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. 2019 Jan 29;15(6):1082–1099. doi: 10.1080/15548627.2019.1569933

Figure 9.

Figure 9.

Lysosomal homeostasis is dysregulated in VCP diseased skeletal muscle. (a) H & E of TA from 13-month-old control (C57) or VCPR155H/+ heterozygous knockin mice. (b) Immunoblot of lysates from the TA from 13-month-old control or VCPR155H/+ knockin mice with antibodies to VCP, SQSTM1, TFEB, p-RPS6, RPS6, LGALS3, HSPA5, TARDBP, ubiquitin and GAPDH. N = 3 per group. (c) Densitometric analysis of TFEB, LGALS3, HSPA5, and TARDBP protein levels in VCPR155H/+ mice as compared to controls. N = 3 per group. (d) Fluorescence microscopy of control or VCPR155H/+ mouse TA muscle electroporated with plasmids expressing LAMP1-GFP (green) or mCherry-LGALS3 (red). (e) Quantification of the average number of LGALS3+ LAMP1 puncta in control or VCPR155H/+ mouse TA muscle electroporated with plasmids expressing LAMP1-GFP or mCherry-LGALS3. N = 30 co-electroporated fibers were counted per condition. (f) Immunofluorescence for TFEB (red) in TA from 13-month-old control or VCPR155H/+ knockin mice. Arrows point to nuclear-localized TFEB. (g) Quantification of the percent of TFEB-positive nuclei in tibialis anterior muscle from 13-month-old control or VCPR155H knockin mice. Quantification was performed by counting the number of nuclei stained with TFEB using ImageJ software. At least 1028 nuclei were counted per group from 3 different mice. Comparison between groups was performed by paired Student t-test. DAPI (blue) stains nuclei. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant. Scale: 100 μm.