Figure 4BMDMs were costimulated with TNF ( 100 ng / ml ) and TAK1i ( 125 nM ) for 4 h and LDH release was quantified Where indicated , cells were treated with the inhibitors Nec - 1s ( 50 μM ) BMDMs were costimulated with TNF ( 100 ng / ml ) and TAK1i ( 125 nM ) for 4 h and LDH release was quantifiedBMDMs were costimulated with TNF ( 100 ng / ml ) and TAK1i ( 125 nM ) for 4 h and mixed supernatant and extracts were analysed by immunoblot . Where indicated , cells were treated with the inhibitors MCC950 ( 10 μM ) , GSK ' 872 ( 1 μM ) , 20 - 30 min prior to cell stimulation . KCl ( 50 mM ) was added together with TNF and TAK1i . To induce necroptosis , BMDMs were primed for 3 h with ultrapure E . coli K12 LPS ( 100 ng / ml ) and Q - VD - OPh ( 10 μM ) was added at the last 20 - 30 min of priming and stimulated with SM ( 500 nM ) for 4 h . BMDMs were costimulated with TNF ( 100 ng / ml ) and TAK1i ( 125 nM ) for 4 h and mixed supernatant and extracts were analysed by immunoblot . Where indicated , cells were treated with the inhibitors GSK ' 872 ( 1 μM ) , 20 - 30 min prior to cell stimulation . BMDMs were costimulated with TNF ( 100 ng / ml ) and TAK1i ( 125 nM ) for 4 h and mixed supernatant and extracts were analysed by immunoblot . Where indicated , cells were treated with the inhibitors probenecid ( 1 mM ) 20 - 30 min prior to cell stimulation . To activate the NLRP3 inflammasome , BMDM were primed with ultrapure E . coli K12 LPS ( 100 ng / ml ) for 4 h and stimulated with nigericin ( 10 μM ) for 1 h . BMDMs were primed for 3 h with ultrapure E . coli K12 LPS ( 100 ng / ml ) and stimulated with SM ( 0 . 5 μM ) for a further 4 h . Probenecid ( 1 mM ) was added 20 - 30 min prior to cell stimulation and mixed supernatant and extracts were analysed by immunoblot . BMDMs were stimulated with TNF ( 100 ng / ml ) and SM ( 0 . 5 μM ) for 6 h and ( G ) mixed supernatant and extracts were analysed by immunoblotBMDMs were stimulated with TNF ( 100 ng / ml ) and SM ( 0 . 5 μM ) for 6 h and LDH release was quantified .

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

Figure 4BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h and LDH release was quantified Where indicated, cells were treated with the inhibitors Nec-1s (50 μM)BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h and LDH release was quantifiedBMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h and mixed supernatant and extracts were analysed by immunoblot. Where indicated, cells were treated with the inhibitors MCC950 (10 μM), GSK'872 (1 μM), 20-30 min prior to cell stimulation. KCl (50 mM) was added together with TNF and TAK1i. To induce necroptosis, BMDMs were primed for 3 h with ultrapure E. coli K12 LPS (100 ng/ml) and Q-VD-OPh (10 μM) was added at the last 20-30 min of priming and stimulated with SM (500 nM) for 4 h.BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h and mixed supernatant and extracts were analysed by immunoblot. Where indicated, cells were treated with the inhibitors GSK'872 (1 μM), 20-30 min prior to cell stimulation.BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h and mixed supernatant and extracts were analysed by immunoblot. Where indicated, cells were treated with the inhibitors probenecid (1 mM) 20-30 min prior to cell stimulation. To activate the NLRP3 inflammasome, BMDM were primed with ultrapure E. coli K12 LPS (100 ng/ml) for 4 h and stimulated with nigericin (10 μM) for 1 h.BMDMs were primed for 3 h with ultrapure E. coli K12 LPS (100 ng/ml) and stimulated with SM (0.5 μM) for a further 4 h. Probenecid (1 mM) was added 20-30 min prior to cell stimulation and mixed supernatant and extracts were analysed by immunoblot.BMDMs were stimulated with TNF (100 ng/ml) and SM (0.5 μM) for 6 h and (G) mixed supernatant and extracts were analysed by immunoblotBMDMs were stimulated with TNF (100 ng/ml) and SM (0.5 μM) for 6 h and LDH release was quantified.


Figure 5BMDMs were stimulated with an increasing dose of S63845 in the presence of ABT - 737 ( 0 . 5 μM ) and LDH release was quantified at 6 h . BMDMs were stimulated with an increasing dose of S63845 in the presence of ABT - 737 ( 0 . 5 μM ) and LDH release was quantified at 6 h . BMDMs were stimulated with ABT - 737 ( 0 . 5 μM ) and S63845 ( 0 . 5 μM ) and LDH release was quantifiedBMDMs were stimulated with ABT - 737 ( 0 . 5 μM ) and S63845 ( 0 . 5 μM ) mixed supernatant and extracts were analysed by immunoblot at 6 hBMDMs were stimulated with ABT - 737 ( 0 . 5 μM ) and S63845 ( 0 . 5 μM ) and LDH release was quantifiedBMDMs were stimulated with ABT - 737 ( 0 . 5 μM ) and S63845 ( 0 . 5 μM ) and mixed supernatant and extracts were analysed by immunoblot at 6 hBMDMs were primed with ultrapure E . coli K12 LPS ( 100 ng / ml ) for 3 h and further stimulated with ABT - 737 ( 1 μM ) and S63845 ( 1 μM ) for 24 h and mixed supernatant and extracts were analysed by immunoblotBMDMs were primed with ultrapure E . coli K12 LPS ( 100 ng / ml ) for 3 h and further stimulated with ABT - 737 ( 1 μM ) and S63845 ( 1 μM ) for 24 h and IL - 1β in cell - free supernatant were quantified by

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Figure 5BMDMs were stimulated with an increasing dose of S63845 in the presence of ABT-737 (0.5 μM) and LDH release was quantified at 6 h.BMDMs were stimulated with an increasing dose of S63845 in the presence of ABT-737 (0.5 μM) and LDH release was quantified at 6 h.BMDMs were stimulated with ABT-737 (0.5 μM) and S63845 (0.5 μM) and LDH release was quantifiedBMDMs were stimulated with ABT-737 (0.5 μM) and S63845 (0.5 μM) mixed supernatant and extracts were analysed by immunoblot at 6 hBMDMs were stimulated with ABT-737 (0.5 μM) and S63845 (0.5 μM) and LDH release was quantifiedBMDMs were stimulated with ABT-737 (0.5 μM) and S63845 (0.5 μM) and mixed supernatant and extracts were analysed by immunoblot at 6 hBMDMs were primed with ultrapure E. coli K12 LPS (100 ng/ml) for 3 h and further stimulated with ABT-737 (1 μM) and S63845 (1 μM) for 24 h and mixed supernatant and extracts were analysed by immunoblotBMDMs were primed with ultrapure E. coli K12 LPS (100 ng/ml) for 3 h and further stimulated with ABT-737 (1 μM) and S63845 (1 μM) for 24 h and IL-1β in cell-free supernatant were quantified by ELISA


Figure 1A , B MAGE - A3 / 6 protein levels decrease upon EBSS treatment of HeLa cells for the indicated times . Note both bands are MAGE - A3 / 6 . Quantitation represents average of n = 3 with standard deviation shown . C Quantitative TMT proteomics of A375 cells grown in complete media ( CM ) or EBSS for 3 hrs . Number of proteins affected are shown . Autophagy related proteins are indicated in grey and MAGE - A proteins in blue . D , E MAGE - A3 / 6 protein levels are actively degraded upon nutrient deprivation . HeLa cells were treated for the indicated times with complete media ( DMEM + 10 % FBS ) or EBSS containing 100 µg / mL translation inhibitor cycloheximide ( CHX ) . Protein degradation rates are shown ( E ) . Quantitation represents average of n = 3 with standard deviation shown . Data information : Asterisks indicate * * p < 0 . 01 . ns indicates p > 0 . 05 using 2 - way ANOVA .

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Figure 1A, B MAGE-A3/6 protein levels decrease upon EBSS treatment of HeLa cells for the indicated times. Note both bands are MAGE-A3/6. Quantitation represents average of n=3 with standard deviation shown.C Quantitative TMT proteomics of A375 cells grown in complete media (CM) or EBSS for 3 hrs. Number of proteins affected are shown. Autophagy related proteins are indicated in grey and MAGE-A proteins in blue.D, E MAGE-A3/6 protein levels are actively degraded upon nutrient deprivation. HeLa cells were treated for the indicated times with complete media (DMEM + 10% FBS) or EBSS containing 100 µg/mL translation inhibitor cycloheximide (CHX). Protein degradation rates are shown (E). Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate ** p<0.01. ns indicates p>0.05 using 2-way ANOVA.


Figure 2FBS ( 10 % ) , but not non - essential amino acids ( NEAA ; 10 mM ) or glucose ( 4 . 5 g / L ) , rescues MAGE - A3 / 6 protein levels upon EBSS treatment . HeLa cells were treated with EBSS and the indicated supplements for the indicated times . Quantitation represents average of n = 3 with standard deviation shown . FBS ( 10 % ) , but not non - essential amino acids ( NEAA ; 10 mM ) or glucose ( 4 . 5 g / L ) , rescues MAGE - A3 / 6 protein levels upon EBSS treatment . HeLa cells were treated with EBSS and the indicated supplements for the indicated times . Quantitation represents average of n = 3 with standard deviation shown . Data information : Asterisks indicate * * p < 0 . 01 ; * * * p < 0 . 001 . ns indicates p > 0 . 05 using 2 - way ANOVA . C Removal of FBS from normal growth conditions ( DMEM + 10 % FBS ) results in MAGE - A3 / 6 protein degradation in HeLa cells . D , E MAGE - A3 / 6 protein stability is controlled by a < 3 kDa , heat stable , lipophilic molecule present in FBS . HeLa cells were treated with EBSS alone , or EBSS containing 10 % complete FBS , 10 % charcoal - dextran stripped FBS , 10 % boiled FBS , 10 % dialyzed > 3 kDa FBS , or 10 % dialyzed < 3 kDa FBS for the indicated times . Quantitation represents average of n = 3 with standard deviation shown . Data information : Asterisks indicate * * p < 0 . 01 ; * * * p < 0 . 001 . ns indicates p > 0 . 05 using 2 - way ANOVA . F MAGE - A3 / 6 protein instability upon nutrient deprivation of HeLa cells is not altered by EGF 0 . 2 ng / mL , testosterone , or bovine pituitary extract 25 µg / mL . Insulin partially rescues MAGE - A3 / 6 protein levels upon nutrient deprivation . HeLa cells were treated for the indicated times with EBSS alone or EBSS containing NEAA ( 10 mM ) or insulin ( 10 µg / mL ) . Quantitation represents average of n = 3 with standard deviation shown . , H Insulin partially rescues MAGE - A3 / 6 protein levels upon nutrient deprivation . HeLa cells were treated for the indicated times with EBSS alone or EBSS containing insulin ( 10 µg / mL ) . Quantitation represents average of n = 3 with standard deviation shown . Data information : Asterisks indicate * * p < 0 . 01 ; * * * p < 0 . 001 . ns indicates p > 0 . 05 using 2 - way ANOVA .

