murine hepatocyte cell line aml12  (ATCC)

Journal: Journal of Extracellular Vesicles

Article Title: The Small Molecule H89 Facilitates Mesenchymal Stem Cell‐derived Extracellular Vesicle Release and Optimizes Therapeutic Efficacy in Liver Regeneration

doi: 10.1002/jev2.70285

Figure Lengend Snippet: H89 promotes extracellular vesicle (EV) production and release in hUCMSCs. (a) hUCMSCs were treated with 1 µM, 5 µM, or 10 µM H89 for 24 h, while cells in the control group received an equal volume of phosphate‐buffered saline (PBS). Immunofluorescence staining was used to visualize CD63 (red), and DAPI was used to stain nuclei (blue). The fluorescence intensity was quantified using ImageJ software ( n = 3). Scale bar: 100 µm. (b) Quantitative analysis of extracellular vesicle (EV) concentration by nanoflow cytometry (NanoFCM) in hUCMSCs pretreated with 10 µM H89 or mTORC1 inhibitors, as shown in Figure . The exact fold‐change for the H89 treatment group was 4.87‐fold compared to the control. (c) NanoFCM quantification of EV secretion by AML12 cells, HepRG cells, MSCs, and PTMSCs after 48 h of treatment with 10 µM H89. (d) Western blot analysis of CD63 and TSG101 expression in cellular lysates and EV fractions isolated from hUCMSCs treated with PBS (negative control) or 10 µM H89 for 48 h. (e, f) NanoFCM quantification of the expression of CD63, CD9, and CD81 in EVs, along with the particle size distribution and concentration. (g) EVs were visualized by transmission electron microscopy (TEM). EV density was quantified in 15 random fields ( n = 3). All the data are presented as the mean ± SD. Unpaired two‐tailed Student's t tests and One‐way ANOVA followed by Dunnett's multiple comparisons test were used to test for statistical significance. ** p < 0.01, *** p < 0.001.

Article Snippet: The murine hepatocyte cell line AML12 was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Control, Saline, Immunofluorescence, Staining, Fluorescence, Software, Concentration Assay, Cytometry, Western Blot, Expressing, Isolation, Negative Control, Transmission Assay, Electron Microscopy, Two Tailed Test

Journal: Journal of Extracellular Vesicles

Article Title: The Small Molecule H89 Facilitates Mesenchymal Stem Cell‐derived Extracellular Vesicle Release and Optimizes Therapeutic Efficacy in Liver Regeneration

doi: 10.1002/jev2.70285

Figure Lengend Snippet: Modulation of the content of hUCMSC‐EVs by H89. (a) Proteomic analysis revealing the percentage of proteins that remained unchanged, were upregulated (>1.5‐fold), or were downregulated (<0.75‐fold) in H‐EVs compared with C‐EVs. (b) Selected differentially expressed proteins associated with “cell cycle regulation,” “energy metabolism,” and “oxidative stress,” along with their fold changes. (c) CCK‐8 assay demonstrating the effect of siRNA knockdown on AML12 cell proliferation. (d) Heatmap illustrating the changes in the expression of differentially expressed miRNAs between H‐EVs and C‐EVs. (e) Volcano plot highlighting the differentially expressed miRNAs. (f) The top 10 highly expressed miRNAs. (g) CCK‐8 assay to assess the effect of miRNA knockdown on AML12 cell proliferation. (h) GO and KEGG pathway analyses to identify the biological functions and pathways regulated by the target genes of miR‐29a. All the data are presented as the mean ± SD ( n = 3). Unpaired two‐tailed Student's t test was used to test for statistical significance. * p < 0.05, ** p < 0.01, ns: not significant.

