representative western blots of histone acetylation of HeLa obtained at indicated time points after UV irradiation ( 16 J / m2 ) , HU / AraC treatment ( 100 mM / 10 μM ) or MMC treatment ( 10 µg / ml ) . Blots were stained with α - acetyl - lysine ( top panel ) , α - histone H4 ( middle panel ) α - tubulin ( bottom panel )

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(A) Representative western blots of histone acetylation levels of HeLa cells obtained at the indicated time points after UV irradiation (16 J/m2), HU/AraC treatment (100 mM/10 μM) or MMC treatment (10 µg/ml). Blots were stained with α-acetyl-lysine (top panel), α-histone H4 (middle panel) and α-tubulin (bottom panel)


( C ) Representative Western blots , stained with indicated antibodies of HeLa pre - treated o / n with an S - phase inhibitor ( PHA 767491 hydrochloride , 10 μM ) lysed at indicated time points after UV ( 16 J / m2 )

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(C) Representative Western blots, stained with the indicated antibodies of HeLa cells pre-treated o/n with an S-phase inhibitor (PHA 767491 hydrochloride, 10 μM) and lysed at indicated time points after UV (16 J/m2)


A Treatment scheme of experimental timeline Immunofluorescence of telogen backskin hair follicles ( HF ) for Integrin α6 ( Itgα6 , red ) , active Caspase 3 ( aCas3 , green ) DAPI ( blue ) in control ( Ctr ) Bcl - 2 inhibitor ( ABT - 199 ) treated mice . B : bulge , CH : club hair , DP : dermal papilla , HF : hair follicle , HG : secondary hair germ . Scale bar , 50 µm B of backskin HF with aCas3 + in bulge , hair germ dermal papilla of Ctr ABT - 199 treated mice ( n = 4 mice biological replicates ; mean ± standard deviation ( SD ) ; * * * p < 0 . 001 , ns = not significant , two - way ANOVA ) . nd = not detected .

O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Treatment scheme of experimental timeline and Immunofluorescence staining of telogen backskin hair follicles (HF) for Integrin α6 (Itgα6, red), active Caspase 3 (aCas3, green) and DAPI (blue) in control (Ctr) and Bcl-2 inhibitor (ABT-199) treated mice. B: bulge, CH: club hair, DP: dermal papilla, HF: hair follicle, HG: secondary hair germ. Scale bar, 50 µm B Quantification of backskin HF with aCas3+ cells in bulge, hair germ and dermal papilla of Ctr and ABT-199 treated mice (n=4 mice biological replicates; mean ± standard deviation (SD); *** p<0.001, ns=not significant, two-way ANOVA test). nd=not detected.


C , D Treatment scheme , FACS plot ( C ) ( D ) of keratinocytes stained for CD34 Itgα6 after 6 days of treatment with vehicle solution ( Ctr ) or ABT - 199 ( n = 6 biological replicates ; mean ± standard deviation ( SD ) ; * * p < 0 . 01 , ns = not significant , two - unpaired Student ' s t - ) . suprabasal ( sbBSC , CD34 + / Itgα6low ) , basal ( bBSC , CD34 + / Itgα6high ) BSCs n - B ( non - bulge epidermal ) . * * p < 0 . 005 .

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

C,D Treatment scheme, FACS plot (C) and quantification (D) of keratinocytes stained for CD34 and Itgα6 after 6 days of treatment with vehicle solution (Ctr) or ABT-199 (n=6 biological replicates; mean ± standard deviation (SD); ** p<0.01, ns=not significant, two-tailed unpaired Student's t-test). suprabasal (sbBSC, CD34+/Itgα6low), basal (bBSC, CD34+/Itgα6high) BSCs and n-B (non-bulge epidermal cells). ** p<0.005.


B Treatment scheme of experimental timeline histology of backskin sections of control ( Ctr ) ABT - 199 treated mice . Scale bar , 200 µm .

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B Treatment scheme of experimental timeline and histology of backskin sections of control (Ctr) and ABT-199 treated mice. Scale bar, 200 µm.


D qRT - PCR for mRNA expression of transcription factors Lef1 Runx1 in epidermis of ABT - 199 treated control ( Ctr ) mice at P77 ( n = 3 biological replicates ; mean ± standard error of mean ( ) ; * * p < 0 . 01 , ns = not significant , two - unpaired Student ' s t - ) .

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D qRT-PCR for mRNA expression of the transcription factors Lef1 and Runx1 in epidermis of ABT-199 treated and control (Ctr) mice at P77 (n=3 biological replicates; mean ± standard error of mean (SEM); ** p<0.01, ns=not significant, two-tailed unpaired Student's t-test).


C qRT - PCR for mRNA expression of anoikis - related markers Bim Bmf in sorted n - B ( non - bulge ) , bBSC ( basal BSCs ) sbBSC ( suprabasal BSCs ) ( n = 3 biological replicates ; mean ± standard error of mean ( ) ; * p < 0 . 05 , ns = not significant , two - way ANOVA ) .

