Figure 2 [ A ] Plants grown with 0 . 05 MS and GMVs ( G 0 . 05 ) showed hyper - induction of IPS1 and PS2 gene expression in both shoots and roots , compared to plants grown with 0 . 05 MS alone ( C 0 . 05 ) . Values are means ± SE of three biological replicates . [ B ] Root acid phosphatase activity as detected by the blue color from the 5 - bromo - 4 - chloro - 3 - indolyl phosphate p - toluidine salt ( BCIP ) treatment . Image contrast enhanced for improved visualization of the blue color in roots Supplementation of Pi to the nutrient - deficient plants ( 0 . 05 + Pi ) significantly reduced hyper - accumulation of anthocyanin ( C ) triggered by GMVs . Data information : The boxplots show representative data from three independent experiments ( n = 12 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test within each group of the same DAT . Supplementation of Pi to the nutrient - deficient plants ( 0 . 05 + Pi ) significantly reduced hyper - induction of IPS1 gene expression ( D ) triggered by GMVs . Data information : The boxplots show representative data from three independent experiments ( n = 12 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test within each group of the same DAT . [ E ] Pi supplementation to the 0 . 05 MS medium partially restored GMV - induced plant vigor , as indicated by increases in total leaf area per plant . [ F ] A low Pi level ( LP ) , which was equal to 1 / 20 of that in the 0 . 5 MS medium , in plant growth medium blocked GMV - induced plant vigor . Data information : The boxplots show representative data from three independent experiments ( n = 12 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test within each group of the same DAT . [ G ] The Arabidopsis phr1phl1 mutant showed substantially decreased growth - promotion , compared to the wild type plants . Data information : The boxplots show representative data from three independent experiments ( n = 12 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test within each group of the same DAT .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 [A] Plants grown with 0.05 MS and GMVs (G 0.05) showed hyper-induction of IPS1 and PS2 gene expression in both shoots and roots, compared to plants grown with 0.05 MS alone (C 0.05). Values are means ± SE of three biological replicates.  [B] Root acid phosphatase activity as detected by the blue color from the 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (BCIP) treatment. Image contrast enhanced for improved visualization of the blue color in roots Supplementation of Pi to the nutrient-deficient plants (0.05 + Pi) significantly reduced hyper-accumulation of anthocyanin (C) triggered by GMVs. Data information: The boxplots show representative data from three independent experiments (n=12). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group of the same DAT.Supplementation of Pi to the nutrient-deficient plants (0.05 + Pi) significantly reduced hyper-induction of IPS1 gene expression (D) triggered by GMVs. Data information: The boxplots show representative data from three independent experiments (n=12). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group of the same DAT. [E] Pi supplementation to the 0.05 MS medium partially restored GMV-induced plant vigor, as indicated by increases in total leaf area per plant. [F] A low Pi level (LP), which was equal to 1/20 of that in the 0.5 MS medium, in plant growth medium blocked GMV-induced plant vigor. Data information: The boxplots show representative data from three independent experiments (n=12). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group of the same DAT. [G] The Arabidopsis phr1phl1 mutant showed substantially decreased growth-promotion, compared to the wild type plants. Data information: The boxplots show representative data from three independent experiments (n=12). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group of the same DAT.


Figure 3 [ A ] Images of Arabidopsis plants exposed to four individual GMV components , including diacetyl ( DA ) , 2 , 3 - butanediol ( BTDL ) , 2 - methyl - 1 - propanol ( MP ) and acetoin ( ATN ) . The compounds were applied at dosages that , when the compounds totally evaporate from the agar - containing solid droplets , would yield in volatile concentrations of 9 . 7 µg ( DA ) , 32 . 5 µg ( BTDL ) , 7 . 9 µg ( MP ) and 28 . 5 µg ( ATN ) per mL free space in the petri dish , which resembled the 1 : 3 . 3 : 0 . 8 : 2 . 9 ratio among the four GMV components in natural GMVs22 . anthocyanin hyper accumulation ( B ) were observed in Pi - deficient plants exposed to DA but not to the other three GMV components . Data information : The boxplots show representative data from three independent experiments ( n = 6 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test . PSR gene hyper induction ( C ) were observed in Pi - deficient plants exposed to DA but not to the other three GMV components . Data information : qPCR results show values of means ± SE of two biological replicates . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test . Compared to the wild type plants , phr1phl1 showed substantially decreased anthocyanin hyper accumulation ( D ) triggered by DA . Data information : The boxplots show representative data from three independent experiments ( n = 6 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test . Compared to the wild type plants , phr1phl1 showed substantially decreased PSR gene hyper induction ( E ) triggered by DA . Data information : qPCR results show values of means ± SE of two biological replicates . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 [A] Images of Arabidopsis plants exposed to four individual GMV components, including diacetyl (DA), 2,3-butanediol (BTDL), 2-methyl-1-propanol (MP) and acetoin (ATN). The compounds were applied at dosages that, when the compounds totally evaporate from the agar-containing solid droplets, would yield in volatile concentrations of 9.7 µg (DA), 32.5 µg (BTDL), 7.9 µg (MP) and 28.5 µg (ATN) per mL free space in the petri dish, which resembled the 1 : 3.3 : 0.8 : 2.9 ratio among the four GMV components in natural GMVs22. anthocyanin hyper accumulation (B) were observed in Pi-deficient plants exposed to DA but not to the other three GMV components. Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.PSR gene hyper induction (C) were observed in Pi-deficient plants exposed to DA but not to the other three GMV components. Data information: qPCR results show values of means ± SE of two biological replicates. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.Compared to the wild type plants, phr1phl1 showed substantially decreased anthocyanin hyper accumulation (D) triggered by DA. Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.Compared to the wild type plants, phr1phl1 showed substantially decreased PSR gene hyper induction (E) triggered by DA. Data information: qPCR results show values of means ± SE of two biological replicates. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.


