E F . Subcellular localization of inositol 1 , 4 , 5 - triphosphate receptor type 1 ( IP3R1 ) type 3 ( IP3R3 ) in HeLa ( F ) . IP3R1 IP3R3 in isolated fractions were identified by immunoblotting with specific antibodies as indicated . Proper fractionation of these samples was confirmed by fraction specific markers as indicated .

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E and F. Subcellular localization of inositol 1,4,5-triphosphate receptor type 1 (IP3R1) and type 3 (IP3R3) in HeLa cells (F). IP3R1 and IP3R3 in the isolated fractions were identified by immunoblotting with the specific antibodies as indicated. Proper fractionation of these samples was confirmed by the fraction specific markers as indicated.


G . Interaction of IP3R3 with wild - type Sig1R or E102Q ALS - linked Sig1R mutant . Sig1R - FLAG variants were transfected in N2a - IP3R3 , IP3R3 was co - immunoprecipitated using an anti - FLAG antibody identified by immunoblotting with specific antibodies as indicated .

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G. Interaction of IP3R3 with wild-type Sig1R or E102Q ALS-linked Sig1R mutant. Sig1R-FLAG variants were transfected in N2a-IP3R3 cells, and IP3R3 was co-immunoprecipitated using an anti-FLAG antibody and identified by immunoblotting with the specific antibodies as indicated.


H . Suppression of Sig1R by siRNA . Lysates of N2a transfected with control siRNA ( siCtrl ) or siRNA against Sig1R ( siSig1R ) for 24 h were blotted . Similar results were obtained from three independent experiments . Paired t - was used for statistical .

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H. Suppression of Sig1R by siRNA. Lysates of N2a cells transfected with control siRNA (siCtrl) or siRNA against Sig1R (siSig1R) for 24 h were blotted. Similar results were obtained from three independent experiments. Paired t-test was used for statistical analysis.


I J . calcium ( Ca2 + ) flux in N2a or human IP3R3 stably expressing N2a ( N2a - IP3R3 ) . siCtrl or siSig1R was transfected to N2a or N2a - IP3R3 , then cytoplasmicCa2 + flux were determined with fluo - 4 Case12 - mito , respectively . was normalized to in resting state at 0 s , plotted as mean ± SD .

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I and J. Cytoplasmic calcium (Ca2+) flux in N2a or human IP3R3 stably expressing N2a (N2a-IP3R3) cells. siCtrl or siSig1R was transfected to N2a or N2a-IP3R3 cells, then cytoplasmicCa2+ flux were determined with fluo-4 and Case12-mito, respectively. The fluorescent intensity was normalized to the intensity in resting state at 0 s, and plotted as mean ± SD.


I J . mitochondrial ( J ) calcium ( Ca2 + ) flux in N2a or human IP3R3 stably expressing N2a ( N2a - IP3R3 ) . siCtrl or siSig1R was transfected to N2a or N2a - IP3R3 , then mitochondrial Ca2 + flux were determined with fluo - 4 Case12 - mito , respectively . was normalized to in resting state at 0 s , plotted as mean ± SD .

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I and J. mitochondrial (J) calcium (Ca2+) flux in N2a or human IP3R3 stably expressing N2a (N2a-IP3R3) cells. siCtrl or siSig1R was transfected to N2a or N2a-IP3R3 cells, then mitochondrial Ca2+ flux were determined with fluo-4 and Case12-mito, respectively. The fluorescent intensity was normalized to the intensity in resting state at 0 s, and plotted as mean ± SD.


K L . ( K ) or mitochondrial ( L ) Ca2 + flux in N2a - IP3R3 . siCtrl or siSig1R was transfected with or without Sig1R - FLAG variants . data are obtained plotted as described above . n = 10 each . * * : p < 0 . 01 , * : p < 0 . 05 ( I - L ) . Two - way ANOVA with subsequent post hoc Tukey ' s ( I - J ) .

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K and L. Cytoplasmic (K) or mitochondrial (L) Ca2+ flux in N2a-IP3R3 cells. siCtrl or siSig1R was transfected with or without the Sig1R-FLAG variants. The data are obtained and plotted as described above. n = 10 each. **: p < 0.01, *: p < 0.05 (I-L). Two-way ANOVA with subsequent post hoc Tukey's test (I-J).


A . of Sig1R in lumbar spinal cord ( LSC ) or brain of Sig1R − / − , Sig1R + / − , or Sig1R + / + mice at 5 months old . Representative immunoblots obtained from three independent experiments are shown . Asterisk denotes non - specific bands .

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A. The levels of Sig1R in the lumbar spinal cord (LSC) or brain of Sig1R−/−, Sig1R+/−, or Sig1R+/+ mice at 5 months old. Representative immunoblots obtained from three independent experiments are shown. Asterisk denotes non-specific bands.


B C . Sig1R - deficient SOD1G85Rmice ( SOD1G85R / Sig1R − / − ) exhibited accelerated onset of disease ( B ) shortened survival time ( C ) compared to SOD1G85Rmice with one or two copies of Sig1R ( SOD1G85R / Sig1R + / − , SOD1G85R / Sig1R + / + ) . Open circles indicate Sig1R knockout mice ( Sig1R − / − ) that never developed motor neuron disease but that were sacrificed at 396 days of ( n = 8 ) . Mean onset or survival times of mice with ± SD are shown on top of Kaplan - Meyer curves . * * : p < 0 . 01 , * : p < 0 . 05 , log - rank . n = 14 ( SOD1G85R / Sig1R + / + ) , 29 ( SOD1G85R / Sig1R + / − ) , 17 ( SOD1G85R / Sig1R − / − ) .

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B and C. Sig1R-deficient SOD1G85Rmice (SOD1G85R/Sig1R−/−) exhibited accelerated onset of the disease (B) and shortened survival time (C) compared to SOD1G85Rmice with one or two copies of Sig1R (SOD1G85R/Sig1R+/−, SOD1G85R/Sig1R+/+). Open circles indicate Sig1R knockout mice (Sig1R−/−) that never developed motor neuron disease but that were sacrificed at 396 days of age (n = 8). Mean onset or survival times of mice with ±SD are shown on the top of Kaplan-Meyer curves. **: p < 0.01, *: p < 0.05, log-rank test. n = 14 (SOD1G85R/Sig1R+/+), 29 (SOD1G85R/Sig1R+/−), 17 (SOD1G85R/Sig1R−/−).


