Fig 2: MYC occupies the promoters of lysosomal genes and that of TFEB and TFE3. a Motif analysis using HDAC2-binding sites present in lysosomal genes. b Left, silencing of HDAC2 downregulated MYC protein expression in HeLa cells. Right, Coomassie stained immunoblot used as the loading control. c Quantification of MYC levels in HDAC2 silenced HeLa cells normalized to loading control (n = 3 independent experiments). d Co-immunoprecipitation of MYC with HDAC2 from lysates obtained from HeLa cells. HDAC2 band is labeled with an asterisk. Heavy chain IgG is labeled with an arrow. e Immunoprecipitation of MYC followed by immunoblot of MYC protein. MYC band is labeled with an asterisk. Heavy chain IgG is labeled with an arrow. f Graph represents HDAC2 binding to the promoter of MYC (n = 5 biologically independent samples) in HeLa cells treated with DMSO and SAHA (20 µM for 24 h); IgG control (n = 3 biologically independent samples). g–i Myc binding to the promoters of (g) Neu1, Ctsa, (h) Lamp1, Mcoln1 and i Tfeb and Tfe3 was analyzed in ChIP-seq datasets performed with anti-Myc antibody in mouse group 3 medulloblastoma cells overexpressing Myc (Trp53–/– overexpressing Myc). Input DNA (Inp) serves as reference. j, k Histograms represent MYC binding to the promoters of (j) NEU1 (n = 3 biologically independent samples), LAMP1 (n = 3 biologically independent samples), MCOLN1 (n = 3 biologically independent samples), TFEB (n = 6 biologically independent samples), TFE3 (n = 3 biologically independent samples) and (k) MYC (n = 3 biologically independent samples) in HeLa cells treated with SAHA (20 µM for 24 h) or DMSO. NEU1 INT oligos were used as negative control for non-specific antibody binding (n = 3 biologically independent samples); IgG control (n = 3 biologically independent samples). l Sequential ChIP experiments were performed from HeLa cells with anti-MYC antibody followed by anti-HDAC2 antibody and analyzed by RT-qPCR at the promoter region of TFEB and TFE3 (n = 3 biologically independent samples); IgG control (n = 3 biologically independent samples). All the graphs are presented as mean ± SD. Statistical analysis was performed using the Student t-test. *p < 0.05, **p < 0.01, ****p < 0.0001

Fig 3: SARS 3a drives TFEB translocation to nucleus via calcineurin.a Immunoblots showing endogenous TFEB after nuclear fractionation of 293T cells transfected with SARS 3a-flag or SARS 3a C133A-flag (SARS 3a* flag). Histone H3 and GAPDH are used as fractionation and loading controls. b Subcellular localization analysis by confocal microscopy of HeLa cells expressing TFEB-mCherry with SARS 3a-GFP or a GFP-vector. c Quantification of immunofluorescence data in b. d Live time-lapse confocal microscopy of SARS 3a-GFP driving TFEB-mCherry nuclear translocation, after co-expression of SARS 3a-GFP and TFEB-mCherry. e Immunoblot showing endogenous TFEB after nuclear fractionation of 293T cell lysates after transfection with SARS 3a-flag and either PPP3CB or control siRNA. f Immunoblot showing endogenous TFEB after nuclear fractionation of 293T cell lysates after transfection with SARS 3a-flag and treatment with cyclosporine. g qRT-PCR expression analysis of TFEB target genes after SARS 3a or SARS 3a C133A overexpression. Bars show the mRNA level fold change normalized to GAPDH and relative to the vector control. h Immunoblot analysis of TFEB target proteins expression level after transfection with SARS 3a-flag or SARS 3a C133A-flag (SARS 3a* flag). All western data are representative of two or three independent experiments. Western bands are quantified and normalized to the loading control then presented relative to the control lane. Confocal imaging done at ×100 electronic zoom, scale bar 10 µm. qRT-PCR data are the average and SEM of n = 3 independent experiments in triplicate (*p < 0.05; **p < 0.001, ***p < 0.0001, ordinary one-way ANOVA with post hoc Tukey’s HSD)

Fig 4: LRRK2G2019S-mediated TFEB nuclear translocation requires Ca2+, NAADP, and TPCN2 (A) Confocal imaging in HeLa cells expressing TFEB-mCherry and a control vector (MT) or His-LRRK2G2019S with or without BAPTA-AM (fast calcium chelator, 10 µM, 1 h), EGTA-AM (slow calcium chelator, 10 µM, 1 h), FK506 (calcineurin inhibitor, 5 nM, 1 h), control siRNA, PPP3CB siRNA, or MCOLN1 siRNA. Scale bar: 10 µm. (B) Quantification TFEB nuclear translocation from (A) (n = 3, = 50 cells/repeat). (C) Confocal imaging in HeLa cells expressing TFEB-mCherry and a control vector (MT), or TFEB-mCherry and His-LRRK2G2019S with or without Ned-19 (NAADP inhibitor, 100 µM, 1 h), PERK inhibitor (PERK-I, 10 µM, 1 h), control siRNA, or TPCN2 siRNA. Scale bar: 10 µm. (D) Quantification of TFEB nuclear translocation from (C) (n = 3, = 50 cells/repeat). (E) Immunoblot of endogenous TFEB from nuclear fractions of HEK293T cells transfected with a control vector or His-LRRK2G2019S (GS) and either control siRNA or PPP3CB siRNA (PPP3CB KD). (F) Immunoblot of endogenous TFEB from nuclear fractions in HEK293T cells transfected with a control vector or His-LRRK2G2019S (GS) and treated with Ned-19 (100 µm, 2 h). (G) Confocal imaging in HeLa cells transfected with TFEB-mCherry and TPC2-EGFP. DAPI and TFEB-mCherry colocalization channel is shown on the right. Scale bar: 10 µm. (H) Quantification of % cells exhibiting TFEB nuclear translocation from (G) (n = 3, = 50 cells/repeat). (I) Immunoblotting of endogenous TFEB following nuclear fractionation of HEK293T cells expressing TPCN2-EGFP. For western data, histone H3 and GAPDH are shown as fractionation and loading controls. Western bands are quantified, normalized to the loading control, and presented relative to control lane. (*p < 0.05; **p < 0.002, ***p < 0.0001, one-way ANOVA with post hoc Tukey’s HSD).

Fig 5: mTOR is activated in TECs under diabetic condition. (a, b) Western blot assay and quantification of p-mTOR level in renal tissues from db/db and m/m mice. Data are presented as mean ± SEM. (c) Immunofluorescence staining of mTOR and LAMP2 in HK-2 cells after exposure to increasing concentrations of AGE-BSA for 12 h. Representative of images is shown. Arrows denote colocalization of mTOR with LAMP2. DAPI was used to stain the nuclei. Scale bar, 10 μm. Boxed areas are magnified and shown in the bottom panel. (d, e) Western blot assay and quantification of p-mTOR level in HK-2 cells after exposure to increasing concentrations of AGE-BSA alone or AGE-BSA (50 μg/ml) plus Torin1 (1.5 μM) for 12 h. Bars represent the mean ± SEM for at least three independent experiments. (f) Immunofluorescence staining of TFEB in HK-2 cells, which were treated with Co-BSA and AGE-BSA (50 μg/ml) and AGE-BSA (50 μg/ml) plus Torin1 (1.5 μM) for 12 h, respectively. ∗P < 0.05 and ∗∗P < 0.01.