Figure 4 [ A ] DA induces genes involved in SA biosynthesis and signaling in Arabidopsis grown with Pi deficiency . Data information : qPCR results show values of means ± SE ( n = 3 ) , two biological replicates were analyzed with similar results . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test . [ B ] DA elevates SA accumulation levels in Pi - deficient Arabidopsis . Means ± SE of three biological replicates . Data information : The boxplots show representative data from three independent experiments ( n = 6 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test . [ C ] Images of plants grown with different treatments using DA ( 9 . 7 µg per mL free space ) or SA ( 100 µM in plant growth medium ) . Exogenous application of SA mimics DA - induced anthocyanin accumulation ( D ) patterns in Pi - deficient plants . Data information : The boxplots show representative data from three independent experiments ( n = 6 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test . Exogenous application of SA mimics DA - induced IPS1 gene induction ( E ) patterns in Pi - deficient plants . Data information : qPCR results show values of means ± SE ( n = 3 ) , two biological replicates were analyzed with similar results . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test . Compared to the wild type plants , the NahG transgenic plants showed altered responses to DA under Pi - deficiency condition , as shown by plant images ( F ) Compared to the wild type plants , the NahG transgenic plants showed altered responses to DA under Pi - deficiency condition and quantification of anthocyanin accumulation levels ( G ) . Data information : The boxplots show representative data from three independent experiments ( n = 6 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 [A] DA induces genes involved in SA biosynthesis and signaling in Arabidopsis grown with Pi deficiency. Data information: qPCR results show values of means ± SE (n=3), two biological replicates were analyzed with similar results. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test. [B] DA elevates SA accumulation levels in Pi-deficient Arabidopsis. Means ± SE of three biological replicates. Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test. [C] Images of plants grown with different treatments using DA (9.7 µg per mL free space) or SA (100 µM in plant growth medium). Exogenous application of SA mimics DA-induced anthocyanin accumulation (D) patterns in Pi-deficient plants. Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.Exogenous application of SA mimics DA-induced IPS1 gene induction (E) patterns in Pi-deficient plants. Data information: qPCR results show values of means ± SE (n=3), two biological replicates were analyzed with similar results. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.Compared to the wild type plants, the NahG transgenic plants showed altered responses to DA under Pi-deficiency condition, as shown by plant images (F)Compared to the wild type plants, the NahG transgenic plants showed altered responses to DA under Pi-deficiency condition and quantification of anthocyanin accumulation levels (G). Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.


Figure 5 [ A ] Clustering of DA - regulated immunity - related genes in Arabidopsis with sufficient Pi , as revealed by RNAseq analysis . [ B ] DA suppresses plant ROS burst induced by flg22 or elf18 . ROS accumulation was represented in relative luminescence units ( RLU ) . Data bar indicate mean ± SE ( n = 24 ) readings for 60 min . Three independent experiments were performed and similar results were observed . * * p < 0 . 01 , student t - test . [ C ] DA induces gene expression of AZI1 in Arabidopsis . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test within each group . Bar indicates mean ± SE ( n = 3 ) , two biological replicates were analyzed with similar results . [ D ] flg22 - induced MPK3 / MPK6 phosphorylation in Arabidopsis is not decreased by DA . Kinase assays were performed at 5 min after flg22 treatment . Three independent experiments were performed and similar results were observed . [ E ] Plant resistance to the pathogen Pst DC3000 is not reduced by DA . The severity of pathogen infection was measured as colony forming units ( CFUs ) per mg FW plants at 0 and 3 days after inoculation . Data bar indicate mean ± SE ( n = 8 ) , two independent experiments were performed . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test within each group . [ F ] Root colonization of GB03 in wild type Arabidopsis ( Col - 0 ) and the mutants / transgenic lines npr1 , NahG , and rbohd . * * * p < 0 . 001compared with Col - 0 without DA treatment , n = 9 biological replicates , student t - test . [ G ] Bacteria chemotaxis with different concentrations of DA . Data point indicates mean ± SE ( n = 6 ) . Two independent experiments were performed and similar results were observed . * * * p < 0 . 001 , student t - test .

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

Figure 5 [A] Clustering of DA-regulated immunity-related genes in Arabidopsis with sufficient Pi, as revealed by RNAseq analysis.  [B] DA suppresses plant ROS burst induced by flg22 or elf18. ROS accumulation was represented in relative luminescence units (RLU). Data bar indicate mean ± SE (n=24) readings for 60 min. Three independent experiments were performed and similar results were observed. **p < 0.01, student t-test.  [C] DA induces gene expression of AZI1 in Arabidopsis. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group. Bar indicates mean ± SE (n=3), two biological replicates were analyzed with similar results.  [D] flg22-induced MPK3/MPK6 phosphorylation in Arabidopsis is not decreased by DA. Kinase assays were performed at 5 min after flg22 treatment. Three independent experiments were performed and similar results were observed.  [E] Plant resistance to the pathogen Pst DC3000 is not reduced by DA. The severity of pathogen infection was measured as colony forming units (CFUs) per mg FW plants at 0 and 3 days after inoculation. Data bar indicate mean ± SE (n=8), two independent experiments were performed. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group.  [F] Root colonization of GB03 in wild type Arabidopsis (Col-0) and the mutants/transgenic lines npr1, NahG, and rbohd. *** p < 0.001compared with Col-0 without DA treatment, n= 9 biological replicates, student t-test.  [G] Bacteria chemotaxis with different concentrations of DA. Data point indicates mean± SE (n=6). Two independent experiments were performed and similar results were observed. *** p < 0.001, student t-test.