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 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 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 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 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

Figure 2FBS (10%), but not non-essential amino acids (NEAA; 10 mM) or glucose (4.5 g/L), rescues MAGE-A3/6 protein levels upon EBSS treatment. HeLa cells were treated with EBSS and the indicated supplements for the indicated times. Quantitation represents average of n=3 with standard deviation shown.FBS (10%), but not non-essential amino acids (NEAA; 10 mM) or glucose (4.5 g/L), rescues MAGE-A3/6 protein levels upon EBSS treatment. HeLa cells were treated with EBSS and the indicated supplements for the indicated times. Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate ** p<0.01; *** p<0.001. ns indicates p>0.05 using 2-way ANOVA.C Removal of FBS from normal growth conditions (DMEM+10% FBS) results in MAGE-A3/6 protein degradation in HeLa cells.D, E MAGE-A3/6 protein stability is controlled by a <3 kDa, heat stable, lipophilic molecule present in FBS. HeLa cells were treated with EBSS alone, or EBSS containing 10% complete FBS, 10% charcoal-dextran stripped FBS, 10% boiled FBS, 10% dialyzed >3 kDa FBS, or 10% dialyzed <3 kDa FBS for the indicated times. Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate ** p<0.01; *** p<0.001. ns indicates p>0.05 using 2-way ANOVA.F MAGE-A3/6 protein instability upon nutrient deprivation of HeLa cells is not altered by EGF 0.2 ng/mL, testosterone, or bovine pituitary extract 25 µg/mL.Insulin partially rescues MAGE-A3/6 protein levels upon nutrient deprivation. HeLa cells were treated for the indicated times with EBSS alone or EBSS containing NEAA (10 mM) or insulin (10 µg/mL). Quantitation represents average of n=3 with standard deviation shown., H Insulin partially rescues MAGE-A3/6 protein levels upon nutrient deprivation. HeLa cells were treated for the indicated times with EBSS alone or EBSS containing insulin (10 µg/mL). Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate ** p<0.01; *** p<0.001. ns indicates p>0.05 using 2-way ANOVA.


Figure 3A MAGE - A3 / 6 mRNA levels do not change upon nutrient deprivation . RNA was isolated from HeLa cells treated with EBSS for the indicated times . RT - QPCR analysis was performed to detect both MAGE - A3 / 6 with a common primer set . Data was normalized to 18S rRNA levels . Quantitation represents average of n = 3 with standard deviation shown . B , Proteasome inhibition rescues MAGE - A3 / 6 protein levels upon nutrient deprivation . HeLa cells were treated for the indicated times in EBSS alone or EBSS containing lysosome inhibitor bafilomycin A1 ( BafA1 , 50 nM ) or proteasome inhibitor MG132 ( 10 µM ) . C Proteasome inhibition rescues MAGE - A3 / 6 protein levels upon nutrient deprivation . HeLa cells were treated for the indicated times in EBSS alone or EBSS containing lysosome inhibitor bafilomycin A1 ( BafA1 , 50 nM ) or proteasome inhibitor MG132 ( 10 µM ) . Quantitation represents average of n = 3 with standard deviation shown . Data information : Asterisks indicate * * * p < 0 . 001 . ns indicates p > 0 . 05 using 2 - way ANOVA . D Nutrient deprivation increases ubiquitination of endogenous MAGE - A3 / 6 . HeLa cells were treated with MG132 ( 10 µM ) in complete media or EBSS for 6 hrs . Cell extracts were incubated with control agarose or agarose - TUBE2 to isolate ubiquitinated proteins . Input and pulldown samples were probed for the indicated proteins . E MAGE - A3 / 6 protein instability upon nutrient deprivation is not rescued by knockdown of its associated E3 ubiquitin ligase , TRIM28 . HeLa cells were treated with control or TRIM28 siRNAs for 6 days before incubation in EBSS for the indicated times . F , MAGE - A3 / 6 degradation upon nutrient starvation is dependent on a Cullin E3 ubiquitin ligase . HeLa cells were treated with EBSS alone or EBSS containing the NAE1 inhibitor MLN4924 ( 1 µM ) that blocks the activity of Cullin E3 ubiquitin ligases for the indicated times . G MAGE - A3 / 6 degradation upon nutrient starvation is dependent on a Cullin E3 ubiquitin ligase . HeLa cells were treated with EBSS alone or EBSS containing the NAE1 inhibitor MLN4924 ( 1 µM ) that blocks the activity of Cullin E3 ubiquitin ligases for the indicated times . Quantitation represents average of n = 3 with standard deviation shown . Data information : Asterisks indicate * * * p < 0 . 001 . ns indicates p > 0 . 05 using 2 - way ANOVA . H Degradation of MAGE - A3 / 6 upon nutrient deprivation is dependent on a Cul4 E3 ubiquitin ligase complex . HeLa cells were transfected with vector control or the indicated dominant negative ( D / N ) Cullin constructs for 48 hrs before harvesting cells after 0 or 4 hrs EBSS treatment . I Degradation of MAGE - A3 / 6 upon nutrient deprivation is dependent on Cul4A / B . HeLa cells were transfected with control siRNAs or siRNAs targeting both Cul4A and Cul4B . 72 hrs after treatment , cells were incubated for the indicated times with EBSS before harvesting and immunoblotting . Note , time 0 samples between two siRNA groups were normalized to more readily discern differences in MAGE - A3 / 6 degradation upon nutrient deprivation .

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

Figure 3A MAGE-A3/6 mRNA levels do not change upon nutrient deprivation. RNA was isolated from HeLa cells treated with EBSS for the indicated times. RT-QPCR analysis was performed to detect both MAGE-A3/6 with a common primer set. Data was normalized to 18S rRNA levels. Quantitation represents average of n=3 with standard deviation shown.B, Proteasome inhibition rescues MAGE-A3/6 protein levels upon nutrient deprivation. HeLa cells were treated for the indicated times in EBSS alone or EBSS containing lysosome inhibitor bafilomycin A1 (BafA1, 50 nM) or proteasome inhibitor MG132 (10 µM).C Proteasome inhibition rescues MAGE-A3/6 protein levels upon nutrient deprivation. HeLa cells were treated for the indicated times in EBSS alone or EBSS containing lysosome inhibitor bafilomycin A1 (BafA1, 50 nM) or proteasome inhibitor MG132 (10 µM). Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate *** p<0.001. ns indicates p>0.05 using 2-way ANOVA.D Nutrient deprivation increases ubiquitination of endogenous MAGE-A3/6. HeLa cells were treated with MG132 (10 µM) in complete media or EBSS for 6 hrs. Cell extracts were incubated with control agarose or agarose-TUBE2 to isolate ubiquitinated proteins. Input and pulldown samples were probed for the indicated proteins.E MAGE-A3/6 protein instability upon nutrient deprivation is not rescued by knockdown of its associated E3 ubiquitin ligase, TRIM28. HeLa cells were treated with control or TRIM28 siRNAs for 6 days before incubation in EBSS for the indicated times.F, MAGE-A3/6 degradation upon nutrient starvation is dependent on a Cullin E3 ubiquitin ligase. HeLa cells were treated with EBSS alone or EBSS containing the NAE1 inhibitor MLN4924 (1 µM) that blocks the activity of Cullin E3 ubiquitin ligases for the indicated times.G MAGE-A3/6 degradation upon nutrient starvation is dependent on a Cullin E3 ubiquitin ligase. HeLa cells were treated with EBSS alone or EBSS containing the NAE1 inhibitor MLN4924 (1 µM) that blocks the activity of Cullin E3 ubiquitin ligases for the indicated times. Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate *** p<0.001. ns indicates p>0.05 using 2-way ANOVA.H Degradation of MAGE-A3/6 upon nutrient deprivation is dependent on a Cul4 E3 ubiquitin ligase complex. HeLa cells were transfected with vector control or the indicated dominant negative (D/N) Cullin constructs for 48 hrs before harvesting cells after 0 or 4 hrs EBSS treatment.I Degradation of MAGE-A3/6 upon nutrient deprivation is dependent on Cul4A/B. HeLa cells were transfected with control siRNAs or siRNAs targeting both Cul4A and Cul4B. 72 hrs after treatment, cells were incubated for the indicated times with EBSS before harvesting and immunoblotting. Note, time 0 samples between two siRNA groups were normalized to more readily discern differences in MAGE-A3/6 degradation upon nutrient deprivation.


