The image contains multiple panels examining TMEM63A function in macrophages. Panel A shows fluorescence lifetime measurements in WT RAW264.7 cells under control conditions and after treatment with LLOMe or GPN. Panel B presents relative gene expression of TMEM63A, TMEM63B, and TMEM63C across RAW264.7 cells, BMDM, and MEF. Panels C and D display TMEM63A-OFP and LAMP1-GFP localization in RAW264.7 cells, highlighting lysosomal structures. Panel E and F show dextran and SRG uptake in WT and TMEM63A KO BMDMs, respectively. Panel G quantifies lysosomal pH in WT and TMEM63A KO BMDMs. Panel H shows immunoblot analysis of Lamp-1, pro-cathepsin C, cathepsin C, and GAPDH. Panel I quantifies relative DQ-BSA fluorescence. Panel J shows phagocytosis assays in WT and TMEM63A KO macrophages, and Panel K provides the corresponding phagocytic index quantification.
Lysosomal membrane tension and TMEM63A expression and targeting in macrophages. (A) Lyso-Flipper FLIM in WT RAW264.7 macrophages incubated with 1 mM LLOMe for 5 or 15 min, or 250 µM GPN for 15 min. Each dot represents one field of 5–10 cells. n = 3. (B) qPCR for TMEM63A, B, and C in RAW264.7, BMDM, or MEF. n = 3. (C and D) TMEM63A-OFP (magenta) and LAMP1-GFP (green) in WT RAW264.7 cells before and after challenge with IgG-coated microspheres. (E and F) 10-kDa dextran or SRG pulsed for 16 h and chased for 1 h in WT and TMEM63A KO BMDM. (G) Lysosomal pH in WT and TMEM63A KO BMDM determined using ratiometric measurements of 10-kDa Oregon Green dextran pulsed for 16 h and chased for 1 h. Each data point represents a field containing >5 cells. n = 3. (H) LAMP-1, cathepsin C expression, and processing by WB. (I) DQ-BSA signal of WT and TMEM63A KO BMDM after pulse (1 h) and chase (1 h). All data points represent a field containing 4–8 cells. n = 3. (J and K) Phagocytosis in WT and TMEM63A KO BMDM, quantified in K. Each data point represents 3–5 fields containing 15–25 cells. n = 3; scale bars 10 µm. Source data are available for this figure: SourceData F2.