Figure 5 ( B ) Bacterial viability of convertase - labeled E . coli MG1655 exposed to Buffer ( Conv ) , preassembled C5b6 ( Conv + C5b6MAC ) or a mixture of C5 and C6 ( Conv - MAC ) , in the presence of C7 , C8 and C9 . Dotted line represents the detection limit of the assay . Data information : ( B - D ) Data represent mean ± SD of 3 independent experiments . ( B ) Statistical analysis was done using a ratio paired two - tailed t - test and displayed only when significant as * * p ≤ 0 . 01 . ( C ) Inner membrane damage ( % Sytox positive ) of convertase - labeled perimCherry / cytoGFP E . coli exposed to a concentration range of preassembled C5b6 or a mixture of C5 and C6 , in the presence of 100 nM C7 . After washing , bacteria were exposed to 20 nM C8 and 100 nM C9 . Data information : ( B - D ) Data represent mean ± SD of 3 independent experiments . ( B ) Statistical analysis was done using a ratio paired two - tailed t - test and displayed only when significant as * * p ≤ 0 . 01 . ( D ) outer membrane damage ( mCherry ) of convertase - labeled perimCherry / cytoGFP E . coli exposed to a concentration range of preassembled C5b6 or a mixture of C5 and C6 , in the presence of 100 nM C7 . After washing , bacteria were exposed to 20 nM C8 and 100 nM C9 . Data information : ( B - D ) Data represent mean ± SD of 3 independent experiments . ( B ) Statistical analysis was done using a ratio paired two - tailed t - test and displayed only when significant as * * p ≤ 0 . 01 .

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

Figure 5(B) Bacterial viability of convertase-labeled E. coli MG1655 exposed to Buffer (Conv), preassembled C5b6 (Conv + C5b6MAC) or a mixture of C5 and C6 (Conv-MAC), in the presence of C7, C8 and C9. Dotted line represents the detection limit of the assay. Data information: (B-D) Data represent mean ± SD of 3 independent experiments. (B) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as ** p≤0.01.(C) Inner membrane damage (% Sytox positive) of convertase-labeled perimCherry/cytoGFP E. coli exposed to a concentration range of preassembled C5b6 or a mixture of C5 and C6, in the presence of 100 nM C7. After washing, bacteria were exposed to 20 nM C8 and 100 nM C9. Data information: (B-D) Data represent mean ± SD of 3 independent experiments. (B) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as ** p≤0.01.(D) outer membrane damage (mCherry) of convertase-labeled perimCherry/cytoGFP E. coli exposed to a concentration range of preassembled C5b6 or a mixture of C5 and C6, in the presence of 100 nM C7. After washing, bacteria were exposed to 20 nM C8 and 100 nM C9. Data information: (B-D) Data represent mean ± SD of 3 independent experiments. (B) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as ** p≤0.01.


Figure 6A , B Step - wise assembly of MAC on convertase - labeled bacteria . Convertase - labeled bacteria were incubated with C5 / C6 or C5 / C6 / C7 for 15 minutes , and subsequently washed ( @ ) or treated with 10 μg / ml Eculizumab ( Ecu ) . Then , the remaining MAC components ( C7 - 9 for C5 / C6 or C8 - 9 for C5 / C6 / C7 respectively ) were added to the incubation mixture . In the control conditions ( Conv - MAC ) , the remaining MAC components were added to the incubation mixture without washing or adding an inhibitor . ( A ) Outer membrane ( mCherry ) and ( B ) inner membrane damage ( % Sytox positive ) were determined . Data information : ( A - B ) Data represent mean ± SD of 3 independent experiments . Statistical analysis was done using a ratio paired two - tailed t - test in which the test conditions were compared to Conv - MAC and displayed only when significant as * p ≤ 0 . 05 .

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

Figure 6A, B Step-wise assembly of MAC on convertase-labeled bacteria. Convertase-labeled bacteria were incubated with C5/C6 or C5/C6/C7 for 15 minutes, and subsequently washed (@) or treated with 10 μg/ml Eculizumab (Ecu). Then, the remaining MAC components (C7-9 for C5/C6 or C8-9 for C5/C6/C7 respectively) were added to the incubation mixture. In the control conditions (Conv-MAC), the remaining MAC components were added to the incubation mixture without washing or adding an inhibitor. (A) Outer membrane (mCherry) and (B) inner membrane damage (% Sytox positive) were determined. Data information: (A-B) Data represent mean ± SD of 3 independent experiments. Statistical analysis was done using a ratio paired two-tailed t-test in which the test conditions were compared to Conv-MAC and displayed only when significant as * p≤0.05.


Figure 7 ( A ) Outer and inner membrane damage of convertase - labeled bacteria exposed to different combinations of MAC components . ' @ ' indicates a washing step . Data information : Data represent mean ± SD of 3 independent experiments . ( A ) Statistical analysis was done using a one - way ANOVA and displayed only when significant as * * p ≤ 0 . 01 or * * * * p ≤ 0 . 0001 . ( B ) Outer and inner membrane damage of convertase - labeled bacteria exposed to C5 - C8 and after washing , to a concentration range of C9 . Data information : Data represent mean ± SD of 3 independent experiments .

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

Figure 7(A) Outer and inner membrane damage of convertase-labeled bacteria exposed to different combinations of MAC components. '@' indicates a washing step. Data information: Data represent mean ± SD of 3 independent experiments. (A) Statistical analysis was done using a one-way ANOVA and displayed only when significant as ** p≤0.01 or **** p≤0.0001.(B) Outer and inner membrane damage of convertase-labeled bacteria exposed to C5-C8 and after washing, to a concentration range of C9. Data information: Data represent mean ± SD of 3 independent experiments.


Figure 8 ( A ) Convertase - labeled bacteria were exposed to a concentration range of either preassembled C5b6 ( C5b6MAC ) or a mixture of C5 and C6 ( Conv - MAC ) , in the presence of 100 nM C7 . After washing , 20 nM C8 and 100 nM C9 - Cy3 was added . Controls at 0 nM C5b6 or C5 - C6 confirm that the detected C9 - Cy3 deposition is specifically related to MAC formation ( solid lines ) . Proper insertion of pores was assessed by a previously described shaving method with trypsin ( Moskovich & Fishelson , 2007 ) . Bacteria were first incubated with MAC components for 30 minutes and subsequently treated with 20 µg / ml trypsin for 15 min at 37 ° C ( dotted lines ) . Data information : ( A ) Data represent mean ± SD of 3 independent experiments . ( B ) Convertase labeled perimCherry / cytoGFP bacteria ( Green ) exposed to C5b6MAC or Conv - MAC . Conditions were similar to those in ( A ) , however C9 - Cy5 was used to detect MAC pores ( Red ) . 100 nM of C5 and C6 or C5b6 were used in combination with 100 nM C7 , 20 nM C8 and 100 nM C9 - Cy5 . Conv + C5b6MAC and conv - MAC images were taken in separate experiments in which laser settings were adjusted to the staining intensity of C9 - Cy5 to properly visualize pore distribution . Scale bars = 3 µm . ( C Atomic force microscopy analysis of E . coli BL21 and MG1655 immobilized using the Poly - L - Lysine protocol . ( C ) Entire bacteria and high - resolution comparisons of untreated and convertase - labeled E . coli BL21 exposed to C5 - C9 ( Conv - MAC ) for 10 minutes . Scale bars : 800 nm ( left ) and 30 nm ( right ) . Height Scales : 1 . 01 µm ( left ) , 8 nm ( top right ) , 22 nm ( bottom right ) . Width of magnification boxes ( left ) : 42 nm , height scales : 8 nm ( top left ) and 13 nm ( bottom left ) . Arrows highlight E . coli porin structures , an asterisk highlights the C5b - 7 stalk . Height profiles ( bottom ) are shown for the white dashed lines in the images . ( D ) Atomic force microscopy ( height and phase images ) of convertase - labeled E . coli MG1655 exposed to a mixture of C5 and C6 ( Conv - MAC ) or preassembled C5b6 ( Conv + C5b6MAC ) , in the presence of C7 , C8 , C9 , FB and FD . Images were generated in the same experiment . Scale bars : 50 nm . Height scales : 15 nm .

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

Figure 8(A) Convertase-labeled bacteria were exposed to a concentration range of either preassembled C5b6 (C5b6MAC) or a mixture of C5 and C6 (Conv-MAC), in the presence of 100 nM C7. After washing, 20 nM C8 and 100 nM C9-Cy3 was added. Controls at 0 nM C5b6 or C5-C6 confirm that the detected C9-Cy3 deposition is specifically related to MAC formation (solid lines). Proper insertion of pores was assessed by a previously described shaving method with trypsin (Moskovich & Fishelson, 2007). Bacteria were first incubated with MAC components for 30 minutes and subsequently treated with 20 µg/ml trypsin for 15 min at 37°C (dotted lines). Data information: (A) Data represent mean ± SD of 3 independent experiments.(B) Convertase labeled perimCherry/cytoGFP bacteria (Green) exposed to C5b6MAC or Conv-MAC. Conditions were similar to those in (A), however C9-Cy5 was used to detect MAC pores (Red). 100 nM of C5 and C6 or C5b6 were used in combination with 100 nM C7, 20 nM C8 and 100 nM C9-Cy5. Conv + C5b6MAC and conv-MAC images were taken in separate experiments in which laser settings were adjusted to the staining intensity of C9-Cy5 to properly visualize pore distribution. Scale bars = 3 µm.(C Atomic force microscopy analysis of E. coli BL21 and MG1655 immobilized using the Poly-L-Lysine protocol. (C) Entire bacteria and high-resolution comparisons of untreated and convertase-labeled E. coli BL21 exposed to C5-C9 (Conv-MAC) for 10 minutes. Scale bars: 800 nm (left) and 30 nm (right). Height Scales: 1.01 µm (left), 8 nm (top right), 22 nm (bottom right). Width of magnification boxes (left): 42 nm, height scales: 8 nm (top left) and 13 nm (bottom left). Arrows highlight E. coli porin structures, an asterisk highlights the C5b-7 stalk. Height profiles (bottom) are shown for the white dashed lines in the images.(D) Atomic force microscopy (height and phase images) of convertase-labeled E. coli MG1655 exposed to a mixture of C5 and C6 (Conv-MAC) or preassembled C5b6 (Conv + C5b6MAC), in the presence of C7, C8, C9, FB and FD. Images were generated in the same experiment. Scale bars: 50 nm. Height scales: 15 nm.