Figure 4A Tandem affinity purification of MAGE - A3 from HEK293 cells identified DDB1 , DCAF5 , and DCAF12 as binding partners . Mascot score , number of peptides identified , and percent coverage is shown . B MAGE - A3 interacts with DCAF12 , but not DCAF5 . HeLa cells were transfected with the indicated constructs ( Myc - MAGE - A3 and HA - DCAF5 or HA - DCAF12 ) . Two days later cells were treated with complete media or EBSS for 4 hrs . Cells were treated in the presence of 1 µM MLN4924 to stabilize MAGE - A3 . Cell extracts were subjected to anti - Myc IP . C Nutrient deprivation increases endogenous DCAF12 binding to MAGE - A3 . HeLa cells were transfect with Myc - vector or Myc - MAGE - A3 for 48 hrs . Cells were then treated with 10 µM MG132 in complete media , EBSS , or EBSS containing 20 µg / mL insulin for 6 hrs . Cell extracts were subjected to anti - Myc IP . Knockdown of DCAF12 , but not DCAF5 , altered MAGE - A3 / 6 protein levels and partially rescued its instability upon nutrient deprivation . HeLa cells were treated with control , DCAF5 siRNAs for 72 hrs before incubation in complete media or EBSS for the indicated times with or without MLN4924 ( 1 µM ) . E Knockdown of DCAF12 , but not DCAF5 , altered MAGE - A3 / 6 protein levels and partially rescued its instability upon nutrient deprivation . HeLa cells were treated with control or DCAF12 siRNAs for 72 hrs before incubation in complete media or EBSS for the indicated times with or without MLN4924 ( 1 µM ) . F Knockdown of DCAF12 with individual siRNAs blocks MAGE - A3 / 6 degradation upon nutrient deprivation . HeLa cells were treated with the indicated siRNAs for 72 hrs before incubation in EBSS for the indicated times . Note , time 0 samples between siRNA groups were normalized to more readily discern differences in MAGE - A3 / 6 degradation upon nutrient deprivation . G Ubiquitination of MAGE - A3 / 6 upon nutrient deprivation is dependent on DCAF12 . A375 parental or DCAF12 KO cells were treated with MG132 ( 10 µM ) in complete media or EBSS for 6 hrs . Cell extracts were incubated with control agarose or agarose - TUBE2 to isolate ubiquitinated proteins . Input and pulldown samples were probed for the indicated proteins .

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Figure 4A Tandem affinity purification of MAGE-A3 from HEK293 cells identified DDB1, DCAF5, and DCAF12 as binding partners. Mascot score, number of peptides identified, and percent coverage is shown.B MAGE-A3 interacts with DCAF12, but not DCAF5. HeLa cells were transfected with the indicated constructs (Myc-MAGE-A3 and HA-DCAF5 or HA-DCAF12). Two days later cells were treated with complete media or EBSS for 4 hrs. Cells were treated in the presence of 1 µM MLN4924 to stabilize MAGE-A3. Cell extracts were subjected to anti-Myc IP.C Nutrient deprivation increases endogenous DCAF12 binding to MAGE-A3. HeLa cells were transfect with Myc-vector or Myc-MAGE-A3 for 48 hrs. Cells were then treated with 10 µM MG132 in complete media, EBSS, or EBSS containing 20 µg/mL insulin for 6 hrs. Cell extracts were subjected to anti-Myc IP.Knockdown of DCAF12, but not DCAF5, altered MAGE-A3/6 protein levels and partially rescued its instability upon nutrient deprivation. HeLa cells were treated with control, DCAF5 siRNAs for 72 hrs before incubation in complete media or EBSS for the indicated times with or without MLN4924 (1 µM).E Knockdown of DCAF12, but not DCAF5, altered MAGE-A3/6 protein levels and partially rescued its instability upon nutrient deprivation. HeLa cells were treated with control or DCAF12 siRNAs for 72 hrs before incubation in complete media or EBSS for the indicated times with or without MLN4924 (1 µM).F Knockdown of DCAF12 with individual siRNAs blocks MAGE-A3/6 degradation upon nutrient deprivation. HeLa cells were treated with the indicated siRNAs for 72 hrs before incubation in EBSS for the indicated times. Note, time 0 samples between siRNA groups were normalized to more readily discern differences in MAGE-A3/6 degradation upon nutrient deprivation.G Ubiquitination of MAGE-A3/6 upon nutrient deprivation is dependent on DCAF12. A375 parental or DCAF12 KO cells were treated with MG132 (10 µM) in complete media or EBSS for 6 hrs. Cell extracts were incubated with control agarose or agarose-TUBE2 to isolate ubiquitinated proteins. Input and pulldown samples were probed for the indicated proteins.


Figure 5A Identification of DCAF12 substrates . Control ( WT ) or DCAF12 knockout ( KO ) A375 cells were subjected to quantitative TMT proteomics to identify potential DCAF12 targets . MAGE - A proteins ( shown in blue ) are stabilized in DCAF12 knockout cells . B Knockout of DCAF12 rescues degradation of MAGE - A proteins in A375 cells . DCAF12 KO A375 cells were treated with complete media or EBSS before quantitative TMT proteomics . Note MAGE - A genes are not significantly altered by EBSS in DCAF12 KO cells . C DCAF12 target proteins are differentially affected by EBSS compared to remainder of the proteome . DCAF12 targets ( n = 33 ) identified show significantly greater decrease upon EBSS treatment compared to non - DCAF12 altered genes ( n = 8243 ) . Average with standard deviation is shown with statistical analysis by student t - test . Asterisks * * * indicate p < 0 . 001 ( student t - test ) . E Overexpression of DCAF12 decreases wild - type MAGE - A3 , but not MAGE - A3 degron mutant . The indicated constructs were expressed for 48 hrs in HeLa cells before immunoblotting . The MAGE - A3 degron mutant was created ] , in which DNYNEPKANQ * were added to the extreme C - term to obscure the DCAF12 degron motif . F Analysis of proteomics indicates that MAGE - A1 , MAGE - A4 , MAGE - A10 , and MAGE - A11 are not regulated by nutrient deprivation or DCAF12 . Average ( n = 3 ) with standard deviation is shown with statistical analysis by student t - test . Asterisks * * * indicate p < 0 . 001 ( student t - test ) . G MAGE - A10 and MAGE - A11 are not altered upon nutrient deprivation or DCAF12 deletion . A375 parental or DCAF12 KO cells were treated with EBSS for the indicated times . Cell lysates were probed for the indicated proteins .

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Figure 5A Identification of DCAF12 substrates. Control (WT) or DCAF12 knockout (KO) A375 cells were subjected to quantitative TMT proteomics to identify potential DCAF12 targets. MAGE-A proteins (shown in blue) are stabilized in DCAF12 knockout cells.B Knockout of DCAF12 rescues degradation of MAGE-A proteins in A375 cells. DCAF12 KO A375 cells were treated with complete media or EBSS before quantitative TMT proteomics. Note MAGE-A genes are not significantly altered by EBSS in DCAF12 KO cells.C DCAF12 target proteins are differentially affected by EBSS compared to remainder of the proteome. DCAF12 targets (n=33) identified show significantly greater decrease upon EBSS treatment compared to non-DCAF12 altered genes (n=8243). Average with standard deviation is shown with statistical analysis by student t-test. Asterisks *** indicate p<0.001 (student t-test).E Overexpression of DCAF12 decreases wild-type MAGE-A3, but not MAGE-A3 degron mutant. The indicated constructs were expressed for 48 hrs in HeLa cells before immunoblotting. The MAGE-A3 degron mutant was created ], in which DNYNEPKANQ* were added to the extreme C-term to obscure the DCAF12 degron motif.F Analysis of proteomics indicates that MAGE-A1, MAGE-A4, MAGE-A10, and MAGE-A11 are not regulated by nutrient deprivation or DCAF12. Average (n=3) with standard deviation is shown with statistical analysis by student t-test. Asterisks *** indicate p<0.001 (student t-test).G MAGE-A10 and MAGE-A11 are not altered upon nutrient deprivation or DCAF12 deletion. A375 parental or DCAF12 KO cells were treated with EBSS for the indicated times. Cell lysates were probed for the indicated proteins.


Figure 6A - B U2OS GFP - LC3 cells were transfected with the indicated siRNAs for 72 hrs before treatment with complete media or EBSS for 6 hrs . Cells were then fixed , imaged , and the number of GFP - LC3 puncta were quantitated per cell . Representative images ( A ) and quantitation of > 50 cells from three independent experiments ( B ) are shown . Scale bar indicates 50 µm . Data information : Asterisks indicate * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . ns indicates p > 0 . 05 using student t - test . C - D A375 parental or DCAF12 KO cells were transfected with the indicated siRNAs for 72 hrs before treatment with complete media or EBSS for 6 hrs . Cells were then fixed , endogenous LC3 was immunostained , and the number of LC3 puncta were quantitated per cell . Representative images ( C ) and quantitation of > 50 cells from three independent experiments ( D ) are shown . Scale bar indicates 20 µm . Data information : Asterisks indicate * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . ns indicates p > 0 . 05 using student t - test . E A375 parental or DCAF12 KO cells were treated Cell lysates were collected and immunoblotted for the indicated antibodies to confirm MAGE - A3 / 6 depletion and monitor the autophagy adaptor / substrate p62 / SQSTM1 .

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Figure 6A-B U2OS GFP-LC3 cells were transfected with the indicated siRNAs for 72 hrs before treatment with complete media or EBSS for 6 hrs. Cells were then fixed, imaged, and the number of GFP-LC3 puncta were quantitated per cell. Representative images (A) and quantitation of >50 cells from three independent experiments (B) are shown. Scale bar indicates 50 µm. Data information: Asterisks indicate * p<0.05; ** p<0.01; *** p<0.001. ns indicates p>0.05 using student t-test.C-D A375 parental or DCAF12 KO cells were transfected with the indicated siRNAs for 72 hrs before treatment with complete media or EBSS for 6 hrs. Cells were then fixed, endogenous LC3 was immunostained, and the number of LC3 puncta were quantitated per cell. Representative images (C) and quantitation of >50 cells from three independent experiments (D) are shown. Scale bar indicates 20 µm. Data information: Asterisks indicate * p<0.05; ** p<0.01; *** p<0.001. ns indicates p>0.05 using student t-test.E A375 parental or DCAF12 KO cells were treated Cell lysates were collected and immunoblotted for the indicated antibodies to confirm MAGE-A3/6 depletion and monitor the autophagy adaptor/substrate p62/SQSTM1.