D E . weights were lost earlier both in ( D ) ( E ) of SOD1G85R / Sig1R − / − mice . Data are shown as mean ± SD . * : p < 0 . 05 in SOD1G85R / Sig1R + / + vs . SOD1G85R / Sig1R − / − , † : p < 0 . 05 in SOD1G85R / Sig1R + / − vs . SOD1G85R / Sig1R − / − . n = 8 ( SOD1G85R / Sig1R + / + ) , 13 ( SOD1G85R / Sig1R + / − ) , 8 ( SOD1G85R / Sig1R − / − ) in ( D ) , n = 6 ( SOD1G85R / Sig1R + / + ) , 16 ( SOD1G85R / Sig1R + / − ) , 9 ( SOD1G85R / Sig1R − / − ) in ( E ) . used were same in panels B C . used in panels D E were same in panels B C . * : p < 0 . 05 in SOD1G85R / Sig1R + / + vs . SOD1G85R / Sig1R − / − , † : p < 0 . 05 in SOD1G85R / Sig1R + / − vs . SOD1G85R / Sig1R − / − . Two - way ANOVA with subsequent post hoc Tukey ' s .

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D and E. Body weights were lost earlier both in male (D) and female (E) of SOD1G85R/ Sig1R−/−mice. Data are shown as mean ± SD. *: p < 0.05 in SOD1G85R/Sig1R+/+ vs. SOD1G85R/Sig1R−/−, †: p < 0.05 in SOD1G85R/Sig1R+/− vs. SOD1G85R/Sig1R−/−. n = 8 (SOD1G85R/Sig1R+/+), 13 (SOD1G85R/Sig1R+/−), 8 (SOD1G85R/Sig1R−/−) in male (D), and n = 6 (SOD1G85R/Sig1R+/+), 16 (SOD1G85R/Sig1R+/−), 9 (SOD1G85R/Sig1R−/−) in female (E). The used animals were the same in the panels B and C. The animals used in the panels D and E were the same in the panels B and C. *: p < 0.05 in SOD1G85R/Sig1R+/+ vs. SOD1G85R/Sig1R−/−, †: p < 0.05 in SOD1G85R/Sig1R+/− vs. SOD1G85R/Sig1R−/−. Two-way ANOVA with subsequent post hoc Tukey's test.


F . Decreased performance on rotarod was observed in SOD1G85R / Sig1R − / − mice . was performed at 0 - 30 rpm with 0 . 1 rpm / s acceleration every week . Data are shown as mean ± SD . * * : p < 0 . 0001 in SOD1G85R / Sig1R + / + vs . SOD1G85R / Sig1R − / − , † † : p < 0 . 0001 in SOD1G85R / Sig1R + / − vs . SOD1G85R / Sig1R − / − . used were same in panels B - E . Two - way ANOVA with subsequent post hoc Tukey ' s .

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F. Decreased performance on the rotarod test was observed in SOD1G85R/ Sig1R−/−mice. The test was performed at 0-30 rpm with 0.1 rpm/s acceleration every week. Data are shown as mean ± SD. **: p < 0.0001 in SOD1G85R/Sig1R+/+ vs. SOD1G85R/Sig1R−/−, † †: p < 0.0001 in SOD1G85R/Sig1R+/− vs. SOD1G85R/Sig1R−/−. The used animals were the same in the panels B-E. Two-way ANOVA with subsequent post hoc Tukey's test.


G H . Motor function of WT ( Sig1R + / + ) Sig1R knockout ( Sig1R − / − ) mice at 5 or 12 months old was evaluated by rotarod ( 0 - 30 rpm , accelerated at 0 . 1 rpm / s ) . Average time on rotating rod was plotted . Error bars denote SD . Unpaired t - .

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G and H. Motor function of WT (Sig1R+/+) and Sig1R knockout (Sig1R−/−) mice at 5 or 12 months old was evaluated by the rotarod test (0-30 rpm, accelerated at 0.1 rpm/s). Average time on the rotating rod was plotted. Error bars denote SD. Unpaired t-tests.


A B . N2a expressing SOD1WT , SOD1G85R or SOD1G93A ( A ) , or spinal cords of WT or mutant SOD1 transgenic mice at end - stage ( B ) were fractionated as described in Materials methods section ( Cyto : cytoplasm , P1 : nuclei debris , Mito : mitochondria , MAM : mitochondria - associate , P3 : microsomal fraction ) . Proper fractionation was confirmed by fraction specific markers as indicated , also shown in Fig EV1 A - E . An asterisk denotes non - specific bands .

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A and B. N2a cells expressing SOD1WT, SOD1G85R or SOD1G93A (A), or spinal cords of WT or mutant SOD1 transgenic mice at end-stage (B) were fractionated as described in Materials and methods section (Cyto: cytoplasm, P1: nuclei and debris, Mito: mitochondria, MAM: mitochondria-associate membrane, P3: microsomal fraction). Proper fractionation was confirmed by the fraction specific markers as indicated, and also shown in Fig EV1 A-E. An asterisk denotes non-specific bands.


C . Tissue - type specific accumulation of mutant SOD1 at MAM . Brain , liver , or astrocytes from SOD1G93Amice were fractionated blotted as in Figures 4A B ( Upper panel ) . of SOD1 at MAM relative to ones in cytoplasm was performed from immunoblotting data with an anti - human SOD1 antibody in ( B ) top panel of ( C ) , data are plotted as mean ± from three independent experiments ( Lower panel ) . * * : p < 0 . 01 vs . SOD1WTspinal cord . A one - way ANOVA with subsequent post hoc Tukey ' s . Proper fractionation of these samples was confirmed in Fig EV 1F - H .

