The image contains multiple panels illustrating the effects of sucrose, alum, LLOMe, and GPN on WT and TMEM63A KO macrophages. Panel A shows SRG staining in WT and TMEM63A KO macrophages treated with 50 mM sucrose. Panels B and C quantify normalized cytosolic SRG intensity per field. Panels D and E quantify galectin-3 puncta per cell after sucrose or alum treatment, respectively. Panels F and G show images and quantification of SRG loaded lysosomes of WT versus TMEM63A KO macrophages treated with 0.25 mM LLOMe. Panel H shows galectin-3 staining after 0.25 mM LLOMe treatment, and Panel I quantifies galectin-3 puncta per cell. Panel J shows ALIX staining following 0.25 mM LLOMe treatment, and Panel K quantifies ALIX puncta per cell. Panel L shows normalized cytosolic SRG intensity after GPN treatment, and Panel M quantifies galectin-3 puncta per cell under the same conditions. Panel N shows propidium iodide (PI) positivity in RAW264.7 cells treated with various concentrations of LLOMe. Panel O shows WT and TMEM63A KO BMDMs challenged with yeast-locked C. albicans or Δece1 C. albicans. Panel Q quantifies the percentage of cells with ruptured phagosomes.
TMEM63A protects cells from lysosomal damage and cell death. (A–E) WT or TMEM63A KO BMDM given SRG and either 50 mM sucrose overnight or 75 µg/ml alum for 4 h. Cytosolic SRG signal (B and C) quantified per field of 5–10 cells or galectin-3 puncta per cell (D and E), each data point representing 5 fields. n = 3. (F–K) WT and TMEM63A KO BMDM treated with 0. 25 mM LLOMe as in Fig. 1. Lysosomal damage was quantified accordingly. In J and K, ALIX was immunostained and puncta above a threshold size per cell quantified. Each dot represents mean of experiment (>5 cells per field, 15 fields total), n = 3. (L and M) Damage in WT and TMEM63A KO BMDM upon GPN. n = 3. (N) Cell death upon LLOMe treatment for 30 min in WT and TMEM63A KO RAW 264.7 cells was determined using propidium iodide. n = 3. (O–Q) BMDM challenged with Δece1 C. albicans-BFP or yeast-locked C. albicans, internalized by phagocytosis, and allowed to grow in the phagolysosome for 2.5 h (O). % of cells with ruptured phagosomes quantified per field of 4–8 cells in Q. n = 3; scale bars 10 µm.