Article Snippet: The murine hepatocyte cell line AML12 was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: CCK-8 Assay, Knockdown, Expressing, Two Tailed Test

Journal: Frontiers in Pharmacology

Article Title: Downregulation of microRNA-300-3p promotes steatosis-to-MASH progression by regulating STX17

doi: 10.3389/fphar.2026.1804415

Figure Lengend Snippet: MiR-300-3p was decreased in the livers of MAFLD mice and FFA-induced HepG2 cells. (A) MiR-300-3p was decreased in the livers of MAFLD mice. (B) MiR-300-3p was downregulated in the FFA-induced HepG2 cells. (C) MiR-300-3p was downregulated in HepG2 cells successfully. (D) MiR-300-3p was overexpressed in HepG2 cells successfully. Data in (A–D) are means±SDs (n = 3). *P < 0.05, **P < 0.01. NC, Healthy control; OC, over-expression control; IC, inhibition control.

Article Snippet: The human hepatocyte cell line HepG2 was procured from the American Type Culture Collection (ATCC, United States) and cultivated in a humidified incubator maintained at 37 °C with a 5% CO 2 atmosphere.

Techniques: Control, Over Expression, Inhibition

Journal: Frontiers in Pharmacology

Article Title: Downregulation of microRNA-300-3p promotes steatosis-to-MASH progression by regulating STX17

doi: 10.3389/fphar.2026.1804415

Figure Lengend Snippet: Downregulation of miR-300-3p promotes FFA-induced lipid accumulation and hepatic inflammation in HepG2 cells. (A) The experimental flow of the MAFLD cell model construction and miR-300-3p mimics, miR-300-3p inhibitors and as well as corresponding scrambled controls transfection in HepG2 cells. (B,C) Intercellular TG contents in miR-300-3p -inhibited and -overexpressing in HepG2 cells. (D) Oil Red O staining (400×) and the relative areas of lipid droplets in miR-300-3p-inhibited and -overexpressing in HepG2 cells. (E,F) mRNA levels of FASN, SREBP-1c, IL-6, IL-2 and TNF-αin miR-300-3p -inhibited and -overexpressing in HepG2 cells. (G) Protein levels of FASN, SREBP-1c and TNF-α in miR-300-3p -inhibited and -overexpressing in HepG2 cells. Data in (B,C) and (E–G) are means±SDs (n = 3). *P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001. OC, over-expression control; IC, inhibition control.

Article Snippet: The human hepatocyte cell line HepG2 was procured from the American Type Culture Collection (ATCC, United States) and cultivated in a humidified incubator maintained at 37 °C with a 5% CO 2 atmosphere.

Techniques: Transfection, Staining, Over Expression, Control, Inhibition

Journal: Frontiers in Pharmacology

Article Title: Downregulation of microRNA-300-3p promotes steatosis-to-MASH progression by regulating STX17

doi: 10.3389/fphar.2026.1804415

Figure Lengend Snippet: Downregulation of miR-300-3p induces apoptosis in HepG2 cells to promote it. (A,B) The apoptosis rate in miR-300-3p -inhibited and -overexpressing in HepG2 cells. (C) mRNA levels of Bax, Bcl-2 and Caspase-3 in miR-300-3p -inhibited and -overexpressing in HepG2 cells. (D) Protein levels of Bax, Bcl-2 and Caspase-3 in miR-300-3p -overexpressing and -inhibited in HepG2 cells. Data in (A–D) are means±SDs (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. OC, over-expression control; IC, inhibition control.

Article Snippet: The human hepatocyte cell line HepG2 was procured from the American Type Culture Collection (ATCC, United States) and cultivated in a humidified incubator maintained at 37 °C with a 5% CO 2 atmosphere.