Figure 1A / B . Hybrid Rab7 / Arl8b compartment . A . Representative confocal images of fixed HeLa cells expressing Arl8b - GFP ( green ) , immunolabeled against endogenous Rab7 ( eRab7 , magenta ) . Zoom insets ( 3x ) highlight select regions of colocalization ( white ) , white arrowheads point to vesicles positive for both GTPases . B . Colocalization ( Mander ' s overlap ) of endogenous Rab7 with Arl8b - GFP versus free GFP ( EV ) , n EV = 4 , n Arl8b = 9 images ( 3 ≥ cells per image ) analysed from 2 independent experiments . Significance : 2 - tailed student ' s t - test , * p < 0 . 05 . C - F . Analysis of Arl8b compartment organization and dynamics as a function of Rab7 activity status . C . Left and middle panels : representative confocal images of live HeLa cells expressing mCherry - Rab7 or its mutants Q67L and T22N ( white ) , together with Arl8b - GFP ( green ) , taken at the start of time - lapse ( t0 ) . Right panels : tracks followed by Arl8b - positive null during the time - lapse lasting 50 s recorded at 0 . 5 s per frame , with highest displacement rates for each track depicted on a rainbow colour scale ( blue : immobile ; red : maximum mobility per time interval ) . Zoom insets ( 2 . 8x ) highlight select peripheral ( PP ) and perinuclear ( PN ) cell regions D . Plot of Arl8b - positive pixel distribution expressed as fractional distance along a straight line from center of nucleus ( 0 ) to the plasma membrane ( 1 . 0 ) , numbers of ( pixels ) plotted given above each scatter , n = 7 cells analysed per condition from 2 independent experiments . E / F . Quantification of mean Arl8b vesicle displacement and maximum speed , respectively , n Rab7 = 21 , n QL = 25 , n TN = 24 images ( 3 ≥ cells per image ) analysed from 2 independent experiments .

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Figure 1A/B. Hybrid Rab7/Arl8b compartment. A. Representative confocal images of fixed HeLa cells expressing Arl8b-GFP (green), immunolabeled against endogenous Rab7 (eRab7, magenta). Zoom insets (3x) highlight select regions of colocalization (white), white arrowheads point to vesicles positive for both GTPases. B. Colocalization (Mander's overlap) of endogenous Rab7 with Arl8b-GFP versus free GFP (EV), n EV=4, n Arl8b=9 images (3≥ cells per image) analysed from 2 independent experiments. Significance: 2-tailed student's t-test, * p<0.05.C-F. Analysis of Arl8b compartment organization and dynamics as a function of Rab7 activity status. C. Left and middle panels: representative confocal images of live HeLa cells expressing mCherry-Rab7 or its mutants Q67L and T22N (white), together with Arl8b-GFP (green), taken at the start of time-lapse (t0). Right panels: tracks followed by Arl8b-positive null during the time-lapse lasting 50 s recorded at 0.5 s per frame, with highest displacement rates for each track depicted on a rainbow colour scale (blue: immobile; red: maximum mobility per time interval). Zoom insets (2.8x) highlight select peripheral (PP) and perinuclear (PN) cell regions D. Plot of Arl8b-positive pixel distribution expressed as fractional distance along a straight line from center of nucleus (0) to the plasma membrane (1.0), numbers of (pixels) plotted given above each scatter, n=7 cells analysed per condition from 2 independent experiments. E/F. Quantification of mean Arl8b vesicle displacement and maximum speed, respectively, n Rab7=21, n QL=25, n TN=24 images (3≥ cells per image) analysed from 2 independent experiments.


Figure 2Effects of select transport route activation on the spatial organization of late compartments . A . Representative confocal images of fixed HeLa cells ectopically expressing Arl8b - GFP or Arl8b - Myc ( green ) , in combination with HA - RILP ( cyan ) and / or Myc - SKIP , PLEKHM1 - FLAG , or GFP - FYCO1 ( red ) , immunolabeled against endogenous Rab7 ( eRab7 , magenta ) and the indicated epitope tags . Cell and nuclear boundaries are demarcated with solid and dashed lines respectively , zoom insets ( 3 . 5x ) highlight select peripheral ( PP ) and perinuclear ( PN ) cell regions , scale bar : 10µm . B . Colocalization ( Mander ' s overlap ) between the indicated pairs of proteins , n ≥ 3 images ( 3 ≥ cells per image ) analysed per condition from 2 independent experiments . C . Plot of eRab7 - or Arl8b - GFP / Myc - Arl8b - positive pixel distribution in response to the indicated effector perturbations , expressed as fractional distance along a straight line from center of nucleus ( 0 ) to the plasma membrane ( 1 . 0 ) , number of ( pixels ) plotted given above each scatter , n ≥ 6 cells per condition analysed from 2 independent experiments . D . Colocalization ( Mander ' s overlap ) between Arl8b and Rab7 as a function of the indicated effector perturbations , n ≥ 5 images ( 3 ≥ cells per image ) analysed per condition from 2 independent experiments .

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Figure 2Effects of select transport route activation on the spatial organization of late compartments. A. Representative confocal images of fixed HeLa cells ectopically expressing Arl8b-GFP or Arl8b-Myc (green), in combination with HA-RILP (cyan) and/or Myc-SKIP, PLEKHM1-FLAG, or GFP-FYCO1 (red), immunolabeled against endogenous Rab7 (eRab7, magenta) and the indicated epitope tags. Cell and nuclear boundaries are demarcated with solid and dashed lines respectively, zoom insets (3.5x) highlight select peripheral (PP) and perinuclear (PN) cell regions, scale bar: 10µm. B. Colocalization (Mander's overlap) between the indicated pairs of proteins, n≥3 images (3≥ cells per image) analysed per condition from 2 independent experiments.C. Plot of eRab7- or Arl8b-GFP / Myc-Arl8b-positive pixel distribution in response to the indicated effector perturbations, expressed as fractional distance along a straight line from center of nucleus (0) to the plasma membrane (1.0), number of (pixels) plotted given above each scatter, n≥6 cells per condition analysed from 2 independent experiments.D. Colocalization (Mander's overlap) between Arl8b and Rab7 as a function of the indicated effector perturbations, n≥5 images (3≥ cells per image) analysed per condition from 2 independent experiments.


Figure 3A - D . Correlative light and electron microscopy ( CLEM ) on A . untransfected ( WT ) HeLa cells harbouring endogenous CD63 labeled with GFP as compared to those ectopically expressing B . HA - RILP , C . HA - SKIP or D . both HA - RILP and HA - SKIP . Left panels : wide field fluorescence images of fixed cells showing endogenous GFP - CD63 ( green ) and SiR - lysosome - stained endosomes and / or lysosomes ( red ) . Cell and nuclear boundaries are demarcated with solid and dashed lines respectively , zoom insets highlight regions selected for EM imaging . Middle panels : overview electron micrographs of perinuclear ( PN ) and peripheral ( PP ) cell regions selected for further analysis . Various endolysosomal subtypes are designated by arrowheads : MVBs ( white ) , lysosomes ( black ) , endolysosomes ( yellow ) and abnormal / enlarged endolysosomes ( red ) . Right panels : graphical representations of endosomal distribution under the indicated conditions based on ultrastructural characterization .

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Figure 3A-D. Correlative light and electron microscopy (CLEM) on A. untransfected (WT) HeLa cells harbouring endogenous CD63 labeled with GFP as compared to those ectopically expressing B. HA-RILP, C. HA-SKIP or D. both HA-RILP and HA-SKIP. Left panels: wide field fluorescence images of fixed cells showing endogenous GFP-CD63 (green) and SiR-lysosome-stained endosomes and/or lysosomes (red). Cell and nuclear boundaries are demarcated with solid and dashed lines respectively, zoom insets highlight regions selected for EM imaging. Middle panels: overview electron micrographs of perinuclear (PN) and peripheral (PP) cell regions selected for further analysis. Various endolysosomal subtypes are designated by arrowheads: MVBs (white), lysosomes (black), endolysosomes (yellow) and abnormal/enlarged endolysosomes (red). Right panels: graphical representations of endosomal distribution under the indicated conditions based on ultrastructural characterization.


Figure 4A . Electron micrograph of sections immunolabelled against RFP - SKIP ( 15nm gold ) and GFP - Rab7 ( 10nm gold ) . Arrowheads and zoom inset ( 1 . 75x ) highlight presence of RFP - SKIP and GFP - Rab7 on the same endosomal membrane , scale bar : 200nm . B . Co - immunoprecipitations ( Co - IP ) of HA - RILP and RFP - SKIP with GFP - Rab7 ( R7 ) versus its mutants Q67L ( QL ) and T22N ( TN ) from HEK293T cells using GFP - trap beads . Representative immunoblots against GFP , HA and RFP are shown , EV : empty vector , IP : immunoprecipitation , TL : total lysate ( see also Fig EV1E ) . C . Quantification of interaction between SKIP and Rab7 mutants expressed as fraction Co - IP relative to wild type Rab7 , n = 3 independent experiments . D . Pull - down ( PD ) of RFP - SKIP or RFP - RILP from HEK293T cell lysates using recombinant GST - Rab7 versus GST - Arl8b and free GST . Representative immunoblots against RFP and GST are shown ( see also EV1F ) . E . In vitro glutathione precipitation assays . E . SKIP truncation analysis by PD against GST - Rab7 . Top panels : representative immunoblots against Myc and GST ( see also Fig EV1G ) . Bottom panels : schematic representation of SKIP domain organization . Regions of SKIP capable of interacting with Arl8b versus Rab7 are demarcated with solid black lines . An alignment of human ( h ) and murine ( m ) SKIP sequences to known effectors of Rab7 surrounding the conserved KML / I effector motif at residues 610 - 612 of SKIP is provided . H . Graphical summary of SKIP as a dual effector of Arl8b and Rab7 . F / G . Co - IP of RFP - SKIP versus its KMI motif mutants AAI and AAA with constitutively active GFP - Rab7 Q67L using RFP - trap beads ( see also Fig EV1H ) . F . Representative immunoblots against GFP and RFP . G . Quantification of interaction between SKIP mutants with Rab7 expressed as fraction Co - IP relative to wild type SKIP , n = 3 independent experiments . I - K . Time - lapse of SKIP - mediated transport of late endosomes . I . Schematic representation of tamoxifen - induced activation of SKIP onto endosomal membranes . J / K . Live HeLa cells co - expressing GFP - ER - SKIP ( green ) and mCherry - Rab7 ( magenta ) together with HA - RILP ( unstained ) expressed at low levels ( cells transfected at 1 : 5 RILP : SKIP ratio ) were imaged in the J . absence or K . presence of tamoxifen , allowing on - demand association of SKIP with endosomal membranes . Confocal frames from time - lapses taken at the indicated time - points following treatment are shown . Cell and nuclear boundaries are demarcated with solid and dashed lines respectively , zoom insets ( 3x ) highlight select peripheral ( PP ) and perinuclear ( PN ) cell regions , scale bars :