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C qRT-PCR for mRNA expression of the anoikis-related markers Bim and Bmf in sorted n-B (non-bulge), bBSC (basal BSCs) and sbBSC (suprabasal BSCs) (n=3 biological replicates; mean ± standard error of mean (SEM); * p<0.05, ns=not significant, two-way ANOVA test).


C FACS plot of keratinocytes stained for CD34 Itgα6 in 8 week old control ( Ctr ) Bcl - 2EOE mice ( n = 3 biological replicates ; mean ± standard deviation ( SD ) ; ns = not significant , two - unpaired Student ' s t - ) . suprabasal ( sbBSC , CD34 + / Itgα6low ) , basal ( bBSC , CD34 + / Itgα6high ) BSCs .

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C FACS plot and quantification of keratinocytes stained for CD34 and Itgα6 in 8 week old control (Ctr) and Bcl-2EOE mice (n=3 biological replicates; mean ± standard deviation (SD); ns=not significant, two-tailed unpaired Student's t-test). suprabasal (sbBSC, CD34+/Itgα6low), basal (bBSC, CD34+/Itgα6high) BSCs.


D Immunofluorescence of telogen backskin HF for BrdU incorporation ( 1 h , green ) DAPI ( blue ) in 8 week old control ( Ctr ) Bcl - 2EOE mice . Scale bar , 50 µm .

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D Immunofluorescence staining of telogen backskin HF for BrdU incorporation (1 h pulse, green) and DAPI (blue) in 8 week old control (Ctr) and Bcl-2EOE mice. Scale bar, 50 µm.


A Histology of P19 catagen backskin HF of control ( Ctr ) Bcl - 2EOE mice . RS : retracting strand , Scale bar , 50 µm . B of width of retracting strands of control ( Ctr ) Bcl - 2EOE mice ( n = 4 ; multiple measurements / biological replicate ; whiskers : min max ; boxes : median ± first third quartile ; * * * * p < 0 . 0001 , two - unpaired Student ' s t - ) .

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A Histology of P19 catagen backskin HF of control (Ctr) and Bcl-2EOE mice. RS: retracting strand, Scale bar, 50 µm. B Analysis of length and width of retracting strands of control (Ctr) and Bcl-2EOE mice (n=4; multiple measurements/biological replicate; whiskers: min and max; boxes: median ± first and third quartile; **** p<0.0001, two-tailed unpaired Student's t-test).


C Itgα6 aCas3 Immunofluorescence of catagen tailskin HF of control ( Ctr ) Bcl - 2EOE mice ( n = 4 biological replicates ) . Dashed lines : retracting strands . Scale bar , 100 µm . D of aCas3 + in RS of P19 Ctr Bcl - 2EOE mice ( n = 4 biological replicates ; mean ± standard deviation ( SD ) ; * * * * p < 0 . 0001 , two - unpaired Student ' s t - ) . HF : hair follicle .

O B-MEASURED_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 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 O O O

C Itgα6 and aCas3 Immunofluorescence staining of catagen tailskin HF of control (Ctr) and Bcl-2EOE mice (n=4 biological replicates). Dashed lines: retracting strands. Scale bar, 100 µm. D Quantification of aCas3+ cells in the RS of P19 Ctr and Bcl-2EOE mice (n=4 biological replicates; mean ± standard deviation (SD); **** p<0.0001, two-tailed unpaired Student's t-test). HF: hair follicle.


F , G qRT - PCR for mRNA expression of quiescence - related markers Bmp6 ( F ) Fgf18 ( G ) in epidermis of control ( Ctr ) Bcl - 2EOE mice ( P27 ) , ( n = 4 biological replicates ; mean ± standard error of mean ( ) ; * p < 0 . 05 , * * * p < 0 . 001 , two - unpaired Student ' s t - ) .

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F,G qRT-PCR for mRNA expression of the quiescence-related markers Bmp6 (F) and Fgf18 (G) in epidermis of control (Ctr) and Bcl-2EOE mice (P27), (n=4 biological replicates; mean ± standard error of mean (SEM); * p<0.05, *** p<0.001, two-tailed unpaired Student's t-test).


B Immunofluorescence of epidermal tail whole mounts of control ( Ctr ) , K15ΔNLef1 K15ΔNLef1 ; Bcl - 2EOE mice , stained for keratin 15 ( Krt15 , green ) , activated Caspase 3 ( aCas3 , red ) DAPI ( blue ) . Note increased number of aCas + in bulge of K15ΔNLef1 mice ( arrows ) . B : bulge , HG : secondary hair germ , SG : sebaceous . Scale bar , 100µm .