f1BrdU incorporation reflecting DNA synthesis and SA - b - gal activity as markers of CTX - induced senescence in Em - myc transgeniccontrol ; Bcl2 versus Suv39h12 ; Bcl2lymphomas in vivo . Numbers represent mean percentages of positive cells6s . d . ( n55 each ) . b , Representative whole - body FLT - and FDG - PET scans of an individual control ; Bcl2 lymphoma - bearing mouse at tumour manifestation ( untreated ) and at day 6 after a single dose of CTX . Red circles , areas of specific tracer retention ; green circles , matching areas with lost retentions . Specific tracer uptake was assessed by calculation of tumour - to - background ratios . Data represent mean ratios ± s . d . ( n = 5 mice for FLT - PET scans and n = 6 mice for FDG - PET scans ) . Insets show representative Ki67 staining and colour - calibrated ( red , highest intensities ; blue , lowest intensities ) bioluminescence images of glucose levels in tissue sections of the respective groups . c , Intracellular glucose concentrations in untreated and ADR - treated control ; Bcl2 ( that is , senescent ) versus Suv39h1 - ; Bcl2 ( that is , non - senescent ) lymphomas in vitro ( as in d - g ) . Results represent means ± s . d . ( n = 5 each ) . d , Extracellular levels of the specified metabolite - to - glucose ratios in the indicated lymphoma cells groups relative to untreated control ; Bcl2 lymphoma cells after 18 h of cultivation . Data represent mean ratios ± s . d . ( n = 4 each , measurements were carried out in duplicate ) . Note that glucose - 6 - phosphate is not detectable ( n . d . ) in the medium . Insets show ( ADR - treated relative to untreated ) intracellular levels of the respective metabolites after labelling with 13C6 - glucose for one representative lymphoma from each group . Data represent mean ratios ( see Supplementary Fig . 5 for an assessment of the heterogeneity of the TIS - associated hypermetabolism and a comparison of the metabolic parameters of individual lymphomas ) . e , Immunoblot analyses of phosphorylated AMP - activated protein kinase ( pAMPK ) , total AMPK , pyruvate kinase isoforms M1 and M2 in ADR - senescent control ; Bcl2 versus ADR - treated Suv39h1 - ; Bcl2 lymphoma cells in vitro ( n = 3 each ; α - tubulin as a loading control . Gels were cropped to ease presentation ; full - length blots are provided in Supplementary Fig . 16 ) . f , Mean OCRs in ADR - senescent control ; Bcl2 versus ADR - treated Suv39h1 − ; Bcl2 lymphoma cells in vitro . Results represent means ± s . d . ( n = 5 each ) . g , Intracellular ATP concentrations in cells as in f . Results represent means ± s . d . ( n = 5 each ) . * P < 0 . 05 .

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

f1BrdU incorporation reflecting DNA synthesis and SA-b-gal activity as markers of CTX-induced senescence in Em-myc transgeniccontrol;Bcl2 versus Suv39h12;Bcl2lymphomas in vivo. Numbers represent mean percentages of positive cells6s.d. (n55 each).b, Representative whole-body FLT- and FDG-PET scans of an individual control;Bcl2 lymphoma-bearing mouse at tumour manifestation (untreated) and at day 6 after a single dose of CTX. Red circles, areas of specific tracer retention; green circles, matching areas with lost retentions. Specific tracer uptake was assessed by calculation of tumour-to-background ratios. Data represent mean ratios ± s.d. (n = 5 mice for FLT-PET scans and n = 6 mice for FDG-PET scans). Insets show representative Ki67 staining and colour-calibrated (red, highest intensities; blue, lowest intensities) bioluminescence images of glucose levels in tissue sections of the respective groups.c, Intracellular glucose concentrations in untreated and ADR-treated control;Bcl2 (that is, senescent) versus Suv39h1-;Bcl2 (that is, non-senescent) lymphomas in vitro (as in d-g). Results represent means ± s.d. (n = 5 each).d, Extracellular levels of the specified metabolite-to-glucose ratios in the indicated lymphoma cells groups relative to untreated control;Bcl2 lymphoma cells after 18 h of cultivation. Data represent mean ratios ± s.d. (n = 4 each, measurements were carried out in duplicate). Note that glucose-6-phosphate is not detectable (n.d.) in the medium. Insets show (ADR-treated relative to untreated) intracellular levels of the respective metabolites after labelling with 13C6-glucose for one representative lymphoma from each group. Data represent mean ratios (see Supplementary Fig. 5 for an assessment of the heterogeneity of the TIS-associated hypermetabolism and a comparison of the metabolic parameters of individual lymphomas).e, Immunoblot analyses of phosphorylated AMP-activated protein kinase (pAMPK), total AMPK, pyruvate kinase isoforms M1 and M2 in ADR-senescent control;Bcl2 versus ADR-treated Suv39h1-;Bcl2 lymphoma cells in vitro (n = 3 each; α-tubulin as a loading control. Gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16).f, Mean OCRs in ADR-senescent control;Bcl2 versus ADR-treated Suv39h1−;Bcl2 lymphoma cells in vitro. Results represent means ± s.d. (n = 5 each).g, Intracellular ATP concentrations in cells as in f. Results represent means ± s.d. (n = 5 each). *P<0.05.


f2a - e , Cell viability by trypan blue dye exclusion in ADR - senescent control ; Bcl2 versus ADR - treated Suv39h1 - ; Bcl2 lymphoma cells at day 5 compared to the untreated condition in various biochemical , pharmacological or genetic interventional settings in vitro . All results in this figure represent means ± s . d . ( n = 5 each ) . a , Inhibition of glucose metabolism by phloretin , cytochalasin B , and sodium oxamate . b , Exposure to 2DG or switch to glucose - free medium with or without glucose replenishment . c , Lentiviral knockdown of hexokinase 2 ( Hk2 ) by short hairpin RNA ( shHk2 ) . d , Inhibition of AMPK by compound C . e , Block of fatty acid oxidation by the carnitine palmitoyltransferase I inhibitor etomoxir . f , Inhibition of the electron transport chain by antimycin A . * P 0 . 01 , except b ( n = 8 each ) and e , in which * P 0 . 05 .

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

f2a-e, Cell viability by trypan blue dye exclusion in ADR-senescent control;Bcl2 versus ADR-treated Suv39h1-;Bcl2 lymphoma cells at day 5 compared to the untreated condition in various biochemical, pharmacological or genetic interventional settings in vitro. All results in this figure represent means ± s.d. (n = 5 each). a, Inhibition of glucose metabolism by phloretin, cytochalasin B, and sodium oxamate.b, Exposure to 2DG or switch to glucose-free medium with or without glucose replenishment.c, Lentiviral knockdown of hexokinase 2 (Hk2) by short hairpin RNA (shHk2). d, Inhibition of AMPK by compound C. e, Block of fatty acid oxidation by the carnitine palmitoyltransferase I inhibitor etomoxir. f, Inhibition of the electron transport chain by antimycin A. *P  0.01, except b (n = 8 each) and e, in which *P  0.05.


f3Proteostasis analysis in TIS control versus senescence - incapable lymphoma cells in vitro , as in Fig . 2 , or versus SASP - attenuated TISlymphoma cells . a , Global protein synthesis rate , measured by incorporation of green - fluorescent methionine . Mean fluorescent intensities reflect newly synthesized protein within 2 h ( DAPI used as a counterstain ) . Data represent means ± s . d . ( n = 5 each ) . b , Rate of global protein synthesis ( measured as in a ) in ADR - senescent relative to untreated ( ut ) control ; Bcl2 lymphoma cells ; either empty vector - infected cells or cells stably expressing the NF - kB super - repressor IkBaDN ( SR , ref . 18 ) . Results represent means ± s . d . ( n = 5 each ) . c , Relative intracellular glucose concentrations in cells as in b . d , Relative OCR in cells as in b . e , Relative intracellular ATP concentrations in cells as in bf , Expression of the transcription factors Xbp1 ( by RT - PCR ( PCR with reverse transcription ; top ) ; note that the lower band reflects the UPR - related splice form ) , ATF4 , CHOP , phospho - Jun N - terminal kinase ( JNK - P - Thr183 / Thr185 ) and total JNK ( by immunoblot analysis , with a - tubulin as a loading control ; bottom ) in lymphoma cells as indicated ( n = 3 each , gels were cropped to ease presentation ; full - length blots are provided in Supplementary Fig . 16 ) . g , Global protein ubiquitination , expression of p62 / SQSTM1 ( whose transcript levels were also selectively induced in TIS cells by approximately 4 . 8 - fold ( data not shown ) ) and V - ATPase subunit A1 , and conversion of MAP1 - LC3 - I to MAP1 - LC3 - II by immunoblot analysis , demonstrating autophagosome formation in lymphoma cells as in f ( α - tubulin was used as a loading control , gels were cropped to ease presentation ; full - length blots are provided in Supplementary Fig . 16 ) . * P < 0 . 05 .

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

f3Proteostasis analysis in TIS control versus senescence-incapable lymphoma cells in vitro, as in Fig. 2, or versus SASP-attenuated TISlymphoma cells. a, Global protein synthesis rate, measured by incorporation of green-fluorescent methionine. Mean fluorescent intensities reflect newly synthesized protein within 2 h (DAPI used as a counterstain). Data represent means ± s.d. (n = 5 each). b, Rate of global protein synthesis (measured as in a) in ADR-senescent relative to untreated (ut) control;Bcl2 lymphoma cells; either empty vector-infected cells or cells stably expressing the NF-kB super-repressor IkBaDN(SR, ref. 18). Results represent means ± s.d. (n = 5 each). c, Relative intracellular glucose concentrations in cells as in b.d, Relative OCR in cells as in b. e, Relative intracellular ATP concentrations in cells as in bf, Expression of the transcription factors Xbp1 (by RT-PCR (PCR with reverse transcription; top); note that the lower band reflects the UPR-related splice form), ATF4, CHOP, phospho-Jun N-terminal kinase (JNK-P-Thr183/Thr185) and total JNK(by immunoblot analysis, with a-tubulin as a loading control; bottom) in lymphoma cells as indicated (n = 3 each, gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16).g, Global protein ubiquitination, expression of p62/SQSTM1 (whose transcript levels were also selectively induced in TIS cells by approximately 4.8-fold (data not shown)) and V-ATPase subunit A1, and conversion of MAP1-LC3-I to MAP1-LC3-II by immunoblot analysis, demonstrating autophagosome formation in lymphoma cells as in f (α-tubulin was used as a loading control, gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16). *P<0.05.