Figure 1A . Endogenous localization of VAPA or VAPB and Syt in hippocampal neurons ( DIV16 ) expressing GFP - Sec61β . Zooms represent ( 1 ) an axonal structure with presynaptic boutons ( arrowheads ) , and ( 2 ) a dendritic structure . Scale bars : 10 µm ( full size ) and 5 µm ( zoom ) . C . Representative image of Syt antibody uptake at axons of hippocampal neurons ( DIV18 ) co - expressing RFP and pSuper empty vector or VAPA / B shRNAs . Yellow and grey arrowheads mark presynaptic boutons with and without internalized Syt , respectively . Zooms represent typical boutons . Scale bars : 5 µm ( full size ) and 2 µm ( zoom ) . D . Quantifications of fluorescence intensity of internalized endogenous Syt ( B , C ) at single presynaptic boutons of hippocampal neurons ( DIV18 ) co - expressing RFP and pSuper empty vector or VAPA / B shRNAs . N = 2 , n = 288 - 541 boutons . Data information : Data represent Mean ± SEM ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U test . E . Western blot of endogenous SCRN1 expression in indicated adult rat neuronal and non - neuronal tissues . Cereb . , cerebellum . Hippoc . , hippocampus . Spin . , spinal . F . Pull down assay of HEK293T cells co - expressing Myc - VAPA with BioGFP or BioGFP - SCRN1 . G . Pull down assay of HEK293T cells co - expressing Myc - VAPB with BioGFP or BioGFP - SCRN1 . Data information : Data represent Mean ± SEM ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U test . H . Scaled representation of SCRN1 - associated proteins identified with pull - down assay followed by mass spectrometry analysis of purified BioGFP or BioGFP - SCRN1 from HEK293T cell lysates with adult rat brain extracts . All candidates showed > 10 enrichment of PSM compared to control . I . Endogenous localization of SCRN1 and Syt in cortical neurons ( DIV18 ) expressing GFP . Zoom represents an axon structure with presynaptic boutons ( arrowheads ) . Scale bars : 10 µm ( full size ) and 5 µm ( zoom ) . J . Representative image of Syt antibody uptake at axons of hippocampal neurons ( DIV18 ) co - expressing RFP and pSuper empty vector , SCRN1 shRNA or SCRN1 shRNA with GFP - SCRN1 . Yellow and grey arrowheads mark presynaptic boutons with and without internalized Syt , respectively . Zooms represent typical boutons . Scale bars : 5 µm ( full size ) and 2 µm ( zoom ) . K . Quantifications of fluorescence intensity of internalized endogenous Syt ( B , C ) at single presynaptic boutons of hippocampal neurons ( DIV18 ) co - expressing RFP and pSuper empty vector , SCRN1 shRNA alone or SCRN1 shRNA with GFP - SCRN1 . N = 2 , n = 201 - 300 boutons . Data information : Data represent Mean ± SEM ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. Endogenous localization of VAPA or VAPB and Syt in hippocampal neurons (DIV16) expressing GFP-Sec61β. Zooms represent (1) an axonal structure with presynaptic boutons (arrowheads), and (2) a dendritic structure. Scale bars: 10 µm (full size) and 5 µm (zoom).C. Representative image of Syt antibody uptake at axons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector or VAPA/B shRNAs. Yellow and grey arrowheads mark presynaptic boutons with and without internalized Syt, respectively. Zooms represent typical boutons. Scale bars: 5 µm (full size) and 2 µm (zoom).D. Quantifications of fluorescence intensity of internalized endogenous Syt (B, C) at single presynaptic boutons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector or VAPA/B shRNAs. N=2, n=288-541 boutons. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.E. Western blot of endogenous SCRN1 expression in indicated adult rat neuronal and non-neuronal tissues. Cereb., cerebellum. Hippoc., hippocampus. Spin., spinal.F. Pull down assay of HEK293T cells co-expressing Myc-VAPA with BioGFP or BioGFP-SCRN1. G. Pull down assay of HEK293T cells co-expressing Myc-VAPB with BioGFP or BioGFP-SCRN1.Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.H. Scaled representation of SCRN1-associated proteins identified with pull-down assay followed by mass spectrometry analysis of purified BioGFP or BioGFP-SCRN1 from HEK293T cell lysates with adult rat brain extracts. All candidates showed >10 enrichment of PSM compared to control.I. Endogenous localization of SCRN1 and Syt in cortical neurons (DIV18) expressing GFP. Zoom represents an axon structure with presynaptic boutons (arrowheads). Scale bars: 10 µm (full size) and 5 µm (zoom).J. Representative image of Syt antibody uptake at axons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector, SCRN1 shRNA or SCRN1 shRNA with GFP-SCRN1. Yellow and grey arrowheads mark presynaptic boutons with and without internalized Syt, respectively. Zooms represent typical boutons. Scale bars: 5 µm (full size) and 2 µm (zoom).K. Quantifications of fluorescence intensity of internalized endogenous Syt (B, C) at single presynaptic boutons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector, SCRN1 shRNA alone or SCRN1 shRNA with GFP-SCRN1. N=2, n=201-300 boutons. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.


Figure 2A . Localization of GFP - SCRN1 and TagRFP - ER in hippocampal neurons ( DIV16 - 18 ) . Scale bars : 10 µm ( bottom panel , full size ) and 5 µm ( top panel ; bottom panel , zoom ) . B . Localization of GFP - SCRN1 and ssRFP - KDEL in COS7 cells . Scale bars : 10 µm ( full size ) and 5 µm ( zoom ) . C . Hippocampal neurons ( DIV16 ) co - expressing GFP - SCRN1 with HA - VAPA or HA - VAPB . Scale bars : 10 µm ( full size ) and 5 µm ( zoom ) . D . COS7 cells co - expressing GFP - SCRN1 with HA - VAPA or HA - VAPB . Scale bars : 10 µm ( full size ) and 5 µm ( zoom ) . F . COS7 cells co - expressing HA - VAPB with GFP - SCRN2 or GFP - SCRN3 . Scale bars : 10 µm ( full size ) and 5 µm ( zoom ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Localization of GFP-SCRN1 and TagRFP-ER in hippocampal neurons (DIV16-18). Scale bars: 10 µm (bottom panel, full size) and 5 µm (top panel; bottom panel, zoom).B. Localization of GFP-SCRN1 and ssRFP-KDEL in COS7 cells. Scale bars: 10 µm (full size) and 5 µm (zoom).C. Hippocampal neurons (DIV16) co-expressing GFP-SCRN1 with HA-VAPA or HA-VAPB. Scale bars: 10 µm (full size) and 5 µm (zoom).D. COS7 cells co-expressing GFP-SCRN1 with HA-VAPA or HA-VAPB. Scale bars: 10 µm (full size) and 5 µm (zoom).F. COS7 cells co-expressing HA-VAPB with GFP-SCRN2 or GFP-SCRN3. Scale bars: 10 µm (full size) and 5 µm (zoom).


Figure 3B . COS7 cells co - expressing GFP - SCRN1 with HA - VAPB - K87D / M89D . Scale bars : 10 µm ( full size ) and 5 µm ( zoom ) . C . COS7 cells co - expressing HA - VAPB with GFP - SCRN1 - Y40A , GFP - SCRN1 - F144A , GFP - SCRN1 - F153A or GFP - SCRN1 - F402A . Scale bars : 10 µm ( full size ) and 5 µm ( zoom ) . D . Hippocampal neurons ( DIV16 ) co - expressing HA - VAPB with GFP - SCRN1 - Y40A , GFP - SCRN1 - F144A , GFP - SCRN1 - F153A or GFP - SCRN1 - F402A . Scale bars : 10 µm ( full size ) and 5 µm ( zoom ) . E . Quantifications of SCRN1 recruitment to VAPB - positive structures in COS7 cells ( % ) . Left graph : co - expression of GFP - SCRN1 with HA - VAPB or HA - VAPB - K87D / M89D ( N = 2 - 3 , n = 44 - 46 ) . Right graph : co - expression of HA - VAPB with GFP or GFP - SCRN1 , GFP - SCRN1 - N , GFP - SCRN1 - C , GFP - SCRN1 - Y40A , GFP - SCRN1 - F144A , GFP - SCRN1 - F153A or GFP - SCRN1 - F402A ( N = 2 - 3 , n = 41 - 64 ) . Data information : Data represent Mean ± SEM ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Chi - square test with post - hoc analysis including Bonferroni correction . F . Pull down of HEK293T cells co - expressing Myc - VAPB with GFP , GFP - SCRN1 , GFP - SCRN1 - Y40A , GFP - SCRN1 - F144A , GFP - SCRN1 - F153A or GFP - SCRN1 - F402A .

