Panel A shows LAMP-1 (green) and Galectin-3 (magenta) staining in WT and TMEM63A KO cells under control and 1 mM LLOMe conditions. Panel B quantifies Galectin-3 puncta per cell after increasing LLOMe concentrations. Panel C and D shows Galectin-3 puncta per cell following Alum and sucrose treatment, respectively. Panel E quantifies Galectin-3 puncta in TMEM63A KO cells re-expressing TMEM63A or indicated variants after 1 mM LLOMe. Panel F shows co-staining of ALIX and Galectin-3 in WT and TMEM63A KO BMDMs treated with 0.25 mM LLOMe.
Lysosomal protection by TMEM63A is lost in patient-variants. (A–D) In all cases, the damage response for WT and TMEM63A KO RAW264.7 cells was determined by staining for galectin-3 puncta along with LAMP-1. (A and B) LLOMe at indicated concentrations was given to the cells for 20 min. (C) 100 μg/ml alum was given for 3 h. (D) 50 mM sucrose was given overnight for 16 h. These experiments were done for multiple TMEM63A KO clones, 3–5 fields per experiment. n = 3. (E) TMEM63A KO RAW264.7 cells re-expressing a TMEM63A-OFP or indicated variants of TMEM63A-OFP and challenged with 1 mM LLOMe for 20 min. Galectin puncta were quantified. n = 3. (F) WT or TMEM63A KO BMDM challenged with 0.25 mM LLOMe for 10 min were immunostained with galectin-3 (magenta) and ALIX (green).