f4a , Cellular viability of super - repressor - or empty - vector - infected control ; Bcl2 lymphomas treated with ADR or untreated for 5 days , then exposed for 2 days to the inhibitors 2DG or bafilomycin A1 , compared to no inhibitor ( ut ) in vitro . Results represent means ± s . d . ( n = 5 each ) . b , Immunoblot analyses of ATF4 , global protein ubiquitination , p62 / SQSTM1 , and MAP1 - LC3 - I to MAP1 - LC3 - II conversion in control ; Bcl2 lymphomas with no ADR treatment , in TIS cells , and in TIS cells after exposure to 2DG or bafilomycin A1in vitro ; representative blot with α - tubulin as a loading control ( gels were cropped to ease presentation ; full - length blots are provided in Supplementary Fig . 16 ) . c , Cleavage ( activation ) of caspase 12 and detection of cleaved caspase 3 by immunoblot analysis of control ; Bcl2 lymphomas as in b ; representative blot with α - tubulin as a loading control ( gels were cropped to ease presentation ; full - length blots are provided in Supplementary Fig . 16 ) . d , Cellular viability of ADR - treated control ; Bcl2 lymphoma cells infected with short - hairpin RNAs against either caspase 12 ( shCasp12 ) or caspase 3 ( shCasp3 ) compared to empty vector - infected lymphoma cells treated as in a . Results represent means ± s . d . ( n = 4 each ) . e , TIS cells in primary human acute myeloid leukaemia ( AML ) samples by SA - b - gal staining of treated ( 0 . 1 µg ml − 1 ADR for 5 days ) versus untreated cells in vitro . Data represent means ± s . d . ( n = 5 each ) . f , Intracellular glucose ( left ) and ATP ( right ) concentrations in senescent AML cells as in e . g , Cellular viability of primary AML samples treated as in e and exposed to inhibitors as in a . Results represent means ± s . d . ( n = 5 each ) . h , Colour - coded whole - body fluorescence imaging to visualize green fluorescent protein ( GFP ) - tagged tumour burden in time - courses of three representative mice harbouring CTX - senescent control ; Bcl2 lymphomas at day 5 ( see Figure 1a ) , and two days later , after the administration of 2DG ( n = 3 mice ) , bafilomycin A1 ( n = 4 mice ) or mock treatment ( n = 4 mice ) starting from day 5 post CTX ( red , highest intensities ; blue , lowest intensities ) . TdT - mediated dUTP nick end labelling ( assay ) ( TUNEL ) staining of lymph node sections in situ to visualize apoptosis at day 7 . Numbers represent means ± s . d . ( n = 3 ) . i , Overall survival of 5 day - CTX - treated control ; Bcl2 or Suv - 39h1 - ; Bcl2 lymphoma - bearing mice randomly assigned to sequential exposure to bafilomycin A1 or mock as in h ( control ; Bcl2 : n = 12 and Suv39h1 - ; Bcl2 : n = 10 ) , presented as matched pair analyses ( P < 0 . 001 for the control ; Bcl2 comparison ; P = 0 . 221 for the Suv39h1 - ; Bcl2 comparison ) . * P < 0 . 05 .

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

f4a, Cellular viability of super-repressor- or empty-vector-infected control;Bcl2 lymphomas treated with ADR or untreated for 5 days, then exposed for 2 days to the inhibitors 2DG or bafilomycin A1, compared to no inhibitor (ut) in vitro. Results represent means ± s.d. (n = 5 each).b, Immunoblot analyses of ATF4, global protein ubiquitination, p62/SQSTM1, and MAP1-LC3-I to MAP1-LC3-II conversion in control;Bcl2 lymphomas with no ADR treatment, in TIS cells, and in TIS cells after exposure to 2DG or bafilomycin A1in vitro; representative blot with α-tubulin as a loading control (gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16). c, Cleavage (activation) of caspase 12 and detection of cleaved caspase 3 by immunoblot analysis of control;Bcl2 lymphomas as in b; representative blot with α-tubulin as a loading control (gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16).d, Cellular viability of ADR-treated control;Bcl2 lymphoma cells infected with short-hairpin RNAs against either caspase 12 (shCasp12) or caspase 3 (shCasp3) compared to empty vector-infected lymphoma cells treated as in a. Results represent means ± s.d. (n = 4 each).e, TIS cells in primary human acute myeloid leukaemia (AML) samples by SA-b-gal staining of treated (0.1 µg ml−1 ADR for 5 days) versus untreated cells in vitro. Data represent means ± s.d. (n = 5 each).f, Intracellular glucose (left) and ATP (right) concentrations in senescent AML cells as in e.g, Cellular viability of primary AML samples treated as in e and exposed to inhibitors as in a. Results represent means ± s.d. (n = 5 each).h, Colour-coded whole-body fluorescence imaging to visualize green fluorescent protein (GFP)-tagged tumour burden in time-courses of three representative mice harbouring CTX-senescent control;Bcl2 lymphomas at day 5 (see Figure 1a), and two days later, after the administration of 2DG (n = 3 mice), bafilomycin A1 (n = 4 mice) or mock treatment (n = 4 mice) starting from day 5 post CTX (red, highest intensities; blue, lowest intensities). TdT-mediated dUTP nick end labelling (assay) (TUNEL) staining of lymph node sections in situ to visualize apoptosis at day 7. Numbers represent means ± s.d. (n = 3).i, Overall survival of 5 day-CTX-treated control;Bcl2 or Suv-39h1-;Bcl2 lymphoma-bearing mice randomly assigned to sequential exposure to bafilomycin A1 or mock as in h (control;Bcl2: n = 12 and Suv39h1-;Bcl2: n = 10), presented as matched pair analyses (P<0.001 for the control; Bcl2 comparison; P = 0.221 for the Suv39h1-;Bcl2 comparison). *P<0.05.


Figure 1A Control animals ( + / w ; UAS - ralIR / + ) , n = 20 , and those with salivary gland - specific knockdown of ral ( fkh - GAL4 / w ; UAS - ralIR / + ) , n = 20 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . B Quantification of data from ( A ) . Data are represented as means . Statistical significance was determined using a Chi - square test . C Control animals ( + / w ; UAS - ralS25N / + ) , n = 20 , and those with salivary gland - specific expression of dominant - negative Ral ( fkh - GAL4 / w ; UAS - ralS25N / + ) , n = 20 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . D Quantification of data from ( C ) . Data are represented as means . Statistical significance was determined using a Chi - square test . E Control animals ( ral35d / + ) , n = 25 , and ral hypomorph mutants ( ral35d / Y ) , n = 23 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . F Quantification of data from ( E ) . Data are represented as means . Statistical significance was determined using a Chi - square test . G Control animals ( + / w ; UAS - rglIR / + ) , n = 19 , and those with salivary gland - specific knockdown of rgl ( fkh - GAL4 / w ; UAS - rglIR / + ) , n = 20 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . H Quantification of data from ( G ) . Data are represented as means . Statistical significance was determined using a Chi - square test .

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

Figure 1A Control animals (+/w; UAS-ralIR/+), n= 20, and those with salivary gland-specific knockdown of ral (fkh-GAL4/w; UAS-ralIR/+), n=20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.B Quantification of data from (A). Data are represented as means. Statistical significance was determined using a Chi-square test.C Control animals (+/w; UAS-ralS25N/+), n= 20, and those with salivary gland-specific expression of dominant-negative Ral (fkh-GAL4/w; UAS-ralS25N/+), n= 20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.D Quantification of data from (C). Data are represented as means. Statistical significance was determined using a Chi-square test.E Control animals (ral35d/+), n= 25, and ral hypomorph mutants (ral35d/Y), n= 23, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.F Quantification of data from (E). Data are represented as means. Statistical significance was determined using a Chi-square test.G Control animals (+/w; UAS-rglIR/+), n= 19, and those with salivary gland-specific knockdown of rgl (fkh-GAL4/w; UAS-rglIR/+), n=20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.H Quantification of data from (G). Data are represented as means. Statistical significance was determined using a Chi-square test.


Figure 2A ral hypomorph mutants ( ral35d / Y ; ; UAS - p35 / + ) , n = 20 , animals with salivary gland - specific expression of p35 ( ral35d / + ; ; UAS - p35 / fkh - GAL4 ) , n = 21 , and ral hypomorph mutants with salivary gland - specific expression of p35 ( ral35d / Y ; ; UAS - p35 / fkh - GAL4 ) , n = 20 , were analyzed by histology for the presence of salivary gland material ( red dotted circles ) 24 hours after puparium formation . B Quantification of data from ( A ) . Data are represented as means . Statistical significance was determined using a Chi - square test comparing the percentages of gland fragments . C Salivary glands dissected from 0 hour after puparium formation control animals ( + / w ; UAS - ralIR / + ) and those with salivary gland - specific knockdown of ral ( fkh - GAL4 / w ; UAS - ralIR / + ) and stained with cleaved Caspase - 3 antibody ( green ) and DAPI ( blue ) . Scale bars represent 20µm . D Salivary glands dissected from 13 hour after puparium formation control animals ( + / w ; UAS - ralIR / + ) and those with salivary gland - specific knockdown of ral ( fkh - GAL4 / w ; UAS - ralIR / + ) and stained with cleaved Caspase - 3 antibody ( green ) and DAPI ( blue ) . Scale bars represent 20µm . E Quantification of data from ( C and D ) . Data are represented as means ± SEM ; n ≥ 10 . Statistical significance was determined using a Student ' s t test .

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

Figure 2A ral hypomorph mutants (ral35d/Y;; UAS-p35/+), n= 20, animals with salivary gland-specific expression of p35 (ral35d/+;; UAS-p35/fkh-GAL4), n= 21, and ral hypomorph mutants with salivary gland-specific expression of p35 (ral35d/Y;; UAS-p35/fkh-GAL4), n= 20, were analyzed by histology for the presence of salivary gland material (red dotted circles) 24 hours after puparium formation.B Quantification of data from (A). Data are represented as means. Statistical significance was determined using a Chi-square test comparing the percentages of gland fragments.C Salivary glands dissected from 0 hour after puparium formation control animals (+/w; UAS-ralIR/+) and those with salivary gland-specific knockdown of ral (fkh-GAL4/w; UAS-ralIR/+) and stained with cleaved Caspase-3 antibody (green) and DAPI (blue). Scale bars represent 20µm.D Salivary glands dissected from 13 hour after puparium formation control animals (+/w; UAS-ralIR/+) and those with salivary gland-specific knockdown of ral (fkh-GAL4/w; UAS-ralIR/+) and stained with cleaved Caspase-3 antibody (green) and DAPI (blue). Scale bars represent 20µm.E Quantification of data from (C and D). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test.