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Figure 3B. COS7 cells co-expressing GFP-SCRN1 with HA-VAPB-K87D/M89D. Scale bars: 10 µm (full size) and 5 µm (zoom).C. COS7 cells co-expressing HA-VAPB with GFP-SCRN1-Y40A, GFP-SCRN1-F144A, GFP-SCRN1-F153A or GFP-SCRN1-F402A. Scale bars: 10 µm (full size) and 5 µm (zoom).D. Hippocampal neurons (DIV16) co-expressing HA-VAPB with GFP-SCRN1-Y40A, GFP-SCRN1-F144A, GFP-SCRN1-F153A or GFP-SCRN1-F402A. Scale bars: 10 µm (full size) and 5 µm (zoom).E. Quantifications of SCRN1 recruitment to VAPB-positive structures in COS7 cells (%). Left graph: co-expression of GFP-SCRN1 with HA-VAPB or HA-VAPB-K87D/M89D ( N=2-3, n= 44-46). Right graph: co-expression of HA-VAPB with GFP or GFP-SCRN1, GFP-SCRN1-N, GFP-SCRN1-C, GFP-SCRN1-Y40A, GFP-SCRN1-F144A, GFP-SCRN1-F153A or GFP-SCRN1-F402A (N=2-3, n=41-64). Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Chi-square test with post-hoc analysis including Bonferroni correction.F. Pull down of HEK293T cells co-expressing Myc-VAPB with GFP, GFP-SCRN1, GFP-SCRN1-Y40A, GFP-SCRN1-F144A, GFP-SCRN1-F153A or GFP-SCRN1-F402A.


Figure 4B . Live hippocampal neurons ( DIV17 - 18 ) co - expressing TagRFP - ER with GFP , GFP - SCRN1 , GFP - SCRN1 - F402A , SCRN1 shRNA or VAPA / B shRNAs . Scale bars : 5 µm ( full size ) and 2 µm ( zoom ) . C . Time - lapse of TagRFP - ER dynamics in hippocampal neurons ( DIV16 - 18 ) co - expressing GFP , GFP - SCRN1 , GFP - SCRN1 - F402A , SCRN1 shRNA or VAPA / B shRNAs . Intact ER structures ( dark arrowheads ) , intact and dynamic ER structures ( light arrowheads ) and impaired an non - dynamic ER structures ( arrows ) are indicated . Scale bars : 5 µm ( full size ) and 2 µm ( zoom ) . E . ER nanostructures visualized with GFP - Sec61β in axons of hippocampal neurons ( DIV18 ) immunostained for α - Tubulin and co - expressed with pSuper empty vector , SCRN1 shRNA or VAPA / B shRNAs , and subjected to expansion microscopy . Scale bars : 2 µm ( full size ) and 500 nm ( zoom ) . F . FRAP experiment of TagRFP - ER in hippocampal neurons ( DIV17 - 18 ) co - expressing GFP , GFP - SCRN1 , GFP - SCRN1 - F402A . Scale bar : 2 µm . G . Average normalized fluorescent TagRFP - ER recovery after photo - bleaching in hippocampal neurons ( DIV17 - 18 ) co - expressing GFP , GFP - SCRN1 , GFP - SCRN1 - F402A . N = 2 , n = 9 neurons . H . Normalized fluorescent TagRFP - ER recovery after photo - bleaching at T = 60 sec in hippocampal neurons ( DIV17 - 18 ) co - expressing GFP , GFP - SCRN1 , GFP - SCRN1 - F402A . N = 2 , n = 9 neurons . Data information : Data represent Mean ± SEM ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U test . I . Average normalized fluorescent TagRFP - ER recovery after photo - bleaching in hippocampal neurons ( DIV18 ) co - expressing pSuper empty vector , SCRN1 shRNA , VAPA / B shRNAs . N = 4 , n = 6 - 12 neurons . J . Normalized fluorescent TagRFP - ER recovery after photo - bleaching at T = 60 sec in hippocampal neurons ( DIV18 ) co - expressing pSuper empty vector , SCRN1 shRNA , VAPA / B shRNAs . N = 4 , n = 6 - 12 neurons . Data information : Data represent Mean ± SEM ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B. Live hippocampal neurons (DIV17-18) co-expressing TagRFP-ER with GFP, GFP-SCRN1, GFP-SCRN1-F402A, SCRN1 shRNA or VAPA/B shRNAs. Scale bars: 5 µm (full size) and 2 µm (zoom).C. Time-lapse of TagRFP-ER dynamics in hippocampal neurons (DIV16-18) co-expressing GFP, GFP-SCRN1, GFP-SCRN1-F402A, SCRN1 shRNA or VAPA/B shRNAs. Intact ER structures (dark arrowheads), intact and dynamic ER structures (light arrowheads) and impaired an non-dynamic ER structures (arrows) are indicated. Scale bars: 5 µm (full size) and 2 µm (zoom).E. ER nanostructures visualized with GFP-Sec61β in axons of hippocampal neurons (DIV18) immunostained for α-Tubulin and co-expressed with pSuper empty vector, SCRN1 shRNA or VAPA/B shRNAs, and subjected to expansion microscopy. Scale bars: 2 µm (full size) and 500 nm (zoom).F. FRAP experiment of TagRFP-ER in hippocampal neurons (DIV17-18) co-expressing GFP, GFP-SCRN1, GFP-SCRN1-F402A. Scale bar: 2 µm.G. Average normalized fluorescent TagRFP-ER recovery after photo-bleaching in hippocampal neurons (DIV17-18) co-expressing GFP, GFP-SCRN1, GFP-SCRN1-F402A. N=2, n=9 neurons. H. Normalized fluorescent TagRFP-ER recovery after photo-bleaching at T=60 sec in hippocampal neurons (DIV17-18) co-expressing GFP, GFP-SCRN1, GFP-SCRN1-F402A. N=2, n=9 neurons. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.I. Average normalized fluorescent TagRFP-ER recovery after photo-bleaching in hippocampal neurons (DIV18) co-expressing pSuper empty vector, SCRN1 shRNA, VAPA/B shRNAs. N=4, n=6-12 neurons. J. Normalized fluorescent TagRFP-ER recovery after photo-bleaching at T=60 sec in hippocampal neurons (DIV18) co-expressing pSuper empty vector, SCRN1 shRNA, VAPA/B shRNAs. N=4, n=6-12 neurons. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.