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B Immunofluorescence staining of epidermal tail whole mounts of control (Ctr), K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice, stained for keratin 15 (Krt15, green), activated Caspase 3 (aCas3, red) and DAPI (blue). Note increased number of aCas+ cells in the bulge of K15ΔNLef1 mice (arrows). B: bulge, HG: secondary hair germ, SG: sebaceous gland. Scale bar, 100µm.


D Immunofluorescence of epidermal tail whole mounts of control ( Ctr ) , K15ΔNLef1 K15ΔNLef1 ; Bcl - 2EOE mice , stained for keratin 15 ( Krt15 , green ) , BrdU ( 1 h , red ) DAPI ( blue ) . Note increased number of BrdU + in bulge of K15ΔNLef1 mice ( arrows ) . B : bulge , HG : secondary hair germ , SG : sebaceous . Scale bar ,

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D Immunofluorescence staining of epidermal tail whole mounts of control (Ctr), K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice, stained for keratin 15 (Krt15, green), BrdU (1 h pulse, red) and DAPI (blue). Note increased number of BrdU+ cells in the bulge of K15ΔNLef1 mice (arrows). B: bulge, HG: secondary hair germ, SG: sebaceous gland. Scale bar, 100µm


B , C Tumour incidence ( B ) frequency ( C ) in Ctr , Bcl - 2EOE , K15ΔNLef1 K15ΔNLef1 ; Bcl - 2EOE mice ( n = 10 biological replicates ; mean ± standard error of mean ( ) ) .

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B,C Tumour incidence (B) and frequency (C) in Ctr, Bcl-2EOE, K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice (n=10 biological replicates; mean ± standard error of mean (SEM)).


D Histology of sebaceous tumours of K15ΔNLef1 K15ΔNLef1 ; Bcl - 2EOE mice . Scale bar , 100µm .

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D Histology of sebaceous tumours of K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice. Scale bar, 100µm.


A ) Hek293T expressing Lamplight - 1D4 ( anti - 1D4 = green , DAPI = blue , Top ) schematic of BRET , measuring interaction of liberated Gβγ - Venus with GRK3 fragment - Nanoluciferase ( Bottom ) . Scale bar = 20µm . B ) Lamplight - driven response to increasing 1s 405nm C ) 405nm irradiance response curve D ) Response to 1s 405nm ( blue ) 525nm ( green ) E ) Response to 405nm ( blue ) followed by 525nm of different intensities ( green )

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 B-MEASURED_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

A) Hek293T cells expressing Lamplight-1D4 (anti-1D4 = green, DAPI = blue, Top) and schematic of BRET assay, measuring interaction of liberated Gβγ-Venus with GRK3 fragment-Nanoluciferase (Bottom). Scale bar = 20µm. B) Lamplight-driven response to increasing intensity 1s 405nm light C) 405nm irradiance response curve D) Response to 1s 405nm (blue line) and 525nm light (green line) E) Response to 405nm (blue line) followed by 525nm light of different intensities (green line)


A ) Hek293T expressing Lamplight - 1D4 ( anti - 1D4 = green , DAPI = blue , Top ) schematic of BRET , measuring interaction of liberated Gβγ - Venus with GRK3 fragment - Nanoluciferase ( Bottom ) . Scale bar = 20µm .

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A) Hek293T cells expressing Lamplight-1D4 (anti-1D4 = green, DAPI = blue, Top) and schematic of BRET assay, measuring interaction of liberated Gβγ-Venus with GRK3 fragment-Nanoluciferase (Bottom). Scale bar = 20µm.


G ) Response to 405nm alone or with 525nm

G) Response to 405nm alone or with 525nm light


H - I ) Representative time courses of Lamplight - driven responses to 8s mixed stimuli ( grey bar ) with different 405nm : 525nm ratios from 1 ( 100 % 405nm ) to 0 ( 100 % 525nm ) from ( H ) or ( I ) after 8s 405nm ( blue bar ) J ) Sustained Lamplight - driven activity ( mean BRET 20 - 80s after stimulus ) after exposure to mixed stimuli from ( ON ) or 405nm - adapted state ( OFF ) . Lamplight data fit with linear trendline . Best fitting parameters for ON , slope = 0 . 311 , Y - intercept = 0 . 04 , R2 = 0 . 85 . For OFF , slope = 0 . 30 , Y - intercept = 0 . 07 , R2 = 0 . 69 ) .

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H-I) Representative time courses of Lamplight-driven responses to 8s mixed stimuli (grey bar) with different 405nm: 525nm ratios from 1 (100% 405nm) to 0 (100% 525nm) from (H) dark or (I) after 8s 405nm light (blue bar) J) Sustained Lamplight-driven activity (mean BRET ratio 20-80s after stimulus) after exposure to mixed stimuli from dark (ON) or 405nm light-adapted state (OFF). Lamplight data fit with linear trendline. Best fitting parameters for ON, slope = 0.311, Y-intercept = 0.04, R2 = 0.85. For OFF, slope = 0.30, Y-intercept = 0.07, R2 = 0.69).