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Figure 4A. Electron micrograph of sections immunolabelled against RFP-SKIP (15nm gold) and GFP-Rab7 (10nm gold). Arrowheads and zoom inset (1.75x) highlight presence of RFP-SKIP and GFP-Rab7 on the same endosomal membrane, scale bar: 200nm. B. Co-immunoprecipitations (Co-IP) of HA-RILP and RFP-SKIP with GFP-Rab7 (R7) versus its mutants Q67L (QL) and T22N (TN) from HEK293T cells using GFP-trap beads. Representative immunoblots against GFP, HA and RFP are shown, EV: empty vector, IP: immunoprecipitation, TL: total lysate (see also Fig EV1E). C. Quantification of interaction between SKIP and Rab7 mutants expressed as fraction Co-IP relative to wild type Rab7, n=3 independent experiments.  D. Pull-down (PD) of RFP-SKIP or RFP-RILP from HEK293T cell lysates using recombinant GST-Rab7 versus GST-Arl8b and free GST. Representative immunoblots against RFP and GST are shown (see also EV1F).  E. In vitro glutathione precipitation assays. E. SKIP truncation analysis by PD against GST-Rab7. Top panels: representative immunoblots against Myc and GST (see also Fig EV1G). Bottom panels: schematic representation of SKIP domain organization. Regions of SKIP capable of interacting with Arl8b versus Rab7 are demarcated with solid black lines. An alignment of human (h) and murine (m) SKIP sequences to known effectors of Rab7 surrounding the conserved KML/I effector motif at residues 610-612 of SKIP is provided. H. Graphical summary of SKIP as a dual effector of Arl8b and Rab7.  F/G. Co-IP of RFP-SKIP versus its KMI motif mutants AAI and AAA with constitutively active GFP-Rab7 Q67L using RFP-trap beads (see also Fig EV1H). F. Representative immunoblots against GFP and RFP. G. Quantification of interaction between SKIP mutants with Rab7 expressed as fraction Co-IP relative to wild type SKIP, n=3 independent experiments. I-K. Time-lapse of SKIP-mediated transport of late endosomes. I. Schematic representation of tamoxifen-induced activation of SKIP onto endosomal membranes. J/K. Live HeLa cells co-expressing GFP-ER-SKIP (green) and mCherry-Rab7 (magenta) together with HA-RILP (unstained) expressed at low levels (cells transfected at 1:5 RILP:SKIP ratio) were imaged in the J. absence or K. presence of tamoxifen, allowing on-demand association of SKIP with endosomal membranes. Confocal frames from time-lapses taken at the indicated time-points following treatment are shown. Cell and nuclear boundaries are demarcated with solid and dashed lines respectively, zoom insets (3x) highlight select peripheral (PP) and perinuclear (PN) cell regions, scale bars: 10µm


Figure 5A - C . Consequences of effector depletion on the endogenous Arl8b / Rab7 hybrid compartment . A . Representative confocal images of fixed HeLa cells harbouring GFP - tagged endogenous Arl8b ( G - eArl8b , green ) , transfected with the indicated siRNAs and immunolabeled against endogenous Rab7 ( eRab7 , magenta ) . B . Colocalization ( Mander ' s overlap ) between endogenous Arl8b and Rab7 in response to effector depletion , n siC = 10 , n siRILP = 9 , n siSKIP = 9 images ( 4 ≥ cells per image ) analysed from 2 independent experiments . C . Immunoblot analysis for depletion efficiency of SKIP and RILP , with actin as loading control . D / E . Effect of Rab7 GTPase activity status on its association with the peripheral SKIP compartment . D . Left panels : Representative confocal images of fixed HeLa cells expressing GFP - Rab7 or its mutants Q67L or T22N ( green ) together with HA - RILP ( red ) and Myc - SKIP ( blue ) , immunolabeled against the indicated epitope tags . Right panels : Schematic overview per condition . E . Colocalization ( Mander ' s overlap ) between the indicated protein pairs , n Rab7 = 5 , n QL = 5 , n TN = 7 images ( 2 ≥ cells per image ) analysed from 2 independent experiments . F . Graphical summary of Rab7 removal from the SKIP compartment .

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Figure 5A-C. Consequences of effector depletion on the endogenous Arl8b/Rab7 hybrid compartment. A. Representative confocal images of fixed HeLa cells harbouring GFP-tagged endogenous Arl8b (G-eArl8b, green), transfected with the indicated siRNAs and immunolabeled against endogenous Rab7 (eRab7, magenta). B. Colocalization (Mander's overlap) between endogenous Arl8b and Rab7 in response to effector depletion, n siC=10, n siRILP=9, n siSKIP=9 images (4≥ cells per image) analysed from 2 independent experiments. C. Immunoblot analysis for depletion efficiency of SKIP and RILP, with actin as loading control.D/E. Effect of Rab7 GTPase activity status on its association with the peripheral SKIP compartment. D. Left panels: Representative confocal images of fixed HeLa cells expressing GFP-Rab7 or its mutants Q67L or T22N (green) together with HA-RILP (red) and Myc-SKIP (blue), immunolabeled against the indicated epitope tags. Right panels: Schematic overview per condition. E. Colocalization (Mander's overlap) between the indicated protein pairs, n Rab7=5, n QL=5, n TN=7 images (2≥ cells per image) analysed from 2 independent experiments. F. Graphical summary of Rab7 removal from the SKIP compartment.


Figure 6B . Representative confocal images of fixed HeLa cells depleted of TBC1D2 , 1D5 , or 1D15 using siRNA oligo pools and immunolabeled against CD63 ( white ) , scale bar : 10µm . C . Plot of CD63 pixel distribution as a function of TBC1D2 / 1D5 / 1D15 depletion expressed as fractional distance along a straight line from center of nucleus ( 0 ) to the plasma membrane ( 1 . 0 ) , number of ( pixels ) plotted given above each scatter , n ≥ 8 cells per condition analysed from 2 independent experiments . D / E . Rescue of TBC1D15 depletion phenotype . D . Representative confocal images of fixed HeLa cells transfected with either control siRNA ( siC ) or oligo # 3 targeting TBC1D15 ( siTBC1D15 ) and ectopically expressing either GFP - EV , GFP - TBC1D5 or siRNA - resistant GFP - TBC1D15res ( green ) , immunolabeled for CD63 ( magenta ) , scale bar : 10µm . E . Quantification of rescue expressed as % cells ( average ) in the population exhibiting one of 3 phenotypes : clustered , dispersed or intermediate ; total numbers of cells analysed per condition appear above each bar , n = 3 independent experiments . Effects of TBC1D15 depletion on the morphology and dynamics of late organelles . F . Representative electron micrographs of fixed HeLa cells transfected with either control siRNA ( siC ) or a pool of oligos targeting TBC1D15 ( siTBC1D15 ) are shown , scale bars as indicated . G . Analysis of late compartment dynamics as a function of TBC1D15 depletion . Tracks followed by SiR - lysosome - positive vesicles during a time - lapse lasting 255 sec ( 5 s per frame ) , with highest displacement rates for each track depicted on a rainbow colour scale ( blue : immobile ; red : maximum mobility per time interval ) . Zoom insets ( 3x ) highlight select peripheral ( PP ) and perinuclear ( PN ) regions , scale bar : 10µm EV10 ) . H . Quantification of high motility fraction ( % tracks with displacement rates above 0 . 9µm / sec ) , n siC = 27 , n si1D15 = 37 images ( 2 ≥ cells per image ) analysed from 2 independent experiments . Effectiveness of TBC1D15 ( si1D15 ) depletion is confirmed by immunoblot against endogenous TBC1D15 ( e1D15 ) following its immunoprecipitation ( IP ) , TL : total lysate . I . FRAP of GFP - Rab7 in live HeLa cells transfected with either control siRNA ( siC , black line ) or a pool of oligos targeting TBC1D15 ( si1D15 , red line ) . Plotted is average GFP - Rab7 signal recovery during 200 s following bleaching , expressed as % of pre - bleach signal , n = 3 bleach regions per sample .

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 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 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 O O 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 6B. Representative confocal images of fixed HeLa cells depleted of TBC1D2, 1D5, or 1D15 using siRNA oligo pools and immunolabeled against CD63 (white), scale bar: 10µm. C. Plot of CD63 pixel distribution as a function of TBC1D2/1D5/1D15 depletion expressed as fractional distance along a straight line from center of nucleus (0) to the plasma membrane (1.0), number of (pixels) plotted given above each scatter, n≥8 cells per condition analysed from 2 independent experiments.D/E. Rescue of TBC1D15 depletion phenotype. D. Representative confocal images of fixed HeLa cells transfected with either control siRNA (siC) or oligo #3 targeting TBC1D15 (siTBC1D15) and ectopically expressing either GFP-EV, GFP-TBC1D5 or siRNA-resistant GFP-TBC1D15res (green), immunolabeled for CD63 (magenta), scale bar: 10µm. E. Quantification of rescue expressed as % cells (average) in the population exhibiting one of 3 phenotypes: clustered, dispersed or intermediate; total numbers of cells analysed per condition appear above each bar, n=3 independent experiments.Effects of TBC1D15 depletion on the morphology and dynamics of late organelles. F. Representative electron micrographs of fixed HeLa cells transfected with either control siRNA (siC) or a pool of oligos targeting TBC1D15 (siTBC1D15) are shown, scale bars as indicated.G. Analysis of late compartment dynamics as a function of TBC1D15 depletion. Tracks followed by SiR-lysosome-positive vesicles during a time-lapse lasting 255 sec (5 s per frame), with highest displacement rates for each track depicted on a rainbow colour scale (blue: immobile; red: maximum mobility per time interval). Zoom insets (3x) highlight select peripheral (PP) and perinuclear (PN) regions, scale bar: 10µm EV10). H. Quantification of high motility fraction (% tracks with displacement rates above 0.9µm/sec), n siC=27, n si1D15=37 images (2≥ cells per image) analysed from 2 independent experiments. Effectiveness of TBC1D15 (si1D15) depletion is confirmed by immunoblot against endogenous TBC1D15 (e1D15) following its immunoprecipitation (IP), TL: total lysate.I. FRAP of GFP-Rab7 in live HeLa cells transfected with either control siRNA (siC, black line) or a pool of oligos targeting TBC1D15 (si1D15, red line). Plotted is average GFP-Rab7 signal recovery during 200 s following bleaching, expressed as % of pre-bleach signal, n=3 bleach regions per sample.