Figure 5A . Representative image of Syt antibody uptake at axons of hippocampal neurons ( DIV18 ) co - expressing RFP and pSuper empty vector , SCRN1 shRNA or SCRN1 shRNA with GFP - SCRN1 - F402A . Yellow and grey arrowheads mark presynaptic boutons with and without internalized Syt , respectively . Zooms represent typical boutons . Scale bars : 5 µm ( full size ) and 2 µm ( zoom ) . B . Quantifications of fluorescence intensity of internalized endogenous Syt at individual presynaptic boutons of hippocampal neurons ( DIV18 ) co - expressing RFP and pSuper empty vector , SCRN1 shRNA or SCRN1 shRNA with GFP - SCRN1 - F402A . N = 2 , n = 201 - 300 boutons . Data information : Data represent Mean ± SEM ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U test . C . Representative time - lapse of cytosolic GCaMP6f upon electric field stimulation ( 50 APs , 20 Hz ) in axons of hippocampal neurons ( DIV21 ) co - expressed with RIM1a - mCherry to visualize presynaptic sites ( arrowheads ) . Scale bar : 5 µm . D . Time - lapses of cytosolic GCaMP6f upon electric field stimulation ( 50 APs , 20 Hz ) in axons of hippocampal neurons ( DIV21 ) co - expressing pSuper control , SCRN1 shRNA or VAPA / B shRNAs . Arrowheads mark presynaptic boutons . Scale bar : 5 µm . E . Average normalized response of GCaMP6f fluorescent intensity at presynaptic boutons upon stimulation ( 50 APs , 20 Hz ) in hippocampal neurons ( DIV21 ) co - expressing pSuper empty vector , SCRN1 shRNA or VAPA / B shRNAs . N = 5 - 6 , n = 15 - 27 . F . Average normalized peak response of GCaMP6f at presynaptic boutons upon stimulation ( 50 APs , 20 Hz ) in hippocampal neurons ( DIV21 ) co - expressing pSuper empty vector , SCRN1 shRNA or VAPA / B shRNAs . N = 5 - 6 , n = 15 - 27 . Data information : Data represent Mean ± SEM ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U test . G . Representative time - lapse of cytosolic GCaMP6f before ( F0 ) and after ( Fmax ) ionomycin treatment at axons of hippocampal neurons ( DIV18 ) co - expressed with mRFP and pSuper empty vector or VAPA / B shRNAs . Arrowheads mark presynaptic boutons . Scale bar : 5 µm . H . Basal GCaMP6f fluorescence ( F ) normalized to the maximum GCaMP6f fluorescence ( Fmax ) after ionomycin treatment at presynaptic boutons of hippocampal neurons ( DIV18 ) co - expressing mRFP with pSuper empty vector or VAPA / B shRNAs . N = 2 , n = 47 - 50 . I . Average basal GCaMP6f fluorescence ( F0 ) normalized to the max GCaMP6f fluorescence intensity ( Fmax ) after ionomycin treatment at presynaptic boutons of hippocampal neurons ( DIV18 ) co - expressing mRFP with pSuper empty vector or VAPA / B shRNAs . N = 2 , n = 47 - 50 . Data information : Data represent Mean ± SEM ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U test . J . Representative time - lapse of cytosolic R - GECO1 before ( F0 ) and after ( Fmax ) ionomycin treatment at axons of hippocampal neurons ( DIV18 ) co - expressing GFP , GFP - SCRN1 or GFP - SCRN1 - F402A . Arrowheads mark presynaptic boutons . Scale bar : 5 µm . K . Basal R - GECO1 fluorescence ( F ) normalized to the maximum R - GECO1 fluorescence ( Fmax ) after ionomycin treatment at presynaptic boutons of hippocampal neurons ( DIV18 ) co - expressing GFP , GFP - SCRN1 or GFP - SCRN1 - F402A . N = 3 , n = 70 - 89 . L . Average basal R - GECO1 fluorescence ( F0 ) normalized to the maximum R - GECO1 fluorescence intensity ( Fmax ) after ionomycin treatment at presynaptic boutons of hippocampal neurons ( DIV18 ) co - expressing GFP , GFP - SCRN1 or GFP - SCRN1 - F402A . N = 3 , n = 70 - 89 . Data information : Data represent Mean ± SEM ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. Representative image of Syt antibody uptake at axons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector, SCRN1 shRNA or SCRN1 shRNA with GFP-SCRN1-F402A. Yellow and grey arrowheads mark presynaptic boutons with and without internalized Syt, respectively. Zooms represent typical boutons. Scale bars: 5 µm (full size) and 2 µm (zoom). B. Quantifications of fluorescence intensity of internalized endogenous Syt at individual presynaptic boutons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector, SCRN1 shRNA or SCRN1 shRNA with GFP-SCRN1-F402A. N=2, n=201-300 boutons. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.C. Representative time-lapse of cytosolic GCaMP6f upon electric field stimulation (50 APs, 20 Hz) in axons of hippocampal neurons (DIV21) co-expressed with RIM1a-mCherry to visualize presynaptic sites (arrowheads). Scale bar: 5 µm.D. Time-lapses of cytosolic GCaMP6f upon electric field stimulation (50 APs, 20 Hz) in axons of hippocampal neurons (DIV21) co-expressing pSuper control, SCRN1 shRNA or VAPA/B shRNAs. Arrowheads mark presynaptic boutons. Scale bar: 5 µm.E. Average normalized response of GCaMP6f fluorescent intensity at presynaptic boutons upon stimulation (50 APs, 20 Hz) in hippocampal neurons (DIV21) co-expressing pSuper empty vector, SCRN1 shRNA or VAPA/B shRNAs. N=5-6, n=15-27. F. Average normalized peak response of GCaMP6f at presynaptic boutons upon stimulation (50 APs, 20 Hz) in hippocampal neurons (DIV21) co-expressing pSuper empty vector, SCRN1 shRNA or VAPA/B shRNAs. N=5-6, n=15-27. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.G. Representative time-lapse of cytosolic GCaMP6f before (F0) and after (Fmax) ionomycin treatment at axons of hippocampal neurons (DIV18) co-expressed with mRFP and pSuper empty vector or VAPA/B shRNAs. Arrowheads mark presynaptic boutons. Scale bar: 5 µm.H. Basal GCaMP6f fluorescence (F) normalized to the maximum GCaMP6f fluorescence (Fmax) after ionomycin treatment at presynaptic boutons of hippocampal neurons (DIV18) co-expressing mRFP with pSuper empty vector or VAPA/B shRNAs. N=2, n=47-50. I. Average basal GCaMP6f fluorescence (F0) normalized to the max GCaMP6f fluorescence intensity (Fmax) after ionomycin treatment at presynaptic boutons of hippocampal neurons (DIV18) co-expressing mRFP with pSuper empty vector or VAPA/B shRNAs. N=2, n=47-50. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.J. Representative time-lapse of cytosolic R-GECO1 before (F0) and after (Fmax) ionomycin treatment at axons of hippocampal neurons (DIV18) co-expressing GFP, GFP-SCRN1 or GFP-SCRN1-F402A. Arrowheads mark presynaptic boutons. Scale bar: 5 µm.K. Basal R-GECO1 fluorescence (F) normalized to the maximum R-GECO1 fluorescence (Fmax) after ionomycin treatment at presynaptic boutons of hippocampal neurons (DIV18) co-expressing GFP, GFP-SCRN1 or GFP-SCRN1-F402A. N=3, n=70-89. L. Average basal R-GECO1 fluorescence (F0) normalized to the maximum R-GECO1 fluorescence intensity (Fmax) after ionomycin treatment at presynaptic boutons of hippocampal neurons (DIV18) co-expressing GFP, GFP-SCRN1 or GFP-SCRN1-F402A. N=3, n=70-89. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.


