Fig 2: Rab7b affects macropinocytosis and lysosome signaling. (A) LPS-DCs were loaded in 5×8 µm micro-fabricated channels. After 16 h, the channels were filled with 10 kDa Alexa Fluor 647-conjugated dextran (magenta) and the cells were imaged 30 min later. Representative images of live WT and Rab7b KO cells are shown. Scale bars: 10 µm. (B) Quantification of the area of internalized dextran in WT and Rab7b KO cells. The graph represents the mean±s.d. of three independent experiments (n=39 and 32 cells for WT and Rab7b-KO, respectively). *P<0.05 (two-tailed unpaired Student's t-test). (C) Distribution of macropinosome numbers for WT and Rab7b KO cells from three independent experiments. Data represents the mean±s.d. (n=39 and 32 cells for WT and Rab7b KO, respectively). **P<0.01 (two-tailed unpaired Student's t-test). (D) Quantification of the lifetime of macropinosomes in WT and Rab7b KO cells. Data represents the mean±s.d. of three independent experiments (>100 tracked macropinosomes per condition, n=23 and 28 cells for WT and Rab7b KO, respectively). ***P<0.001 (two-tailed unpaired Student's t-test). (E) Spinning disk images of live WT and Rab7b KO LPS-DCs stained with Alexa Fluor 594-conjugated WGA to label lysosomes (cyan) and loaded in 5×8 µm micro-fabricated channels filled with 10 kDa Alexa Fluor 647-conjugated dextran (magenta). Representative images from one out of three independent experiments are shown. Scale bar: 10 µm. The magnified area shows a lysosome (arrow) moving towards a macropinosome in a control cell. Image contrast has been increased to improve visualization. (F) Quantification of the number of lysosomes with area <0.5 µm2 per cell. Data represents the mean±s.d. of three independent experiments (n=29 cells for WT and Rab7b-KO). *P<0.05 (two-tailed unpaired Student's t-test). (G) BMDCs from WT and Rab7b KO mice were pulsed with 100 ng/ml LPS for 30 min and lysed after 6 h. Cytosolic and nuclear fractions were subjected to immunoblotting analysis with the indicated antibodies. Histone 3 was used as control of the cytosolic and nuclear fraction separation. (H) The graph shows the quantification of the nucleus-to-cytosol ratio for TFEB in Rab7b-KO DCs relative to WT. Data represents the mean±s.d. of four independent experiments. **P<0.01 (two-tailed unpaired Student's t-test).

Fig 3: Immunofluorescence examination of endogenous transcription factor EB (TFEB) localization in: (i) HaCaT cells activated with “cytokine mix” and cultured in medium containing Ca2+ = 0.1 mM; (ii) non-activated keratinocyte control cultured in medium containing Ca2+ = 0.1 mM; (iii) “cytokine mix”-activated HaCaT cells cultured in medium containing 2 mM Ca2+; and (iv) non-activated keratinocyte control cultured in medium containing 2 mM Ca2+. Representative microscopy images from randomly selected ten microscopic fields containing 50–100 cells, each from three independent experiments (n = 3) of the tested keratinocyte cultures demonstrate the level of TFEB translocation from the cytoplasm to the nucleus. Scale bar represents 0.25 µm.

Fig 4: CO enhances mitophagy through TFEB activation.a–c Primary hepatocytes were treated with CORM2 for 6 h at the indicated concentrations (a) or for the indicated times at 20 µM (b and c). The levels of LC3B-II and TOM20 were determined by immunoblotting. d Primary hepatocytes were treated with CORM2 (20 µM) in the presence of bafilomycin A1 (Baf A1), autophagy flux inhibitor. The cells were collected and cell lysates were subjected to immunoblotting to detect the expression of LC3B. e Primary hepatocytes were transfected with mcherry-GFP-LC3 for 24 h. After transfection, cells were treated with CORM2 (20 µM) or Torin 1 (1 µM) for 6 h, and analyzed by confocal fluorescence microscopy. Scale bar, 20 µm. The number of autolysosomes (red puncta) per cell (n = 10) was counted. Data are shown as mean ± SEM. ***P < 0.001 vs. control. f, g Primary hepatocytes were transfected with scrambled RNA (scRNA) or siRNA against TFEB (siTFEB) for 24 h, and then treated with CORM2 (20 µM) for the indicated times (f) or at the indicated doses for 6 h (g). The levels of LC3B-II, TOM20, and TFEB were determined by immunoblotting. h Mitochondrial fractions isolated from primary hepatocytes treated with CORM2 (20 µM) for the indicated times were analyzed by immunoblotting using antibodies against PINK1 and Parkin. i HepG2 were transfected with scRNA or siTFEB for 24 h and then treated with CORM2 (20 µM) for 6 h. Cells were fractionated into cytosol and mitochondria, and the levels of PINK1 and Parkin were analyzed by immunoblotting. Fraction quality was verified by immunoblotting with markers for the cytoplasm (a-tubulin) and mitochondria (cytochrome IV oxidase, COX IV). j Left panel, primary hepatocytes were transfected with GFP-mitoTS and RFP-LC3 for 24 h. After transfection, cells were treated with CORM2 (20 µM) for 3 h and analyzed by confocal fluorescence microscopy. Scale bar, 10 µm. Right graph, GFP-LC3 punctate structures associated with mitochondria were quantified. Values represent mean number of puncta counts per cell, n = 20 cells from two independent experiments. ***P < 0.001 vs. control

Fig 5: TFEB and TFE3 promote transcription of the retromer complex in a CLEAR element-dependent manner. (A) Representative IF images of endogenous TFEB in control and VPS35 knockout (KO) HeLa cells. Scale bar, 20 µm. (B) Schematic of the promoter-luciferase reporter constructs used in C and D indicating the location (relative to TSS) and sequence of CLEAR elements in the retromer genes VPS35 and VPS26A and the sequence of the mutated CLEAR elements that were generated. (C and D) Luciferase activity measured in HEK293T cells lentivirally transduced with GFP, TFEB-GFP, or TFE3-GFP and transiently expressing the indicated wild type or mutated CLEAR element VPS35 (C) or VPS26A or VPS26B (D) promoter-luciferase reporter construct. Bars represent a fold increase in luciferase activity relative to cells infected with GFP from three independent experiments; two-way ANOVA followed by Dunnett’s multiple comparison. (E) Quantitative RT-PCR analysis of the retromer complex gene expression in HeLa cells lentivirally transduced with GFP or TFEB-GFP. Data were normalized to ACTB. Bars represent a fold change relative to GFP-transduced cells; n = three independent experiments; two-way ANOVA followed by Sidak’s multiple comparison. (F) mRNA levels of TFEB and VPS35 in HeLa cells treated with L-glutamine starvation for 4 h. Values normalized to ACTB and expressed as a fold change relative to untreated cells (complete media); n = three independent experiments; Student’s t test (unpaired). (G and H) HeLa cells were treated with nontarget siRNA or siRNA to TFEB and/or TFE3 for 72 h. Cells were then maintained in complete media (control) or in EBSS (starved) for 4 h. Total RNAs were extracted, and mRNA transcript abundance was assessed using primers specific for VPS35 (G) or VPS26A (H). Data were normalized to ACTB. Bars represent a fold change relative to nontarget siRNA control cells from at least three experiments; one-way ANOVA followed by Dunnett’s multiple comparison. (C–H) Data represent mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. NT, nontarget.