Journal: Molecular Medicine Reports

Article Title: High glucose-induced upregulation of CD36 promotes inflammation stress via NF-κB in H9c2 cells

doi: 10.3892/mmr.2021.12404

Figure Lengend Snippet: HG-induced CD36 expression in H9c2 cells is dependent on reactive oxygen species. CD36 expression was determined by western blotting (A) and reverse transcription-quantitative PCR (B) in HG-treated cells. (C) CD36 levels in the cell membrane were measured by immunofluorescence staining in HG-treated cells (magnification, ×400). (D) CD36 levels in the cell membrane were measured by flow cytometry. (E) Quantitative analysis of DCFDA fluorescence intensity using flow cytometry. (F and G) CD36 expression levels were analyzed by western blotting. (H) mRNA expression levels of CD36 were measured by reverse transcription-quantitative PCR. H9c2 cells were incubated with NG (5.6 mM, NG group), NG plus mannitol (24.4 mM, M group), HG (30 mM, HG group), HG plus N-acetylcysteine (5 mM, HG + N group) and HG plus MitoTEMPO (10 µM, HG + T group), NG plus LV3 containing CD36 (NG + HC group), NG plus LV3 containing CD36 knockout (NG + KC group), HG plus LV3 containing CD36 knockout (HG + KC group), NG plus LV3 empty vector (NG + C group) and HG plus LV3 empty vector (HG + C group) for 72 h. Data are expressed as the means ± SD. n=4. **P<0.01 vs. NG or 0 h; # P<0.05 vs. HG. HG, high glucose; NG, normal glucose; DCFDA, 2′, 7′-dichlorodihydrofluorescein diacetate.

Article Snippet: The cells were incubated with normal glucose (NG; 5.6 mM), NG plus mannitol (M; 24.4 mM), HG (30 mM), HG plus NAC (HG + N; 5 mM), and/or MitoTEMPO (HG + T; 10 µM) and HG plus PA (HG + 100 µM PA) for 72 h. The H9c2 cells were infected with LV3 empty vector (LV3-NC), LV3 containing CD36 (LV3-CD36), CD36 knockout short hairpin (sh)RNA (LV3-shRNA) (Shanghai GeneChem Co. Ltd.) lentivirus expression vectors (viral titer: 1×10 9 transducing U/ml; multiplicity of infection: 10) for 6 h to knockout or overexpress CD36.

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Flow Cytometry, Fluorescence, Incubation, Knock-Out, Plasmid Preparation

Journal: Molecular Medicine Reports

Article Title: High glucose-induced upregulation of CD36 promotes inflammation stress via NF-κB in H9c2 cells

doi: 10.3892/mmr.2021.12404

Figure Lengend Snippet: Knockout of CD36 prevents NF-κB-mediated inflammatory responses and ROS generation in HG-induced H9c2 cells. H9c2 cells were incubated with NG (5.6 mM, NG group), NG plus mannitol (24.4 mM, M group), HG (30 mM, HG group), HG plus LV3 empty vector (HG + C group) or HG plus LV3 containing CD36 mutant (HG + KC group) for 72 h. (A) The TNF-α, IL-6 and IL-1β levels in the culture supernatant were measured by enzyme-linked immunosorbent assay. (B) The mRNA levels of TNF-α, IL-6 and IL-1β were measured by reverse transcription-quantitative PCR and normalized to β-actin. (C and D) CD36 in the nucleus and cytosol and nuclear translocation of NF-κB p65 protein were detected by western blotting. (C and E) IκBα protein in cytosolic extracts was measured by western blotting. (F) Immunofluorescence labeling with anti–NF-κB p65 (magnification, ×400). (G) Intracellular ROS were detected by flow cytometry. Data are expressed as the means ± SD. n=4. **P<0.01 vs. NG; # P<0.05 vs. HG. NF-κB, nuclear factor-κB; ROS, reactive oxygen species; HG, high glucose; NG, normal glucose; TNF, tumor necrosis factor; IL, interleukin.