Figure 2A . Immunoblotting analysis of Neuro2a ( N2a ) cells expressing Sig1R - FLAG variants . B . Cycloheximide chase analysis on N2a cells expressing Sig1R - FLAG . Quantitative data of immunoblotting for the levels of Sig1R - FLAGprotein and its variants during the cyclohexamide chase were plotted as mean ± SEM of three independent experiments ( upper panel ) . Representative immunoblots for the levels of Sig1R - FLAGproteins were shown ( lower panel ) . * * : p < 0 . 01 , * : p = 0 . 0216 in E102Q vs . wild - type ( WT ) ; # # : p < 0 . 01 , # : p < 0 . 05 in L95fs vs . WT . A one - way ANOVA with subsequent post hoc Tukey ' s test . C . N2a cells transfected with Sig1R - FLAG variants were incubated with MG - 132 ( 10 µM ) or the combination of E64d and pepstatin A ( 5 µg / mL each ) ( E64d / PepA ) for 8 h . The relative mean levels of Sig1R - FLAG variants determined by immunoblotting from three independent experiments were plotted as mean ± SEM ( upper panel ) . The representative immunoblots were shown ( lower panel ) . * : p < 0 . 05 vs . no inhibitor control . A one - way ANOVA with subsequent post hoc Tukey ' s test . E and F . Subcellular localization of inositol 1 , 4 , 5 - triphosphate receptor type 1 ( IP3R1 ) and type 3 ( IP3R3 ) in N2a cells stably expressing human IP3R3 ( N2a - IP3R3 cells , E ) . 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 . 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 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 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 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 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 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Immunoblotting analysis of Neuro2a (N2a) cells expressing Sig1R-FLAG variants.B. Cycloheximide chase analysis on N2a cells expressing Sig1R-FLAG. Quantitative data of immunoblotting for the levels of Sig1R-FLAGprotein and its variants during the cyclohexamide chase were plotted as mean ± SEM of three independent experiments (upper panel). Representative immunoblots for the levels of Sig1R-FLAGproteins were shown (lower panel). **: p < 0.01, *: p = 0.0216 in E102Q vs. wild-type (WT); ##: p < 0.01, #: p < 0.05 in L95fs vs. WT. A one-way ANOVA with subsequent post hoc Tukey's test.C. N2a cells transfected with Sig1R-FLAG variants were incubated with MG-132 (10 µM) or the combination of E64d and pepstatin A (5 µg/mL each) (E64d/PepA) for 8 h. The relative mean levels of Sig1R-FLAG variants determined by immunoblotting from three independent experiments were plotted as mean ± SEM (upper panel). The representative immunoblots were shown (lower panel). *: p < 0.05 vs. no inhibitor control. A one-way ANOVA with subsequent post hoc Tukey's test.E and F. Subcellular localization of inositol 1,4,5-triphosphate receptor type 1 (IP3R1) and type 3 (IP3R3) in N2a cells stably expressing human IP3R3 (N2a-IP3R3 cells, E). 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.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 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 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 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 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 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).


Figure 3A . 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 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 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. 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 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 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 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 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.


Figure 4A 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 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 ) 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 .

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Figure 4A 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 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) 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.


Figure 5A 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 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 proteins at MAM and in the whole lysates of SOD1G93Amousespinal cords .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O

Figure 5A 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 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 proteins at MAM and in the whole lysates of SOD1G93Amousespinal cords.


Figure 6A - 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 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 . 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 .

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Figure 6A-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 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. 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.


Figure 7A . 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 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 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 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 .

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Figure 7A. 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 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 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 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.


Figure 8A 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 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 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 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 .

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Figure 8A 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 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 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 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.


Figure 1U2OS cells modified to expressed endogenous GFP - tagged ATG2A ( green ) were grown in complete media ( CM ) or starved ( EBSS ) for 2 h before fixation and immunostaining with antibodies against ATG2B ( red ) and LC3B ( magenta ) and analysed by confocal microscopy . Arrows mark ATG2A / LC3B / ATG2B positive structures . Scale bar 10µm . ( B - D ) Cells were treated as in ( A ) and stained with anti - WIPI2 ( red ) or ( C ) anti - ATG16L1 ( red ) or ( D ) GABARAP - L1 ( red ) . Arrows mark structures of interest . Scale bar 10µm . U2OS WT or U2OS GFP - ATG2A knock - in ( KI ) cells were grown in CM or starvation media for 2 h , lysed and incubated with anti - GFP nanobody beads coupled to agarose to immunoprecipitate ( IP ) GFP - ATG2A . IP samples and 2 % input lysates were run on 4 - 12 % gradient gel and processed for western blotting . Anti - ATG2A , anti - WIPI4 and anti - LC3B , anti - GABARAP ( pan ) were used to probe for the presence / absence of autophagy proteins in the immunoprecipitated samples . p - p70S6K ( T389 ) was used as a marker for starved cells and total p70S6K as loading control .

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Figure 1U2OS cells modified to expressed endogenous GFP-tagged ATG2A (green) were grown in complete media (CM) or starved (EBSS) for 2 h before fixation and immunostaining with antibodies against ATG2B (red) and LC3B (magenta) and analysed by confocal microscopy. Arrows mark ATG2A/LC3B/ATG2B positive structures. Scale bar 10µm. (B-D) Cells were treated as in (A) and stained with anti-WIPI2 (red) or (C) anti-ATG16L1 (red) or (D) GABARAP-L1 (red). Arrows mark structures of interest. Scale bar 10µm. U2OS WT or U2OS GFP-ATG2A knock-in (KI) cells were grown in CM or starvation media for 2 h, lysed and incubated with anti-GFP nanobody beads coupled to agarose to immunoprecipitate (IP) GFP-ATG2A. IP samples and 2% input lysates were run on 4-12% gradient gel and processed for western blotting. Anti-ATG2A, anti-WIPI4 and anti-LC3B, anti-GABARAP (pan) were used to probe for the presence/absence of autophagy proteins in the immunoprecipitated samples. p-p70S6K (T389) was used as a marker for starved cells and total p70S6K as loading control.