Figure 7Effects of depleting known GAPs for Rab7 on late compartment segregation mediated by RILP and SKIP . A . Representative confocal images of fixed HeLa cells transfected with either control siRNA ( siC ) , two different siRNA oligos targeting TBC1D15 ( # 1 and # 3 ) or oligo pool targeting TBC1D17 and ectopically expressing GFP - Arl8b ( green ) in combination with HA - RILP ( cyan ) and Myc - SKIP ( red ) , immunolabeled against endogenous Rab7 ( eRab7 , magenta ) and the indicated epitope tags . Cell and nuclear boundaries are demarcated with solid and dashed lines respectively , zoom insets ( 3 . 5x ) highlight select peripheral ( PP ) and perinuclear ( PN ) cell regions , scale bar : 10µm . B . Colocalization of SKIP with endogenous Rab7 ( Mander ' s overlap ) in response to depletion of TBC - containing proteins , n ≥ 4 images ( 3 ≥ cells per image ) analysed per condition from 2 or more independent experimentsC . Plot of Rab7 - positive pixel distribution expressed as fractional distance along a straight line from center of nucleus ( 0 ) to the plasma membrane ( 1 . 0 ) , number of ( pixels ) plotted given above each scatter , n ≥ 8 cells analysed per condition from 2 independent experiments . D . Validation of depletion efficiencies expressed as fraction of the indicated mRNA remaining relative to siC assayed by qPCR , n = 3 independent experiments . E . Effect of TBC1D15 depletion on Rab7 - dependent MHC - II receptor trafficking to the SKIP - positive compartment , n siC = 21 , n si1D15 = 21 cells analysed from 3 independent

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Figure 7Effects of depleting known GAPs for Rab7 on late compartment segregation mediated by RILP and SKIP. A. Representative confocal images of fixed HeLa cells transfected with either control siRNA (siC), two different siRNA oligos targeting TBC1D15 (#1 and #3) or oligo pool targeting TBC1D17 and ectopically expressing GFP-Arl8b (green) in combination with HA-RILP (cyan) and Myc-SKIP (red), immunolabeled against endogenous Rab7 (eRab7, magenta) and the indicated epitope tags. Cell and nuclear boundaries are demarcated with solid and dashed lines respectively, zoom insets (3.5x) highlight select peripheral (PP) and perinuclear (PN) cell regions, scale bar: 10µm. B. Colocalization of SKIP with endogenous Rab7 (Mander's overlap) in response to depletion of TBC-containing proteins, n≥4 images (3≥ cells per image) analysed per condition from 2 or more independent experimentsC. Plot of Rab7-positive pixel distribution expressed as fractional distance along a straight line from center of nucleus (0) to the plasma membrane (1.0), number of (pixels) plotted given above each scatter, n≥8 cells analysed per condition from 2 independent experiments.D. Validation of depletion efficiencies expressed as fraction of the indicated mRNA remaining relative to siC assayed by qPCR, n=3 independent experiments.E. Effect of TBC1D15 depletion on Rab7-dependent MHC-II receptor trafficking to the SKIP-positive compartment, n siC=21, n si1D15=21 cells analysed from 3 independent experiments


Figure 8Representative confocal images of either control ( siC ) Hela cells harbouring endogenous TBC1D15 tagged with GFP ( Ge1D15 , green ) or those depleted of FIS1 using a pool of siRNA oligos , fixed and immunolabeled for the mitochondrial marker TOM20 ( blue ) in combination with either endogenous A . Rab7 Zoom insets ( 2 . 5x ) highlight select cell regions , arrowheads point to vesicles positive for TBC1D15 and negative for TOM20 ( yellow ) . Representative confocal images of either control ( siC ) Hela cells harbouring endogenous TBC1D15 tagged with GFP ( Ge1D15 , green ) or those depleted of FIS1 using a pool of siRNA oligos , fixed and immunolabeled for the mitochondrial marker TOM20 ( blue ) in combination with either endogenous B . CD63 ( red ) . Zoom insets ( 2 . 5x ) highlight select cell regions , arrowheads point to vesicles positive for TBC1D15 and negative for TOM20 ( yellow ) . Effect of FIS1 on late compartment distribution . C . Representative confocal images of fixed HeLa cells depleted of FIS1 using a siRNA oligo pool and immunolabeled against CD63 ( white ) . D . Plot of CD63 pixel distribution as a function of FIS1 depletion expressed as fractional distance along a straight line from center of nucleus ( 0 ) to the plasma membrane ( 1 . 0 ) , number of ( pixels ) plotted given above each scatter , n siC = 14 , n siFis1 = 17 cells analysed from 2 independent experiments . E . Colocalization of SKIP with endogenous Rab7 ( Mander ' s overlap ) in response to FIS1 depletion , n siC = 13 , n siFis1 = 9 images ( 3 ≥ cells per image ) analysed from 2 independent experimentsF . Validation of FIS1 depletion efficiency assayed by qPCR and expressed as fraction FIS1 mRNA remaining relative to siC , n = 3 independent experiments . G - J . Recruitment of endogenous TBC1D15 to the SKIP compartment . G . Left panels : representative confocal images of fixed HeLa cells harbouring endogenous TBC1D15 tagged with GFP ( G - e1D15 , green ) and ectopically expressing either empty vector , HA - SKIP or PLEKHM1 - HA ( red ) , immunolabeled against HA . Zoom insets ( 2 . 7x ) highlight select peripheral ( PP ) and perinuclear ( PN ) cell regions . Right panels : pixel plots of endogenous GFP - TBC1D15 ( green line ) and HA signals ( red line ) corresponding to the dashed white lines in G . H - J . Colocalization ( Mander ' s overlap ) between the indicated pairs of proteins , n ≥ 5 images , 3 ≥ cells per image , analysed per condition from 2 independent

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 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 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 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 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

Figure 8Representative confocal images of either control (siC) Hela cells harbouring endogenous TBC1D15 tagged with GFP (Ge1D15, green) or those depleted of FIS1 using a pool of siRNA oligos, fixed and immunolabeled for the mitochondrial marker TOM20 (blue) in combination with either endogenous A. Rab7 Zoom insets (2.5x) highlight select cell regions, arrowheads point to vesicles positive for TBC1D15 and negative for TOM20 (yellow).Representative confocal images of either control (siC) Hela cells harbouring endogenous TBC1D15 tagged with GFP (Ge1D15, green) or those depleted of FIS1 using a pool of siRNA oligos, fixed and immunolabeled for the mitochondrial marker TOM20 (blue) in combination with either endogenous B. CD63 (red). Zoom insets (2.5x) highlight select cell regions, arrowheads point to vesicles positive for TBC1D15 and negative for TOM20 (yellow).Effect of FIS1 on late compartment distribution. C. Representative confocal images of fixed HeLa cells depleted of FIS1 using a siRNA oligo pool and immunolabeled against CD63 (white).D. Plot of CD63 pixel distribution as a function of FIS1 depletion expressed as fractional distance along a straight line from center of nucleus (0) to the plasma membrane (1.0), number of (pixels) plotted given above each scatter, n siC=14, n siFis1=17 cells analysed from 2 independent experiments.E. Colocalization of SKIP with endogenous Rab7 (Mander's overlap) in response to FIS1 depletion, n siC=13, n siFis1=9 images (3≥ cells per image) analysed from 2 independent experimentsF. Validation of FIS1 depletion efficiency assayed by qPCR and expressed as fraction FIS1 mRNA remaining relative to siC, n=3 independent experiments.G-J. Recruitment of endogenous TBC1D15 to the SKIP compartment. G. Left panels: representative confocal images of fixed HeLa cells harbouring endogenous TBC1D15 tagged with GFP (G-e1D15, green) and ectopically expressing either empty vector, HA-SKIP or PLEKHM1-HA (red), immunolabeled against HA. Zoom insets (2.7x) highlight select peripheral (PP) and perinuclear (PN) cell regions. Right panels: pixel plots of endogenous GFP-TBC1D15 (green line) and HA signals (red line) corresponding to the dashed white lines in G. H-J. Colocalization (Mander's overlap) between the indicated pairs of proteins, n≥5 images, 3≥ cells per image, analysed per condition from 2 independent experiments


Figure 9Effects of SKIP KMI motif on HOPS complex recruitment . A . Analysis of SKIP / HOPS interactions by co - IP of RFP / mCherry - HOPS subunits with Myc - SKIP versus the KMI motif mutant AAA from HEK293T cells using RFP - trap beads . Representative immunoblots against RFP and Myc are shown , IP : immunoprecipitation , TL : total lysate . B . Quantification of SKIP / HOPS interactions , n = 3 independent experiments . Top graph : fold change in binding of HOPS subunits to AAA mutant relative to wild type ( WT ) SKIP ( 1 . 0 ) , significance : one - way ANOVA test ( relative to VPS41 ) . Bottom graph : Co - IP per HOPS subunit relative to VPS39 , significance : one - way ANOVA test ( relative to VPS39 ) , * p < 0 . 05 , * * * p < 0 . 001 , ns : not significant . C . Colocalization ( Mander ' s overlap ) between SKIP and RFP - VPS39 ( images or mCherry - VPS41 ( images , n ≥ 10 images ( 2 ≥ cells per image ) analysed per condition from 2 independent experiments . D . Representative confocal images of fixed HeLa cells ectopically expressing Myc - SKIP , KMI motif mutant ( AAA ) or RUN domain truncation mutant ( ∆ RUN ) ( red ) together with RFP - VPS39 ( green ) , immunolabeled for endogenous VPS11 ( eVPS11 , blue ) . Effect of HOPS over - expression on recruitment of TBC1D15 to the SKIP compartment . E . Representative confocal images of fixed HeLa cells harbouring endogenous GFP - TBC1D15 ( Ge1D15 , green ) and ectopically expressing HA - SKIP ( red ) together with mCh - VPS18 or RFP - VPS39 ( white ) , immunolabeled for endogenous VPS18 or VPS11 ( blue ) , as indicatedF - I . Colocalization of SKIP and HOPS complex with TBC1D15 as a function of HOPS subunit over - expression . F / H . Mander ' s overlap between F . SKIP or H . eVPS18 and endogenous TBC1D15 , n ≥ 5 images ( 2 ≥ cells per image ) analysed per condition from 2 independent experiments , significance : one - way ANOVA test ( relative to EV ) , * * p < 0 . 01 , * * * p < 0 . 001 , ns : not significant . G / I . Mander ' s overlap between G . SKIP or I . eVPS11 and endogenous TBC1D15 as a function VPS18 overabundance , with or without FIS1 depletion ( siFIS1 ) , n ≥ 3 images ( 2 ≥ cells per image ) analysed per condition from 2 or more independent experiments .