Figure 3A ral hypomorph mutants ( ral35d / Y ; ; UAS - Atg6IR / + ) , n = 27 , animals with salivary gland - specific expression of Atg6IR ( ral35d / + ; ; UAS - Atg6IR / fkh - GAL4 ) , n = 20 , and ral hypomorph mutants with salivary gland - specific expression of Atg6IR ( ral35d / Y ; ; UAS - Atg6IR / fkh - GAL4 ) , n = 24 , were analyzed by histology for the presence of salivary gland material ( red dotted circles ) 24 hours after puparium formation . B Quantification of data from ( A ) . Data are represented as means . Statistical significance was determined using a Chi - square test . C Salivary glands dissected from 14 hours after puparium formation animals expressing mCherry - Atg8a in all cells , and ralIR specifically in GFP - marked clone cells ( yw , hsflp / w ; pmCherry - Atg8a / UAS - ralIR ; act < FRT , cd2 , FRT > GAL4 , UAS - GFP / + ) analyzed for mCherry - Atg8a puncta . GFP - marked clone ralIR - expressing cells contain a yellow asterisk . Scale bars represent 20µm . D Quantification of data from ( C ) . Data are represented as means ± SEM ; n ≥ 10 . Statistical significance was determined using a Student ' s t test . E Salivary glands dissected from 14 hours after puparium formation animals that have ral loss of function ( ralPG89 ) specifically in non - RFP cells , and that express GFP - Atg8a in all cells ( yw , hs - Flp , FRT19A , Ubi - RFP / ralPG89 , FRT19A ; pGFP - Atg8a ) analyzed for GFP - Atg8a puncta . ralPG89 mutant cells are marked with a yellow asterisk . Scale bars represent 20µm . F Quantification of data from ( E ) . Data are represented as means ± SEM ; n ≥ 10 . Statistical significance was determined using a Student ' s t test . G Salivary glands dissected from 14 hours after puparium formation animals that have ral loss of function ( ralPG89 ) specifically in non - RFP cells ( yw , hs - Flp , FRT19A , Ubi - RFP / ralPG89 , FRT19A ) and are stained for Ref ( 2 ) p ( green ) and DAPI ( blue ) . ralPG89 mutant cells are marked with a yellow asterisk . Scale bars represent 20µm . H Quantification of data from ( G ) . Data are represented as means ± SEM ; n ≥ 10 . Statistical significance was determined using a Student ' s t test . I Fat bodies dissected from 4 hour starved animals that have ral loss of function ( ralPG89 ) specifically in non - RFP cells , and that express GFP - Atg8a in all cells ( yw , hs - Flp , FRT19A , Ubi - RFP / ralPG89 , FRT19A ; pGFP - Atg8a ) analyzed for GFP - Atg8a puncta . ralPG89 mutant cells are marked with a yellow asterisk . Scale bars represent 20µm . J Quantification of data from ( I ) . Data are represented as means ± SEM ; n ≥ 10 . Statistical significance was determined using a Student ' s t test .

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

Figure 3A ral hypomorph mutants (ral35d/Y;; UAS-Atg6IR/+), n= 27, animals with salivary gland-specific expression of Atg6IR (ral35d/+;; UAS-Atg6IR/fkh-GAL4), n= 20, and ral hypomorph mutants with salivary gland-specific expression of Atg6IR (ral35d/Y;; UAS-Atg6IR/fkh-GAL4), n= 24, were analyzed by histology for the presence of salivary gland material (red dotted circles) 24 hours after puparium formation.B Quantification of data from (A). Data are represented as means. Statistical significance was determined using a Chi-square test.C Salivary glands dissected from 14 hours after puparium formation animals expressing mCherry-Atg8a in all cells, and ralIR specifically in GFP-marked clone cells (yw, hsflp/w; pmCherry-Atg8a/UAS-ralIR; act< FRT, cd2, FRT> GAL4, UAS-GFP/+) analyzed for mCherry-Atg8a puncta. GFP-marked clone ralIR-expressing cells contain a yellow asterisk. Scale bars represent 20µm.D Quantification of data from (C). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test.E Salivary glands dissected from 14 hours after puparium formation animals that have ral loss of function (ralPG89) specifically in non-RFP cells, and that express GFP-Atg8a in all cells (yw, hs-Flp, FRT19A, Ubi-RFP/ ralPG89,FRT19A; pGFP-Atg8a) analyzed for GFP-Atg8a puncta. ralPG89 mutant cells are marked with a yellow asterisk. Scale bars represent 20µm.F Quantification of data from (E). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test.G Salivary glands dissected from 14 hours after puparium formation animals that have ral loss of function (ralPG89) specifically in non-RFP cells (yw, hs-Flp, FRT19A, Ubi-RFP/ ralPG89,FRT19A) and are stained for Ref(2)p (green) and DAPI (blue). ralPG89 mutant cells are marked with a yellow asterisk. Scale bars represent 20µm.H Quantification of data from (G). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test.I Fat bodies dissected from 4 hour starved animals that have ral loss of function (ralPG89) specifically in non-RFP cells, and that express GFP-Atg8a in all cells (yw, hs-Flp, FRT19A, Ubi-RFP/ ralPG89,FRT19A; pGFP-Atg8a) analyzed for GFP-Atg8a puncta. ralPG89 mutant cells are marked with a yellow asterisk. Scale bars represent 20µm.J Quantification of data from (I). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test.


Figure 4A , C , E , G , I Fat bodies dissected from 4 hour starved animals expressing mCherry - Atg8a in all cells , and ( A ) sec5IR , ( C ) sec15IR , ( E ) sec3IR , ( G ) sec8IR , ( I ) exo84IR specifically in GFP - marked clone cells ( yw , hsflp / w ; pmCherry - Atg8a / + ; act < FRT , cd2 , FRT > GAL4 , UAS - GFP / + ) analyzed for mCherry - Atg8a puncta . Scale bars represent 20µm . B , D , F , H , J Quantification of data from ( A , C , E , G , I ) . Data are represented as means ± SEM ; n ≥ 10 . Statistical significance was determined using a Student ' s t test .

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

Figure 4A, C, E, G, I Fat bodies dissected from 4 hour starved animals expressing mCherry-Atg8a in all cells, and (A) sec5IR, (C) sec15IR, (E) sec3IR, (G) sec8IR, (I) exo84IR specifically in GFP-marked clone cells (yw, hsflp/w; pmCherry-Atg8a/+; act< FRT, cd2, FRT> GAL4, UAS-GFP/+) analyzed for mCherry-Atg8a puncta. Scale bars represent 20µm.B, D, F, H, J Quantification of data from (A, C, E, G, I). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test.


Figure 5A Salivary glands dissected from 14 hours after puparium formation ( top left ) control ( fkh - GAL4 / + ; pmCherry - Atg8a / + ) , and those with salivary gland - specific knockdown of ( middle left ) sec5 ( fkh - GAL4 / w ; pmCherry - Atg8a / + ; UAS - sec5IR / + ) , ( bottom left ) sec15 ( fkh - GAL4 / w ; pmCherry - Atg8a / + ; UAS - sec15IR / + ) , ( top right ) sec3 ( fkh - GAL4 / w ; pmCherry - Atg8a / UAS - sec3IR ) , ( middle right ) sec8 ( fkh - GAL4 / w ; pmCherry - Atg8a / UAS - sec8IR ) , ( bottom right ) exo84 ( fkh - GAL4 / w ; pmCherry - Atg8a / UAS - exo84IR ) analyzed for mCherry - Atg8a puncta . Scale bars represent 20µm . B Quantification of data from ( A ) . Data are represented as means ± SEM ; n ≥ 10 . Statistical significance was determined using a Student ' s t test ( * * p < 0 . 001 , * * * p < 0 . 0001 ) .

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

Figure 5A Salivary glands dissected from 14 hours after puparium formation (top left) control (fkh-GAL4/+; pmCherry-Atg8a/+), and those with salivary gland-specific knockdown of (middle left) sec5 (fkh-GAL4/w; pmCherry-Atg8a/+; UAS-sec5IR/+), (bottom left) sec15 (fkh-GAL4/w; pmCherry-Atg8a/+; UAS-sec15IR/+), (top right) sec3 (fkh-GAL4/w; pmCherry-Atg8a/UAS-sec3IR), (middle right) sec8 (fkh-GAL4/w; pmCherry-Atg8a/UAS-sec8IR), (bottom right) exo84 (fkh-GAL4/w; pmCherry-Atg8a/UAS-exo84IR) analyzed for mCherry-Atg8a puncta. Scale bars represent 20µm.B Quantification of data from (A). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test (** p< 0.001, *** p< 0.0001).


Figure 6A Control animals ( + / w ; UAS - sec5IR / + ) , n = 19 , and those with salivary gland - specific knockdown of sec5 ( fkh - GAL4 / w ; UAS - sec5IR / + ) , n = 20 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . B Quantification of data from ( A ) . Data are represented as means . Statistical significance was determined using a Chi - square test . C Control animals ( + / w ; UAS - sec15IR / + ) , n = 20 , and those with salivary gland - specific expression sec15 ( fkh - GAL4 / w ; UAS - sec15IR / + ) , n = 20 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . D Quantification of data from ( C ) . Data are represented as means . Statistical significance was determined using a Chi - square test . E Control animals ( + / w ; UAS - sec3IR / + ) , n = 20 , and those with salivary gland - specific expression sec3 ( fkh - GAL4 / w ; UAS - sec3IR / + ) , n = 20 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . F Quantification of data from ( E ) . Data are represented as means . Statistical significance was determined using a Chi - square test . G Control animals ( + / w ; UAS - sec8IR / + ) , n = 20 , and those with salivary gland - specific knockdown of sec8 ( fkh - GAL4 / w ; UAS - sec8IR / + ) , n = 19 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . H Quantification of data from ( G ) . Data are represented as means . Statistical significance was determined using a Chi - square test . I Control animals ( + / w ; UAS - exo84IR / + ) , n = 19 , and those with salivary gland - specific knockdown of exo84 ( fkh - GAL4 / w ; UAS - exo84IR / + ) , n = 20 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . J Quantification of data from ( I ) . Data are represented as means . Statistical significance was determined using a Chi - square test .

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

Figure 6A Control animals (+/w; UAS-sec5IR/+), n= 19, and those with salivary gland-specific knockdown of sec5 (fkh-GAL4/w; UAS-sec5IR/+), n=20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.B Quantification of data from (A). Data are represented as means. Statistical significance was determined using a Chi-square test.C Control animals (+/w; UAS-sec15IR/+), n= 20, and those with salivary gland-specific expression sec15 (fkh-GAL4/w; UAS-sec15IR/+), n= 20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.D Quantification of data from (C). Data are represented as means. Statistical significance was determined using a Chi-square test.E Control animals (+/w; UAS-sec3IR/+), n= 20, and those with salivary gland-specific expression sec3 (fkh-GAL4/w; UAS-sec3IR/+), n= 20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.F Quantification of data from (E). Data are represented as means. Statistical significance was determined using a Chi-square test.G Control animals (+/w; UAS-sec8IR/+), n= 20, and those with salivary gland-specific knockdown of sec8 (fkh-GAL4/w; UAS-sec8IR/+), n=19, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.H Quantification of data from (G). Data are represented as means. Statistical significance was determined using a Chi-square test.I Control animals (+/w; UAS-exo84IR/+), n= 19, and those with salivary gland-specific knockdown of exo84 (fkh-GAL4/w; UAS-exo84IR/+), n=20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.J Quantification of data from (I). Data are represented as means. Statistical significance was determined using a Chi-square test.