H - I ) Representative time courses of Lamplight - driven responses to 8s mixed stimuli ( grey bar ) with different 405nm : 525nm ratios from 1 ( 100 % 405nm ) to 0 ( 100 % 525nm ) from ( H ) or ( I ) after 8s 405nm ( blue bar )

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H-I) Representative time courses of Lamplight-driven responses to 8s mixed stimuli (grey bar) with different 405nm: 525nm ratios from 1 (100% 405nm) to 0 (100% 525nm) from (H) dark or (I) after 8s 405nm light (blue bar)


B ) Representative trace of hyperpolarisation to 405nm in a Lamplight - expressing SCN neuron C ) Hyperpolarisation to 405nm was observed over a range of intensities . Recordings from 3 different across 2 SCN slices

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B) Representative trace of hyperpolarisation to 405nm light in a Lamplight-expressing SCN neuron C) Hyperpolarisation to 405nm was observed over a range of intensities. Recordings from 3 different cells across 2 SCN slices


B ) Representative trace of hyperpolarisation to 405nm in a Lamplight - expressing SCN

B) Representative trace of hyperpolarisation to 405nm light in a Lamplight-expressing SCN neuron


D ) Representative trace showing hyperpolarisation was sustained during after 405nm ( 5s ) reversed by 525nm ( 15s )

D) Representative trace showing hyperpolarisation was sustained during dark after 405nm light (5s) and reversed by 525nm light (15s)


E ) Hyperpolarisation depolarisation of a Lamplight - expressing neuron by 405nm 525nm , respectively , across multiple stimulus presentations

E) Hyperpolarisation and depolarisation of a Lamplight-expressing neuron by 405nm and 525nm light, respectively, across multiple stimulus presentations


E ) Hyperpolarisation depolarisation of a Lamplight - expressing neuron by 405nm 525nm , respectively , across multiple stimulus


F ) Three example traces from a single exposed to 405nm followed by 525nm . traces were fit with exponential decay ( 405nm , blue ) or association curves ( 525nm , green ) for 6 trials across 2

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F) Three example traces from a single cell exposed to 405nm followed by 525nm light. Individual traces were fit with exponential decay (405nm, blue) or association curves (525nm, green) for 6 trials across 2 cells


G , H ) Parameters from best - fit curves used to calculate ( G ) latency ( ms ) ( H ) half - life ( ms ) .

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G, H) Parameters from best-fit curves used to calculate (G) latency (ms) and (H) half-life (ms).


I ) Simultaneous 525nm 405nm prevents spike silencing J ) Constant 405nm ( 12 . 9 log photons ) presented with 525nm of decreasing from 14 . 9 - 12 . 9log photons in 6 steps of 10s ( left ) or 20s ( right ) . 525nm antagonised 405nm - driven hyperpolarisation for intensities except 12 . 9log photons

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I) Simultaneous 525nm and 405nm light prevents spike silencing J) Constant 405nm light (12.9 log photons) presented with 525nm light of decreasing intensity from 14.9 - 12.9log photons in 6 steps of 10s (left) or 20s (right). 525nm light antagonised the 405nm-driven hyperpolarisation for all intensities except 12.9log photons


I ) Simultaneous 525nm 405nm prevents spike silencing

I) Simultaneous 525nm and 405nm light prevents spike silencing


A ) L7Cre rd1 retina expressing Lamplight - T2A - mCherry AAV . Anti - mCherry ( ) was widespread ( Left , scale bar = 500µm ) . bodies found in different focal planes consistent with expression in ganglion ( Top Right , scale bar = 50µm ) bipolar ( Bottom Right , scale bar = 50µm ) .

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A) L7Cre rd1 retina expressing Lamplight-T2A-mCherry AAV. Anti-mCherry staining (white) was widespread (Left, scale bar = 500µm). Fluorescent cell bodies found in different focal planes consistent with expression in ganglion (Top Right, scale bar = 50µm) and bipolar cells (Bottom Right, scale bar = 50µm).


B ) Representative single unit showing characteristic ipRGC response .

B) Representative single unit showing characteristic ipRGC response.


C ) Three Lamplight response categories - transient excitation ( N = 35 biological replicates ) , transient inhibition ( N = 6 biological replicates ) , sustained inhibition ( N = 8 biological replicates ) . Left panel shows representative single unit response to 2s 405nm or 525nm . Mean baseline normalised firing ( grey shading shows ) is shown in centre left ( for entire recording , 500ms bins ) , centre right far right panels ( during 405nm 525nm stimuli , respectively 100ms bins , N as above , except N = 5 biological replicates for sustained response to 525nm ) .

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C) Three Lamplight response categories - transient excitation (N = 35 biological replicates), transient inhibition (N = 6 biological replicates), and sustained inhibition (N = 8 biological replicates). Left panel shows representative single unit response to 2s 405nm or 525nm light. Mean baseline normalised firing (grey shading shows SEM) is shown in centre left (for entire recording, 500ms bins), centre right and far right panels (during 405nm and 525nm stimuli, respectively 100ms bins, N as above, except N = 5 biological replicates for sustained response to 525nm).


