Fig 2: 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).

Fig 3: CQ-reset macrophages ameliorate tumor immunosuppression. a, b, c, d C57BL/6 mice bearing subcutaneous B16 melanoma and BALB/c mice bearing H22 hepatocarcinoma ascites that received clodronate liposomes or control treatment were given i.p. injections with PBS or CQ. Representative flow cytometric analysis and quantification of MDSCs number in melanoma a (n = 6) and hepatocarcinoma b (n = 6). Representative flow cytometric analysis and quantification of Treg number in melanoma c (n = 6) and hepatocarcinoma d (n = 6). e, f, g, h C57BL/6 mice bearing subcutaneous B16 melanoma and BALB/c mice bearing H22 hepatocarcinoma ascites received intratumor injection with either PBS, CQ-treated BMDM-M2, BMDM-M2, or BMDM-M1 cells. MDSCs were quantified in melanoma e (n = 6) and hepatocarcinoma f (n = 6); Tregs were quantified in melanoma g (n = 6) and hepatocarcinoma h (n = 6). i BALB/c mice bearing H22 hepatocarcinoma ascites received i.p. injection with PBS or cell therapy with Mcoln1-SGGFP or Mcoln1-SGRNA Raw264.7 macrophages that had been pretreated with CQ or PBS. Quantification of MDSCs (left) and Tregs (right) were done (n = 3). j BALB/c mice bearing H22 hepatocarcinoma ascites received i.p. injection with PBS or cell therapy with Tfeb or control (NC) siRNA Raw264.7 macrophages that had been pretreated with CQ or PBS. Quantification of MDSCs (left) and Tregs (right) were analyzed (n = 3). k C57BL/6 mice (n = 9) with subcutaneous B16 melanoma were treated with PBS or CQ by intratumor injection along with i.p. administration of 0.5 mg CY or not. The tumor growth was followed (left). After 2 weeks, the mice were killed and the weight of the tumors was recorded (right). Black asterisk, PBS group compared with CQ, CY, and CY + CQ group; red asterisk, CQ group compared with CY + CQ group. l BALB/c mice (n = 6) bearing H22 hepatocarcinoma ascites were i.p. treated with PBS or CQ together with i.p. administration of 0.5 mg CY or not. Tumor ascites volume (left) and tumor cell number (right) were measured after one week treatment. Black asterisk, CQ group compared with PBS group; red asterisk, CQ group compared with CY + CQ group. Data shown are representative of three independent experiments and error bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not statistically significant (Student’s t-test)

Fig 4: Cd decreases TFEB expression in cultured Neuro-2a cells.(a) Representative western blot and quantification analysis for LAMP1 protein in Neuro-2a cells treated with 50 µM Cd for different times (0, 6, 12, or 24 h). ACTB was used as an internal standard for protein loading. (b) qRT-PCR analysis revealing fold expression of TFEB in Neuro-2a cells treated with 50 µM Cd for different times (0, 6, 12, 24 h). (c) Immunofluorescence of Neuro-2a cells incubated with anti-TFEB antibody and DAPI after Cd (50 µM) treatment for 24 h; scale bar: 20 µm. (d) Relative mRNA levels of TFEB target genes were determined after Neuro-2a cells were treated with 50 µM Cd for 24 h. The results are expressed as fold changes compared with the control. The values are presented as the means ± SEM. **p < 0.01 versus the control group. (n = 6).

Fig 5: Transcription factor EB (TFEB) is required for compound C1 to degrade MAPT/Tau in vitro. (a) N2a cells were transfected with P301L-Tau plasmid or empty vector (V) for 24 hr and then treated with C1 (0–1.2 µM) for 16 hr. EBSS treatment for 4 hr was used as a positive control. Data are presented as the average ± SEM from three independent experiments. *p < .05, **p < .01, ***p < .001 vs. DMSO control. (b) N2a cells expressing P301L-Tau were pretreated with C1 (1.2 µM) for 12 hr and then co-treated with bafilomycin A1 (Baf, 100 nM) for an additional 4 hr. Data are presented as the average ± SEM from three independent experiments. # p < .05, ## p < .01, ### p < .001 vs. vehicle control (DMSO); *p < .05, **p < .01, ***p < .001 vs. C1 treatment alone. (c) N2a cells with/without stable Tfeb knockdown were transfected with P301L-Tau plasmid for 24 hr and then treated with C1 (1.2 µM) for 16 hr. Data are presented as the average ± SEM from three independent experiments. ### p < .001 vs. shNT-Veh; ***p < .001 vs. shNT-C1