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Figure 9Effects of SKIP KMI motif on HOPS complex recruitment. A. Analysis of SKIP/HOPS interactions by co-IP of RFP/mCherry-HOPS subunits with Myc-SKIP versus the KMI motif mutant AAA from HEK293T cells using RFP-trap beads. Representative immunoblots against RFP and Myc are shown, IP: immunoprecipitation, TL: total lysate. B. Quantification of SKIP/HOPS interactions, n=3 independent experiments. Top graph: fold change in binding of HOPS subunits to AAA mutant relative to wild type (WT) SKIP (1.0), significance: one-way ANOVA test (relative to VPS41). Bottom graph: Co-IP per HOPS subunit relative to VPS39, significance: one-way ANOVA test (relative to VPS39), * p<0.05, *** p<0.001, ns: not significant.C. Colocalization (Mander's overlap) between SKIP and RFP-VPS39 (images or mCherry-VPS41 (images , n≥10 images (2≥ cells per image) analysed per condition from 2 independent experiments.D. Representative confocal images of fixed HeLa cells ectopically expressing Myc-SKIP, KMI motif mutant (AAA) or RUN domain truncation mutant (∆RUN) (red) together with RFP-VPS39 (green), immunolabeled for endogenous VPS11 (eVPS11, blue).Effect of HOPS over-expression on recruitment of TBC1D15 to the SKIP compartment. E. Representative confocal images of fixed HeLa cells harbouring endogenous GFP-TBC1D15 (Ge1D15, green) and ectopically expressing HA-SKIP (red) together with mCh-VPS18 or RFP-VPS39 (white), immunolabeled for endogenous VPS18 or VPS11 (blue), as indicatedF-I. Colocalization of SKIP and HOPS complex with TBC1D15 as a function of HOPS subunit over-expression. F/H. Mander's overlap between F. SKIP or H. eVPS18 and endogenous TBC1D15, n≥5 images (2≥ cells per image) analysed per condition from 2 independent experiments, significance: one-way ANOVA test (relative to EV), ** p<0.01, *** p<0.001, ns: not significant. G/I. Mander's overlap between G. SKIP or I. eVPS11 and endogenous TBC1D15 as a function VPS18 overabundance, with or without FIS1 depletion (siFIS1), n≥3 images (2≥ cells per image) analysed per condition from 2 or more independent experiments.


Figure 10A / B . In situ SKIP / TBC1D15 complex formation assayed using proximity - based biotin ligation ( BioID ) . A . Neutravidin precipitates ( PD ) from biotin - treated HEK293T cells ectopically expressing GFP - TBC1D15 ( GFP - 1D15 ) together with HA - BioID - SKIP or HA - BioID - EV . Representative immunoblots against GFP , HA and VPS18 , TL : total lysate . B . Quantification of biotinylation of GFP - TBC1D15 ( top graph ) and endogenous VPS18 ( eVPS18 , bottom graph ) by BioID - SKIP , expressed relative to BioID - EV ( 1 . 0 ) , n = 4 independent experiments . C . Analysis of TBC1D15 / HOPS interactions by co - IP of RFP / mCherry - HOPS subunits with GFP - TBC1D15 from HEK293T cells using GFP - trap beads . Left panel : schematic of HOPS complex composition , with subunits tested marked in yellow . Right panel : representative immunoblots against GFP and RFP ; IP : immunoprecipitation , TL : total lysate . Effects of VPS18 depletion on the recruitment of TBC1D15 to the SKIP complex . D . Representative confocal images of fixed HeLa cells harbouring endogenous GFP - TBC1D15 ( G - e1D15 , green ) , transfected with either control siRNA ( siC ) or oligo pool targeting VPS18 ( siVPS18 ) and ectopically expressing HA - SKIP ( red ) , immunolabeled against endogenous VPS18 ( eVPS18 , blue ) and HA , scale bar : 10µm . Cell and nuclear boundaries are demarcated with solid and dashed lines respectively , zoom insets ( 3 . 5x ) highlight select peripheral ( PP ) and perinuclear ( PN ) cell regions . E . Colocalization ( Mander ' s overlap ) between SKIP and endogenous TBC1D15 as a function of VPS18 depletion , n siC = 9 , n siVPS18 = 11 images ( 2 ≥ cells per image ) analysed from 2 independent experiments . F . Neutravidin precipitates ( PD ) from biotin - treated HeLa cells ectopically expressing GFP - TBC1D15 ( GFP - 1D15 ) together with BioID - SKIP or BioID - EV . Representative immunoblots against GFP , HA and VPS18 are shown , TL : total lysate . G . Quantification of GFP - TBC1D15 biotinylation by BioID - SKIP over BioID - EV in response to VPS18 depletion expressed as % of control ( siC ) , n = 4 independent experiments performed in HeLa or HEK293T cells . H . Quantification of VPS18 protein abundance from experiments in G , n = 4 . . Effect of TBC1D15 activity on Rab7 displacement from the SKIP compartment . Colocalization ( Mander ' s overlap ) between Rab7 and TBC1D15 ( WT or R417A mutant ) in the presence of either SKIP or PLEKHM1 combined with the indicated VPS proteins , n ≥ 5 images ( 2 ≥ cells per image ) analysed from 2 independent

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Figure 10A/B. In situ SKIP/TBC1D15 complex formation assayed using proximity-based biotin ligation (BioID). A. Neutravidin precipitates (PD) from biotin-treated HEK293T cells ectopically expressing GFP-TBC1D15 (GFP-1D15) together with HA-BioID-SKIP or HA-BioID-EV. Representative immunoblots against GFP, HA and VPS18 , TL: total lysate. B. Quantification of biotinylation of GFP-TBC1D15 (top graph) and endogenous VPS18 (eVPS18, bottom graph) by BioID-SKIP, expressed relative to BioID-EV (1.0), n=4 independent experiments.C. Analysis of TBC1D15/HOPS interactions by co-IP of RFP/mCherry-HOPS subunits with GFP-TBC1D15 from HEK293T cells using GFP-trap beads. Left panel: schematic of HOPS complex composition, with subunits tested marked in yellow. Right panel: representative immunoblots against GFP and RFP; IP: immunoprecipitation, TL: total lysate.Effects of VPS18 depletion on the recruitment of TBC1D15 to the SKIP complex. D. Representative confocal images of fixed HeLa cells harbouring endogenous GFP-TBC1D15 (G-e1D15, green), transfected with either control siRNA (siC) or oligo pool targeting VPS18 (siVPS18) and ectopically expressing HA-SKIP (red), immunolabeled against endogenous VPS18 (eVPS18, blue) and HA, scale bar: 10µm. Cell and nuclear boundaries are demarcated with solid and dashed lines respectively, zoom insets (3.5x) highlight select peripheral (PP) and perinuclear (PN) cell regions. E. Colocalization (Mander's overlap) between SKIP and endogenous TBC1D15 as a function of VPS18 depletion, n siC=9, n siVPS18=11 images (2≥ cells per image) analysed from 2 independent experiments.F. Neutravidin precipitates (PD) from biotin-treated HeLa cells ectopically expressing GFP-TBC1D15 (GFP-1D15) together with BioID-SKIP or BioID-EV. Representative immunoblots against GFP, HA and VPS18 are shown, TL: total lysate. G. Quantification of GFP-TBC1D15 biotinylation by BioID-SKIP over BioID-EV in response to VPS18 depletion expressed as % of control (siC), n=4 independent experiments performed in HeLa or HEK293T cells. H. Quantification of VPS18 protein abundance from experiments in G, n=4.. Effect of TBC1D15 activity on Rab7 displacement from the SKIP compartment. Colocalization (Mander's overlap) between Rab7 and TBC1D15 (WT or R417A mutant) in the presence of either SKIP or PLEKHM1 combined with the indicated VPS proteins, n≥ 5 images (2≥ cells per image) analysed from 2 independent experiments


Figure 1 ( C ) Expression profile of the indicated ion channel analyzed by RT - PCR in the WM266 . 4 and 451Lu metastatic melanoma cell lines ( middle and bottom panels , respectively ) . Expected amplicons sizes ( positive controls in top panel ) are in base pairs ( bp ) ; TRPV1 = 120 , TRPV2 = 199 , TRPV3 = 226 , TRPV4 = 190 / 370 , TRPV6 = 208 , TRPC1 = 201 , TRPC6 = 121 , TRPM2 = 303 , TRPM7 = 226 , TRPA1 = 140 , Orai1 = 161 , STIM1 = 109 , STIM2 = 114 ) . ( F ) TRPV2 immunoblotting in 36 out of the 60 NCI - 60 cell lines . Tubulin was used as a loading control . Note that after longer exposure times , TRPV2 expression was also detected in some breast and prostate cancer cell lines , as it has been previously described ( Elbaz et al , 2018 ; Gambade et al , 2016 ; Monet et al , 2010 ) . ( G ) TRPV2 protein expression in melanoma cell lines harboring either B - RAF ( black ) or N - Ras ( blue ) mutations . β - Actin was used as a loading control .

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

Figure 1(C) Expression profile of the indicated ion channel analyzed by RT-PCR in the WM266.4 and 451Lu metastatic melanoma cell lines (middle and bottom panels, respectively). Expected amplicons sizes (positive controls in top panel) are in base pairs (bp); TRPV1=120, TRPV2=199, TRPV3=226, TRPV4=190/370, TRPV6=208, TRPC1=201, TRPC6=121, TRPM2=303, TRPM7=226, TRPA1=140, Orai1=161, STIM1=109, STIM2=114).(F) TRPV2 immunoblotting in 36 out of the 60 NCI-60 cell lines. Tubulin was used as a loading control. Note that after longer exposure times, TRPV2 expression was also detected in some breast and prostate cancer cell lines, as it has been previously described (Elbaz et al, 2018; Gambade et al, 2016; Monet et al, 2010).(G) TRPV2 protein expression in melanoma cell lines harboring either B-RAF (black) or N-Ras (blue) mutations. β-Actin was used as a loading control.


