Panels A to C show the effects of increasing sucrose concentrations on primary WT macrophages. Panel A quantifies galectin-3 puncta per cell, Panel B measures LysoTracker intensity per cell, and Panel C shows normalized SRG cytosolic intensity. Each dot represents an individual experiment. Panel D illustrates the TMEM63a targeting strategy, including LoxP sites and generation of conditional and recombined alleles. Panel E shows PCR genotyping results for TMEM63a alleles and LysM-Cre, with expected band sizes indicated. Panel F presents normalized TMEM63a mRNA expression by qPCR in wildtype and TMEM63A KO bone marrow-derived macrophages.
TMEM63A conditional KO mice and responses to the slow accumulation of solutes in macrophages. (A–C) Primary macrophages with indicated concentrations of sucrose for 16 h. In each case, the damage to lysosomes was determined by fixing and staining for galectin puncta (each dot represents the mean of the experiment), quantifying the LysoTracker intensity per cell (>5 cells per field, 15 fields, each dot represents the mean of the experiment) or SRG (>5 cells per field, 9 fields, each dot representing a field). n = 3. (D) Diagram indicating the LoxP sites flanking the 6–8 exons of Tmem63a. (E) Genotyping of Tmem63aflox/flox and LysM-Cre. (F) qPCR for WT and TMEM63A KO BMDM. n = 3. qPCR, quantitative RT-PCR. Source data are available for this figure: SourceData FS1.