Techniques: Over Expression, Control, Inhibition

Journal: Frontiers in Pharmacology

Article Title: Downregulation of microRNA-300-3p promotes steatosis-to-MASH progression by regulating STX17

doi: 10.3389/fphar.2026.1804415

Figure Lengend Snippet: MiR-300-3p regulates STX17 in HepG2 cells. (A,B) GO enrichment analysis (Biological Process category) and KEGG enrichment analysis diagrams of sixteen predicted target genes of miR-300-3p. (C) The predicted binding sites between miR-300-3p and STX17 were putatively identified via the TargetScan database. (D) Dual-luciferase reporter assay (DLRA) in HepG2 cells validated that STX17 is a direct target gene of miR-300-3p. (E,F) The mRNA and protein expression levels of STX17 in HepG2 cells with miR-300-3p inhibition or overexpression. Data in (A–D) are means±SDs (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. OC, overexpression control; IC, inhibition control.

Article Snippet: The human hepatocyte cell line HepG2 was procured from the American Type Culture Collection (ATCC, United States) and cultivated in a humidified incubator maintained at 37 °C with a 5% CO 2 atmosphere.

Techniques: Binding Assay, Luciferase, Reporter Assay, Expressing, Inhibition, Over Expression, Control

Journal: Frontiers in Pharmacology

Article Title: Downregulation of microRNA-300-3p promotes steatosis-to-MASH progression by regulating STX17

doi: 10.3389/fphar.2026.1804415

Figure Lengend Snippet: P62 and LC3-II in miR-300-3p -inhibited and -overexpressing in HepG2 cells. (A) The protein levels of P62 and LC3-II in miR-300-3p -inhibited and -overexpressing in HepG2 cells. (B) P62 and LC3-II in miR-300-3p -inhibited and -overexpressing by Immunofluorescence assay. Data in (A-D) are means±SDs (n = 3). *P < 0.05, **P < 0.01,***P < 0.001,****P < 0.0001. OC, overexpression control; IC, inhibition control.

Article Snippet: The human hepatocyte cell line HepG2 was procured from the American Type Culture Collection (ATCC, United States) and cultivated in a humidified incubator maintained at 37 °C with a 5% CO 2 atmosphere.

Techniques: Immunofluorescence, Over Expression, Control, Inhibition

Journal: Frontiers in Pharmacology

Article Title: Downregulation of microRNA-300-3p promotes steatosis-to-MASH progression by regulating STX17

doi: 10.3389/fphar.2026.1804415

Figure Lengend Snippet: MiR-300-3p promoted autophagy by regulating STX17, reduces lipid accumulation, inflammatory response, and decreases hepatocyte apoptosis. (A) STX17 was inhibited or overexpressed in HepG2 cells successfully. (B) Protein levels of FASN, SREBP-1c and TNF-αin STX17 -inhibited and -overexpressing in HepG2 cells. (C,D) Intercellular TG contents in STX17-inhibited and -overexpressing in HepG2 cells. (E) Oil Red O staining (400×) and relative areas of lipid droplets in STX17-inhibited and -STX17 in HepG2 cells. (F) Protein levels of Bax, Capase-3 and Bcl-2 in STX17 -inhibited and -overexpressing in HepG2 cells. Data in (A–D) and (F) are means±SDs (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. OC, miR-300-3p overexpression control; IC,miR-300-3p inhibition control; Control, STX17-overexpression control or STX17-inhibition control; Si-STX17, STX17-inhibition; Over-STX17, STX17-overexpression.

Article Snippet: The human hepatocyte cell line HepG2 was procured from the American Type Culture Collection (ATCC, United States) and cultivated in a humidified incubator maintained at 37 °C with a 5% CO 2 atmosphere.

Techniques: Staining, Over Expression, Control, Inhibition

Journal: Frontiers in Pharmacology

Article Title: Downregulation of microRNA-300-3p promotes steatosis-to-MASH progression by regulating STX17

doi: 10.3389/fphar.2026.1804415

Figure Lengend Snippet: P62, LC3-II in STX17-inhibited and -overexpressing in HepG2 cells. (A) Protein levels of P62, LC3-II in STX17-inhibited and -overexpressing in HepG2 cells. (B) P62 and LC3-II in STX17-inhibited and -overexpressing by Immunofluorescence assay. Data in (A-D) are means±SDs (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. OC, overexpression control; IC, inhibition control.