Figure 2 ( A ) Analysis for total and plasma membrane ( PM ) TRPV2 expression in melanoma cell lines . β - actin was used as loading and cell integrity control . PM fractions were isolated using the surface protein biotinylation technique . The higher molecular weight bands of TRPV2 correspond to the glycosylated and mature channel ( C ) TRPV2 overexpression in the non - invasive 501mel cell line ( GFP - TRPV2 ) or downregulation by lentiviral - delivery of TRPV2 specific shRNAs ( shRNA V2 - 1 and V2 - 2 ) in the WM266 . 4 and 451Lu metastatic melanoma cell lines assessed by western - blot . β - actin was used as a loading control . ( E ) Average normalized quenching rates ( as in D ) presented as mean ± SEM ( n > 3 biological replicates ) . * * P = 0 . 0095 , the Mann - Whitney test ( left panel ) ; * P = 0 . 0399 - 0 . 0336 ( middle panel ) and * P = 0 . 0438 , * * P = 0 . 0033 ( right panel ) the Kruskal - Wallis test followed by Dunn ' s multiple comparisons test .

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Figure 2(A) Analysis for total and plasma membrane (PM) TRPV2 expression in melanoma cell lines. β-actin was used as loading and cell integrity control. PM fractions were isolated using the surface protein biotinylation technique. The higher molecular weight bands of TRPV2 correspond to the glycosylated and mature channel(C) TRPV2 overexpression in the non-invasive 501mel cell line (GFP-TRPV2) or downregulation by lentiviral-delivery of TRPV2 specific shRNAs (shRNA V2-1 and V2-2) in the WM266.4 and 451Lu metastatic melanoma cell lines assessed by western-blot. β-actin was used as a loading control.(E) Average normalized quenching rates (as in D) presented as mean ± SEM (n>3 biological replicates). **P = 0.0095, the Mann-Whitney test (left panel); *P = 0.0399-0.0336 (middle panel) and *P = 0.0438, **P = 0.0033 (right panel) the Kruskal-Wallis test followed by Dunn's multiple comparisons test.


Figure 3 ( A ) Impact of TRPV2 genetic manipulation in 501mel , WM266 . 4 and 451Lu on serum - induced migration and matrigel invasion . Representative pictures show crystal violet - stained cells that have migrated through 8 µm pore size membranes . Histograms illustrate the average numbers of migrating / invading cells normalized to control ( presented as mean ± SEM of n = 3 independent experiments ) . For 501mel the Mann - Whitney test was used for statistical analysis ( * * P = 0 . 0022 ) ; For WM266 . 4 & 451Lu one - way ANOVA followed by Dunnett ' s multiple comparisons tests was used ( * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ; See appendix Table S2 for exact P values ) . ( D ) TRPV2 - silencing effect on 3D invasion . Representative images were taken every day for 3 days after collagen embedding of spheroids from WM266 . 4 cells expressing either shRNA ctrl or TRPV2 - targeting shRNAs ( scale bar = 0 . 5 mm ) . ( E ) Quantification of collagen invasion . For each spheroid , the cell - covered area at day 2 was normalized to the starting area of the collagen - embedded spheroids . Histograms represent the invasion relative to control WM266 . 4 spheroids ( shRNA Ctrl ) from at least 12 spheroids from three independent experiments ( See also appendix Fig S3C ) . M . * * * * P < 0 . 0001 and * * P = 0 . 0012 the Kruskal - Wallis test followed by Dunn ' s multiple comparisons test .

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Figure 3(A) Impact of TRPV2 genetic manipulation in 501mel, WM266.4 and 451Lu on serum-induced migration and matrigel invasion. Representative pictures show crystal violet-stained cells that have migrated through 8 µm pore size membranes. Histograms illustrate the average numbers of migrating/invading cells normalized to control (presented as mean ± SEM of n=3 independent experiments). For 501mel the Mann-Whitney test was used for statistical analysis (**P = 0.0022); For WM266.4&451Lu one-way ANOVA followed by Dunnett's multiple comparisons tests was used (**P < 0.01; ***P < 0.001; ****P < 0.0001 ; See appendix Table S2 for exact P values).(D) TRPV2-silencing effect on 3D invasion. Representative images were taken every day for 3 days after collagen embedding of spheroids from WM266.4 cells expressing either shRNA ctrl or TRPV2-targeting shRNAs (scale bar=0.5 mm). (E) Quantification of collagen invasion. For each spheroid, the cell-covered area at day 2 was normalized to the starting area of the collagen-embedded spheroids. Histograms represent the invasion relative to control WM266.4 spheroids (shRNA Ctrl) from at least 12 spheroids from three independent experiments (See also appendix Fig S3C). M. ****P <0.0001 and **P = 0.0012 the Kruskal-Wallis test followed by Dunn's multiple comparisons test.


Figure 4 ( B ) Representative confocal images of the in situ detection of endogenous TRPV2 interactions with paxillin and vinculin by proximity - ligation assays ( PLA ) . Low confluency WM266 . 4 and 451Lu metastatic melanoma cells plated on fibronectin - coated coverslips were stained for F - actin ( green ) , cell nuclei ( blue ) and PLA reaction using antibodies specific of the indicated proteins ( red ) ( scale bar = 20 µm ; identical for all pictures ) . Red fluorescent spots indicate the association of the two proteins of interest , close to 40 nm . Scatter plots represent the quantification of the number of PLA spots per cell ( bars indicate the medians ) between TRPV2 and a control antibody ( IgG ) , paxillin ( PAX ) or vinculin ( VCL ) . ( n = 29 - 51 cells from at least three independent experiments ) . ( C ) Super - resolution ( dSTORM ) imaging of the clustering of TRPV2 channels with paxillin , but not with vinculin , in migrating WM266 . 4 cells ( Scale bar = 5µm ) . a and b insets show expanded views of a region of the cell and arrows highlight TRPV2 channel and paxillin co - clusters .

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Figure 4(B) Representative confocal images of the in situ detection of endogenous TRPV2 interactions with paxillin and vinculin by proximity-ligation assays (PLA). Low confluency WM266.4 and 451Lu metastatic melanoma cells plated on fibronectin-coated coverslips were stained for F-actin (green), cell nuclei (blue) and PLA reaction using antibodies specific of the indicated proteins (red) (scale bar = 20 µm ; identical for all pictures). Red fluorescent spots indicate the association of the two proteins of interest, close to 40 nm. Scatter plots represent the quantification of the number of PLA spots per cell (bars indicate the medians) between TRPV2 and a control antibody (IgG), paxillin (PAX) or vinculin (VCL). (n=29-51 cells from at least three independent experiments).(C) Super-resolution (dSTORM) imaging of the clustering of TRPV2 channels with paxillin, but not with vinculin, in migrating WM266.4 cells (Scale bar = 5µm). a and b insets show expanded views of a region of the cell and arrows highlight TRPV2 channel and paxillin co-clusters.


Figure 5 ( B ) Comparison of calpain activity in control ( GFP ) or overexpressing TRPV2 ( GFP - TRPV2 ) 501mel cells , and in control ( shRNA Ctrl ) or TRPV2 - silenced ( shRNA V2 - 1 , - 2 ) WM266 . 4 cells or 451Lu cells . Bar graphs show mean normalized calpain activity ± SEM ( n = 3 biological replicates ) . For 501mel the Mann - Whitney test was used for statistical analysis ( * P = 0 . 05 ) ; For WM266 . 4 & 451Lu one - way ANOVA followed by Dunnett ' s multiple comparisons tests was used ( * * P < 0 . 01 ; See appendix Table S2 for exact P values ) . ( C ) Representative immunoblots showing the full - length ( 230 kDa ; Black Arrows ) and the calpain - mediated degradation product ( 190 kDa ; Grey Arrows ) of talin after overexpression or repression of TRPV2 , in the corresponding cell lines . Tubulin was used as a loading control .

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Figure 5(B) Comparison of calpain activity in control (GFP) or overexpressing TRPV2 (GFP-TRPV2) 501mel cells, and in control (shRNA Ctrl) or TRPV2-silenced (shRNA V2-1, -2) WM266.4 cells or 451Lu cells. Bar graphs show mean normalized calpain activity ± SEM (n=3 biological replicates). For 501mel the Mann-Whitney test was used for statistical analysis (*P = 0.05); For WM266.4&451Lu one-way ANOVA followed by Dunnett's multiple comparisons tests was used (**P < 0.01; See appendix Table S2 for exact P values).(C) Representative immunoblots showing the full-length (230 kDa ; Black Arrows) and the calpain-mediated degradation product (190 kDa ; Grey Arrows) of talin after overexpression or repression of TRPV2, in the corresponding cell lines. Tubulin was used as a loading control.


Figure 6A - D Representative bioluminescence imaging ( BLI ) data of mice injected intravenously with the non - metastatic melanoma cell line 501mel - Luc transfected with either GFP - TRPV2 or GFP control ( A - B ) , or with the invasive 451Lu - Luc melanoma cell line expressing either control shRNA or TRPV2 targeting shRNA ( C - D ) . Tumor growth and metastasis formation were monitored for 64 days ( 501mel - Luc ) or 35 days ( 451Lu - Luc ) after injection . Graphs presented in A and C show in vivo normalized photon flux quantification at the end time point ( ns P = 0 . 0903 ( A ) and * * P = 0 . 0064 ( C ) , the Mann - Withney test ) . Graphs presented in B and D show the number of metastatic foci per animal ( B ) or per lungs ( D ) counted at necropsy ( * P = 0 . 0397 ( B ) and ns P = 0 . 0691 ( D ) , unpaired t - tests ) . For ( D ) Representative ex vivo BLI images of lung metastasis are

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

Figure 6A-D Representative bioluminescence imaging (BLI) data of mice injected intravenously with the non-metastatic melanoma cell line 501mel-Luc transfected with either GFP-TRPV2 or GFP control (A-B), or with the invasive 451Lu-Luc melanoma cell line expressing either control shRNA or TRPV2 targeting shRNA (C-D). Tumor growth and metastasis formation were monitored for 64 days (501mel-Luc) or 35 days (451Lu-Luc) after injection. Graphs presented in A and C show in vivo normalized photon flux quantification at the end time point (ns P = 0.0903 (A) and **P = 0.0064 (C), the Mann-Withney test). Graphs presented in B and D show the number of metastatic foci per animal (B) or per lungs (D) counted at necropsy (*P = 0.0397 (B) and ns P = 0.0691 (D), unpaired t-tests). For (D) Representative ex vivo BLI images of lung metastasis are shown