A . 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 ) .

A. 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 ) .

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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 ) .

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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 ) .

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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 ) .

F. COS7 cells co-expressing HA-VAPB with GFP-SCRN2 or GFP-SCRN3. Scale bars: 10 µm (full size) and 5 µm (zoom).


B . COS7 cells co - expressing GFP - SCRN1 with HA - VAPB - K87D / M89D . Scale bars : 10 µm ( full size ) and 5 µm ( zoom ) .

B. 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 ) .

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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 ) .

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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 .

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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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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.


B . 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 ) .

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B. 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 ) .

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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 ) .

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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 .

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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 .

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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 .

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.


A . 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 .

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A. 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 .

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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 .

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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 .

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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 .

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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 .

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

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 .

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

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.


A . Immunoblotting analysis of Neuro2a ( N2a ) cells expressing Sig1R - FLAG variants .

A. 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 .

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

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 .

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

C. 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 .

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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 .

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


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

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

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 .

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

H. 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 .

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

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 .

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

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

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


A . 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 .

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

A. The 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 − / − ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

D 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 .

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

F. 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


A 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 .

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

A and B. 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 .

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

C. 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 .

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

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


A 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 .

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

A and B. 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 .

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

C and D. 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 O O O O O O O O O O O O O O O O O O O O

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


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

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

A-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 .

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

J 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 .

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

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


A . N2a 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 .

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

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


B - G . Calpain activity ( B - D ) were measured in N2a - IP3R3 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 .

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

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


B - G . intracellular ATP 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 .

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

B-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 .

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

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


A 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 .

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

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


C 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 .

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

C 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 .

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

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


E 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 .

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

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.


U2OS 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 .

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

U2OS 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 .

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

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.


Myc - 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 .

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

Myc-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 .

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

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 .

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

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 .

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

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.


U2OS 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 .

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

U2OS 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 .

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

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 ) .

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

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 .

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

D, 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.


U2OS 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 .

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

U2OS 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 .

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

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.


( A - C ) U2OS ATG2A / B double knock out ( DKO ) cells or DKO reconstituted with ATG2A - WT , - mLIR ( FCIL / AAAA ) or - mYFS ( YFS / AAA ) were starved for 2 h , fixed and stained for LC3B ( green ) or WIPI2 ( magenta ) , ( B ) ATG9A ( magenta ) or ( C ) GABARAP ( Magenta ) and imaged using a Zeiss 880 Airyscan super - resolution confocal microscope . Images are representatives of n = 3 independent experiments . Scale bars 10µm .

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

(A-C) U2OS ATG2A/B double knock out (DKO) cells or DKO reconstituted with ATG2A-WT, -mLIR (FCIL/AAAA) or -mYFS (YFS/AAA) were starved for 2 h, fixed and stained for LC3B (green) or WIPI2 (magenta), (B) ATG9A (magenta) or (C) GABARAP (Magenta) and imaged using a Zeiss 880 Airyscan super-resolution confocal microscope. Images are representatives of n=3 independent experiments. Scale bars 10µm.


Transmission electron micrographs of ATG2A / B DKO cells ( upper left ) , DKO + ATG2A - WT ( upper right ) , DKO + ATG2A - mLIR ( lower left ) and DKO + ATG2A - mYFS ( lower right ) starved ( EBSS ) for 2 h . Clustered small vesicles ( open arrowheads ; DKO and DKO - mLIR ) and autophagosomes ( closed arrow heads , DKO + ATG2A - WT and - mYFS ) indicated . Images are representative of n = 3 independent experiments . Scale bars 500nm . ER = endoplasmic reticulum ; N = Nucleus ; Mt = Mitochondria ; G = Golgi ; AP = Autophagosome .

