Figure 5. The PI4K2A‐mediated lysosome repair pathway is activated independently of ESCRTs.

1. HeLa cells were co‐transfected with siRNAs targeting ESCRT proteins TSG101 and ALIX (siTSG101 + siALIX) or with non‐targeting siRNA control (siControl) and incubated for 24 h before transfection with the PtdIns4P binding probe 2xSidM‐eGFP. Twenty‐four‐hour post‐transfection, lysosomes were damaged with 250‐μM LLOMe and PtdIns4P recruitment to damaged lysosomes was monitored using the 2xSidM‐eGFP probe in live‐cell imaging experiments. The graph shows the quantification of 2xSidM‐eGFP foci per cell in cells co‐depleted of TSG101 + ALIX and control (siControl) cells. Error bars denote ± SD from n = 2 independent live‐cell imaging experiments, > 27 cells were analyzed per experiment for each condition.
2. Representative fluorescence micrographs of HeLa‐PI4K2A knockout cells treated with 250‐μM LLOMe or an equal volume of DMSO (Pre) for 5 min, 15 min, and 1 h before fixation and immunostained with Hoechst (blue), anti‐CHMP4B (red), anti‐GAL3 (green), and anti‐ALIX (white).