Article Snippet: The cells were incubated with normal glucose (NG; 5.6 mM), NG plus mannitol (M; 24.4 mM), HG (30 mM), HG plus NAC (HG + N; 5 mM), and/or MitoTEMPO (HG + T; 10 µM) and HG plus PA (HG + 100 µM PA) for 72 h. The H9c2 cells were infected with LV3 empty vector (LV3-NC), LV3 containing CD36 (LV3-CD36), CD36 knockout short hairpin (sh)RNA (LV3-shRNA) (Shanghai GeneChem Co. Ltd.) lentivirus expression vectors (viral titer: 1×10 9 transducing U/ml; multiplicity of infection: 10) for 6 h to knockout or overexpress CD36.

Techniques: Knock-Out, Incubation, Plasmid Preparation, Mutagenesis, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Translocation Assay, Western Blot, Immunofluorescence, Labeling, Flow Cytometry

Journal: Molecular Medicine Reports

Article Title: High glucose-induced upregulation of CD36 promotes inflammation stress via NF-κB in H9c2 cells

doi: 10.3892/mmr.2021.12404

Figure Lengend Snippet: Knockout of CD36 prevents NF-κB by inhibiting ROS in H9c2 cells. H9c2 cells were incubated with NG (5.6 mM, NG group), HG (30 mM, HG group), HG plus LV3 empty vector (HG + C group), HG plus LV3 containing CD36 (HG + HC group), HG + HC plus MitoTEMPO (100 nM, HG + HC + T group), HG plus LV3 containing the CD36 short hairpin RNA (HG + KC group), and HG plus MitoTEMPO (10 µM, HG + T group) for 72 h. (A and B) The nuclear translocation of NF-κB p65 protein in the nucleus and cytosol was detected by western blotting. (A and C) The IκBα protein expression in cytosolic extracts was measured by western blotting. (D) Immunofluorescence labeling with anti-NF-κB p65 (magnification, ×400). (E) Expression levels of mitochondrial ROS were detected by FACS using MitoSOX reagent. Data are expressed as the mean ± SD. n=4. **P<0.01 vs. NG; # P<0.05 vs. HG. NF-κB, nuclear factor-κB; ROS, reactive oxygen species; HG, high glucose; NG, normal glucose.

Article Snippet: The cells were incubated with normal glucose (NG; 5.6 mM), NG plus mannitol (M; 24.4 mM), HG (30 mM), HG plus NAC (HG + N; 5 mM), and/or MitoTEMPO (HG + T; 10 µM) and HG plus PA (HG + 100 µM PA) for 72 h. The H9c2 cells were infected with LV3 empty vector (LV3-NC), LV3 containing CD36 (LV3-CD36), CD36 knockout short hairpin (sh)RNA (LV3-shRNA) (Shanghai GeneChem Co. Ltd.) lentivirus expression vectors (viral titer: 1×10 9 transducing U/ml; multiplicity of infection: 10) for 6 h to knockout or overexpress CD36.

Techniques: Knock-Out, Incubation, Plasmid Preparation, shRNA, Translocation Assay, Western Blot, Expressing, Immunofluorescence, Labeling

Journal: Molecular Medicine Reports

Article Title: High glucose-induced upregulation of CD36 promotes inflammation stress via NF-κB in H9c2 cells

doi: 10.3892/mmr.2021.12404

Figure Lengend Snippet: Knockout of CD36 ameliorates metabolism reprogramming and lipid accumulation caused by HG in H9c2 cells. H9c2 cells were incubated with NG (5.6 mM, NG group), HG (30 mM, HG group), HG plus LV3 containing CD36 mutant (HG + KC group), HG plus PA (100 µM HG + PA group), or HG + PA plus LV3 containing CD36 mutant (HG + PA + KC group) for 72 h. (A) Protocol used in data collection and calculations for evaluating mitochondrial respiration. (B) Representative OCR curve. (C) Quantitative analysis of non-mitochondrial respiration, basal respiration, ATP production, proton leak, maximal respiration and spare respiratory capacity. (D) Quantitative analysis of glycolysis, glycolytic capacity and glycolytic reserve. All measured OCR and ECAR were normalized by total cellular protein. (E) The triglyceride content in the cells. (F) The p-AMPK and AMPK expression levels were analyzed by western blotting. Data are expressed as the mean ± SD. n=4. **P<0.01 vs. NG, # P<0.05 vs. HG. HG, high glucose; OCR, oxygen consumption rate; ECAR, extracellular acidification rate; p-, phosphorylated-.