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C. Tissue and cell-type specific accumulation of mutant SOD1 at the MAM. Brain, liver, or primary astrocytes from SOD1G93Amice were fractionated and blotted as in Figures 4A and B (Upper panel). Quantification of SOD1 proteins at the MAM relative to ones in the cytoplasm was performed from immunoblotting data with an anti-human SOD1 antibody in (B) and the top panel of (C), and the data are plotted as mean ± SEM from three independent experiments (Lower panel). **: p < 0.01 vs. SOD1WTspinal cord. A one-way ANOVA with subsequent post hoc Tukey's test.Proper fractionation of these samples was confirmed in Fig EV 1F-H.


D . Mutant SOD1 prevented association of ER with mitochondria . Isolated ER ( P3 : microsomal fraction ) mitochondria of N2a expressing wild - type ( WT ) or G85R ( 85 ) SOD1 for 48 h were mixed incubated . Mitochondrial pellets after centrifugation were analyzed by immunoblotting using anti - PDI ( ER marker ) anti - VDAC ( mitochondrial marker ) , respectively . these results were confirmed by three independent experiments .

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D. Mutant SOD1 prevented association of ER with mitochondria. Isolated ER (P3: microsomal fraction) and mitochondria of N2a cells expressing wild-type (WT) or G85R (85) SOD1 for 48 h were mixed and incubated. Mitochondrial pellets after centrifugation were analyzed by immunoblotting using anti-PDI (ER marker) and anti-VDAC (mitochondrial marker), respectively. All these results were confirmed by three independent experiments.


A B . Time course analyses of mutant SOD1 at MAM in SOD1G93Amousespinal cords ( A ) brains ( B ) . Immunoblots show of mutant SOD1 in indicated fractions at various time points ( left panels ) . Quantitative data at right were plotted as mean ± of three independent experiments . Arrows indicate a rapid reduction in SOD1 level at MAM just following onset of disease .

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A and B. Time course analyses of mutant SOD1 levels at the MAM in SOD1G93Amousespinal cords (A) and brains (B). Immunoblots show levels of mutant SOD1 protein in the indicated fractions at various time points (left panels). Quantitative data at the right were plotted as mean ± SEM of three independent experiments. Arrows indicate a rapid reduction in SOD1 level at the MAM just following the onset of disease.


C D . Time course analyses of mutant SOD1 accumulation at MAM in SOD1G85R ( C ) SOD1G37R ( D ) mousespinal cords . Quantitative data from three independent experiments were plotted ( bottom ) . arrows in bottom panel are same as in A B .

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C and D. Time course analyses of mutant SOD1 accumulation at the MAM in SOD1G85R (C) and SOD1G37R (D) mousespinal cords. Quantitative data from three independent experiments were plotted (bottom). The arrows in the bottom panel are the same as in A and B.


E . Time course analyses of MAM - specific at MAM in whole lysates of SOD1G93Amousespinal cords .

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E. Time course analyses of MAM-specific proteins at MAM and in the whole lysates of SOD1G93Amousespinal cords.


A - I . Immunofluorescence of spinal cords brains from non - transgenic ( Non - Tg ) , SOD1 transgenic , or Sig1R − / − mice . Transverse sections of mousespinal cords ( A , D - I ) or sagittal sections of mouse brains ( B , C ) were stained using anti - Sig1R ( ) , βIII - tubulin ( red ) IP3R3 ( green ) antibodies . Note that Sig1R IP3R3 are co - localized in motor neurons of anterior horn ( A ) hypoglossal nucleus ( B ) , IP3R3 was not expressed in hippocampal neurons ( C ) . Mutant SOD1 induced aggregation of Sig1R mislocalization of IP3R3 in anterior horn neurons ( D - G ) , while their abnormalities were not observed in SOD1WTmotor neurons ( H ) . Mislocalization of IP3R3 was also observed in Sig1R − / − mouse spinal cords ( I ) . Scale bars : 50 µm .

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A-I. Immunofluorescence staining of spinal cords and brains from non-transgenic (Non-Tg), SOD1 transgenic, or Sig1R−/−mice. Transverse sections of mousespinal cords (A, D-I) or sagittal sections of mouse brains (B, C) were stained using anti-Sig1R (white), βIII-tubulin (red) and IP3R3 (green) antibodies. Note that Sig1R and IP3R3 are co-localized in the motor neurons of the anterior horn (A) and the hypoglossal nucleus (B), and IP3R3 was not expressed in hippocampal neurons (C). Mutant SOD1 induced aggregation of Sig1R and mislocalization of IP3R3 in anterior horn neurons (D-G), while their abnormalities were not observed in SOD1WTmotor neurons (H). Mislocalization of IP3R3 was also observed in Sig1R−/−mouse spinal cords (I). Scale bars: 50 µm.


J K . Representative electron of MAM ( J ) ( double - headed arrows ) in motor neurons of 12 months - old Non - Tg , SOD1G85R or Sig1R − / − mice . Note that ER - mitochondria contacted areas were reduced in both SOD1G85R or Sig1R − / − mice . of mitochondria surface associated to ER was calculated in K . For , 13 - 19 motor neurons 224 - 316 mitochondria with MAM in each ( n = 2 ) were analyzed . Data are expressed as mean ± . Double asterisk means p < 0 . 0001 vs Non - Tg . A one - way ANOVA with subsequent post hoc Tukey ' s . Scale bars : 300 nm .

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J and K. Representative electron micrographs of the MAM (J) (double-headed arrows) in motor neurons of 12 months-old Non-Tg, SOD1G85R or Sig1R−/−mice. Note that ER-mitochondria contacted areas were reduced in both SOD1G85R or Sig1R−/−mice. Quantification of the mitochondria surface associated to ER was calculated in K. For quantification, 13-19 motor neurons and 224-316 mitochondria with MAM in each animal (n = 2) were analyzed. Data are expressed as mean ± SEM. Double asterisk means p < 0.0001 vs Non-Tg. A one-way ANOVA with subsequent post hoc Tukey's test. Scale bars: 300 nm.