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

Transmission electron micrographs of ATG2A/B DKO cells (upper left), DKO + ATG2A-WT (upper right), DKO+ ATG2A-mLIR (lower left) and DKO+ATG2A-mYFS( lower right) starved (EBSS) for 2 h. Clustered small vesicles (open arrowheads; DKO and DKO-mLIR) and autophagosomes (closed arrow heads, DKO+ATG2A-WT and -mYFS) indicated. Images are representative of n=3 independent experiments. Scale bars 500nm. ER= endoplasmic reticulum; N = Nucleus; Mt = Mitochondria; G = Golgi; AP = Autophagosome.


A Immunoblot analysis of overexpressed and ALFA - tagged CNX in CNX knockout cell lines clones # 1 and # 2 , which were used for mass spectrometry . Endogenous CNX level of wildtype HEK293T cells is shown as a control .

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

A Immunoblot analysis of overexpressed and ALFA-tagged CNX in CNX knockout cell lines clones #1 and #2, which were used for mass spectrometry. Endogenous CNX level of wildtype HEK293T cells is shown as a control.


C Volcano plots derived from LC - MS / MS analyses of ALFA - tagged CNX , immunoprecipitated in 1 % NP - 40 buffer from transfected CNX deficient HEK293T cells after DSSO crosslinking and compared to empty vector ( EV ) control co - IPs . Depicted is an enlarged section of the associated complete volcano plot in the upper right . Among the significantly enriched proteins , annotations are Uniprot gene names . CNX is shown in green . ER proteins with the GO - term annotation " endoplasmic reticulum " , which are localized to the ER lumen , are highlighted in blue and among those several known CNX interaction partners are indicated with their names . Membrane proteins are shown in red and those which are non - glycosylated are additionally labeled with their gene names .

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C Volcano plots derived from LC-MS/MS analyses of ALFA-tagged CNX, immunoprecipitated in 1% NP-40 buffer from transfected CNX deficient HEK293T cells after DSSO crosslinking and compared to empty vector (EV) control co-IPs. Depicted is an enlarged section of the associated complete volcano plot in the upper right. Among the significantly enriched proteins, annotations are Uniprot gene names. CNX is shown in green. ER proteins with the GO-term annotation "endoplasmic reticulum", which are localized to the ER lumen, are highlighted in blue and among those several known CNX interaction partners are indicated with their names. Membrane proteins are shown in red and those which are non-glycosylated are additionally labeled with their gene names.


E Co - immunoprecipitations of endogenous CNX with FLAG - tagged Cx32 WT and its mutants including quantification and statistical analysis . Constructs were expressed in HEK293T cells . One representative immunoblot is shown . Monomers and dimers of Cx32 are indicated with brackets . All samples were normalized to

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E Co-immunoprecipitations of endogenous CNX with FLAG-tagged Cx32 WT and its mutants including quantification and statistical analysis. Constructs were expressed in HEK293T cells. One representative immunoblot is shown. Monomers and dimers of Cx32 are indicated with brackets. All samples were normalized to WT


F Immunofluorescence images of Cx32 and its mutants . COS - 7 cells were transiently transfected with the indicated FLAG - tagged Cx32 constructs and immunofluorescence microscopy was performed using anti PDI ( green ) as an ER marker . Detection of Cx32 was performed using anti FLAG antibodies and suitable labeled secondary antibodies ( red ) . Nuclei were stained with DAPI ( blue ) . All three channels are overlayed . Images are representative of cells from at least three different biological replicates . GJ denotes gap junctions observed at cell - cell junctions of cells transfected with WT Cx32 .

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F Immunofluorescence images of Cx32 and its mutants. COS-7 cells were transiently transfected with the indicated FLAG-tagged Cx32 constructs and immunofluorescence microscopy was performed using anti PDI (green) as an ER marker. Detection of Cx32 was performed using anti FLAG antibodies and suitable labeled secondary antibodies (red). Nuclei were stained with DAPI (blue). All three channels are overlayed. Images are representative of cells from at least three different biological replicates. GJ denotes gap junctions observed at cell-cell junctions of cells transfected with WT Cx32.


