Fig 1: Effects of CD147 silencing on HG- and CML- mediated changes in MMP2 and MMP9. Cells were treated with HG (25 mM) or CML (2 µg/mL) with and without CD147 siRNA for 24 h. Control (non-specific) siRNA was used as a negative control for CD147 siRNA. (A,B) Quantitative assessment of MMP2 and MMP9 mRNA levels normalized to the house keeping gene, GAPDH using real-time PCR. (C) Western blot analysis and quantification of the normalized signal intensity of MMP2 and MMP9 proteins. (D) Percentage reduction in soluble MMP2 and MMP9 proteins relative to the corresponding control (HG or CML). (E) Percentage reduction in MMP2/9 activity relative to the corresponding control (HG or CML). Results represent the means ± SD for three independent experiments. (* p < 0.05) for MMP2 (MMP activity in (E)) comparison with the corresponding control (HG vs. control; CML vs. BSA; CD147 siRNA + HG vs. HG; and CD147 siRNA + CML vs. CML). († p < 0.05) for MMP9 comparison with the corresponding control.
Fig 2: miR-124 suppresses cell viability in U87 cells. (A) miR-124 expression levels in U87 cells after transient transfection with a miR-124 mimic or the NC. (B) miR-124 inhibited cell proliferation, as assessed by the MTT assay. (C) The expression of EMMPRIN in U87 cells transfected with EMMPRIN plasmids. (D) EMMPRIN overexpression reversed the effect of the miR-124 on cell proliferation, as assessed by the MTT assay. (E and F) miR-124 suppressed cell proliferation, as assessed by flow cytometry. (G) miR-124 expression levels in U87 cells after transient transfection with an miR-124 inhibitor or the NC. (H) miR-124 inhibitor increased cell proliferation, as assessed by the MTT assay. Data are presented as the mean ± SEM. The experiments were performed in triplicate. *P<0.05, **P<0.01 vs. NC group, #P<0.05 vs. miR-124 group. miR, microRNA; NC, negative control; EMMPRIN, extracellular matrix metalloproteinase inducer; Con, untreated control.
Fig 3: CD147 promotes Nrf2 expression through blocking its protein degradation (A) Nrf2 protein levels were determined in U251 or T98G cells after CD147 knockdown. (B) The indicated protein levels were determined in cells with or without CD147 knockdown. (C-E) NQO-1 (C), HO-1(D) and GCLC-1 (E) mRNA levels were determined by qCPR respectively. (F-G) Luciferase reporter gene assay was performed to determine CD147 knockdown could suppress the ARE activity. (H-I) Nrf2 protein stability were determined by western blotting in cells treated with CHX (10 μg/ml) for indicated time or MG-132 (10 μM) for 4 h, respectively.
Fig 4: CD147 dictates Nrf2 stability through the suppression of GSK3ß/ß-TrCP dependent protein degradation (A-B) The indicated proteins were determined in U251 cells with CD147 knockdown (A) or overexpression (B). (C) Nrf2 and GSK3ß protein levels were determined in U251 cells following Capivasertib (10nM) treatment. (D-E) Immunoprecipitation was performed to determine the interaction between ß-TrCP and Nrf2 in indicated U251 cells. After the ß-TrCP protein was immunoprecipitated with an anti-ß-TrCP antibody, indicated proteins were detected by western blotting. (F) Suppresion of CD147 decreased the total and nuclear Nrf2 expression. ß-Actin and Lamin B served as internal controls. WCL, whole cell lysate; N, nuclear; C, cytoplasmic.
Fig 5: Effects of inhibiting NF?B and RAGE on HG- and CML-mediated changes in differentiated adipocytes. Cells were treated with HG (25 mM) or CML (2 µg/mL) with and without NF?B inhibitory peptide (15 µg/mL) or RAGE antibody (20 µg/mL) for 24 h. (A) Quantitative assessment of CD147 mRNA levels normalized to the house keeping gene, GAPDH using real-time PCR. (B) Western blot analysis and quantification of the normalized signal intensity of CD147 proteins. (C) Measurements of soluble CD147 protein in cell culture media using CD147 Quantikine ELISA assay. (D) Representative image and quantification of CD147 protein glycosylation using Pierce™ Glycoprotein Stain. Mouse IgG was used as a control for the immunoprecipitation step. The graphs represent the means ± SD for three independent experiments. (* p < 0.05) for comparison with the corresponding control (HG vs. control; CML vs. BSA; NF?B + HG vs. HG; and NF?B + CML vs. CML; RAGE Ab + HG vs. HG; and RAGE Ab+CML vs. CML). († p < 0.05) for comparison with the corresponding control in low glycosylated protein (B).
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