Figure 2Myc - tagged ATG2A - wild type ( WT ) and ATG2A - mLIR ( orange ) were co - expressed with GFP - alone , GFP - LC3B or GFP - GABARAP in HEK293T cells , lysed and anti - GFP nanobodies coupled to agarose were used to immunoprecipitate GFP - tagged proteins . Samples were subjected to western blotting and probed for the presence of Myc - ATG2A in immunoprecipitated samples . Anti - p62 / SQSTM1 was used as an internal control for the immunoprecipitated samples . As in ( B ) but using Myc - tagged ATG2B - WT or ATG2B - mLIR ( orange ) co - expressed with GFP - alone , GFP - LC3B or GFP - GABARAP . Samples were subjected to western blotting and probed for the presence of Myc - ATG2B in immunoprecipitated samples . Anti - p62 / SQSTM1 was used as an internal control for the immunoprecipitated samples . HA - tagged ATG2A - WT , - mLIR ( orange ) and - mYFS ( YFS / AAA ; purple ) were stably expressed in U2OS ATG2A / B double knock - out cells using retrovirus transduction . Cells were transfected with GFP - GABARAP and 24 h later grown in complete medium ( CM ) or starved for 2 h ( EBSS ) . Cells were lysed and anti - HA beads were used to immunoprecipitate HA - tagged ATG2A and processed for western blot . Blots were then probed with antibodies against HA - tag ( ATG2A ) , anti - WIPI4 , and anti - GFP for the presence / absence in immunoprecipitated samples . All blots are representative of at least n = 3 independent experiments . Quantification of WIPI4 and GFP - GABARAP - ii co - precipitation with HA - ATG2A from ( D ) . bands were normalized against HA - ATG2A immunoprecipitate and expressed as a fold change compared to HA - ATG2A - WT ( complete media ; CM ) . Each symbol represents an independent experiment ( n = 3 ) and error bars mean ± SD . ATG2A / B do8uble knock - out cells ( DKO ) stably expressing HA - tagged ATG2A - WT , - mLIR ( orange ) or - mYFS ( YFS / AAA ; purple ) were transfected with GFP - GABARAP and 24 h later grown in complete medium ( CM ) or starved for 2 h ( EBSS ) . Cells were lysed and GFP - GABARAP was immunoprecipitated using anti - GFP trap beads . Samples were processed for western blot and probed with anti - HA - tag ( ATG2A ) , anti - WIPI4 , and anti - GFP for their presence / absence in immunoprecipitated samples . All blots are representative of at least n = 3 independent experiments . Quantification of HA - ATG2A and WIPI4 co - precipitation with GFP - GABARAP from ( F ) . Results were expressed as a fold change compared to HA - ATG2A - WT ( complete media ; CM ) . Each symbol represents an independent experiment ( n = 3 ) with error bars mean ± SD . GFP alone , GFP - GABARAP or GFP - GABARAP with increasing concentrations of mCherry - WIPI4 were expressed in HEK293T cells for 24 h , lysed and GFP - trap beads used to immunoprecipitate GFP alone or GFP - GABARAP . Samples were then probed with antibodies to detect endogenous ATG2A or ATG2B in immunoprecipitated samples . Blots are representative of n = 3 independent experiments . Quantification of ATG2A ( green line , round symbols ) and ATG2B ( grey line and squares ) co - precipitation with GFP - GABARAP in the presence of increasing concentrations of mCherry - WIPI4 from ( H ) . Co - precipitation was normalised to GFP - GABARAP alone . Line and error bars are mean ± SD of n = 3 independent experiments .

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Figure 2Myc-tagged ATG2A-wild type (WT) and ATG2A-mLIR (orange) were co-expressed with GFP-alone, GFP-LC3B or GFP-GABARAP in HEK293T cells, lysed and anti-GFP nanobodies coupled to agarose were used to immunoprecipitate GFP-tagged proteins. Samples were subjected to western blotting and probed for the presence of Myc-ATG2A in immunoprecipitated samples. Anti-p62/SQSTM1 was used as an internal control for the immunoprecipitated samples.   As in (B) but using Myc-tagged ATG2B-WT or ATG2B-mLIR (orange) co-expressed with GFP-alone, GFP-LC3B or GFP-GABARAP. Samples were subjected to western blotting and probed for the presence of Myc-ATG2B in immunoprecipitated samples. Anti-p62/SQSTM1 was used as an internal control for the immunoprecipitated samples.   HA-tagged ATG2A-WT, -mLIR (orange) and -mYFS (YFS/AAA; purple) were stably expressed in U2OS ATG2A/B double knock-out cells using retrovirus transduction. Cells were transfected with GFP-GABARAP and 24 h later grown in complete medium (CM) or starved for 2 h (EBSS). Cells were lysed and anti-HA beads were used to immunoprecipitate HA-tagged ATG2A and processed for western blot. Blots were then probed with antibodies against HA-tag (ATG2A), anti-WIPI4, and anti-GFP for the presence/absence in immunoprecipitated samples. All blots are representative of at least n=3 independent experiments.   Quantification of WIPI4 and GFP-GABARAP-ii co-precipitation with HA-ATG2A from (D). bands were normalized against HA-ATG2A immunoprecipitate and expressed as a fold change compared to HA-ATG2A-WT (complete media; CM). Each symbol represents an independent experiment (n=3) and error bars mean ± SD.   ATG2A/B do8uble knock-out cells (DKO) stably expressing HA-tagged ATG2A-WT, -mLIR (orange) or -mYFS (YFS/AAA; purple) were transfected with GFP-GABARAP and 24 h later grown in complete medium (CM) or starved for 2 h (EBSS). Cells were lysed and GFP-GABARAP was immunoprecipitated using anti-GFP trap beads. Samples were processed for western blot and probed with anti-HA-tag (ATG2A), anti-WIPI4, and anti-GFP for their presence/absence in immunoprecipitated samples. All blots are representative of at least n=3 independent experiments.   Quantification of HA-ATG2A and WIPI4 co-precipitation with GFP-GABARAP from (F). Results were expressed as a fold change compared to HA-ATG2A-WT (complete media; CM). Each symbol represents an independent experiment (n=3) with error bars mean ± SD.   GFP alone, GFP-GABARAP or GFP-GABARAP with increasing concentrations of mCherry-WIPI4 were expressed in HEK293T cells for 24 h, lysed and GFP-trap beads used to immunoprecipitate GFP alone or GFP-GABARAP. Samples were then probed with antibodies to detect endogenous ATG2A or ATG2B in immunoprecipitated samples. Blots are representative of n=3 independent experiments.   Quantification of ATG2A (green line, round symbols) and ATG2B (grey line and squares) co-precipitation with GFP-GABARAP in the presence of increasing concentrations of mCherry-WIPI4 from (H). Co-precipitation was normalised to GFP-GABARAP alone. Line and error bars are mean ±SD of n=3 independent experiments.