D ) Representative single unit showing responses over 20 stimulus repeats .

D) Representative single unit showing light responses over 20 stimulus repeats.


E ) Distribution of response latencies for 3 Lamplight - driven responses to 405nm , as well as reversal caused by 525nm in sustained inhibition units . Two outliers with slow latency ( > 2 . 5s ) in transient excitation group are likely responding to stimulus offset ( N = 35 for Transient Excitation , N = 6 for Transient Inhibition , N = 7 for Sustained Inhibition N = 5 for Sustained Excitation , biological replicates ) .

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E) Distribution of response latencies for 3 Lamplight-driven responses to 405nm, as well as reversal caused by 525nm in sustained inhibition units. Two outliers with slow latency (>2.5s) in transient excitation group are likely responding to stimulus offset (N = 35 for Transient Excitation, N = 6 for Transient Inhibition, N = 7 for Sustained Inhibition and N = 5 for Sustained Excitation, all biological replicates).


F ) Inhibition responses were smaller less sustained for mixed stimuli ( 50 % 405nm , 50 % 525nm ) compared to 405nm only ( N = 14 biological replicates ) . G ) Excitation responses were attenuated for mixed stimuli compared to 405nm only . Left panel shows representative retinal single unit ( N = 35 biological replicates ) . Data information : For ( F - G ) Left panel shows representative single unit . Right panel shows distribution of difference in firing rate for 405nm vs mixed stimulus . representative single units show perievent rasters ( first trial at top ) firing rate histograms ( Bin = 500ms ) . Timing of stimuli shown by shaded vertical bars . stimuli are 16 log total Lamplight effective photons / cm2 / s . Error bars show . * p < 0 . 05 , * * * p < 0 . 001 ( One - sample t - compared to zero ) .

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F) Inhibition responses were smaller and less sustained for mixed stimuli (50% 405nm, 50% 525nm) compared to 405nm only (N = 14 biological replicates). G) Excitation responses were attenuated for mixed stimuli compared to 405nm only. Left panel shows representative retinal single unit (N = 35 biological replicates). Data information: For (F-G) Left panel shows representative single unit. Right panel shows distribution of difference in firing rate for 405nm vs mixed stimulus. All representative single units show perievent rasters (first trial at top) and firing rate histograms (Bin size = 500ms). Timing of light stimuli shown by shaded vertical bars. All light stimuli are 16 log total Lamplight effective photons/cm2/s. Error bars show SEM. * p<0.05, *** p < 0.001 (One-sample t-test compared to zero).


F ) Inhibition responses were smaller less sustained for mixed stimuli ( 50 % 405nm , 50 % 525nm ) compared to 405nm only ( N = 14 biological replicates ) .

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F) Inhibition responses were smaller and less sustained for mixed stimuli (50% 405nm, 50% 525nm) compared to 405nm only (N = 14 biological replicates).


( E - G ) cortical neurons from E15 cerebral cortices were transfected with indicated plasmids , incubated for two days in vitro treated with Tf - 555 for 30 minutes before fixation . were immunostained with indicated antibodies . are obtained with high - resolution microscopy ( Nikon ) . Blue alone channels are shown in black . Lower panels in ( E ) ( F ) are high magnification indicated by or blue rectangles in upper panels . graph in ( G ) shows Pearson ' s correlation coefficient of Tf - 555 Rab11 or APPL1 . Each score represents mean with points . Control : n = 23 , Rab21 - sh115 : n = 18 , Rab5 - sh232 ( Tf - Rab11 ) : n = 23 , Rab5 - sh232 ( Tf - APPL1 ) : n = 22 .

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(E-G) Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids, incubated for two days in vitro and treated with Tf-555 for 30 minutes before fixation. Cells were immunostained with the indicated antibodies. The images are obtained with high-resolution microscopy (Nikon). Blue alone channels are shown in black and white images. Lower panels in (E) and (F) are high magnification images indicated by white or blue rectangles in upper panels. The graph in (G) shows the Pearson's correlation coefficient of Tf-555 and Rab11 or APPL1. Each score represents the mean with the individual points. Control: n = 23 cells, Rab21-sh115: n = 18 cells, Rab5-sh232 (Tf - Rab11): n = 23 cells, Rab5-sh232 (Tf - APPL1): n = 22 cells.


Cerebral cortices at E17 , electroporated with indicated plasmids plus pCAG - EGFP at E14 . ( C ) of locomoting neurons with more than three neurites in IZ . Each score represents mean with points . Control : n = 4 brains ( 60 ) , Rab21 - sh115 : n = 3 brains ( 65 ) , Rab5 - sh232 : n = 4 brains ( 111 ) .