Article Snippet: The cells were incubated with normal glucose (NG; 5.6 mM), NG plus mannitol (M; 24.4 mM), HG (30 mM), HG plus NAC (HG + N; 5 mM), and/or MitoTEMPO (HG + T; 10 µM) and HG plus PA (HG + 100 µM PA) for 72 h. The H9c2 cells were infected with LV3 empty vector (LV3-NC), LV3 containing CD36 (LV3-CD36), CD36 knockout short hairpin (sh)RNA (LV3-shRNA) (Shanghai GeneChem Co. Ltd.) lentivirus expression vectors (viral titer: 1×10 9 transducing U/ml; multiplicity of infection: 10) for 6 h to knockout or overexpress CD36.

Techniques: Knock-Out, Incubation, Mutagenesis, Expressing, Western Blot

Journal: iScience

Article Title: MiR-4769-3p suppresses adipogenesis in systemic sclerosis by negatively regulating the USP18/VDAC2 pathway

doi: 10.1016/j.isci.2024.110483

Figure Lengend Snippet: USP18, a novel target of miR-4769-3p, promotes adipogenic differentiation of 3T3-L1 cells (A) The 2D structure of miR-4769-3p bound to the 3′UTR of USP18 was obtained from the miRWalk database. (B) USP18 expression was evaluated by immunohistochemical staining in skin samples from SSc patients and healthy controls (dark brown indicates USP18) (100×). (C and D) After inducing adipogenic differentiation of 3T3-L1 cells transfected with Antago-miR-4769-3p or miR-4769-3p mimics for 8 days, the mRNA and protein levels of USP18 were analyzed using real-time qPCR (C) and western blot analysis (D), respectively. (E and F) The validation of the direct interaction between miR-4769-3p and USP18 was confirmed through RIP (E) and dual luciferase reporter assay (F). (G and H) The mRNA and protein levels of USP18 in 3T3-L1 cells during adipogenic differentiation were analyzed using real-time qPCR (G) and western blot analysis (H). (I and J) After inducing adipogenic differentiation of 3T3-L1 cells infected with si-USP18 for 8 days, lipid droplet formation was assessed using oil red O staining (I) (red indicates stained lipid droplets) (200×), and the levels of adipogenic proteins (PPAR-γ, FABP4, SREBP-1, and Lipin1) were measured using western blot analysis (J). Data are presented as mean ± SEM (n = 3); statistical analysis by ANOVA: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 versus the indicated group.

Article Snippet: 3T3-L1 cells were transfected with Antago-NC/Antago-miR-4769-3p (purchased from RiboBio) using a riboFECT CP Transfection Kit (RiboBio), and infected with Empty-vector/USP18-HA-OE (constructed by RiboBio) using Lipofectamine 2000 (Invitrogen).

Techniques: Expressing, Immunohistochemical staining, Staining, Transfection, Western Blot, Luciferase, Reporter Assay, Infection

Journal: iScience

Article Title: MiR-4769-3p suppresses adipogenesis in systemic sclerosis by negatively regulating the USP18/VDAC2 pathway

