Journal: Cellular & Molecular Biology Letters

Article Title: Lipotoxic hepatocyte derived LIMA1 enriched small extracellular vesicles promote hepatic stellate cells activation via inhibiting mitophagy

doi: 10.1186/s11658-024-00596-4

Figure Lengend Snippet: LTH-sEV derived LIMA1 inhibits mitophagy in LX2. A Immunofluorescence staining showing LC3B (green) and TOM20 (red) in LX2 treated with LTH-sEV shCtr or LTH-sEV shLIMA1 . Scale bar = 10 μm. B LTH-sEV shCtr or LTH-sEV shLIMA1 treated LX2 were transfected with mitochondrially targeted mKeima and excitation at 550 nm (red) and 438 nm (green) by microscopy. Scale bar = 10 µm. C Autophagic microstructures in LX2 mitochondria were examined by transmission electron microscopy. Scale bars, 500 nm. Black arrowheads, mitophagy. D ATP content in LX2 treated with LTH-sEV shCtr or LTH-sEV shLIMA1 were measured. E The mitochondrial membrane potential of LX2 was determined by JC-1 staining. Scale bar = 20 μm. F Different treated LX2 were transfected with mitochondrially targeted mKeima and excitation at 550 nm (red) and 438 nm (green) by microscopy. Scale bar = 20 µm. G ATP levels of LX2 were determined using an ATP determination kit. H Western blot of COL1A1, COL3A1 and α-SMA in LX2 treated with Liensinine or Urolithin A. I qRT-PCR of COL1A1, COL3A1 and α-SMA mRNA in LX2 treated with Liensinine or Urolithin A. All data were expressed as the means ± SD of at least 3 independent experiments, * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Article Snippet: To investigate the role of mitophagy in HSCs activation, we conducted experiments using mitophagy inhibitors Liensinine (Solarbio, Beijing, China, IL0640, 50 μM) and mitophagy promoters Urolithin A (Solarbio, Beijing, China, IU0200, 50 μM) in co-cultivation with LX2 for 48 h. Subsequently, protein and RNA were collected for further analysis.

Techniques: Derivative Assay, Immunofluorescence, Staining, Transfection, Microscopy, Transmission Assay, Electron Microscopy, Membrane, Western Blot, Quantitative RT-PCR

Journal: Cellular & Molecular Biology Letters

Article Title: Lipotoxic hepatocyte derived LIMA1 enriched small extracellular vesicles promote hepatic stellate cells activation via inhibiting mitophagy

doi: 10.1186/s11658-024-00596-4

Figure Lengend Snippet: LIMA1 overexpression and knockdown promote or inhibit HSCs mitophagy. A Mitochondrial membrane potential of LX2 overexpressing LIMA1 was measured by JC-1 staining. Scale bar = 25 μm. B Immunofluorescence staining shows LC3B (green) and TOM20 (red) fluorescence of LX2 overexpressing LIMA1. Scale bar = 10 μm. C LX2 overexpressing LIMA1 was transfected with mitochondria-targeted mKeima and microscopically excited at 550 nm (red) and 438 nm (green). Scale bar = 10 µm. D Autophagic microstructures in LX2 overexpressing LIMA1 mitochondria were examined by transmission electron microscopy. Scale bars = 500 nm. Black arrowheads, mitophagy. E ATP levels of LX2 overexpressing LIMA1 were determined using an ATP determination kit. F The mitochondrial membrane potential of LIMA1-knockdown LX2 was determined by JC-1 staining. Scale bar = 25 μm. G Autophagic microstructures in LIMA1-knockdown LX2 mitochondria were examined by transmission electron microscopy. Scale bars = 500 nm. H Immunofluorescence staining showing LC3B (green) and TOM20 (red) in LIMA1-knockdown LX2. Scale bar = 10 μm. I LIMA1-knockdown LX2 were transfected with mitochondrially targeted mKeima and excitation at 550 nm (red) and 438 nm (green) by microscopy. Scale bar = 10 µm. J ATP levels of LIMA1-knockdown LX2 were determined using an ATP determination kit. All data were expressed as the means ± SD of at least 3 independent experiments, * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: To investigate the role of mitophagy in HSCs activation, we conducted experiments using mitophagy inhibitors Liensinine (Solarbio, Beijing, China, IL0640, 50 μM) and mitophagy promoters Urolithin A (Solarbio, Beijing, China, IU0200, 50 μM) in co-cultivation with LX2 for 48 h. Subsequently, protein and RNA were collected for further analysis.

Techniques: Over Expression, Knockdown, Membrane, Staining, Immunofluorescence, Fluorescence, Transfection, Transmission Assay, Electron Microscopy, Microscopy