B Co - immunoprecipitations of endogenous CNX with the different CL - TMD constructs ( NVT glycosylation site mutated to QVT ) including quantification and statistical analysis . One representative immunoblot is shown . The asterisk ( * ) denotes a fraction of cleaved species as observed previously for similar constructs ( Behnke et al . , 2016 ) . All samples were normalized to the construct containing

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B Co-immunoprecipitations of endogenous CNX with the different CL-TMD constructs (NVT glycosylation site mutated to QVT) including quantification and statistical analysis. One representative immunoblot is shown. The asterisk (*) denotes a fraction of cleaved species as observed previously for similar constructs (Behnke et al., 2016). All samples were normalized to the construct containing TMD1


C Interaction analysis of Calnexin with the first transmembrane domain of Cx32 . Intracellular BMH crosslinking of transiently transfected V5 - tagged CNX with the transiently transfected R26C mutant of Cx32 - TMD1 . In the AA mutant of CNX , the two cysteines within the CNX TMD were replaced by alanines . The species at approx . 250 kDa are possibly CNX dimers .

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C Interaction analysis of Calnexin with the first transmembrane domain of Cx32. Intracellular BMH crosslinking of transiently transfected V5-tagged CNX with the transiently transfected R26C mutant of Cx32-TMD1. In the AA mutant of CNX, the two cysteines within the CNX TMD were replaced by alanines. The species at approx. 250 kDa are possibly CNX dimers.


E COS - 7 cells were transiently transfected with the indicated constructs and immunofluorescence microscopy was performed using anti PDI ( green ) as an ER marker . Detection of minCNXTMD and both control constructs was carried out by mScarlet - I fluorescence ( red ) . Nuclei were stained with DAPI ( blue ) . Images are representative of cells from at least three different biological replicates .

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E COS-7 cells were transiently transfected with the indicated constructs and immunofluorescence microscopy was performed using anti PDI (green) as an ER marker. Detection of minCNXTMD and both control constructs was carried out by mScarlet-I fluorescence (red). Nuclei were stained with DAPI (blue). Images are representative of cells from at least three different biological replicates.


F Interaction of Cx32 - TMD1 with the minCNX system . MinCNXTMD constructs and controls ( see ( D ) ) were co - transfected with Cx32 - TMD1 into HEK293T cells . Interaction between the proteins was analyzed by co - immunoprecipitation experiments followed by immunoblotting .

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F Interaction of Cx32-TMD1 with the minCNX system. MinCNXTMD constructs and controls (see (D)) were co-transfected with Cx32-TMD1 into HEK293T cells. Interaction between the proteins was analyzed by co-immunoprecipitation experiments followed by immunoblotting.


B HEK293T cells were transiently transfected with plasmids expressing ConMem or the indicated variants , where either the first ( N1 ) or the second ( N2 ) NVT glycosylation motif was ablated ( N to Q mutation ) . Lysates were treated with or without EndoH or PNGaseF as indicated , and analyzed by immunoblotting . N - glycosylation occurs in the ER , complex glycosylation in the Golgi .

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B HEK293T cells were transiently transfected with plasmids expressing ConMem or the indicated variants, where either the first (N1) or the second (N2) NVT glycosylation motif was ablated (N to Q mutation). Lysates were treated with or without EndoH or PNGaseF as indicated, and analyzed by immunoblotting. N-glycosylation occurs in the ER, complex glycosylation in the Golgi.


C COS - 7 cells were transfected with the indicated constructs and fluorescence microscopy was performed using Sec61 mCherry ( red ) as an ER marker . ConMem , detected by sfGFP fluorescence ( green ) , localizes to the plasma membrane . Nuclei were stained with DAPI ( blue ) . Images are representative of cells from at least three different biological replicates .

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C COS-7 cells were transfected with the indicated constructs and fluorescence microscopy was performed using Sec61 mCherry (red) as an ER marker. ConMem, detected by sfGFP fluorescence (green), localizes to the plasma membrane. Nuclei were stained with DAPI (blue). Images are representative of cells from at least three different biological replicates.


