Extended Data Fig. 2: Selective PtdIns4P production on damaged lysosomes in different cell lines.
From: A phosphoinositide signalling pathway mediates rapid lysosomal repair
a, b, OSBP-PH-GFP is recruited to damaged lysosomes in U2OS cells as shown by its colocalization with the lysosomal markers LAMP2 (a) and CD63 (b) upon lysosome damage. U2OS cells stably expressing OSBP-PH-GFP were treated with 1 mM LLOME for 30 min and then fixed for immunostaining of the endogenous indicated lysosomal markers. c, OSBP-PH-GFP is recruited to damaged lysosomes in human PC3 prostate adenocarcinoma cells. Cells stably expressing OSBP-PH-GFP were treated with 1 mM LLOME for 30 min and then fixed for immunostaining of endogenous IST1, an ESCRT-III subunit and marker for damaged lysosomes. Pearson’s correlation coefficient of OSBP-PH-GFP and IST1 were quantified; mean ± sem; n = 11 cells from 3 trials for each condition. d, An alternative PtdIns4P probe GFP-P4M is recruited to damaged lysosomes in COS7 cells. Cells were transiently transfected with GFP-P4M. After 24 h, cells were treated with 1 mM LLOME for 30 min and then fixed for immunostaining of endogenous IST1 and LAMP1. Pearson’s correlation coefficient of GFP-P4M and IST1 were quantified; mean ± sem; n = 9 (− LLOME) and 24 (+ LLOME) cells over 3 trials. e, f, Probes for PI(4,5)P2 (e) or PI3P (f) are not recruited to damaged lysosomes. U2OS cells stably expressing the indicated probe were treated with 1 mM LLOME for indicated time periods and then fixed for immunostaining of LAMP1 or IST1. g, Schematic illustration of the dispensability for an intact Golgi in the recruitment of OSBP-PH-GFP to damaged lysosomes. h, Disassembly of the Golgi complex by Brefeldin A does not affect the recruitment of the PtdIns4P probe OSBP-PH-GFP to damaged lysosomes. Cells were pretreated or not with 2 μM Brefeldin A for 10 min followed by the addition of 1 mM LLOME for 30 min. Cells were then fixed for the staining of endogenous LAMP1. i, Lysosomal damage by LLOME does not affect the overall morphologies of the cis- (GM130) and trans-Golgi (Golgin-97) complex. U2OS cells were treated as indicated and stained for endogenous markers of the Golgi complex. DAPI stains the nuclei. Bar, 10 μm. Source data for graphs in this Figure are provided.