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Cerebral cortices at E17, electroporated with the indicated plasmids plus pCAG-EGFP at E14. (C) The ratio of locomoting neurons with more than three primary neurites in the IZ. Each score represents the mean with the individual points. Control: n = 4 brains (60 cells), Rab21-sh115: n = 3 brains (65 cells), Rab5-sh232: n = 4 brains (111 cells).


( E ) Time - lapse imaging of control Rab21 - sh115 - electroporated in cortical slices from E16 cerebral cortices , electroporated with indicated plasmids at E14 . After formation of leading process , control neurons rapidly eliminated their immature neurites ( yellow arrows ) , whereas Rab21 - knockdown neurons retained immature neurites ( arrows ) for long periods . Time interval of each frame is 30 min .

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(E) Time-lapse imaging of control and Rab21-sh115-electroporated cells in cortical slices from E16 cerebral cortices, electroporated with the indicated plasmids at E14. After formation of the leading process, control neurons rapidly eliminated their immature neurites (yellow arrows), whereas the Rab21-knockdown neurons retained the immature neurites (white arrows) for long periods. Time interval of each frame is 30 min.


cortical neurons from E15 cerebral cortices were transfected with pCAG - PM - mAG1 ( green ) incubated for two days in vitro . were immunostained with indicated antibodies ( red blue / ) . transfected PM - mAG1 is a marker for plasma . are obtained with high - resolution microscopy ( Nikon ) lower panels are high magnification near plasma . Blue alone channels are shown in black . Each score represents mean of ratios with points . Rab5 : n = 18 ( middle right ) , Rab21 : n = 31 ( left middle ) or 20 ( right ) , Caveolin - 1 : n = 43 ( left ) .

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Primary cortical neurons from E15 cerebral cortices were transfected with pCAG-PM-mAG1 (green) and incubated for two days in vitro. Cells were immunostained with the indicated antibodies (red and blue/white). The transfected PM-mAG1 is a marker for the plasma membrane. The images are obtained with high-resolution microscopy (Nikon) and the lower panels are high magnification images near the plasma membrane. Blue alone channels are shown in black and white images. Each score represents the mean of ratios with the individual points. Rab5: n = 18 cells (middle and right), Rab21: n = 31 cells (left and middle) or 20 cells (right), Caveolin-1: n = 43 cells (left).


( H - I ) HeLa were immunostained with indicated antibodies . are obtained with high - resolution microscopy ( Nikon ) . graph in ( I ) shows Pearson ' s correlation coefficient between caveolin - 1 Rab5 or Rab21 . Each score represents mean of ratios with points . Rab5 : n = 80 , Rab21 : n = 62 .

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(H-I) HeLa cells were immunostained with the indicated antibodies. The images are obtained with high-resolution microscopy (Nikon). The graph in (I) shows the Pearson's correlation coefficient between caveolin-1 and Rab5 or Rab21. Each score represents the mean of ratios with the individual points. Rab5: n = 80 cells, Rab21: n = 62 cells.


cortical neurons from E15 cerebral cortices were transfected with indicated plasmids plus pCAG - EGFP , incubated for two days in vitro treated with BODIPY - LacCer ( LacCer ) ( green ) ( A - B ) for 30 minutes before fixation . arrows in ( A ) indicate perinuclear accumulation of LacCer . Blue alone channels are shown in black . graphs in ( B ) show of with perinuclear accumulation of LacCer ( B ) which was quantified in a blinded counting . Each score represents mean with points . B : n = 5 biological replicates ( Control : 115 , Cav1 - sh490 : 127 , Rab21 - sh115 : 136 , Rab5 - sh232 : 216 , Rab21 - sh115 + wt - Rab21 : 132 )

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Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids plus pCAG-EGFP , incubated for two days in vitro and treated with BODIPY-LacCer (LacCer) (green) (A-B) for 30 minutes before fixation. White arrows in (A) indicate the perinuclear accumulation of LacCer. Blue alone channels are shown in black and white images. The graphs in (B) show the ratio of cells with perinuclear accumulation of LacCer (B) which was quantified in a blinded counting. Each score represents the mean with the individual points. B: n = 5 biological replicates (Control: 115 cells, Cav1-sh490: 127 cells, Rab21-sh115: 136 cells, Rab5-sh232: 216 cells, Rab21-sh115 + wt-Rab21: 132 cells)


cortical neurons from E15 cerebral cortices were transfected with pCAG - PM - mAG1 , incubated for two days in vitro stained with indicated antibodies . Blue alone channels are shown in black . Arrowheads indicate accumulation of N - cadherin at plasma . Lower panels in ( D ) are high magnification indicated by or blue rectangles . are obtained with high - resolution microscopy ( Nikon ) .

