Fig. 7

HDAC7 regulates TFEB acetylation and lysosomal biogenesis. A Representative blots showing HDAC7-TFEB interaction and TFEB acetylation level by immunoprecipitation conducted in primary astrocytes overexpressing HDAC7 or vector. B Quantification of TFEB acetylation in vector and HDAC7-overexpressing astrocytes. n = 5 per group. C Representative immunostaining images of TFEB in vector and HDAC7-overexpressing astrocytes treated with Torin1 (100 nM) or vehicle for 4 h. Scale bar:10 μm. D Quantification of nuclear/cytoplasmic TFEB ratio in C. n = 31 (Vec), 32 (HDAC7), 38 (Vec+Torin1), 38 (HDAC7+Torin1) cell per group. E Analysis of TFEB transcriptional activity in HEK293T cells transfected with HDAC7 or vector and then treated with Torin1 (100 nM) or vehicle for 4 h. n = 4 per group. F mRNA levels of Lamp1, Ctsb, Ctsd, Ctsf and Map1lc3b measured by RT-qPCR in cultured astrocytes treated as described in C. n = 4 per group. G, H Immunoprecipitation assay showing the acetylation level of TFEB in WT and HDAC7 knockout astrocytes. n = 3 per group. I Representative immunostaining images of TFEB in control and HDAC7 knockout astrocytes. Scale bar: 10 μm. J Quantification of nuclear/cytoplasmic TFEB ratio in I. n = 31 (HDAC7^+/+), 37 (HDAC7^-/-). K Analysis of TFEB transcriptional activity in HEK293T cells transfected with shRNA targeting HDAC7 (shHD7) or control scramble. n = 4 per group. L mRNA levels of Lamp1, Ctsb, Ctsd, Ctsf, Map1lc3b and Atg9b quantified by RT-qPCR in control and HDAC7 knockout astrocytes. n = 4 per group. M, N Western blotting analysis and quantification of LAMP1, CTSB and CTSD in control and HDAC7 knockout astrocytes. n = 6 per group. Statistical significance was determined by unpaired Student’s t test (B, H, J-L and N) or one-way ANOVA with Tukey’s post hoc analysis (D-F). Data are shown as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001