Figure 7A Salivary glands dissected from late 3rd instar larvae ( Gbe Su ( H ) - lac - Z / w - ; + ; Fkh - GAL4 / + ) show less notch activity ( left ) compared to glands from 14 hours after puparium formation ( APF ) animals ( middle ) . Down - regulation of ral in salivary glands from 14 hours APF animals ( Gbe Su ( H ) - lac - Z / w - ; UAS - ralIR / + ; Fkh - GAL4 / + ) reduces the levels of notch activity ( right ) . Scale bar represents 20µm . B Quantification of data from ( A ) . Data are represented as means ± SEM ; n ≥ 4 . Statistical significance was determined using a Student ' s t test . C Salivary glands dissected from 14 hours after puparium formation animals expressing mCherry - Atg8a in all cells , and notchIR specifically in GFP - marked clone cells ( yw , hsflp / w , UAS - notchIR ; pmCherry - Atg8a / + ; act < FRT , cd2 , FRT > GAL4 , UAS - GFP / + ) analyzed for mCherry - Atg8a puncta . Scale bar represents 20µm . D Quantification of data from ( C ) . Data are represented as means ± SEM ; n > 10 . Statistical significance was determined using a Student ' s t test .

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

Figure 7A Salivary glands dissected from late 3rd instar larvae (Gbe Su(H)-lac-Z/w-;+;Fkh-GAL4/+) show less notch activity (left) compared to glands from 14 hours after puparium formation (APF) animals (middle). Down-regulation of ral in salivary glands from 14 hours APF animals (Gbe Su(H)-lac-Z/w-;UAS-ralIR/+;Fkh-GAL4/+) reduces the levels of notch activity (right). Scale bar represents 20µm.B Quantification of data from (A). Data are represented as means ± SEM; n≥ 4. Statistical significance was determined using a Student's t test.C Salivary glands dissected from 14 hours after puparium formation animals expressing mCherry-Atg8a in all cells, and notchIR specifically in GFP-marked clone cells (yw, hsflp/w,UAS-notchIR; pmCherry-Atg8a/+; act< FRT, cd2, FRT> GAL4, UAS-GFP/+) analyzed for mCherry-Atg8a puncta. Scale bar represents 20µm.D Quantification of data from (C). Data are represented as means ± SEM; n> 10. Statistical significance was determined using a Student's t test.


Figure 1 ( A ) Inducible GFP ‐ S ‐ Ubqln1 293 cell line was treated with 1 μg / ml of doxycycline for 24 h . GFP ‐ S ‐ Ubqln1 was immunoprecipitated and sent for mass spectrometry analysis . Red sequences indicate Ubqln4 peptides identified by mass spectrometry . ( B ) 293 cells were co ‐ transfected with GFP ‐ S or GFP ‐ S ‐ Ubqln1 and HA ‐ Ubqln4 . GFP was immunoprecipitated and analysed by western blot using anti ‐ GFP and anti ‐ HA antibodies . Lower molecular weight GFP ‐ tag ‐ containing bands that appear in the GFP ‐ S ‐ Ubqln1 lane might represent degradation products . ( C ) Endogenous Ubqln1 in 293 cells was immunoprecipitated using a rabbit Ubqln1 antibody or a control rabbit IgG . The immunoprecipitates were analysed by western blotting using anti ‐ Ubqln1 and anti ‐ Ubqln4 antibodies . ( D ) Confocal images of Hela cells transfected with GFP ‐ S ‐ Ubqln1 and RFP ‐ Ubqln4 . Linescan of the white line in the merged image indicates the intensities of the fluorescent molecules in the cross ‐ section . GFP , green fluorescent protein ; HA , haemagglutinin ; IP , immunoprecipitation ; Ubqln , Ubiquilin ; WCL , whole ‐ cell lysate .

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

Figure 1(A) Inducible GFP‐S‐Ubqln1 293 cell line was treated with 1 μg/ml of doxycycline for 24 h. GFP‐S‐Ubqln1 was immunoprecipitated and sent for mass spectrometry analysis. Red sequences indicate Ubqln4 peptides identified by mass spectrometry.(B) 293 cells were co‐transfected with GFP‐S or GFP‐S‐Ubqln1 and HA‐Ubqln4. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐HA antibodies. Lower molecular weight GFP‐tag‐containing bands that appear in the GFP‐S‐Ubqln1 lane might represent degradation products.(C) Endogenous Ubqln1 in 293 cells was immunoprecipitated using a rabbit Ubqln1 antibody or a control rabbit IgG. The immunoprecipitates were analysed by western blotting using anti‐Ubqln1 and anti‐Ubqln4 antibodies.(D) Confocal images of Hela cells transfected with GFP‐S‐Ubqln1 and RFP‐Ubqln4. Linescan of the white line in the merged image indicates the intensities of the fluorescent molecules in the cross‐section. GFP, green fluorescent protein; HA, haemagglutinin; IP, immunoprecipitation; Ubqln, Ubiquilin; WCL, whole‐cell lysate.


Figure 2Ubqln4 interacts with LC3 . Confocal images of ( A ) Hela cells transfected with GFP ‐ LC3 and RFP ‐ Ubqln4 and starved for 1 h or ( B ) Hela cells starved for 1 h and stained for endogenous LC3 and Ubqln4 . The squares show colocalization . The insets show the enlargement of the indicated areas . Linescan of the white line in the merged image indicates the intensities of the fluorescent molecules in the cross ‐ section . ( C ) 293 cells were transfected with GFP ‐ S , GFP ‐ S ‐ Ubqln1 or GFP ‐ S ‐ Ubqln4 and treated with 50 μM chloroquine for 14 h . GFP was immunoprecipitated and analysed by western blot using anti ‐ GFP and anti ‐ LC3 antibodies . ( D ) Recombinant His ‐ Ubqln1 , His ‐ Ubqln4 , His ‐ S5a or His ‐ p62 was mixed with LC3 covalently coupled to agarose beads . The beads were washed , eluted and detected by western blot using anti ‐ His antibody . The extent of Ubqln1 , Ubqln4 , s5a or p62 co ‐ immunoprecipitation with LC3 was measured . Bars for Ubqln1 , Ubqln4 and s5a represents the average and s . e . m . of four independent experiments . * P ‐ value = 0 . 0094 . ( E ) 293 cells were transfected with GFP ‐ S ‐ Ubqln4 WT , GFP ‐ S ‐ Ubqln4 ΔSTI1 12 , GFP ‐ S ‐ Ubqln4 Δ between STI1 or GFP ‐ S ‐ Ubqln4 ΔSTI1 34 and treated with 50 μM chloroquine for 14 h . GFP was immunoprecipitated and analysed by western blot using anti ‐ GFP and anti ‐ LC3 antibodies . GFP , green fluorescent protein ; IP , immunoprecipitation ; LC3 , microtubule ‐ associated protein light chain 3 ; Ubqln , Ubiquilin ; WCL , whole ‐ cell lysate ; WT , wild ‐ type .

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

Figure 2Ubqln4 interacts with LC3. Confocal images of (A) Hela cells transfected with GFP‐LC3 and RFP‐Ubqln4 and starved for 1 h or(B) Hela cells starved for 1 h and stained for endogenous LC3 and Ubqln4. The squares show colocalization. The insets show the enlargement of the indicated areas. Linescan of the white line in the merged image indicates the intensities of the fluorescent molecules in the cross‐section.(C) 293 cells were transfected with GFP‐S, GFP‐S‐Ubqln1 or GFP‐S‐Ubqln4 and treated with 50 μM chloroquine for 14 h. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐LC3 antibodies.(D) Recombinant His‐Ubqln1, His‐Ubqln4, His‐S5a or His‐p62 was mixed with LC3 covalently coupled to agarose beads. The beads were washed, eluted and detected by western blot using anti‐His antibody. The extent of Ubqln1, Ubqln4, s5a or p62 co‐immunoprecipitation with LC3 was measured. Bars for Ubqln1, Ubqln4 and s5a represents the average and s.e.m. of four independent experiments. *P‐value=0.0094.(E) 293 cells were transfected with GFP‐S‐Ubqln4 WT, GFP‐S‐Ubqln4 ΔSTI1 12, GFP‐S‐Ubqln4 Δ between STI1 or GFP‐S‐Ubqln4 ΔSTI1 34 and treated with 50 μM chloroquine for 14 h. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐LC3 antibodies. GFP, green fluorescent protein; IP, immunoprecipitation; LC3, microtubule‐associated protein light chain 3; Ubqln, Ubiquilin; WCL, whole‐cell lysate; WT, wild‐type.


Figure 3 ( A ) Confocal images of Hela cells transfected with GFP ‐ S ‐ Ubqln1 and also transfected with either control or Ubqln4 siRNA . The cells were either untreated or were transferred to starvation medium for 1 h . ( B ) Quantification of the number of GFP ‐ S ‐ Ubqln1 WT puncta per cell in ( A ) . Each bar represents the average and s . e . m . of 30 cells per condition in three independent experiments . * P ‐ value = 0 . 011 , * * P ‐ value = 0 . 0005 and * * * P ‐ value = 0 . 0001 . ( C ) Depletion of Ubqln4 with siRNA in ( A ) was verified by western blot using anti ‐ Ubqln1 , anti ‐ Ubqln4 and anti ‐ tubulin antibodies . ( D ) Hela cells transfected with GFP ‐ S ‐ Ubqln1 and also transfected with either control or Ubqln4 siRNA were starved for 1 h . GFP was immunoprecipitated and analysed by western blot using anti ‐ GFP and anti ‐ LC3 antibodies . ( E ) Confocal images of GFP ‐ S ‐ Ubqln1 WT in Hela cells transfected with RFP , RFP ‐ Ubqln4 or RFP ‐ Ubqln4 ΔUBL . ( F ) Quantification of the number of GFP ‐ S ‐ Ubqln1 WT puncta per cell in ( E ) . Each bar represents the average and s . e . m . of 30 cells per condition in three independent experiments . * P ‐ value = 0 . 0001 and * * P ‐ value = 0 . 0003 . GFP , green fluorescent protein ; IP , immunoprecipitation ; LC3 , microtubule ‐ associated protein light chain 3 ; UBL , ubiquitin ‐ like ; Ubqln , Ubiquilin ; WCL , whole ‐ cell lysate ; WT , wild ‐ type .