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Primary cortical neurons from E15 cerebral cortices were transfected with pCAG-PM-mAG1, incubated for two days in vitro and stained with the indicated antibodies. Blue alone channels are shown in black and white images. Arrowheads indicate accumulation of N-cadherin at the plasma membrane. Lower panels in (D) are high magnification images indicated by white or blue rectangles. The images are obtained with high-resolution microscopy (Nikon).


Immature neurons in IZ of cerebral cortices at E17 , electroporated with indicated plasmids plus pCAG - PM - mAG1 pCAG - HA - N - cadherin at E14 . Frozen sections were immunostained with anti - mAG1 anti - HA antibodies . Arrow arrowheads indicate accumulation of HA - N - cadherin at perinuclear regions or plasma , respectively . Control : n = 77 from 3 biological replicates , Rab21 - sh115 : n = 108 from 3 biological replicates .

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Immature neurons in the IZ of the cerebral cortices at E17, electroporated with the indicated plasmids plus pCAG-PM-mAG1 and pCAG-HA-N-cadherin at E14. Frozen sections were immunostained with anti-mAG1 and anti-HA antibodies. Arrow and arrowheads indicate the accumulation of HA-N-cadherin at the perinuclear regions or the plasma membrane, respectively. Control: n = 77 cells from 3 biological replicates, Rab21-sh115: n = 108 cells from 3 biological replicates.


( A - B ) cortical neurons from E15 cerebral cortices were transfected with indicated plasmids plus pCAG - PM - mAG1 ( green ) incubated for two days in vitro . Immunocytochemical analyses with anti - caveolin - 1 ( red ) antibody were performed . are obtained with high - resolution microscopy ( Nikon ) . graph in ( B ) indicates of caveolin - 1 signals in plasma to total intensities of caveolin - 1 in each immature neuron . Each score represents mean of ratios with points . Control : n = 20 , Rab21 - sh115 : n = 22 , Rab21 - sh115 + wt - Rab21 : n = 23 . Overexpression of wt - Rab21 resulted in neuronal , which making it difficult to determine accurate of wt - Rab21 in rescue experiments .

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(A-B) Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids plus pCAG-PM-mAG1 (green) and incubated for two days in vitro. Immunocytochemical analyses with anti-caveolin-1 (red) antibody were performed. The images are obtained with high-resolution microscopy (Nikon). The graph in (B) indicates the ratio of the caveolin-1 staining signals in the plasma membrane to total fluorescence intensities of caveolin-1 in each immature neuron. Each score represents the mean of ratios with the individual points. Control: n = 20 cells, Rab21-sh115: n = 22 cells, Rab21-sh115 + wt-Rab21: n = 23 cells. Overexpression of wt-Rab21 resulted in neuronal cell death, which making it difficult to determine the accurate DNA concentration of wt-Rab21 in the rescue experiments.


( H - I ) cortical neurons from E15 cerebral cortices were transfected with indicated plasmids , incubated for two days in vitro , treated with Bafilomycin A1 ( Baf A1 ) for 6 h stained with indicated antibodies . are obtained with high - resolution microscopy ( Nikon ) lower panels are high magnification of lysosomes , indicated by or blue rectangles . Blue alone channels are shown in black . graph in ( I ) shows Pearson ' s correlation coefficient between caveolin - 1 Lamp1 , a lysosomal marker , in control Rab21 - sh115 - transfected neurons . Each score represents mean of ratios with points . Control : n = 31 , Rab21 - sh115 : n = 22 .

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(H-I) Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids, incubated for two days in vitro, treated with Bafilomycin A1 (Baf A1) for 6 h and stained with the indicated antibodies. The images are obtained with high-resolution microscopy (Nikon) and the lower panels are high magnification images of the lysosomes, indicated by white or blue rectangles. Blue alone channels are shown in black and white images. The graph in (I) shows the Pearson's correlation coefficient between caveolin-1 and Lamp1, a lysosomal marker, in control and Rab21-sh115-transfected neurons. Each score represents the mean of ratios with the individual points. Control: n = 31 cells, Rab21-sh115: n = 22 cells.


( A ) Locomoting neurons in upper IZ of cerebral cortices at E17 , electroporated with indicated plasmids plus pCAG - EGFP at E14 . ( B ) of locomoting neurons with more than three neurites . Each score represents mean of ratios with points . Control : n = 4 brains , Rab21 - sh115 : n = 5 brains , Rab21 - sh115 + Caveolin - 1 : n = 6 brains . ( C ) box - - whisker plot shows average leading process of locomoting neurons in IZ . Control : n = 115 , Rab21 - sh115 : n = 188 , Rab21 - sh115 + Caveolin - 1 : n = 185 . (

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(A) Locomoting neurons in the upper IZ of the cerebral cortices at E17, electroporated with the indicated plasmids plus pCAG-EGFP at E14. (B) The ratio of locomoting neurons with more than three primary neurites. Each score represents the mean of ratios with the individual points. Control: n = 4 brains, Rab21-sh115: n = 5 brains, Rab21-sh115+Caveolin-1: n = 6 brains. (C) The box-and-whisker plot shows average leading process length of the locomoting neurons in the IZ. Control: n = 115 cells, Rab21-sh115: n = 188 cells, Rab21-sh115+Caveolin-1: n = 185 cells. (