Figure 3U2OS ATG2A / B double knock - out ( DKO ) CRISPR / Cas9 cells stably expressing Tandem tagged LC3B ( mCherry - GFP - LC3B ) were retrovirally transduced to express vector , or HA - tagged ATG2A - WT , - mLIR ( FCIL / AAAA ) or - mYFS ( YFS / AAA ) . Cells were grown in complete medium ( CM ) or starved for 2 h ( EBSS ) or treated with CM plus BafilomycinA1 ( 200nM , 4 h ) , fixed and analysed by confocal microscopy . Merged images of GFP ( green ) and mCherry ( red ) channels show the presence of autophagosomes / phagophores ( GFP and mCherry positive , yellow puncta ) or autolysosomes ( mCherry only , red puncta ) Scale bar 10µm . Images are representative of n = 3 independent experiments . Quantification using flow cytometry of measuring GFP and mCherry fluorescence . Cells were gated based on GFP and mCherry fluorescence and % mCherry positive cells gated used as an indication of autolysosome formation due to GFP quenching . Each symbol represents 1 independent experiment with 10 , 000 cells analysed per condition . A total of n = 3 independent experiments were performed , and horizontal bar indicates mean ± SD . U2OS ATG2A / B DKO cells reconstituted with vector only , HA - ATG2A - WT , - mLIR and - mYFS were stimulated with complete medium , ( CM ) , 2 h starvation ( EBSS ) or 4 h BafilomycinA1 ( BafA1 , 200nM ) , lysed in total cell lysis buffer and subjected to western blot analysis . Blots were probed for the presence of HA - tag ( ATG2A ) , p62 / SQSTM1 , LC3B , pan - GABARAP ( GABARAP , GABARAP - L1 and GABARAP - L2 ) and vinculin ( loading control ) . D , E p62 / SQSTM1 ( D ) and LC3B - II ( E ) levels were normalized to loading control and quantified as fold change of DKO proteins levels . Each symbol represents an independent experiment . Quantification of at least n = 3 independent experiments is shown . Horizontal bar represents mean ± SD .

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Figure 3U2OS ATG2A/B double knock-out (DKO) CRISPR/Cas9 cells stably expressing Tandem tagged LC3B (mCherry-GFP-LC3B) were retrovirally transduced to express vector, or HA-tagged ATG2A-WT, -mLIR (FCIL/AAAA) or -mYFS (YFS/AAA). Cells were grown in complete medium (CM) or starved for 2 h (EBSS) or treated with CM plus BafilomycinA1 (200nM, 4 h), fixed and analysed by confocal microscopy. Merged images of GFP (green) and mCherry (red) channels show the presence of autophagosomes/phagophores (GFP and mCherry positive, yellow puncta) or autolysosomes (mCherry only, red puncta) Scale bar 10µm. Images are representative of n=3 independent experiments.Quantification using flow cytometry of measuring GFP and mCherry fluorescence. Cells were gated based on GFP and mCherry fluorescence and % mCherry positive cells gated used as an indication of autolysosome formation due to GFP quenching. Each symbol represents 1 independent experiment with 10,000 cells analysed per condition. A total of n=3 independent experiments were performed, and horizontal bar indicates mean ± SD.U2OS ATG2A/B DKO cells reconstituted with vector only, HA-ATG2A-WT, -mLIR and -mYFS were stimulated with complete medium, (CM), 2 h starvation (EBSS) or 4 h BafilomycinA1 (BafA1, 200nM), lysed in total cell lysis buffer and subjected to western blot analysis. Blots were probed for the presence of HA-tag (ATG2A), p62/SQSTM1, LC3B, pan-GABARAP (GABARAP, GABARAP-L1 and GABARAP-L2) and vinculin (loading control).D, E p62/SQSTM1 (D) and LC3B-II (E) levels were normalized to loading control and quantified as fold change of DKO proteins levels. Each symbol represents an independent experiment. Quantification of at least n=3 independent experiments is shown. Horizontal bar represents mean ± SD.


Figure 4U2OS ATG2A / B DKO cells reconstituted with vector only , HA - ATG2A - WT , - mLIR and - mYFS were stimulated with complete medium , ( CM ) , 4 h starvation ( EBSS ) plus BafilomycinA1 ( BafA1 , 200nM ) treatment . Cells were centrifuged and resuspended in PBS digitonin , spun , washed and the membrane fractions incubated with proteinase K with and without 0 . 1 % TritonX - 100 . Sampels were then subjected to western blotting using anti - p62 / SQSTM1 and anti - HA ( ATG2A ) antibodies . Percentage p62 / SQSTM1 remaining was calculated using densitometry analysis . Blots are representative of n = 3 independent experiments . U2OS ATG2A / B DKO cells reconstituted with vector only , HA - ATG2A - WT , - mLIR and - mYFS and stably expressing GFP - Syntaxin17 ( STX17 ) were stimulated starvation ( EBSS ) plus BafilomycinA1 ( BafA1 , 200nM ) for 4 hours to stimulate autophagosome generation and prevent their degradation in the lysosome . Cells were fixed and immune - stained for LC3B ( magenta ) . DAPI was included ( blue ) to mark the DNA / nucleus . Confocal analysis of LC3B and GFP - STX17 ( green ) localization was performed . Closed arrows ( ATG2A / B DKO and DKO + ATG2A mLIR ) highlight aggregate structures . Open arrows ( DKO + ATG2A - WT and ATG2A - mYFS ) highlight STX17 / LC3B positive vesicles . Scale bar 10µm . Quantification of ( C ) expressed as a percentage of cells with STX17 / LC3B positive vesicles . Each symbol represents a single field of cells with 5 - 10 cells per field . A total of 300 - 600 cells were analysed over n = 3 independent experiments . Data is shown as mean ± SD .

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Figure 4U2OS ATG2A/B DKO cells reconstituted with vector only, HA-ATG2A-WT, -mLIR and -mYFS were stimulated with complete medium, (CM), 4 h starvation (EBSS) plus BafilomycinA1 (BafA1, 200nM) treatment. Cells were centrifuged and resuspended in PBS digitonin, spun, washed and the membrane fractions incubated with proteinase K with and without 0.1% TritonX-100. Sampels were then subjected to western blotting using anti-p62/SQSTM1 and anti-HA (ATG2A) antibodies. Percentage p62/SQSTM1 remaining was calculated using densitometry analysis. Blots are representative of n=3 independent experiments.U2OS ATG2A/B DKO cells reconstituted with vector only, HA-ATG2A-WT, -mLIR and -mYFS and stably expressing GFP-Syntaxin17 (STX17) were stimulated starvation (EBSS) plus BafilomycinA1 (BafA1, 200nM) for 4 hours to stimulate autophagosome generation and prevent their degradation in the lysosome. Cells were fixed and immune-stained for LC3B (magenta). DAPI was included (blue) to mark the DNA/nucleus. Confocal analysis of LC3B and GFP-STX17 (green) localization was performed. Closed arrows (ATG2A/B DKO and DKO + ATG2A mLIR) highlight aggregate structures. Open arrows (DKO + ATG2A-WT and ATG2A-mYFS) highlight STX17/LC3B positive vesicles. Scale bar 10µm.   Quantification of (C) expressed as a percentage of cells with STX17/LC3B positive vesicles. Each symbol represents a single field of cells with 5-10 cells per field. A total of 300-600 cells were analysed over n=3 independent experiments. Data is shown as mean ± SD.