Article Snippet: The human hepatocyte cell line HepG2 was procured from the American Type Culture Collection (ATCC, United States) and cultivated in a humidified incubator maintained at 37 °C with a 5% CO 2 atmosphere.

Techniques: Immunofluorescence, Over Expression, Control, Inhibition

Journal: bioRxiv

Article Title: Inhibition of p107 alleviates liver steatosis by reducing de novo fatty acid synthesis

doi: 10.64898/2026.04.14.718271

Figure Lengend Snippet: (A) p107 protein levels in THLE2 cells transfected with siRNA p107 or siRNA control for 48 hours (n=3 per group). (B) Representative microphotographs of Oil Red O staining (left pannel) of THLE2 cells downregulating p107 (sip107) for 48 hours (n = 3 per group) and Oil Red O semiquantification (right pannel) (n = 3 per group). (C) Quantification of immunoblot analysis of de novo lipogenesis markers (n = 3 per group) and a representative immunoblot. (D) RNA expression of de novo lipogenesis markers (n = 3 per group. (E) De novo synthesis of free fatty acids (FFA) and triglycerides (TG) in THLE2 (n = 6 per group). (F) FAO activity in THLE2 cells transfected with siRNA p107 or siRNA control for 48 hours (n=5 per group). (G) Oxidation rate of palmitic acid (n = 6 per group). (H and I)) OCR and (I) ECAR of p107-silenced THLE2 (n = 17-19 per group). (J) Basal energetic metabolic states, based on quantification of ECAR and OCR during basal metabolism. (K) p107 protein levels in THLE2 after overexpressing p107 (n = 3 per group). (L) Representative microphotographs (left pannel) and semiquantification (right pannel) of Oil Red O staining of THLE2 cells overexpressing p107 (plasmid p107) for 24 hours. Oil Red O staining was quantified using ImageJ and normalized to the total number of nuclei per field (n = 4 per group). (M) Quantification of immunoblot analysis of de novo lipogenesis markers (n = 3 per group) and a representative immunoblot. (N) OCR of p107-overexpressed THLE2 (n =10 per group). GAPDH was used to normalize protein levels. Data are expressed as mean ±SEM. *p < 0.05, **p < 0.01, ***p <0.001, using a Student’s t test .

Article Snippet: THLE2 human hepatic cell line (American Type Culture Collection, ATCC) was cultured in bronchial epithelial cell basal medium (BEBM) supplemented with a growth factors BulleKit (Lonza/Clonetics Corporation), 70ng/mL phosphoethanolamine, 5 ng/mL epidermal growth factor, 10% (v/v) FBS and 1% (v/v) Glutamine-Penicillin-Streptomycin solution (MERCK).

Techniques: Transfection, Control, Staining, Western Blot, RNA Expression, Activity Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: Inhibition of p107 alleviates liver steatosis by reducing de novo fatty acid synthesis

doi: 10.64898/2026.04.14.718271

Figure Lengend Snippet: (A) Volcano plot of total protein expression in p107 liver KO mice compared to shLucif controls (n = 7). Red and blue points indicate significantly up- and downregulated proteins (p < 0.05). (B) Volcano plot of phosphosite abundance in p107 liver KO mice compared to shLucif controls (n = 7). Points represent individual phosphosites—annotated by their parent protein name—with red and blue indicating significant changes (p < 0.05). (C) Heatmap of total protein expression differences grouped by GO Biological Process terms, filtered by significance (p < 0.001). (D) Heatmap of robust total protein expression differences grouped by GO terms, utilizing strict filtering (> 3 combined razor and unique peptides, p < 0.001, absolute t-test difference > 0.58). (E) Quantification of p107 (left panel) and FAS (right panel) in THLE2 after overexpressing p107 (plasmid p107) and silencing FAS (siRNA FAS) during 24h (n = 3 per group) and representative immunoblot. (F) Representative microphotographs of Oil Red O staining of THLE2 cells overexpressing p107 (plasmid p107) and silencing FAS (siRNA FAS) for 24 hours. (G) Semiquantification of Oil Red O staining. Vinculin was used to normalize protein levels. Data are expressed as mean ±SEM. *p < 0.05, **p < 0.01, ***p <0.001, using a Student’s t test .