( D - E ) Cerebral cortices at P0 , electroporated with indicated plasmids plus pCAG - EGFP at E14 . lower graphs in ( D ) graph in ( E ) show estimation of migration , as measured by EGFP intensities in distinct regions of cerebral cortices using Leica SP5 software . Each bar in graph in ( E ) represents mean of relative intensities . Rab21 - sh115 : n = 4 brains , Rab21 - sh115 + CAG - wt - caveolin - 1 : n = 6 brains . II - IV : layers II - IV of cortical plate , V - VI : layers V - VI of cortical plate , IZ : intermediate zone , WM : matter , SVZ / VZ : subventricular zone / ventricular zone .

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(D-E) Cerebral cortices at P0, electroporated with the indicated plasmids plus pCAG-EGFP at E14. The lower graphs in (D) and the graph in (E) show the estimation of cell migration, as measured by EGFP fluorescence intensities in distinct regions of the cerebral cortices using Leica SP5 software. Each bar in the graph in (E) represents the mean percentage of relative intensities. Rab21-sh115: n = 4 brains, Rab21-sh115+CAG-wt-caveolin-1: n = 6 brains. II-IV: layers II-IV of the cortical plate, V-VI: layers V-VI of the cortical plate, IZ: intermediate zone, WM: white matter, SVZ/VZ: subventricular zone/ventricular zone.


B . Analyses of Hog1 phosphorylation by immunoblotting with anti - phospho - p38 ( Hog1 - P ) anti - Hog1 ( total Hog1 ) antibodies . of indicated genotypes were stimulated with indicated concentrations of NaCl for indicated time . Strains used are TM257 , KT207 KY594 - 1 .

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B. Analyses of Hog1 phosphorylation by immunoblotting with anti-phospho-p38 (Hog1-P) and anti-Hog1 (total Hog1) antibodies. Cells of the indicated genotypes were stimulated with the indicated concentrations of NaCl for the indicated time. Strains used are TM257, KT207 and KY594-1.


C . Analyses of Hog1 phosphorylation by Phos - tag band - shift . Yeast KY594 - 1 was stimulated with indicated concentrations of NaCl for 5 min . percentages of phosphorylated Hog1 [ Hog1 - P ( % ) ] were calculated as explained in Materials Methods , are shown beneath panel . Data information : Representative results from three independent experiments .

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C. Analyses of Hog1 phosphorylation by Phos-tag band-shift assay. Yeast strain KY594-1 was stimulated with the indicated concentrations of NaCl for 5 min. The percentages of phosphorylated Hog1 [Hog1-P (%)] were calculated as explained in Materials and Methods, and are shown beneath the panel. Data information: Representative results from three independent experiments.


D . Analyses of Hog1 phosphorylation by immunoblotting with anti - phospho - p38 ( Hog1 - P ) anti - Hog1 ( total Hog1 ) antibodies . Yeast KT219 was transformed with indicated STE11 mutant gene carried by a single - copy plasmid that is expressed from STE11 promoter : vec , vector ; WT , wild - type ; DDD , S281D / S285D / T286D . were incubated with ( + ) or without ( - ) 1 M NaCl for 5 min .

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D. Analyses of Hog1 phosphorylation by immunoblotting with anti-phospho-p38 (Hog1-P) and anti-Hog1 (total Hog1) antibodies. Yeast strain KT219 was transformed with the indicated STE11 mutant gene carried by a single-copy plasmid that is expressed from the STE11 promoter: vec, vector; WT, wild-type; DDD, S281D/S285D/T286D. Cells were incubated with (+) or without (-) 1 M NaCl for 5 min.


E - H . Analyses of Hog1 phosphorylation by Phos - tag band - shift . Yeast strains ( E ) KY603 - 3 ; ( F ) TM142 ; ( G ) TM257 ; ( H ) FP54 were stimulated with indicated concentrations of NaCl for 5 min . Data information : Representative results from three independent experiments .

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E-H. Analyses of Hog1 phosphorylation by Phos-tag band-shift assay. Yeast strains (E) KY603-3; (F) TM142; (G) TM257; and (H) FP54 were stimulated with the indicated concentrations of NaCl for 5 min. Data information: Representative results from three independent experiments.


I . Comparison of NaCl dose - responses of Hog1 activation by various strains . Phos - tag band - shift assays were independently repeated three times , average values were plotted . Data information : ( I ) Error bars are ( n = 3 ) .

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I. Comparison of the NaCl dose-responses of Hog1 activation by various strains. Phos-tag band-shift assays were independently repeated three times, and average values were plotted. Data information: (I) Error bars are SEM (n=3).