Journal: Stem Cell Research & Therapy

Article Title: Human umbilical cord-derived mesenchymal stem cells ameliorate non-alcoholic fatty liver disease via activating TFEB-mediated autophagy in male mice

doi: 10.1186/s13287-025-04855-9

Figure Lengend Snippet: hUC-MSCs induced autophagy by regulating the AMPK-mTOR-TFEB signaling axis in the liver of mice and HepG2 cells. The protein levels of p-AMPK, AMPK, p-mTOR, and mTOR in the livers of mice ( A ) ( n = 3 per group) and HepG2 hepatocytes ( B ) were detected by western blot. Expression levels were normalised by GAPDH. C Representative Oil Red O staining images of HepG2 cells in the indicated groups. After release from PA/OA treatment, HepG2 cells were treated with or without hUCMSCs-CM, 10 µM Compound C or 5 µM MHY1485 for 12 h. Compound C, an AMPK inhibitor; MHY1485, a mTOR activator. The protein levels of TFEB in the nuclei of mouse liver cells ( D ) and HepG2 cells ( E ) were measured after nucleo-cytoplasmic fractionation. Expression levels were normalised by Histone-H3. All full-length western blots are presented in Supplementary material 1: Fig. 6. F . Fluorescence microscopy images of HepG2 hepatocytes transfected with TFEB-GFP plasmids for each condition. Nuclei were stained with DAPI. Scale bar, 5 μm. Data were expressed as mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: To further validate the TFEB knockdown phenotype in HepG2 cells, a synthetic human TFEB shRNA plasmid was purchased from Shanghai Genechem.

Techniques: Western Blot, Expressing, Staining, Fractionation, Fluorescence, Microscopy, Transfection

Journal: Stem Cell Research & Therapy

Article Title: Human umbilical cord-derived mesenchymal stem cells ameliorate non-alcoholic fatty liver disease via activating TFEB-mediated autophagy in male mice

doi: 10.1186/s13287-025-04855-9

Figure Lengend Snippet: TFEB knockdown blocked hUC-MSCs-induced autophagy and impaired ameliorative effects on lipid metabolism, inflammation and fibrosis in NAFLD models. A Representative Oil Red O staining images of HepG2 cells in the indicated groups. B The TG concentrations in HepG2 cells in each group were measured. C The levels of lipid metabolism-associated protein (FASN and SREBP1c) and fibrosis-associated protein (α-SMA and COL1A1) in HepG2 hepatocytes. D The secretion of the inflammatory marker IL-1β in the culture medium was detected using ELISA. E The levels of autophagy-associated proteins in HepG2 cells exposed to PA/OA. All full-length western blots are presented in Supplementary material 1: Fig. 7. F Representative images showing mCherry-LC3 puncta in HepG2 cells after TFEB knockdown. Nuclei were stained with DAPI. Scale bar: 10 μm. G mCherry-LC3 (red) puncta per cell were counted. Data were expressed as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001

Article Snippet: To further validate the TFEB knockdown phenotype in HepG2 cells, a synthetic human TFEB shRNA plasmid was purchased from Shanghai Genechem.

Techniques: Knockdown, Staining, Marker, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Stem Cell Research & Therapy

Article Title: Human umbilical cord-derived mesenchymal stem cells ameliorate non-alcoholic fatty liver disease via activating TFEB-mediated autophagy in male mice

doi: 10.1186/s13287-025-04855-9

Figure Lengend Snippet: Schematic diagram of hUC-MSCs alleviating NAFLD. hUC-MSCs alleviated steatosis, inflammation and fibrosis in NAFLD via inducing autophagy, which was mediated through AMPK-mTOR-TFEB signaling pathway

Article Snippet: To further validate the TFEB knockdown phenotype in HepG2 cells, a synthetic human TFEB shRNA plasmid was purchased from Shanghai Genechem.

Techniques:

Journal: Cell death discovery

Article Title: CO confers neuroprotection via activating the PERK-calcineurin pathway and inhibiting necroptosis.