Article Snippet: THLE2 human hepatic cell line (American Type Culture Collection, ATCC) was cultured in bronchial epithelial cell basal medium (BEBM) supplemented with a growth factors BulleKit (Lonza/Clonetics Corporation), 70ng/mL phosphoethanolamine, 5 ng/mL epidermal growth factor, 10% (v/v) FBS and 1% (v/v) Glutamine-Penicillin-Streptomycin solution (MERCK).

Techniques: Expressing, Phospho-proteomics, Plasmid Preparation, Western Blot, Staining

Journal: bioRxiv

Article Title: Discovery and Preclinical Validation of a Clinically Optimized Mitochondrial Complex I Modulator for Alzheimer’s Disease

doi: 10.64898/2026.04.10.717554

Figure Lengend Snippet: a A drug discovery funnel used for the identification of C273. b C273 protects MC65 Tet-Off cells from Aβ toxicity (red line). # indicates cell death in Tet-Off cells treated with vehicle. No toxicity was observed in MC65 Tet-On cells (Aβ not expressed, black line). EC 50 values were calculated using nonlinear regression in GraphPad Prism 10. c Treatment with C273 for 24 h does not induce cytotoxicity in HepG2 human hepatic cells at concentrations up to 100 μM. d C273 increases NADH levels in MC65 Tet-On cells, consistent with inhibition of mtCI. NADH levels were measured over 24 h. e Inhibition of mtCI activity in isolated mouse brain mitochondria by C273 measured using an NADH oxidation assay. f Activities of mitochondrial ETC complexes I–IV were assessed in permeabilized MC65 cells using an electron flow assay in the Seahorse XFe96 Extracellular Flux Analyzer in the presence of C273 (25 and 50 μM). g Injection of rotenone or C273 to monitor OCR kinetics in MC65 Tet-On cells. h Pretreatment with rotenone blocks the protective effect of C273 in MC65 Tet-Off cells (Aβ expressed). Open orange squares: Tet-On cells, no Aβ expression, treated with vehicle (positive control, 100% viability). Blue triangles: Tet-Off cells (Aβ expressed), which undergo extensive cell death after 3 days. Tet-Off cells treated with C273 (2 nM - 5 μM; blue line) show complete protection from Aβ toxicity. Solid red triangles: Tet-On cells treated with 32 nM rotenone, which show no toxicity. Open red triangles: Tet-Off cells treated with 32 nM rotenone alone, resulting in complete cell death. Pretreatment with 32 nM rotenone blocks the protective effect of C273 in Tet-Off cells (red line, grey box), supporting a competitive interaction at mtCI. Partial rescue at higher C273 concentrations reflects incomplete occupancy of the mtCI binding site by rotenone, which inhibits ∼50% of mtCI activity at this dose. The grey box indicates the EC 50 concentration range for C273. P values were calculated using unpaired two-tailed Student’s t -tests; ** P < 0.01. Data are presented as mean ± SD ( n = 4 technical replicates). All experiments were replicated at least twice.

Article Snippet: NF-κB Reporter (Luc) HEK293 cell line and the antioxidant response element (ARE) Luciferase Reporter HepG2 hepatic cell line were purchased from BPS Bioscience (CA, USA) and were cultured in accordance of manufacturer instructions.

Techniques: Drug discovery, Inhibition, Activity Assay, Isolation, Oxidation Assay, Injection, Expressing, Positive Control, Binding Assay, Concentration Assay, Two Tailed Test