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. 2021 Oct 28;11:21268. doi: 10.1038/s41598-021-99958-7

Figure 5.

Figure 5

Flcn loss activates autophagy in a mouse model of liver fibrosis. (A-B) Unsupervised hierarchical clustering following RNA-seq analysis in livers of mice fed either on chow or methionine/choline deficient diet (MCD) over a period of 6 weeks. (C) Relative quantitative real‐time PCR analysis of autophagy-related genes mRNA transcript levels in livers of mice fed as described in (A) (mean ± SEM of the RNA fold change of indicated mRNAs; two-way ANOVA, *P < 0.05, **P < 0.01, ****P < 0.0001; n = 8 mice per condition). (D) Immunoblot of liver protein lysates extracted from mice fed as described in (A). Data are representative of 6 mice per condition. Full-length blots are presented in Supplementary Fig. 5. (E) Quantification of the relative amount of p62 immunoblot. Data normalized to tubulin levels (mean ± SEM of the relative fold change; two-way ANOVA, *P < 0.05, **P < 0.01, ****P < 0.0001; n = 6 mice per condition). (F) Relative RNAseq analysis of p62 mRNA transcript levels in livers of mice fed as described in (A) (mean ± SEM of p62 RNA fold change; one-way ANOVA, ***P < 0.001; n = 5 mice per condition). (G) Immunoblot of protein lysates of WT and Flcn KO MEFs incubated in presence of DMSO, BafA1 (100 nM), or Chloroquine (CQ; 100 μM) for 6 h. Data are representative of three independent experiments. Full-length blots are presented in Supplementary Fig. 5. (H) Quantification of the relative amount of LC3-II/LC3-I immunoblot of MEFs treated as described in (G) (mean ± SEM of three independent experiment, one-way ANOVA, *P < 0.05). (I) TFE3 Immunohistochemistry (IHC) staining in liver sections of mice fed as described in (A) Data are representative of 6 mice per condition. (J) Quantification of TFE3 IHC staining in liver sections of mice fed as described in (A) (mean ± SEM, two-way ANOVA, ***P < 0.001; n = 4 mice per condition). (K) TFEB Immunohistochemistry (IHC) staining in liver sections of mice fed as described in (A) Data are representative of 4 mice per condition. (L) Quantification of TFEB IHC staining in liver sections of mice fed as described in (A) (mean ± SEM, two-way ANOVA, ***P < 0.001; n = 4 mice per condition).