B Representative blots from co - immunoprecipitation experiments from HEK293T cells transfected with the indicated ConMem constructs with or without ER - lumenal glycosylation site ( N1 ) ( mean ± SEM , N = 3 , * P value < 0 . 05 , two - tailed Student ' s t tests ) . Individual values were normalized to ConMem N1Q ( Val ) values that were set to 1 .

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B Representative blots from co-immunoprecipitation experiments from HEK293T cells transfected with the indicated ConMem constructs with or without ER-lumenal glycosylation site (N1) (mean ± SEM, N = 3, *P value < 0.05, two-tailed Student's t tests). Individual values were normalized to ConMem N1Q (Val) values that were set to 1.


C Interaction of ConMem N1Q variants as described in ( A ) with endogenous CNX ( mean ± SEM , N ≥ 5 , * P value < 0 . 05 , two - tailed Student ' s t tests ) . Individual values were normalized to ConMem N1Q ( Val ) values that were set to 1 . D Same as in ( C ) only that selected amino acids ( proline and arginine ) where shifted through the ConMem N1Q TMD segment to five different positions as shown in the illustration , revealing positional binding dependencies ( mean ± SEM , N ≥ 4 , * P value < 0 . 05 , two - tailed Student ' s t tests ) . Individual values were normalized to ConMem N1Q ( Val ) values that were set to 1 .

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C Interaction of ConMem N1Q variants as described in (A) with endogenous CNX (mean ± SEM, N ≥ 5, *P value < 0.05, two-tailed Student's t tests ). Individual values were normalized to ConMem N1Q (Val) values that were set to 1. D Same as in (C) only that selected amino acids (proline and arginine) where shifted through the ConMem N1Q TMD segment to five different positions as shown in the illustration, revealing positional binding dependencies (mean ± SEM, N ≥ 4, *P value < 0.05, two-tailed Student's t tests). Individual values were normalized to ConMem N1Q (Val) values that were set to 1.


D Interaction of non - glycosylated ConMem N1Q constructs with V5 - tagged CNX . In CNX variants , amino acids important for interaction as revealed in ( A ) were replaced by valine residues . HEK293T cells were transiently transfected with the indicated constructs and cell lysates and HA - immunoprecipitates were analyzed for HA - tagged ConMem N1Q and co - immunoprecipitating CNX mutants . E The same as in ( D ) for Cx32 - TMD1 F Analysis of mutations in the CNX TMD region and their effect on binding glycosylated ConMem . G The same as in ( D ) for Cx32 full -

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D Interaction of non-glycosylated ConMem N1Q constructs with V5-tagged CNX. In CNX variants, amino acids important for interaction as revealed in (A) were replaced by valine residues. HEK293T cells were transiently transfected with the indicated constructs and cell lysates and HA-immunoprecipitates were analyzed for HA-tagged ConMem N1Q and co-immunoprecipitating CNX mutants. E The same as in (D) for Cx32-TMD1 F Analysis of mutations in the CNX TMD region and their effect on binding glycosylated ConMem. G The same as in (D) for Cx32 full-length


H Interaction of Cx32 - TMD1 with CNX mutants , where amino acids not predicted to be important for interaction as shown in ( A ) were exchanged by valine residues . HEK293T cells were transiently transfected with the indicated constructs .

H Interaction of Cx32-TMD1 with CNX mutants, where amino acids not predicted to be important for interaction as shown in (A) were exchanged by valine residues. HEK293T cells were transiently transfected with the indicated constructs.


I Shifting of the residues Y , T and L , most important vor CNX substrate interaction , and replacing these against valine residues , impacts interactions with Cx32 - TMD1 . HEK293T cells were transiently transfected with the indicated constructs .

I Shifting of the residues Y, T and L, most important vor CNX substrate interaction, and replacing these against valine residues, impacts interactions with Cx32-TMD1. HEK293T cells were transiently transfected with the indicated constructs.


