Figure 3HeLa cells stably expressing CHMP4B-eGFP were transfected with siRNAs against: (A) HR 48 h post-transfection cells were used for live-cell imaging experiments. LLOMe was added using perfusion system. A. Depletion of HRS does not alter the dynamics of CHMP4B-eGFP recruitment as compared to siCtrl during treatment with LLOMe Data information: The quantification graphs represent average CHMP4B foci per cell quantified from three independent live-cell imaging experiments per condition. Error bars correspond to 95% confidence intervals. Data from >56 cells per condition were analyzed in each experiment. Right panels show knockdown efficiency of siRNA oligos as detected by Western blot (*, nonspecific immunoreactivity) HeLa cells stably expressing CHMP4B-eGFP were transfected with siRNAs agains (B) TSG10 48 h post-transfection cells were used for live-cell imaging experiments. LLOMe was added using perfusion system B. Downregulation of TSG101, with two independent siRNAs causes a delay in CHMP4B-eGFP recruitment as compared to siCtrl, indicating an important role of the ESCRT-I complex in recruiting the downstream components Data information: The quantification graphs represent average CHMP4B foci per cell quantified from three independent live-cell imaging experiments per condition. Error bars correspond to 95% confidence intervals. Data from >56 cells per condition were analyzed in each experiment. Right panels show knockdown efficiency of siRNA oligos as detected by Western blot (*, nonspecific immunoreactivity) HeLa cells stably expressing CHMP4B-eGFP were transfected with siRNAs agains (C) CHMP2 48 h post-transfection cells were used for live-cell imaging experiments. LLOMe was added using perfusion system C. CHMP2A depletion accumulates and stabilizes CHMP4B-eGFP positive foci Data information: The quantification graphs represent average CHMP4B foci per cell quantified from three independent live-cell imaging experiments per condition. Error bars correspond to 95% confidence intervals. Data from >56 cells per condition were analyzed in each experiment. Right panels show knockdown efficiency of siRNA oligos as detected by Western blot (*, nonspecific immunoreactivity) HeLa cells stably expressing CHMP4B-eGFP were transfected with siRNAs against (D) ALI 48 h post-transfection cells were used for live-cell imaging experiments. LLOMe was added using perfusion system D. siRNA mediated depletion of ALIX shows no significant change on dynamics of CHMP4B recruitment Data information: The quantification graphs represent average CHMP4B foci per cell quantified from three independent live-cell imaging experiments per condition. Error bars correspond to 95% confidence intervals. Data from >56 cells per condition were analyzed in each experiment. Right panels show knockdown efficiency of siRNA oligos as detected by Western blot (*, nonspecific immunoreactivity) HeLa cells stably expressing CHMP4B-eGFP were transfected with siRNAs against (E) both TSG101 and ALIX 48 h post-transfection cells were used for live-cell imaging experiments. LLOMe was added using perfusion system E. Simultaneous depletion of TSG101 and ALIX caused almost no recruitment of CHMP4B upon induction of endolysosomal damage Data information: The quantification graphs represent average CHMP4B foci per cell quantified from three independent live-cell imaging experiments per condition. Error bars correspond to 95% confidence intervals. Data from >56 cells per condition were analyzed in each experiment. Right panels show knockdown efficiency of siRNA oligos as detected by Western blot (*, nonspecific immunoreactivity)