L . for IP3R3 calreticulin were decreased in MAM fractions of Sig1R − / − mouse brains . MAM fractions whole tissue lysates of Sig1R + / + or Sig1R − / − mouse brains were immunoblotted . Representative blots from three independent experiments are shown .

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L. The levels for IP3R3 and calreticulin were decreased in the MAM fractions of Sig1R−/−mouse brains. MAM fractions and whole tissue lysates of Sig1R+/+ or Sig1R−/−mouse brains were immunoblotted. Representative blots from three independent experiments are shown.


A . N2a were transfected with WT or mutant SOD1 in presence or absence of IP3R3 . Plotted viability of measured by a neurotoxicity revealed that IP3R3 is involved in vulnerability against mutant SOD1 . Data are expressed as mean ± from three independent experiments , triplicated in each experiment . * * : p < 0 . 01 , * : p < 0 . 05 .

O O B-CELL_LINE O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O 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. N2a cells were transfected with WT or mutant SOD1 in the presence or absence of IP3R3. Plotted viability of the cells measured by a neurotoxicity assay revealed that IP3R3 is involved in cell vulnerability against mutant SOD1. Data are expressed as mean ± SEM from three independent experiments, triplicated in each experiment. **: p < 0.01, *: p < 0.05.


B - G . Calpain activity ( B - D ) were measured in N2a - IP3R3 expressing Sig1R - FLAG ( B E ) , SOD1 ( C F ) , or both ( D G ) . Mean ± from three independent experiments is plotted . * : p < 0 . 05 .

O O O O B-GENEPROD O O O O O O O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O 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-G. Calpain activity (B-D) were measured in N2a-IP3R3 cells expressing Sig1R-FLAG (B and E), SOD1 (C and F), or both (D and G). Mean ± SEM from three independent experiments is plotted. *: p < 0.05.


B - G . intracellular ATP ( E - G ) were measured in N2a - IP3R3 expressing Sig1R - FLAG ( B E ) , SOD1 ( C F ) , or both ( D G ) . Mean ± from three independent experiments is plotted . * : p < 0 . 05 .

O O O O O B-SMALL_MOLECULE O O O O O O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O 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-G. intracellular ATP levels (E-G) were measured in N2a-IP3R3 cells expressing Sig1R-FLAG (B and E), SOD1 (C and F), or both (D and G). Mean ± SEM from three independent experiments is plotted. *: p < 0.05.


H I . Calpain activity in vivo was determined by cleavage of spectrin αII . Calpain - 150 kDa fragment of spectrin αII in mouselumbar spinal cord or brain ( H ) was normalized to β - actin ( I ) . Quantitative data in immunoblotting analyses are plotted as mean ± from three independent experiments . * * : p < 0 . 01 .

O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O B-ORGANISM B-TISSUE I-TISSUE O B-TISSUE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O 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 and I. Calpain activity in vivo was determined by the cleavage of spectrin αII. Calpain-cleaved 150 kDa fragment of spectrin αII in mouselumbar spinal cord or brain (H) was normalized to β-actin (I). Quantitative data in immunoblotting analyses are plotted as mean ± SEM from three independent experiments. **: p < 0.01.


A B . ( A ) or mitochondrial ( B ) Ca2 + flux was measured in N2a - IP3R3 treated with PRE - 084 ( 5 µM ) or NE - 100 ( 5 µM ) for 1 h prior to imaging . mitochondrialCa2 + flux were detected by fluo - 4 Case12 - mito , respectively . was normalized by resting state at 0 s , plotted as mean ± SD . n = 10 each . * * : p < 0 . 01 , * : p < 0 . 05 . Two - way ANOVA with subsequent post hoc Tukey ' s .

O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-CELL_LINE O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and B. Cytoplasmic (A) or mitochondrial (B) Ca2+ flux was measured in N2a-IP3R3 cells treated with PRE-084 (5 µM) or NE-100 (5 µM) for 1 h prior to fluorescent imaging. Cytoplasmic and mitochondrialCa2+ flux were detected by fluo-4 and Case12-mito, respectively. The fluorescent intensity was normalized by the resting state at 0 s, and plotted as mean ± SD. n = 10 each. **: p < 0.01, *: p < 0.05. Two-way ANOVA with subsequent post hoc Tukey's test.


C D . Calpain activity were measured in N2a - IP3R3 treated with PRE - 084 ( 5 µM ) or NE - 100 ( 5 µM ) for 24 h . Data are plotted as mean ± from three independent experiments . * : p < 0 . 05 vs . mock control . A one - way ANOVA with subsequent post hoc Tukey ' s .

O O O B-GENEPROD O O O O B-CELL_LINE O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O 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 and D. Calpain activity were measured in N2a-IP3R3 cells treated with PRE-084 (5 µM) or NE-100 (5 µM) for 24 h. Data are plotted as mean ± SEM from three independent experiments. *: p < 0.05 vs. mock control. A one-way ANOVA with subsequent post hoc Tukey's test.


C D . intracellular ATP were measured in N2a - IP3R3 treated with PRE - 084 ( 5 µM ) or NE - 100 ( 5 µM ) for 24 h . Data are plotted as mean ± from three independent experiments . * : p < 0 . 05 vs . mock control . A one - way ANOVA with subsequent post hoc Tukey ' s .

O O O O B-SMALL_MOLECULE O O O B-CELL_LINE O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O 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 and D. intracellular ATP levels were measured in N2a-IP3R3 cells treated with PRE-084 (5 µM) or NE-100 (5 µM) for 24 h. Data are plotted as mean ± SEM from three independent experiments. *: p < 0.05 vs. mock control. A one-way ANOVA with subsequent post hoc Tukey's test.


E F . SOD1G93Amice were intraperitoneally administered with saline or PRE - 084 ( 0 . 25 mg / kg , 3 times per week ) from postnatal day 35 to 95 . Lumbar spinal cord sections were stained by using anti - Sig1R ( ) , IP3R3 ( green ) , βIII - tubulin ( red ) at 95 days old . Arrowheads indicate neurons with normal distribution of Sig1R IP3R3 . Scale bars : 50 µm .

