Skip to main content
. 2023 May 5;9(18):eadd2676. doi: 10.1126/sciadv.add2676

Fig. 4. TMEM106B deficiency results in lysosomal abnormalities in microglia.

Fig. 4.

(A) Western blot analysis of lysosomal proteins in the control and Tmem106b−/− BV2 cells using the indicated antibodies. The levels of each protein were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data from three independent experiments were plotted. (B) Reduced CathD/E activities in TMEM106B-deficient BV2 cells. CathD/E activities were measured in control and Tmem106b−/− BV2 cells using fluorogenic substrates. Data represent the means ± SEM. Statistical significance was analyzed by unpaired one-tailed Student’s t test (n = 3). *P < 0.05. (C) Lysosomal pH measurement in control and Tmem106b−/− BV2 cells. BV2 cells were incubated with dextran conjugated with pH-sensitive fluorescein (green) and pH-insensitive tetramethylrhodamine (red). Representative images were shown. Scale bar, 10 μm. FITC and TRITC intensity per cell were quantified and the ratio (FITC/TRITC) was calculated. Lysosomal pH was determined from the calibration curve generated from the FTIC/TRITC value of permeabilized cells in various calibration standard solutions of different pH values. Data represent the means ± SEM. Statistical significance was analyzed by unpaired one-tailed Student’s t test (n = 3). *P < 0.05. (D and E) WT and KO mice were fed with CPZ-containing chow (CPZ 5w) for 5 weeks or fed with CPZ for 5 weeks and a normal diet for additional 3 weeks after CPZ removal (Rec 3w). Brain sections were stained with LAMP1, CathD, and IBA1 antibodies. Representative confocal images in the corpus callosum region were shown (A). Scale bars, 10 μm. The LAMP1 or CathD levels in microglia were quantified (B). Data represent the means ± SEM. Statistical significance was analyzed by unpaired two-tailed Student’s t test (n = 3 mice per group). *P < 0.05 and **P < 0.01.