doi: 10.1016/j.isci.2024.110483

Figure Lengend Snippet: USP18 inhibits the ubiquitination and degradation of VDAC2, synergistically promoting adipogenesis in 3T3-L1 cells (A) The levels of USP18 and HA in 3T3-L1 cells transfected with USP18-HA-OE fusion vector were measured using western blot analysis. (B) 3T3-L1 cells co-immunoprecipitated with HA antibody were analyzed by mass spectrometry for the binding proteins of USP18, and finally VDAC2 was focalized. (C) VDAC2 expression was evaluated by immunohistochemical staining in skin samples from SSc patients and healthy controls (dark brown indicates VDAC2) (100×). (D) 3T3-L1 cells co-immunoprecipitated with HA or VDAC2 antibodies were analyzed by coIP assay to verify the direct binding between USP18 and VDAC2. (E) VDAC2 expression in 3T3-L1 cells transfected with USP18-HA-OE fusion vector were measured using western blot analysis. (F and G) The mRNA and protein levels of VDAC2 in 3T3-L1 cells infected with si-USP18 or si-VDAC2 were analyzed using real-time qPCR (F) and western blot analysis (G). (H and I) After inducing adipogenic differentiation of 3T3-L1 cells infected with si-VDAC2 for 8 days, lipid droplet formation was assessed using oil red O staining (H) (red indicates stained lipid droplets) (200×), and the levels of adipogenic proteins (PPAR-γ, FABP4, SREBP-1, and Lipin1) were measured using western blot analysis (I). (J and K) The ubiquitination level of VDAC2 was analyzed by coIP assay in USP18-overexpressing cells (J) or skin lesions of bleomycin-induced SSc mice (K) after immunoprecipitation with VDAC2. Data are presented as mean ± SEM (n = 3); statistical analysis by ANOVA or Student’s t test: ∗∗ p < 0.01 and ∗∗∗∗ p < 0.0001 versus the indicated group.

Article Snippet: 3T3-L1 cells were transfected with Antago-NC/Antago-miR-4769-3p (purchased from RiboBio) using a riboFECT CP Transfection Kit (RiboBio), and infected with Empty-vector/USP18-HA-OE (constructed by RiboBio) using Lipofectamine 2000 (Invitrogen).

Techniques: Transfection, Plasmid Preparation, Western Blot, Immunoprecipitation, Mass Spectrometry, Binding Assay, Expressing, Immunohistochemical staining, Staining, Co-Immunoprecipitation Assay, Infection

Journal: iScience

Article Title: MiR-4769-3p suppresses adipogenesis in systemic sclerosis by negatively regulating the USP18/VDAC2 pathway

doi: 10.1016/j.isci.2024.110483

Figure Lengend Snippet: Overexpression of miR-4769-3p abolished USP18/VDAC2-mediated adipogenesis After transfection with miR-4769-3p mimics, OE-VDAC2, or both, 3T3-L1 cells were induced to undergo adipogenic differentiation for 8 days. (A and B) The mRNA and protein levels of VDAC2 in these cells were analyzed using real-time qPCR (A) and western blot analysis (B). (C) The lipid droplet formation was assessed using oil red O staining (red indicates stained lipid droplets) (200×). (D) The levels of adipogenic proteins (PPAR-γ, FABP4, SREBP-1, and Lipin1) were measured using western blot analysis. Data are presented as mean ± SEM (n = 3); statistical analysis by ANOVA: ∗ p < 0.05, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 versus the indicated group.

Article Snippet: 3T3-L1 cells were transfected with Antago-NC/Antago-miR-4769-3p (purchased from RiboBio) using a riboFECT CP Transfection Kit (RiboBio), and infected with Empty-vector/USP18-HA-OE (constructed by RiboBio) using Lipofectamine 2000 (Invitrogen).

Techniques: Over Expression, Transfection, Western Blot, Staining

Journal: iScience

Article Title: MiR-4769-3p suppresses adipogenesis in systemic sclerosis by negatively regulating the USP18/VDAC2 pathway

doi: 10.1016/j.isci.2024.110483

Figure Lengend Snippet:

Article Snippet: 3T3-L1 cells were transfected with Antago-NC/Antago-miR-4769-3p (purchased from RiboBio) using a riboFECT CP Transfection Kit (RiboBio), and infected with Empty-vector/USP18-HA-OE (constructed by RiboBio) using Lipofectamine 2000 (Invitrogen).