doi: 10.1038/s41420-025-02530-9

Figure Lengend Snippet: Fig. 5 TFEB nuclear export is prevented by CO-induced GSK-3β inhibition. a To assess the effect of CO on inhibition of TFEB nuclear export, SH-SY5Y cells transiently expressing TFEB-GFP were starved for amino acids for 2 h and then refed with complete DMEM for 2 h in the presence or absence of 20 μM CORM-A1 and the nuclear export inhibitor LMB (20 nM) and analyzed by confocal microscopy. b SH-SY5Y cells were co-transfected with TFEB-GFP, CA-GSK-3β, or KD-GSK-3β. The transfected cells were treated with 20 μM CORM-A1 for 2 h after cells were refed with complete DMEM. Fluorescence images of SH-SY5Y cells were obtained by confocal microscopy. S (Starved), S + R (Starved + Refed), S + R + C (Starved + Refed + CORM-A1). c, d SH-SY5Y cells were placed in HBSS medium for 2 h and then refed with complete DMEM medium for 2 h in the presence or absence of 20 μM CORM-A1. Images of TFEB-GFP were detected by confocal microscopy in SH-SY5Y cells transfected with siRNA against GSK-3β (c) or treated with LiCl (d). The percentage of cells showing nuclear TFEB localization was calculated. n > 20 cells per condition. Data were expressed as mean ± SD; ***p < 0.001 and NS not significant.

Article Snippet: To knockdown mRNA levels of TFEB, GSK-3β, PERK, BECN-1, and PP2BAα, SHSY5Y cells were transfected with siRNA against TFEB, GSK-3β, PERK, BECN-1, and PP2BAα (Santa Cruz, CA, USA), or with scramble siRNA (scRNA) (Ambion, Austin, TX, USA) as the control, using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. α-syn-A53T, pCAX-APP-Swe/Ind, pEGFP-N1-TFEB, pcDNA3.1, pcDNA3.1GSK-3β-S9A, and pcDNA3.1-GSK-3β-K85A, and hMLKL-Venus were from Addgene (Watertown, MA, USA) and were transfected into cells via Lipofectamine 2000 (Invitrogen). qRT-PCR Total RNA from cells and midbrain tissues was isolated by QIAzol Lysis reagent (QIAGEN, Valencia, CA, USA).

Techniques: Inhibition, Expressing, Confocal Microscopy, Transfection, Fluorescence

Journal: Cell death discovery

Article Title: CO confers neuroprotection via activating the PERK-calcineurin pathway and inhibiting necroptosis.

doi: 10.1038/s41420-025-02530-9

Figure Lengend Snippet: Fig. 6 Regulation of GSK-3β and PERK by CO sustain nuclear TFEB levels, leading to suppressed amyloidogenesis. a–d SH-SY5Y cells were treated with 20 μM CORM-A1 after the cells were transfected with CA-GSK-3β or treated with 1 μM GSK2606414. Nuclear and cytoplasmic fractions extracted from SH-SY5Y cells were analyzed by western blotting using TFEB antibody (left), and quantification of nuclear TFEB and cytosol TFEB was analyzed (right) (a). The levels of p-GS, GS, p-eIF2α, and eIF2α expression were assessed by western blotting (b). Quantification of p-GS and p-eIF2α (c). The protein expression of autophagy protein LC3-I/-II and lysosomal protein LAMP1 was detected by western blotting (left panel) (d), and quantification of LC3-II conversion and LAMP1 is shown in the right panels. e SH-SY5Y cells were transfected with APP-Swe/Ind and then treated with 20 μM CORM-A1 for 12 h in the presence or absence of 1 μM GSK2606414. The levels of APP, LAMP1, LC3-I/-II, p-eIF2α, and eIF2α expression were detected by western blotting. f SH-SY5Y cells were co-transfected with APP-Swe/Ind and siTfeb for 36 h and were treated with 20 μM CORM-A1 for 12 h. The levels of APP, LAMP1, LC3-I/-II, and TFEB were analyzed by western blotting. g SH-SY5Y cells were treated with 20 μM CORM-A1 after the cells were co-transfected with CA-GSK-3β and APP-Swe/Ind or treated with 1 μM GSK2606414. The protein expression of FL-APP was determined by western blotting (left), and quantification of FL-APP was analyzed (right). Data were expressed as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, and NS not significant.

Article Snippet: To knockdown mRNA levels of TFEB, GSK-3β, PERK, BECN-1, and PP2BAα, SHSY5Y cells were transfected with siRNA against TFEB, GSK-3β, PERK, BECN-1, and PP2BAα (Santa Cruz, CA, USA), or with scramble siRNA (scRNA) (Ambion, Austin, TX, USA) as the control, using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. α-syn-A53T, pCAX-APP-Swe/Ind, pEGFP-N1-TFEB, pcDNA3.1, pcDNA3.1GSK-3β-S9A, and pcDNA3.1-GSK-3β-K85A, and hMLKL-Venus were from Addgene (Watertown, MA, USA) and were transfected into cells via Lipofectamine 2000 (Invitrogen). qRT-PCR Total RNA from cells and midbrain tissues was isolated by QIAzol Lysis reagent (QIAGEN, Valencia, CA, USA).

Techniques: Transfection, Western Blot, Expressing