A Analysis of the interaction of the binding deficient CNX YTL TMD variant with Rhodopsin . CNX knockout HEK293T cells were transiently transfected with either N - terminal ALFA - tagged Rhodopsin alone or in combination with the indicated V5 - tagged CNX constructs . In the absence of any CNX , Rho is hardly detectable ( long exposure shown ) . Cell lysates and ALFA - immunoprecipitates were analyzed for Rhodopsin and co - immunoprecipitating CNX .

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A Analysis of the interaction of the binding deficient CNX YTL TMD variant with Rhodopsin. CNX knockout HEK293T cells were transiently transfected with either N-terminal ALFA-tagged Rhodopsin alone or in combination with the indicated V5-tagged CNX constructs. In the absence of any CNX, Rho is hardly detectable (long exposure shown). Cell lysates and ALFA-immunoprecipitates were analyzed for Rhodopsin and co-immunoprecipitating CNX.


B Analysis of Rhodopsin degradation and its dependency on CNX . CNX knockout HEK293T cells that were transfected with the indicated constructs were incubated with cycloheximide and lysates were collected at different timepoints . CNX WT and CNX YTL immunoblots are from the same blot ( dashed line ) , showing similar expression levels . Hsc70 was used as a loading control ( also from the same blot , dashed line ) . Quantifications are shown below representative

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B Analysis of Rhodopsin degradation and its dependency on CNX. CNX knockout HEK293T cells that were transfected with the indicated constructs were incubated with cycloheximide and lysates were collected at different timepoints. CNX WT and CNX YTL immunoblots are from the same blot (dashed line), showing similar expression levels. Hsc70 was used as a loading control (also from the same blot, dashed line). Quantifications are shown below representative immunoblots


( B ) Histogram displaying the SILAC ratios ( log2 ) of stress - induced phosphorylation sites in setup cdc55Δ ( upper panel ) and setup rts1Δ ( lower panel ) ( Hollenstein et al . , 2020 ) , stratified by their Hog1 - dependence ( grey area versus thick black line ) .

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(B) Histogram displaying the SILAC ratios (log2) of stress-induced phosphorylation sites in setup cdc55Δ (upper panel) and setup rts1Δ (lower panel) (Hollenstein et al., 2020), stratified by their Hog1-dependence (grey area versus thick black line).


( D ) Line plot monitoring growth rate ( y - axis ) of indicated strains ( colored lines ) under different osmolarity conditions ( x - axis ) .

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(D) Line plot monitoring growth rate (y-axis) of indicated strains (colored lines) under different osmolarity conditions (x-axis).


B ) Western - blot illustrating the depletion of AID - Cdc55 at various time points after addition of auxin ( upper panel ) ; Pgk2 serves as a negative control ( lower panel ) .

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B) Western-blot illustrating the depletion of AID-Cdc55 at various time points after addition of auxin (upper panel); Pgk2 serves as a negative control (lower panel).


( D ) Transcriptional analysis of stress - induced expression of ESR genes CTT1 ( upper panel ) and PGM2 ( lower panel ) under different strain conditions ( i . e . presence / absence of IGO1 / 2 and induction of iAID - Cdc55 depletion ) . Measured time points after stress application are depicted on the x - axis , target gene mRNA levels are illustrated on the y - axis as fold over reference gene IPP1 , values are normalized relative to wild type without induction of iAID - Cdc55 depletion at 30 minutes post - stress .

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(D) Transcriptional analysis of stress-induced expression of ESR genes CTT1 (upper panel) and PGM2 (lower panel) under different strain conditions (i.e. presence/absence of IGO1/2 and induction of iAID-Cdc55 depletion). Measured time points after stress application are depicted on the x-axis, target gene mRNA levels are illustrated on the y-axis as fold over reference gene IPP1, values are normalized relative to wild type without induction of iAID-Cdc55 depletion at 30 minutes post-stress.