Techniques: Virus, Over Expression, Plasmid Preparation, Recombinant, Staining, Modification, Transfection, Reverse Transcription, SYBR Green Assay, Luciferase, Reporter Gene Assay, Silver Staining, Mass Spectrometry, Software

Journal: Clinical and Translational Medicine

Article Title: Smad3‐mediated lncRNA HSALR1 enhances the non‐classic signalling pathway of TGF‐β1 in human bronchial fibroblasts by binding to HSP90AB1

doi: 10.1002/ctm2.1292

Figure Lengend Snippet: HSALR1 mice showed noticeable pathological differences of chronic obstructive pulmonary disease than wild‐type mice. (A) Schematic structures of AAV‐based negative control vector (AAV‐NC) and AAV‐based HSALR1 gene vector (AAV‐HSALR). (B) qRT‐PCR analysis of the expression of HSALR1 between AAV‐NC mice and AAV‐ HSALR1 mice (Number of mice in each group = 7, Student's t ‐test). (C) Workflow of AAV intratracheal instillation and cigarette smoke exposure. (D) Lung FEV20/FVC, RI, Cchord, Cydn and FRC were performed in 4 groups of mice. Cydn, lung dynamic compliance; Cchord, lung compliance; FRC, functional residual capacity; RI, respiratory index (Number of mice in each group = 7, Student's t ‐test). (E) Sections of the lung tissues from 4 groups mice were analysed by HE staining (Number of mice in each group = 7, Student's t ‐test). (F) Sections of the lung tissues from 4 groups mice were analysed by Masson staining (Number of mice in each group = 7, Student's t ‐test). (G) Sections of the lung tissues from 4 groups mice were analysed by IHC staining of α‐SMA (Number of mice in each group = 7, Student's t ‐test). (H) Sections of the lung tissues from 4 groups mice were analysed by IHC staining of FN1 (Number of mice in each group = 7, Student's t ‐test). Data information: Error bars represent means ± SD. * p < .05, ** p < .01 and *** p < .05.

Article Snippet: Adeno‐associated virus encoding HSALR1 (AAV‐ HSALR1 ) and AAV‐empty vector (AAV‐NC) were purchased from PackGene Biotech (Guangzhou, China).

Techniques: Negative Control, Plasmid Preparation, Quantitative RT-PCR, Expressing, Functional Assay, Staining, Immunohistochemistry

Journal: Molecular Therapy. Nucleic Acids

Article Title: ac4C acetylation of RUNX2 catalyzed by NAT10 spurs osteogenesis of BMSCs and prevents ovariectomy-induced bone loss

doi: 10.1016/j.omtn.2021.06.022

Figure Lengend Snippet: Overexpression of NAT10 prevents bone loss in OVX mice (A) Illustration of adenoviruses overexpressing NAT10 or empty vector for OVX mice or sham mice, n = 6. (B and C) Confirmation of the efficiency of adenoviruses overexpressing NAT10 by western blotting. (C) Quantification of band intensities (B), n = 6. (D) The ac4C content of total RNA was increased in adenoviruses overexpressing NAT10 group compared with empty vector adenoviruses group, as determined by dot blot, n = 6. (E) Densitometry quantitation of (D), n = 6. (F) Representative μCT images of four groups of mice, n = 6. (G–K) Quantification of bone microstructural parameters of BV/TV (G), Tb.N (H), Tb.Th (I), Tb.Sp (J), and BSA/BV (K), n = 6. (L and M) OVX mice treated with adenoviruses overexpressing NAT10 demonstrated aggravated bone loss, but the bone mass of sham mice treated with Remodelin had no difference from sham mice treated with empty vector adenoviruses (scale bars: 100 μm), as determined by H&E staining, n = 6. (N) Quantification of the trabecular area from four groups of mice, n = 6. All data are expressed as the means ± SD. ∗p < 0.05, ∗∗p < 0.01 (n = 3 independent experiments).

Article Snippet: NAT10 overexpression adenovirus (NAT10-OE) and empty vector adenovirus (NC-OE) were constructed by OBiO Technology (Shanghai, China).

Techniques: Over Expression, Plasmid Preparation, Western Blot, Dot Blot, Quantitation Assay, Staining