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

Figure 3(A) Confocal images of Hela cells transfected with GFP‐S‐Ubqln1 and also transfected with either control or Ubqln4 siRNA. The cells were either untreated or were transferred to starvation medium for 1 h.(B) Quantification of the number of GFP‐S‐Ubqln1 WT puncta per cell in (A). Each bar represents the average and s.e.m. of 30 cells per condition in three independent experiments. *P‐value=0.011, **P‐value=0.0005 and ***P‐value=0.0001.(C) Depletion of Ubqln4 with siRNA in (A) was verified by western blot using anti‐Ubqln1, anti‐Ubqln4 and anti‐tubulin antibodies.(D) Hela cells transfected with GFP‐S‐Ubqln1 and also transfected with either control or Ubqln4 siRNA were starved for 1 h. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐LC3 antibodies.(E) Confocal images of GFP‐S‐Ubqln1 WT in Hela cells transfected with RFP, RFP‐Ubqln4 or RFP‐Ubqln4 ΔUBL.(F) Quantification of the number of GFP‐S‐Ubqln1 WT puncta per cell in (E). Each bar represents the average and s.e.m. of 30 cells per condition in three independent experiments. *P‐value=0.0001 and **P‐value=0.0003. GFP, green fluorescent protein; IP, immunoprecipitation; LC3, microtubule‐associated protein light chain 3; UBL, ubiquitin‐like; Ubqln, Ubiquilin; WCL, whole‐cell lysate; WT, wild‐type.


Figure 4 ( A ) 293 cells were co ‐ transfected with GFP ‐ S ‐ Ubqln1 , GFP ‐ S ‐ Ubqln1 ΔUBL ( deletion of UBL domain ) or GFP ‐ S ‐ Ubqln1 ΔUBA ( deletion of UBA domain ) and HA ‐ Ubqln4 . GFP was immunoprecipitated and analysed by western blot using anti ‐ GFP and anti ‐ HA antibodies . ( B ) The extent of Ubqln4 co ‐ immunoprecipitation with Ubqln1 was measured and the ratio of Ubqln4 to Ubqln1 was calculated for WT Ubqln1 , Ubqln1ΔUBL and Ubqln1ΔUBA . The numbers represent the ratio of Ubqln4 to Ubqln1 relative to WT . Each bar represents the average and s . e . m . of four independent experiments . * P ‐ value = 0 . 0041 . ( C ) 293 cells were co ‐ transfected with GFP ‐ S or GFP ‐ S ‐ Ubqln1 and HA ‐ Ubqln4 , HA ‐ Ubqln4 ΔUBL or HA ‐ Ubqln4 ΔUBA . GFP was immunoprecipitated and analysed by western blot using anti ‐ GFP and anti ‐ HA antibodies . ( D ) The extent of Ubqln4 co ‐ immunoprecipitation with Ubqln1 was measured and the ratio of Ubqln4 to Ubqln1 was calculated for WT Ubqln4 , Ubqln4ΔUBL and Ubqln4ΔUBA . The numbers represent the ratio of Ubqln4 to Ubqln1 relative to WT . Each bar represents the average and s . e . m . of four independent experiments . * P ‐ value = 0 . 0003 and * * P ‐ value = 0 . 0001 . ( E ) 293 cells were co ‐ transfected with GFP ‐ S ‐ Ubqln1 and HA ‐ Ubqln4 , HA ‐ Ubqln4 ΔUBA or HA ‐ Ubqln4 ΔUBA I55A ( I55 in the UBL domain of Ubqln4 was mutated to A ) . GFP was immunoprecipitated and analysed by western blot using anti ‐ GFP and anti ‐ HA antibodies . ( F ) The extent of Ubqln4 co ‐ immunoprecipitation with Ubqln1 was measured and the ratio of Ubqln4 to Ubqln4 was calculated for WT Ubqln4 , Ubqln4ΔUBA and Ubqln4ΔUBA I55A . The numbers represent the ratio of Ubqln4 to Ubqln1 relative to WT . Each bar represents the average of two independent experiments . GFP , green fluorescent protein ; HA , haemagglutinin ; IP , immunoprecipitation ; UBA , ubiquitin ‐ associated ; UBL , ubiquitin ‐ like ; Ubqln , Ubiquilin ; WCL , whole ‐ cell lysate ; WT , wild ‐ type .

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

Figure 4(A) 293 cells were co‐transfected with GFP‐S‐Ubqln1, GFP‐S‐Ubqln1 ΔUBL (deletion of UBL domain) or GFP‐S‐Ubqln1 ΔUBA (deletion of UBA domain) and HA‐Ubqln4. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐HA antibodies.(B) The extent of Ubqln4 co‐immunoprecipitation with Ubqln1 was measured and the ratio of Ubqln4 to Ubqln1 was calculated for WT Ubqln1, Ubqln1ΔUBL and Ubqln1ΔUBA. The numbers represent the ratio of Ubqln4 to Ubqln1 relative to WT. Each bar represents the average and s.e.m. of four independent experiments. *P‐value=0.0041.(C) 293 cells were co‐transfected with GFP‐S or GFP‐S‐Ubqln1 and HA‐Ubqln4, HA‐Ubqln4 ΔUBL or HA‐Ubqln4 ΔUBA. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐HA antibodies.(D) The extent of Ubqln4 co‐immunoprecipitation with Ubqln1 was measured and the ratio of Ubqln4 to Ubqln1 was calculated for WT Ubqln4, Ubqln4ΔUBL and Ubqln4ΔUBA. The numbers represent the ratio of Ubqln4 to Ubqln1 relative to WT. Each bar represents the average and s.e.m. of four independent experiments. *P‐value=0.0003 and **P‐value=0.0001.(E) 293 cells were co‐transfected with GFP‐S‐Ubqln1 and HA‐Ubqln4, HA‐Ubqln4 ΔUBA or HA‐Ubqln4 ΔUBA I55A (I55 in the UBL domain of Ubqln4 was mutated to A). GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐HA antibodies.(F) The extent of Ubqln4 co‐immunoprecipitation with Ubqln1 was measured and the ratio of Ubqln4 to Ubqln4 was calculated for WT Ubqln4, Ubqln4ΔUBA and Ubqln4ΔUBA I55A. The numbers represent the ratio of Ubqln4 to Ubqln1 relative to WT. Each bar represents the average of two independent experiments. GFP, green fluorescent protein; HA, haemagglutinin; IP, immunoprecipitation; UBA, ubiquitin‐associated; UBL, ubiquitin‐like; Ubqln, Ubiquilin; WCL, whole‐cell lysate; WT, wild‐type.


Figure 5 ( A ) Confocal images of Hela cells transfected with mCherry ‐ GFP ‐ LC3 and also transfected with control , Ubqln1 or Ubqln4 siRNA . The cells were either untreated or were transferred to starvation medium for 8 h . ( B ) Quantification of the number of mCherry / GFP double ‐ positive and mCherry single ‐ positive puncta per cell in ( A ) . Each bar represents the average and s . e . m . of 30 cells per condition in three independent experiments . The number of mCherry single ‐ positive puncta of Ubqln1 and Ubqln4 siRNA was compared with that of control siRNA . * P ‐ value = 0 . 0475 and * * P ‐ value = 0 . 0373 . GFP , green fluorescent protein ; LC3 , microtubule ‐ associated protein light chain 3 ; Ubqln , Ubiquilin .

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Figure 5(A) Confocal images of Hela cells transfected with mCherry‐GFP‐LC3 and also transfected with control, Ubqln1 or Ubqln4 siRNA. The cells were either untreated or were transferred to starvation medium for 8 h.(B) Quantification of the number of mCherry/GFP double‐positive and mCherry single‐positive puncta per cell in (A). Each bar represents the average and s.e.m. of 30 cells per condition in three independent experiments. The number of mCherry single‐positive puncta of Ubqln1 and Ubqln4 siRNA was compared with that of control siRNA. *P‐value=0.0475 and **P‐value=0.0373. GFP, green fluorescent protein; LC3, microtubule‐associated protein light chain 3; Ubqln, Ubiquilin.


Figure 2 ( B ) Structures of S . cerevisiae Hrr251 - 394 K38R bound to CK1 - 7 ( Chijiwa et al , 1989 ; Xu et al , 1996 ) and C . glabrata Hrr251 - 403 K38R bound to ADP , with domains colored as in ( A ) . Bound PO4 - / SO4 - ions are shown as spheres . See Figure EV2A for additional C . glabrataHrr25 structures , and Figure EV3A for SO4 - ion electron density . ( C ) Overlay of the kinase domain of C . glabrataHrr25 ( Apo form , crystallized with 1 . 2 M PO4 - ) with ratCK1δ crystallized in the presence of tungstate ions ( WO4 ; PDB ID 1CKJ , ( Longenecker et al , 1996 ) ) See Figure EV2B for detailed views of ion binding . ( D ) Stereo view of CK1 - 7 binding to S . cerevisiae Hrr25 . Bound drug is positioned identically to a previous structure of S . pombe Cki1 bound to CK1 - 7 ( Xu et al , 1996 ) . ( E ) Stereo view of ADP binding to C . glabrata Hrr25 ( formate structure ; SO4 - structure is equivalent , but the GxGxxG motif is disordered in that structure ) . All active - site residues shown in panels ( D ) and ( E ) are conserved between S . cerevisiae and C . glabrata Hrr25 .

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Figure 2(B) Structures of S. cerevisiae Hrr251-394 K38R bound to CK1-7 (Chijiwa et al, 1989; Xu et al, 1996) and C. glabrata Hrr251-403 K38R bound to ADP, with domains colored as in (A). Bound PO4-/SO4- ions are shown as spheres. See Figure EV2A for additional C. glabrataHrr25 structures, and Figure EV3A for SO4- ion electron density.(C) Overlay of the kinase domain of C. glabrataHrr25 (Apo form, crystallized with 1.2 M PO4-) with ratCK1δ crystallized in the presence of tungstate ions (WO4; PDB ID 1CKJ, (Longenecker et al, 1996)) See Figure EV2B for detailed views of ion binding.(D) Stereo view of CK1-7 binding to S. cerevisiae Hrr25. Bound drug is positioned identically to a previous structure of S. pombe Cki1 bound to CK1-7 (Xu et al, 1996).(E) Stereo view of ADP binding to C. glabrata Hrr25 (formate structure; SO4- structure is equivalent, but the GxGxxG motif is disordered in that structure). All active-site residues shown in panels (D) and (E) are conserved between S. cerevisiae and C. glabrata Hrr25.