E ) Transcriptional analysis of stress - induced expression of ESR genes CTT1 ( upper panel ) and PGM2 ( lower panel ) at 30 minutes post - stress . Target gene mRNA levels are illustrated on the y - axis as fold over reference gene IPP1 , values are normalized relative to wild type without induction of iAID - Cdc55 depletion . dep . : Cdc55 depleted , n . d . : Cdc55 non - depleted .

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E) Transcriptional analysis of stress-induced expression of ESR genes CTT1 (upper panel) and PGM2 (lower panel) at 30 minutes post-stress. Target gene mRNA levels are illustrated on the y-axis as fold over reference gene IPP1, values are normalized relative to wild type without induction of iAID-Cdc55 depletion. dep.: Cdc55 depleted, n.d.: Cdc55 non-depleted.


( C ) Mobility shift assays monitoring phosphorylation - induced mobility changes of Gis1 ( two upper rows ) and Rph1 ( two lower rows ) upon hyperosmotic stress in wild type , igo1 ∆ igo2 ∆ and Cdc55 - depleted cells ( right - hand side ) .

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(C) Mobility shift assays monitoring phosphorylation-induced mobility changes of Gis1 (two upper rows) and Rph1 (two lower rows) upon hyperosmotic stress in wild type, igo1∆igo2∆ and Cdc55-depleted cells (right-hand side).


( E ) Western blot phosphorylation shift assays of wild type Gis1 - myc ( upper panel ) and Rph1 - Flag ( lower panel ) , and mutated forms Gis1 - 5A - myc ( upper panel ) and Rph1 - 5A - Flag ( lower panel ) . The measurement was done using Phos - tag ( Kinoshita et al . , 2006 ) and at the indicated time points after exposure to hyperosmotic stress ( 0 . 5 M NaCl ) using Pgk1 protein levels as a negative control . The location of the mutated residues is shown in the respective schematics . Red boxes : demethylase associated domains ( JmjN and JmjC ) , blue box : zinc finger domain .

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(E) Western blot phosphorylation shift assays of wild type Gis1-myc (upper panel) and Rph1-Flag (lower panel), and mutated forms Gis1-5A-myc (upper panel) and Rph1-5A-Flag (lower panel). The measurement was done using Phos-tag (Kinoshita et al., 2006) and at the indicated time points after exposure to hyperosmotic stress (0.5 M NaCl) using Pgk1 protein levels as a negative control. The location of the mutated residues is shown in the respective schematics. Red boxes: demethylase associated domains (JmjN and JmjC), blue box: zinc finger domain.


( F ) Transcriptional analysis of stress - induced expression of ESR genes CTT1 ( upper panel ) and PGM2 ( lower panel ) at 30 minutes post - stress across indicated strains expressing either wild type or point - mutated forms of Gis1 and Rph1 in a gis1 ∆ rph1 ∆ deletion background , with the empty vector as control .

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(F) Transcriptional analysis of stress-induced expression of ESR genes CTT1 (upper panel) and PGM2 (lower panel) at 30 minutes post-stress across indicated strains expressing either wild type or point-mutated forms of Gis1 and Rph1 in a gis1∆rph1∆ deletion background, with the empty vector as control.


A . Unified cell cycle map of unperturbed ( gray ) and serum - starved cells ( 1d : light blue , 7d : dark blue , N = 3007 cells ) . The proliferative cell cycle ( dotted gray line ) and the hypomitogenic arrest trajectory ( black dotted line ) are indicated on the map . Inset : Each treatment condition is shown individually on the unified map ( other conditions are shown in lighter gray ) .

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A. Unified cell cycle map of unperturbed (gray) and serum-starved cells (1d: light blue, 7d: dark blue, N = 3007 cells). The proliferative cell cycle (dotted gray line) and the hypomitogenic arrest trajectory (black dotted line) are indicated on the map. Inset: Each treatment condition is shown individually on the unified map (other conditions are shown in lighter gray).