O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O

E and F. SOD1G93Amice were intraperitoneally administered with saline or PRE-084 (0.25 mg/kg, 3 times per week) from postnatal day 35 to 95. Lumbar spinal cord sections were stained by using anti-Sig1R (white), IP3R3 (green), and βIII-tubulin (red) at 95 days old. Arrowheads indicate the neurons with normal distribution of Sig1R and IP3R3. Scale bars: 50 µm.


A , B , Immunostaining for CD31 + blood vessels in control ( ctrl , A ) Gpr124KO ( B ) forebrains . Note normal vasculature in cortical hem ( asterisk ) of Gpr124KO mice .

O O O O B-EXP_ASSAY O B-GENEPROD O B-TISSUE I-TISSUE O O O O O O O B-GENEPROD O O O B-TISSUE O O O B-TISSUE O B-TISSUE I-TISSUE O O O O B-GENEPROD B-ORGANISM O

A,B, Immunostaining for CD31+ blood vessels in control (ctrl, A) and Gpr124KO (B) forebrains. Note the normal vasculature in the cortical hem (asterisk) of Gpr124KO mice.


C , D , Immunostaining for Pax6 in control ( C ) Gpr124KO ( D ) forebrains to reveal lateral extension of neocortex ( dashed ) between cortical hem ( arrowhead ) lateral ganglionic eminence ( arrow ) . E , of lateral extension of cortex in control Gpr124KO brains ( mean ± ; N = 10 ; * * * p < 0 . 001 ) .

O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O B-TISSUE O O O O O B-TISSUE O O O O B-TISSUE I-TISSUE O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O B-TISSUE O O B-GENEPROD B-TISSUE O O O O O O O O O O O O O O O O O O

C,D, Immunostaining for Pax6 in control (C) and Gpr124KO (D) forebrains to reveal the lateral extension of the neocortex (dashed line) between the cortical hem (arrowhead) and the lateral ganglionic eminence (arrow). E, Quantification of the lateral extension of the cortex in control and Gpr124KO brains (mean±SEM; N=10; *** p<0.001).


F , G for EdU ( red ) Ki67 ( green ) in control ( F ) Gpr124KO ( G ) cortices at E13 . 5 , 24 hours after EdU injection . H , of Ki67 + NPCs in VZ ( blue ) , SVZ / CP ( grey ) of Ki67 - neurons ( cyan ) generated from EdU - labeled NPCs in control Gpr124KO ( mean ± ; N = 4 ( control ) N = 3 ( Gpr124KO ) ; * p < 0 . 05 , * * 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 B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-TISSUE O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-CELL_TYPE O B-TISSUE O O O O B-TISSUE O B-TISSUE O O O O B-GENEPROD O B-CELL_TYPE O O O O O B-EXP_ASSAY O O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O 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-TISSUE O B-TISSUE I-TISSUE O O O O O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O

F,G Staining for EdU (red) and Ki67 (green) in control (F) and Gpr124KO (G) cortices at E13.5, 24 hours after EdU injection. H, Quantification of Ki67+ NPCs in the VZ (blue), SVZ/CP (grey) and of Ki67- neurons (cyan) generated from EdU-labeled NPCs in control and Gpr124KO embryos (mean±SEM; N=4 (control) and N=3 (Gpr124KO); * p<0.05, ** 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


I , J , Immunostaining for Tbr2 ( red ) Pax6 ( green ) in control ( I ) Gpr124KO ( J ) cortices . K , of newborn BPs in VZ of control Gpr124KO cortices ( mean ± ; N = 4 ; * p < 0 . 05 ) . 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 B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-TISSUE O O O O O O O B-TISSUE O O B-GENEPROD B-TISSUE O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O

I,J, Immunostaining for Tbr2 (red) and Pax6 (green ) in control (I) and Gpr124KO (J) cortices. K, Quantification of newborn BPs in the VZ of control and Gpr124KO cortices (mean±SEM; N=4; * p<0.05). 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


L , M , Immunostaining for Tbr2 ( red ) Ngn1 ( green ) in control ( L ) Gpr124KO ( M ) cortices . N , O of neurogenic ( Ngn1 + ) RGs BPs ( N ) or expanding ( Tbr2 - Ngn1 - ) RGs ( O ) in control Gpr124KO cortices ( mean ± ; N = 4 ; * p < 0 . 05 , * * * p < 0 . 001 ) . Note that fraction of total BPs is significantly reduced ( Fig 1K ) while fraction of Ngn1 + BPs is insignificantly reduced ( Fig 1N ) , because proportion of Ngn1 + Tbr2 + within BPs is slightly , though statisically insignificantly higher in Gpr124KObrains ( see Fig S2M ) . 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 B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-TISSUE O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O B-GENEPROD B-TISSUE O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O 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-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O

L,M, Immunostaining for Tbr2 (red) and Ngn1 (green) in control (L) and Gpr124KO (M) cortices. N,O Quantification of neurogenic (Ngn1+) RGs and BPs (N) or expanding (Tbr2- Ngn1-) RGs (O) in control and Gpr124KO cortices (mean±SEM; N=4; * p<0.05, *** p<0.001). Note that the fraction of total BPs is significantly reduced (Fig 1K) while the fraction of Ngn1+BPs is insignificantly reduced (Fig 1N), because the proportion of Ngn1+Tbr2+ cells within all BPs is slightly, though statisically insignificantly higher in the Gpr124KObrains (see Fig S2M). 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


P , Q Stainings for Tbr2 ( red ) for Tis21 - GFP expression ( green ) in control ( P ) Gpr124KO ( Q ) cortices . R , S of neurogenic ( Tis21 - GFP + ) RGs BPs ( R ) or expanding ( Tbr2 - Tis21 - GFP - ) RGs ( S ) in control Gpr124KO cortices ( mean ± ; N = 4 ; * * * 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 B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-TISSUE O O O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O B-GENEPROD B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O

P,Q Stainings for Tbr2 (red) and for Tis21-GFP expression (green) in control (P) and Gpr124KO (Q) cortices. R,S Quantification of neurogenic (Tis21-GFP+) RGs and BPs (R) or expanding (Tbr2- Tis21-GFP-) RGs (S) in control and Gpr124KO cortices (mean±SEM; N=4; *** 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 , B , for Isolectin B4 + blood vessels in control ( A ) Gpr124ECcKO ( B ) forebrains . Asterisks mark lateral ventricles .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O B-GENEPROD O O O B-TISSUE O O O B-TISSUE I-TISSUE O

A,B, Staining for Isolectin B4+ blood vessels in control (A) and Gpr124ECcKO (B) forebrains. Asterisks mark the lateral ventricles.