Figure 3 ( A ) Two views of C . glabrata Hrr25 ( Apo form ) with domains colored as in Figure 1 , showing bound PO4 - ions and the previously - identified Mam1 - binding residues His25 and Glu34 ( Petronczki et al , 2006 ) . ( B ) Two views , roughly equivalent to the views in ( A ) , of the Hrr25 central domain , colored as a rainbow according to the schematic at top . Dotted lines in the schematic indicate disordered loops . See Figure EV1A for sequence alignment of this domain .

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Figure 3(A) Two views of C. glabrata Hrr25 (Apo form) with domains colored as in Figure 1, showing bound PO4- ions and the previously-identified Mam1-binding residues His25 and Glu34 (Petronczki et al, 2006).(B) Two views, roughly equivalent to the views in (A), of the Hrr25 central domain, colored as a rainbow according to the schematic at top. Dotted lines in the schematic indicate disordered loops. See Figure EV1A for sequence alignment of this domain.


Figure 4 ( B ) Overall structure of the S . cerevisiae Hrr251 - 394 K38R : Mam187 - 191 complex , colored as in ( A ) . View is equivalent to Figure 2B . ( C ) Close - up view of Mam187 - 191 , colored as in the schematic in ( A ) . ( D ) Close - up of the variant zinc knuckle motif of Mam1 , with zinc anomalous difference electron density shown at 3 σ ( gray ) and 10 σ ( red ) . ( E ) Geometry of zinc binding in Mam1 . Zn - S bonds and the Zn - N bond were restrained to ~ 2 . 3 Å and ~ 2 . 0 Å , respectively , during refinement .

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Figure 4(B) Overall structure of the S. cerevisiae Hrr251-394 K38R:Mam187-191 complex, colored as in (A). View is equivalent to Figure 2B.(C) Close-up view of Mam187-191, colored as in the schematic in (A). (D) Close-up of the variant zinc knuckle motif of Mam1, with zinc anomalous difference electron density shown at 3 σ (gray) and 10 σ (red).(E) Geometry of zinc binding in Mam1. Zn-S bonds and the Zn-N bond were restrained to ~2.3 Å and ~2.0 Å, respectively, during refinement.


Figure 5 ( A ) The Hrr25 : Mam1 complex with Mam1 shown as orange ribbons , and Hrr25 shown as a white surface with Mam1 - binding interface # 1 shown in green and # 2 in magenta . ( B ) Stereo view of interface # 1 , which involves hydrophobic interactions by Mam1 L92 , L97 , L100 , and I104 , and a hydrogen - bond network between Mam1 Q101 / E108 / R131 and Hrr25 residues 12 - 15 . ( C ) Stereo view of interface # 2 . Hrr25 residues H25 and E34 participate in a buried hydrogen - bond network with Mam1 residues R149 and Y158 , and are surrounded by hydrophobic residues on both proteins . For clarity , Mam1 helix 4 , on which R149 , Y153 , and Y158 are located , is not shown . ( D ) Pulldown assay with in vitro - translated Mam187 - 191 ( wild - type and alanine mutants ) and Hrr251 - 394 K38R . Mam1 mutations also disrupt Hrr25 binding when the two are co - expressed in E . coli ( Figure EV6B ) . Bottom : Sporeviability of MAM1 mutant strains ( see Table EV2 for strains ; strains tested are KC549 , KC560 , KC566 , KC552 , KC554 , KC556 , and KC558 ; 46 - 48 tetrads were dissected for each strain ) . " - - " indicates that this mutation was not tested for sporeviability .

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Figure 5(A) The Hrr25:Mam1 complex with Mam1 shown as orange ribbons, and Hrr25 shown as a white surface with Mam1-binding interface #1 shown in green and #2 in magenta.(B) Stereo view of interface #1, which involves hydrophobic interactions by Mam1 L92, L97, L100, and I104, and a hydrogen-bond network between Mam1 Q101/E108/R131 and Hrr25 residues 12-15. (C) Stereo view of interface #2. Hrr25 residues H25 and E34 participate in a buried hydrogen-bond network with Mam1 residues R149 and Y158, and are surrounded by hydrophobic residues on both proteins. For clarity, Mam1 helix 4, on which R149, Y153, and Y158 are located, is not shown.(D) Pulldown assay with in vitro-translated Mam187-191 (wild-type and alanine mutants) and Hrr251-394 K38R. Mam1 mutations also disrupt Hrr25 binding when the two are co-expressed in E. coli (Figure EV6B). Bottom: Sporeviability of MAM1 mutant strains (see Table EV2 for strains; strains tested are KC549, KC560, KC566, KC552, KC554, KC556, and KC558; 46-48 tetrads were dissected for each strain). "--" indicates that this mutation was not tested for sporeviability.


Figure 6 ( A - C ) Ribbon views of the S . cerevisiae Hrr251 - 394 : Mam187 - 191 structure ( form 1 ) , CK1 - 7 bound S . cerevisiae Hrr251 - 394 , and ADP - bound C . glabrata Hrr251 - 403 ( formate condition ) , with Hrr25 colored according to main - chain B - factor from low ( purple ) to cyan ( high ) . For each , coloring is normalized to correspond to the average of the 20 lowest ( purple ) or highest ( cyan ) main - chain B - factors in Hrr25 . Mam1 ( panel A ) is colored in light gray . ( D ) Overall main - chain B - factors for the N - lobe ( residues 1 - 85 ) of four structures of Hrr25 ( both C . glabrata and S . cerevisiae ) and the three crystallographically - unique views of the S . cerevisiae Hrr25 : Mam1 complex . Values are normalized to the average main - chain B - factor for the entire Hrr25 chain ( dotted line at 1 . 0 ) . P - value = 0 . 004 ( student ' s T - test ) . See Figure EV4 for residue - by - residue B - factor plots for each structure .

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Figure 6(A-C) Ribbon views of the S. cerevisiae Hrr251-394:Mam187-191 structure (form 1), CK1-7 bound S. cerevisiae Hrr251-394, and ADP-bound C. glabrata Hrr251-403 (formate condition), with Hrr25 colored according to main-chain B-factor from low (purple) to cyan (high). For each, coloring is normalized to correspond to the average of the 20 lowest (purple) or highest (cyan) main-chain B-factors in Hrr25. Mam1 (panel A) is colored in light gray.(D) Overall main-chain B-factors for the N-lobe (residues 1-85) of four structures of Hrr25 (both C. glabrata and S. cerevisiae) and the three crystallographically-unique views of the S. cerevisiae Hrr25:Mam1 complex. Values are normalized to the average main-chain B-factor for the entire Hrr25 chain (dotted line at 1.0). P-value=0.004 (student's T-test). See Figure EV4 for residue-by-residue B-factor plots for each structure.


Figure 7 ( A ) ADP - Glo ATPase assay showing stimulation of S . cerevisiae Hrr251 - 394 ( blue circles ) , Hrr251 - 394 K38R ( red diamonds ) , Hrr25 full - length : Mam187 - 191 ( black triangles ) , Hrr251 - 394 : Mam187 - 191 ( orange squares ) , and Hrr251 - 394 : Mam187 - 191 R131A ( green triangles ) by bovine casein . See Figure EV6A for SDS - PAGE analysis of purified proteins . ( B ) Stimulation of Hrr251 - 394 , Hrr251 - 394 : Mam187 - 191 , and Hrr251 - 394 : Mam187 - 191 R131A by ATP , measured using an enzyme - coupled ATPase assay . ( C ) ADP - Glo assay showing the effect of added CK1 - 7 on Hrr251 - 394 , Hrr251 - 394 : Mam187 - 191 , and Hrr251 - 394 : Mam187 - 191 R131A . ( D ) Km , Vmax , and IC50 values for tested enzymes . Vmax was calculated from curves in ( A ) at 100 M ATP .

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Figure 7(A) ADP-Glo ATPase assay showing stimulation of S. cerevisiae Hrr251-394 (blue circles), Hrr251-394 K38R (red diamonds), Hrr25 full-length:Mam187-191 (black triangles), Hrr251-394:Mam187-191 (orange squares), and Hrr251-394:Mam187-191 R131A (green triangles) by bovine casein. See Figure EV6A for SDS-PAGE analysis of purified proteins.(B) Stimulation of Hrr251-394, Hrr251-394:Mam187-191, and Hrr251-394:Mam187-191 R131A by ATP, measured using an enzyme-coupled ATPase assay.(C) ADP-Glo assay showing the effect of added CK1-7 on Hrr251-394, Hrr251-394:Mam187-191, and Hrr251-394:Mam187-191 R131A.(D) Km, Vmax, and IC50 values for tested enzymes. Vmax was calculated from curves in (A) at 100 M ATP.


Figure 8 ( A ) Phos - tag gels showing phosphorylation of purified S . cerevisiae Mtw1 complex by Hrr251 - 394 and Hrr251 - 394 : Mam187 - 191 . Asterisks denote N - terminal proteolytic cleavage products of Dsn1 , with the red asterisk denoting a previously - characterized product lacking the first 171 residues ( Hornung et al , 2011 ) .

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Figure 8(A) Phos-tag gels showing phosphorylation of purified S. cerevisiae Mtw1 complex by Hrr251-394 and Hrr251-394:Mam187-191. Asterisks denote N-terminal proteolytic cleavage products of Dsn1, with the red asterisk denoting a previously-characterized product lacking the first 171 residues (Hornung et al, 2011).


Figure 1Pre - enriched thymocytes from WT BALB / c thymi as in ( a ) were labeled by CFSE ( CFSE + ) , mixed with pre - enriched thymocytes from SKG thymi ( CFSE - ) , loaded with Indo - 1 , and stimulated as in ( a ) . Ratio of Indo ( Violet ) and Indo ( Blue ) was analyzed by flow cytometry . Comparison of cytoplasmic Ca2 + profiles between WT and SKG NKT1 ( c ) and NKT2 ( d ) cells in the same test tube are shown . Data are representative from one of at least three independent experiments . Graphs represent the average Ca2 + levels .

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Figure 1Pre-enriched thymocytes from WT BALB/c thymi as in (a) were labeled by CFSE (CFSE+), mixed with pre-enriched thymocytes from SKG thymi (CFSE-), loaded with Indo-1, and stimulated as in (a). Ratio of Indo (Violet) and Indo (Blue) was analyzed by flow cytometry. Comparison of cytoplasmic Ca2+ profiles between WT and SKG NKT1 (c) and NKT2 (d) cells in the same test tube are shown. Data are representative from one of at least three independent experiments. Graphs represent the average Ca2+ levels.