( B ) Diffusion pseudotime , ( C ) p21 , ( D ) cyclin D1 , ( E ) DNA content of unperturbed ( left panels ) or serum - starved cells ( right panels ) are plotted on the map .

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(B) Diffusion pseudotime, (C) p21, (D) cyclin D1, (E) DNA content of unperturbed (left panels) or serum-starved cells (right panels) are plotted on the map.


( F ) phospho / total RB of unperturbed ( left panels ) or serum - starved cells ( right panels ) are plotted on the map .

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(F) phospho/total RB of unperturbed (left panels) or serum-starved cells (right panels) are plotted on the map.


G . Time - lapse imaging of cyclinD1 - mVenus intensity in unperturbed ( control , green ) and serum - starved cells ( blue ) . Cells were serum - starved for at least 8h prior to imaging . The solid line represents the population median and the shaded area indicates the 95 % confidence interval . N = 105 control cells and N = 111 starved cells .

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G. Time-lapse imaging of cyclinD1-mVenus intensity in unperturbed (control, green) and serum-starved cells (blue). Cells were serum-starved for at least 8h prior to imaging. The solid line represents the population median and the shaded area indicates the 95% confidence interval. N = 105 control cells and N = 111 starved cells.


I . Median nuclear p27 abundance in serum - starved cells is plotted on the map .

I. Median nuclear p27 abundance in serum-starved cells is plotted on the map.


Unified cell cycle map of unperturbed ( gray ) and etoposide - treated cells ( 1 μM ; 1d : light green , 2d : green , 3d : dark green , 4d : darker green - see inset , N = 4315 cells ) . The unperturbed cell cycle trajectory ( dotted gray line ) and two arrest trajectories ( into G02C and G04C ; black dotted lines ) are indicated on the map . Inset : Each condition is shown individually on the map ( other conditions are shown in lighter gray ) .

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Unified cell cycle map of unperturbed (gray) and etoposide-treated cells (1 μM; 1d: light green, 2d: green, 3d: dark green, 4d: darker green - see inset, N = 4315 cells). The unperturbed cell cycle trajectory (dotted gray line) and two arrest trajectories (into G02C and G04C; black dotted lines) are indicated on the map. Inset: Each condition is shown individually on the map (other conditions are shown in lighter gray).


B - E . ( B ) Diffusion pseudotime , ( C ) DNA content , ( D ) phospho / total RB and ( E ) p21 of unperturbed ( left panels ) or etoposide - treated cells ( right panels ) are plotted on the arrest architecture . Median nuclear values are shown .

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B-E. (B) Diffusion pseudotime, (C) DNA content, (D) phospho/total RB and (E) p21 of unperturbed (left panels) or etoposide-treated cells (right panels) are plotted on the arrest architecture. Median nuclear values are shown.


F . Time - lapse imaging of CDK2 activity ( DHB - mCherry , gray ) and p21 - YPet ( green ) intensity in etoposide - treated cells . Schematic shows the two arrest trajectories observed following etoposide treatment . Cells that successfully complete G2 ( " Mothers " , N = 32 cells ) but arrest following cell division ( " Daughters " , N = 45 cells ) are shown in the two leftmost panels . Cells that arrest in G2 ( N = 40 cells ) are shown in the rightmost panel . The solid lines represent population medians and the shaded area indicates the 95 % confidence interval .

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F. Time-lapse imaging of CDK2 activity (DHB-mCherry, gray) and p21-YPet (green) intensity in etoposide-treated cells. Schematic shows the two arrest trajectories observed following etoposide treatment. Cells that successfully complete G2 ("Mothers", N = 32 cells) but arrest following cell division ("Daughters", N = 45 cells) are shown in the two leftmost panels. Cells that arrest in G2 (N = 40 cells) are shown in the rightmost panel. The solid lines represent population medians and the shaded area indicates the 95% confidence interval.