C , D , for pimonidazole ( PIMO , green ) Isolectin B4 + blood vessels ( red ) in ctrl ( C ) Gpr124ECcKO ( D ) cortices .

O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O O O O B-GENEPROD O O O B-TISSUE O

C,D, Staining for pimonidazole (PIMO, green) and Isolectin B4+ blood vessels (red) in ctrl (C) and Gpr124ECcKO (D) cortices.


E , F , Immunostaining for Pax6 in control ( E ) Gpr124ECcKO ( F ) forebrain sections to reveal lateral extension of neocortex ( dashed ) between cortical hem ( arrowhead ) lateral ganglionic eminence ( arrow ) . G , lateral extension of cortex in control Gpr124ECcKObrains ( mean ± ; N = 6 for ctrl N = 8 for Gpr124ECcKO ; * p < 0 . 05 ) .

O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O B-TISSUE O O O O B-TISSUE I-TISSUE O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O B-TISSUE O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O

E,F, Immunostaining for Pax6 in control (E) and Gpr124ECcKO (F) forebrain sections to reveal the lateral extension of the neocortex (dashed line) between the cortical hem (arrowhead) and the lateral ganglionic eminence (arrow). G, Quantification the lateral extension of the cortex in control and Gpr124ECcKObrains (mean±SEM; N=6 for ctrl and N=8 for Gpr124ECcKO; * p<0.05).


H , I , for EdU ( red ) Ki67 ( green ) in control ( H ) Gpr124ECcKO ( I ) cortices at E13 . 5 , 24 hours after EdU injection . J , of Ki67 + NPCs in in VZ ( blue ) , SVZ / CP ( grey ) of Ki67 - neurons ( cyan ) generated from EdU - labeled NPCs in control Gpr124ECcKO ( mean ± ; N = 4 ; * p < 0 . 05 , * * 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 B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-TISSUE O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-CELL_TYPE O O B-TISSUE O O O O B-TISSUE O B-TISSUE O O O O B-GENEPROD O B-CELL_TYPE O O O O O B-EXP_ASSAY O O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O 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-TISSUE O B-TISSUE I-TISSUE O O O O O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O

H,I, Staining for EdU (red) and Ki67 (green) in control (H) and Gpr124ECcKO (I) cortices at E13.5, 24 hours after EdU injection. J, Quantification of Ki67+ NPCs in the in VZ (blue), SVZ/CP (grey) and of Ki67- neurons (cyan) generated from EdU-labeled NPCs in control and Gpr124ECcKO embryos (mean±SEM; N=4; * p<0.05, ** 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


K , L , Immunostaining for Tbr2 ( red ) Ngn1 ( green ) in control ( K ) Gpr124KO ( L ) cortices . M - O , of neurogenic ( Ngn1 + ) RGs BPs ( M ) , newborn BPs ( N ) or expanding ( Tbr2 - Ngn1 - ) RGs ( O ) in control Gpr124ECcKO cortices ( mean ± ; N = 4 ; * p < 0 . 05 , * * 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 B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-TISSUE O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O B-GENEPROD B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O

K,L, Immunostaining for Tbr2 (red) and Ngn1 (green) in control (K) and Gpr124KO (L) cortices. M-O, Quantification of neurogenic (Ngn1+) RGs and BPs (M), newborn BPs (N) or expanding (Tbr2- Ngn1-) RGs (O) in control and Gpr124ECcKO cortices (mean±SEM; N=4; * p<0.05, ** 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 NSC - specific markers ( Fabp7 , Hes5 ) neuronal markers ( Dcx , Tubb3 ) in Prom1 + fraction , presented as fold change compared to Prom1 - fraction ) from WT cortices at E14 . 5 ( mean ± ; N = 3 for Dcx ; N = 6 for other genes ) .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O O O O O O O O O

A, qRT-PCR analysis of NSC-specific markers (Fabp7, Hes5) and neuronal markers (Dcx, Tubb3) in the Prom1+ fraction, presented as fold change compared to the Prom1- fraction) from WT cortices at E14.5 (mean±SEM; N=3 for Dcx; N=6 for all other genes).


F , Western blot for HIF - 1 ( top ) , HIF - 2 ( middle ) a - tubulin as loading control ( bottom ) using forebrain lysates from E13 . 5 control ( left ) or Gpr124KOembryos ( middle ) . HIF - 1 / 2 - transfected HEK293 served as positive control ( right ) . G , of HIF - 1 band intensities shown in panel F ( mean ± ; N = 3 ; * * p < 0 . 01 ) .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-TISSUE O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

F, Western blot for HIF-1 (top), HIF-2 (middle) and a-tubulin as loading control (bottom) using forebrain lysates from E13.5 control (left) or Gpr124KOembryos (middle). HIF-1/2-transfected HEK293 cells served as positive control (right). G, Quantification of HIF-1 band intensities shown in panel F (mean±SEM; N=3; ** p<0.01).