Figure 2Sorted thymic NKT1 and NKT2 cells were labeled with CellTrace Violet , and activated by anti - CD3ε / CD28 Dynabeads for 2 days , in the presence of different [ EGTA ] in the medium . CellTrace Violet / CTV signal in living cells at the end of the culture is shown . Cell proliferation was quantified as division index based on CTV signals in ( a ) . Thymic lobes from new born ( day 0 ) BALB / c mice were cultured in vitro for 6 days . EGTA was added 48h after the start of culture ( day 2 ) , and the expression of CXCR3 ( g ) , PLZF ( h ) and PD1 ( i ) in total iNKT cells are shown .

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Figure 2Sorted thymic NKT1 and NKT2 cells were labeled with CellTrace Violet, and activated by anti-CD3ε/CD28 Dynabeads for 2 days, in the presence of different [EGTA] in the medium. CellTrace Violet/CTV signal in living cells at the end of the culture is shown. Cell proliferation was quantified as division index based on CTV signals in (a).Thymic lobes from new born (day 0) BALB/c mice were cultured in vitro for 6 days. EGTA was added 48h after the start of culture (day 2), and the expression of CXCR3 (g), PLZF (h) and PD1 (i) in total iNKT cells are shown.


Figure 3oxygen consumption rate ( OCR ) from sorted thymic T cell populations measured in a Seahorse Mitostress test ;

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Figure 3oxygen consumption rate (OCR) from sorted thymic T cell populations measured in a Seahorse Mitostress test;


Figure 4Confocal images of Tim 23 ( Alexa Fluor 488 , green ) and MitoTracker deep red ( Alexa Fluor 647 , red ) in freshly isolated thymic NKT1 ( c1 , 2 ) and NKT2 ( c5 , 6 ) cells using Airyscan technology . 3D images reconstructed by Imaris software in NKT1 ( c3 , 4 ) and NKT2 cells ( c7 , 8 ) . Dotted line in panel ( 5 - 8 ) indicates juxtaposition of two NKT2 cell images taken with the same resolution and magnification as NKT1 cells . Representative images of electron microscopy in NKT1 and NKT2 cells ; ( f ) individual mitochondria size and ( g ) cristae perimeters normalized to mitochondria perimeter based on EM in ( e ) .

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Figure 4Confocal images of Tim 23 (Alexa Fluor 488, green) and MitoTracker deep red (Alexa Fluor 647, red) in freshly isolated thymic NKT1 (c1, 2) and NKT2 (c5, 6) cells using Airyscan technology. 3D images reconstructed by Imaris software in NKT1 (c3, 4) and NKT2 cells(c7, 8). Dotted line in panel (5-8) indicates juxtaposition of two NKT2 cell images taken with the same resolution and magnification as NKT1 cells.Representative images of electron microscopy in NKT1 and NKT2 cells; (f) individual mitochondria size and (g) cristae perimeters normalized to mitochondria perimeter based on EM in (e).


Figure 5Maximal cytoplasmic [ Ca2 + ] in single , freshly sorted NKT1 and NKT2 cells following TCR stimulation in the presence or absence of BTP2 ( 1uM ) ; BTP2 ( 1uM ) was added 48h after the start of culture , and the expression of Tbet in total iNKT cells is shown . Data are representative from one of three independent experiments . Graphs represent mean ± SD with symbols representing individual cells ( a ) , or individual thymic lobes ( b - f ) . * * p < 0 . 01 ; * * * * p < 0 . 0001 ; n . s . not significant ( unpaired two - tailed Student ' s t test ) . BTP2 ( 1uM ) was added 48h after the start of culture , and the expression of CXCR3 ( d ) , PLZF ( e ) , and PD1 ( f ) in total iNKT cells is shown . Data are representative from one of three independent experiments . Graphs represent mean ± SD with symbols representing individual cells ( a ) , or individual thymic lobes ( b - f ) . * * p < 0 . 01 ; * * * * p < 0 . 0001 ; n . s . not significant ( unpaired two - tailed Student ' s t test ) .

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

Figure 5Maximal cytoplasmic [Ca2+] in single, freshly sorted NKT1 and NKT2 cells following TCR stimulation in the presence or absence of BTP2 (1uM);BTP2 (1uM) was added 48h after the start of culture, and the expression of Tbet in total iNKT cells is shown. Data are representative from one of three independent experiments. Graphs represent mean ± SD with symbols representing individual cells (a), or individual thymic lobes (b-f). **p < 0.01; ****p < 0.0001; n.s. not significant (unpaired two-tailed Student's t test).BTP2 (1uM) was added 48h after the start of culture, and the expression of CXCR3 (d), PLZF (e), and PD1 (f) in total iNKT cells is shown. Data are representative from one of three independent experiments. Graphs represent mean ± SD with symbols representing individual cells (a), or individual thymic lobes (b-f). **p < 0.01; ****p < 0.0001; n.s. not significant (unpaired two-tailed Student's t test).


Figure 6Profiles showing average Ca2 + influx induced by Thapsigargin ( TG ) in freshly sorted NKT1 ( Red ) and NKT2 ( Black ) cells . The timing of TG ( 1uM ) , ionomycin ( Iono ) ( 2uM ) and CCCP ( 1uM ) addition , and extracellular [ Ca2 + ] in mM are shown .

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Figure 6Profiles showing average Ca2+ influx induced by Thapsigargin (TG) in freshly sorted NKT1 (Red) and NKT2 (Black) cells. The timing of TG (1uM), ionomycin (Iono) (2uM) and CCCP (1uM) addition, and extracellular [Ca2+] in mM are shown.


Figure 1A , B . HeLa cells treated with non - targeting siRNA ( Ctrl ) or C9orf72 siRNA and transfected with mCherry - EGFP - LC3 were treated with vehicle ( Ctrl ) , Torin1 ( 250 nM ; 3 h ) , bafilomycin A1 ( 100 nM ; 6 h - BafA1 ) or combinations thereof as indicated . Autophagosomes ( green + red ) and autolysosomes ( red only ) were quantified per cell ( Mean ± SEM from 3 independent experiments ; one - way ANOVA with Fisher ' s LSD test : ns , not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 ; N ( cells ) = Ctrl / Ctrl : 120 ; Ctrl / Torin1 : 101 ; C9orf72 / Ctrl : 99 ; C9orf72 / Torin1 : 106 ; Ctrl / BafA1 : 116 ; Ctrl / Torin1 / BafA1 : 118 ; C9orf72 / BafA1 : 109 ; C9orf72 / Torin1 / BafA1 : 106 ) . Scale bar = 20 µm . C9orf72 knockdown was confirmed by RT - qPCR ( Appendix Fig S2 ) . C , D . HEK293 cells treated with non - targeting ( Ctrl ) or C9orf72 siRNA were incubated with BafA1 , BafA1 + Torin1 ( C ) or BafA1 + rapamycin ( D ) and levels of LC3 - I and II were determined on immunoblots . Levels of LC3 - II were normalized against α - tubulin and are shown relative to the BafA1 - treated sample ( Mean ± SEM ; one - way ANOVA with Fisher ' s LSD test : ns , not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 ; N = 3 experiments ) . E , F . Primary cortical neurons ( DIV5 / 6 ) were transfected with ECFP non - targeting ( Ctrl ) or C9orf72 miRNA ( cyan ) and EGFP - LC3 ( green ) . 3 days post transfection neurons were treated with vehicle ( Ctrl ) , Torin1 ( 250 nM ; 3 h ) , BafA1 ( 100 nM ; 5 h ) or combinations thereof as indicated . Autophagosomes were quantified as the number of EGFP - LC3 positive puncta per soma from 2 independent experiments ( Mean ± SEM ; one - way ANOVA with Fisher ' s LSD test : ns , not significant , * p ≤ 0 . 05 ; * * * P ≤ 0 . 001 ; * * * * P ≤ 0 . 0001 ; N ( cells ) = Ctrl miRNA / Ctrl : 77 ; Ctrl miRNA / Torin1 : 68 ; C9orf72 miRNA / Ctrl : 57 ; C9orf72 miRNA / Torin1 : 69 ; Ctrl miRNA / BafA1 : 35 ; Ctrl miRNA / Torin1 / BafA1 : 57 ; C9orf72 miRNA / BafA1 : 66 ; C9orf72 miRNA / Torin1 / BafA1 : 64 ) . Scale bar = 5 µm .

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

Figure 1A, B. HeLa cells treated with non-targeting siRNA (Ctrl) or C9orf72 siRNA and transfected with mCherry-EGFP-LC3 were treated with vehicle (Ctrl), Torin1 (250 nM; 3 h), bafilomycin A1 (100 nM; 6 h - BafA1) or combinations thereof as indicated. Autophagosomes (green+red) and autolysosomes (red only) were quantified per cell (Mean ± SEM from 3 independent experiments; one-way ANOVA with Fisher's LSD test: ns, not significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; N (cells) = Ctrl/Ctrl: 120; Ctrl/Torin1: 101; C9orf72/Ctrl: 99; C9orf72/Torin1: 106; Ctrl/BafA1: 116; Ctrl/Torin1/BafA1: 118; C9orf72/BafA1: 109; C9orf72/Torin1/BafA1: 106). Scale bar = 20 µm. C9orf72 knockdown was confirmed by RT-qPCR (Appendix Fig S2).C, D. HEK293 cells treated with non-targeting (Ctrl) or C9orf72 siRNA were incubated with BafA1, BafA1+Torin1 (C) or BafA1+rapamycin (D) and levels of LC3-I and II were determined on immunoblots. Levels of LC3-II were normalized against α-tubulin and are shown relative to the BafA1-treated sample (Mean ± SEM; one-way ANOVA with Fisher's LSD test: ns, not significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; N = 3 experiments).E, F. Primary cortical neurons (DIV5/6) were transfected with ECFP non-targeting (Ctrl) or C9orf72 miRNA (cyan) and EGFP-LC3 (green). 3 days post transfection neurons were treated with vehicle (Ctrl), Torin1 (250 nM; 3 h), BafA1 (100 nM; 5 h) or combinations thereof as indicated. Autophagosomes were quantified as the number of EGFP-LC3 positive puncta per soma from 2 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test: ns, not significant, * p ≤ 0.05; *** P ≤ 0.001; **** P ≤ 0.0001; N (cells) = Ctrl miRNA/Ctrl: 77; Ctrl miRNA/Torin1: 68; C9orf72 miRNA/Ctrl: 57; C9orf72 miRNA/Torin1: 69; Ctrl miRNA/BafA1: 35; Ctrl miRNA/Torin1/BafA1: 57; C9orf72 miRNA/BafA1: 66; C9orf72 miRNA/Torin1/BafA1: 64). Scale bar = 5 µm.