H , I , Immunostaining for HIF - 1α ( red ) DAPI ( blue ) in control ( H , H ' , H ' ' ) Gpr124KO ( I , I ' , I ' ' ) cortices . Panels H ' , H ' ' , I ' I ' ' are magnifications of boxed areas in panels I J , showing HIF - 1α signal alone ( H ' , I ' ) or together with DAPI ( H ' ' , I ' ' ) . Asterisks indicate autofluorescent blood . dashed indicates basal boundary of VZ . Scale bar = 100 µm . 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 B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-TISSUE O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-CELL_TYPE O O O O O O B-TISSUE O O 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-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O

H,I, Immunostaining for HIF-1α (red) and DAPI (blue) in control (H, H', H'') and Gpr124KO (I,I',I'') cortices. Panels H',H'',I' and I'' are magnifications of the boxed areas in panels I and J, showing HIF-1α signal alone (H',I') or together with DAPI (H'',I''). Asterisks indicate autofluorescent blood cells. The dashed line indicates the basal boundary of the VZ. Scale bar = 100 µm. 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


J , qRT - PCR of HIF - 1 target gene expression in VZ from cortex of control Gpr124KO E13 . 5 ( mean ± ; N = 4 ; * * * p < 0 . 001 ) . SVZ , subventricular zone ; VZ , ventricular zone .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE O B-TISSUE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O

J, qRT-PCR analysis of HIF-1 target gene expression in VZ cells from the cortex of control and Gpr124KO E13.5 embryos (mean±SEM; N=4; *** p<0.001). SVZ, subventricular zone; VZ, ventricular zone.


A - F , for Isolectin B4 ( A - C ) oxidized thiols ( D - F ) in WT cortices at E10 . 5 ( A , D ) , E11 . 5 ( B , E ) E12 . 5 ( C , F ) . Panels D - F are pseudocolor conversions of ( from low [ purple ] to high [ red ] ) . Arrowheads denote position of vascular front .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Staining for Isolectin B4 (A-C) and oxidized thiols (D-F) in WT cortices at E10.5 (A,D), E11.5 (B,E) and E12.5 (C,F). Panels D-F are pseudocolor conversions of staining intensity (from low [purple] to high [red]). Arrowheads denote the position of the vascular front.


G - I , Immunostaining for HIF - 1α ( red ) DAPI ( blue ) in WT cortices at E10 . 5 ( G - G ' ' ) , E11 . 5 ( H - H ' ' ) E12 . 5 ( I - I ' ' ) . Panels G ' , G ' ' , H ' , H ' ' , I ' I ' ' are magnifications of boxed areas in their respective original panels . Arrows point to border of lateral dorsal cortex .

O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O

G-I, Immunostaining for HIF-1α (red) and DAPI (blue) in WT cortices at E10.5 (G-G''), E11.5 (H-H'') and E12.5 (I-I''). Panels G',G'',H',H'',I' and I'' are magnifications of the boxed areas in their respective original panels. Arrows point to the border of lateral and dorsal cortex.


J - L , Immunostaining for Glut1 of WT cortices at E10 . 0 ( J ) , E11 . 5 ( K ) E12 . 5 ( L ) . Dashed lines indicate apical border of cortex . Scale bar : 100 µm ( D , F , G , H , I , J - L ) or 50 µm ( I ' ' ) , respectively .

O O O O B-EXP_ASSAY O B-GENEPROD O O B-TISSUE O O O 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-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J-L, Immunostaining for Glut1 of WT cortices at E10.0 (J), E11.5 (K) and E12.5 (L). Dashed lines indicate the apical border of the cortex. Scale bar: 100 µm (D,F,G,H,I, J-L) or 50 µm (I''), respectively.


A , qRT - PCR of HIF - 1 HIF - 1 target gene expression in cortices from E13 . 5 WT HIF - 1αCC + / - ( mean ± ; N = 8 for WT N = 5 for HIF - 1αCC + / - ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O 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, qRT-PCR analysis of HIF-1 and HIF-1 target gene expression in cortices from E13.5 WT and HIF-1αCC+/-embryos (mean±SEM; N=8 for WT and N=5 for HIF-1αCC+/-; * p<0.05, ** p<0.01, *** p<0.001).


B , C , for EdU ( red ) Ki67 ( green ) in WT ( B ) HIF - 1αCC + / - ( C ) cortices at E13 . 5 , 24 hours after EdU injection . D , of Ki67 + NPCs in in VZ ( blue ) , SVZ / CP ( grey ) of Ki67 - neurons ( cyan ) generated from EdU - labeled NPCs in control HIF - 1αCC + / - brains ( mean ± ; N = 4 ; * p < 0 . 05 ) . 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 B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-TISSUE O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-CELL_TYPE O O B-TISSUE O O O O B-TISSUE O B-TISSUE O O O O B-GENEPROD O B-CELL_TYPE O O O O O B-EXP_ASSAY O O B-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE O O O O O O O O O O O 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-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O

B,C, Staining for EdU (red) and Ki67 (green) in WT (B) and HIF-1αCC+/- (C) cortices at E13.5, 24 hours after EdU injection. D, Quantification of Ki67+NPCs in the in VZ (blue), SVZ/CP (grey) and of Ki67-neurons (cyan) generated from EdU-labeled NPCs in control and HIF-1αCC+/-brains (mean±SEM; N=4; * p<0.05). 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


E , F , Immunostaining for Tbr2 ( red ) Ngn1 ( green ) in WT ( E ) or HIF - 1αCC + / - ( F ) cortices at E13 . 5 . G - I of neurogenic ( Ngn1 + ) RGs BPs ( G ) , newborn BPs ( H ) or expanding ( Tbr2 - Ngn1 - ) RGs ( I ) in control HIF - 1αCC + / - cortices at E13 . 5 ( 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 B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-TISSUE O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O

E,F, Immunostaining for Tbr2 (red) and Ngn1 (green) in WT (E) or HIF-1αCC+/- (F) cortices at E13.5. G-I Quantification of neurogenic (Ngn1+) RGs and BPs (G), newborn BPs (H) or expanding (Tbr2-Ngn1-) RGs (I) in control and HIF-1αCC+/-cortices at E13.5 (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


J , Whole mount image of brains from WT or HIF - 1αCC + / - mice at P5 . K of cortex in brain sections from P5 WT or HIF - 1αCC + / - mice ( mean ± ; N = 8 ( WT ) or N = 6 ( HIF - 1αCC + / - ) ; * p < 0 . 05 ) .

O O O O O O B-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O

J, Whole mount image of brains from WT or HIF-1αCC+/-mice at P5. K Quantification of cortex area in brain sections from P5 WT or HIF-1αCC+/-mice (mean±SEM; N=8 (WT) or N=6 (HIF-1αCC+/-); * p<0.05).


L , M , with Hoechst 33248 in P5 WT ( L ) or HIF - 1αCC + / - ( M ) cortices . N , of cortical thickness of WT HIF - 1αCC + / - cortices . double arrow denotes measured thickness of cortex . N , of cortical thickness of WT HIF - 1αCC + / - cortices ( mean ± ; N = 8 ( WT ) or N = 6 ( HIF - 1αCC + / - ) ; * * * p < 0 . 001 ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-TISSUE O O O O B-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O

L,M, Staining with Hoechst 33248 in P5 WT (L) or HIF-1αCC+/- (M) cortices. N, Quantification of cortical thickness of WT and HIF-1αCC+/-cortices. The double arrow denotes the measured thickness of the cortex. N, Quantification of cortical thickness of WT and HIF-1αCC+/-cortices (mean±SEM; N=8 (WT) or N=6 (HIF-1αCC+/-); *** p<0.001).


O - Q , Epifluorescent of E16 . 5 cortex , 3 days after in utero electroporation ( IUE ) with EGFP alone ( ctrl , O ) , wild type HIF - 1 + EGFP ( P ) or mutant HIF - 1ΔC + EGFP ( Q ) . O ' , P ' Q ' are magnifications of boxed in their respective original panel . R , of EGFP + in cortical zones VZ / SVZ , IZ CP shown in O - Q ( mean ± ; N = 5 ; * * p < 0 . 01 , * p < 0 . 05 ) . 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 B-EXP_ASSAY O O O O B-TISSUE O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD 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-GENEPROD O O O O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O 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-TISSUE O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O

O-Q, Epifluorescent images of the E16.5 cortex, 3 days after in utero electroporation (IUE) with EGFP alone (ctrl, O), wild type HIF-1+EGFP (P) or mutant HIF-1ΔC+EGFP (Q). O',P' and Q' are magnifications of the boxed area in their respective original panel. R, Quantification of EGFP+ cells in the cortical zones VZ/SVZ, IZ and CP shown in O-Q (mean±SEM; N=5; ** p< 0.01,* p<0.05). 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


S - U , qRT - PCR of NSC marker ( S ; mean ± ; N = 5 ; * P < 0 . 05 ) , neurogenic markers ( T ; mean ± ; N = 5 ; * * * p < 0 . 001 , * p < 0 . 05 ) , HIF target genes ( U ; mean ± ; N = 5 ; * * p < 0 . 01 , * p < 0 . 05 ) in expanding NPCs ( eNPCs ) neurogenic NPCs ( nNPCs ) . Gene expression was normalized to in eNPCs .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_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 O O O O B-GENEPROD O O O O O 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-CELL_TYPE O O O O B-CELL_TYPE O O O O O O O O O O O O

S-U, qRT-PCR analysis of NSC marker (S; mean±SEM; N=5; * P<0.05), neurogenic markers (T; mean±SEM; N=5; *** p< 0.001, * p<0.05), and HIF target genes (U; mean±SEM; N=5; ** p< 0.01,* p<0.05) in expanding NPCs (eNPCs) and neurogenic NPCs (nNPCs). Gene expression was normalized to the levels in eNPCs.


B - E , for pimonodazole ( PIMO , green ) Isolectin B4 ( IB4 , red ) in Gpr124KO ( B , C ) control ( D , E ) cortices after exposure of pregnant dams to 21 % O2 ( B , D ) or 80 % O2 ( C , E ) . Compared to pimonidazole in panels B , C , of this is much weaker in panels D , E . For reasons of clarity , pimonidazole in panels D , E was therefore enhanced , equally in both panels .

O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O B-TISSUE O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O

B-E, Staining for pimonodazole (PIMO, green) and Isolectin B4 (IB4,red) in Gpr124KO (B,C) and control (D,E) cortices after exposure of the pregnant dams to 21% O2 (B,D) or 80% O2 (C,E). Compared to the pimonidazole staining in panels B,C, the intensity of this staining is much weaker in panels D,E. For reasons of clarity, the pimonidazole staining in panels D,E was therefore enhanced, equally in both panels.


F , G , Immunostaining for HIF - 1 ( red ) DAPI ( blue ) in Gpr124KOcortices after exposure of pregnant dams to 21 % O2 ( F ) or 80 % O2 ( G ) . 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 B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O 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-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O

F,G, Immunostaining for HIF-1 (red) and DAPI (blue) in Gpr124KOcortices after exposure of the pregnant dams to 21% O2 (F) or 80% O2 (G). 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


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 B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD B-TISSUE O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O B-GENEPROD B-TISSUE O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O 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-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O B-TISSUE O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O B-TISSUE O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-TISSUE O B-GENEPROD O O O O O B-CELL_TYPE O O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD 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 B-SMALL_MOLECULE O O O B-CELL_TYPE O O B-CELL_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

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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-CELL_TYPE O O O O O B-GENEPROD 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 B-SMALL_MOLECULE O O O B-CELL_TYPE O O O O B-GENEPROD 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 B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-CELL_TYPE O O O O O B-GENEPROD 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 B-EXP_ASSAY O O O O B-TISSUE O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O 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-EXP_ASSAY O O B-TISSUE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE 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 B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O 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-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O 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-EXP_ASSAY 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 B-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O 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 .

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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

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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

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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 .

B-EXP_ASSAY O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-TISSUE O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM 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 B-GENEPROD O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O B-EXP_ASSAY O O 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-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE 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 B-GENEPROD O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE 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

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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-GENEPROD O B-CELL_LINE B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O B-CELL_LINE B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O 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

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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 )

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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

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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 )

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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

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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-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY 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 B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY 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 )

O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

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

O B-CELL_LINE O B-GENEPROD O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O

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).