Journal: Cells
Article Title: WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts’ Activation and Collagen Processing
doi: 10.3390/cells13110989
Figure Lengend Snippet: WISP-1 protein promoted Akt phosphorylation via integrin β1/FAK/ILK in human cardiac fibroblasts (HCFs). HCFs were cultured in supplemented fibroblast growth medium for 24 h and then starved in serum-free medium (SFM) for 48 h. The medium was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL) for 30 min before cell lysis. Cell lysate samples were analysed by Western blotting using phosphorylated Akt (p-Akt) (Ser473) and total Akt (t-Akt) antibodies. ( A ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to the control. Data shown as mean ± SEM (n = 9). Statistical analysis was performed using Mann–Whitney U test. * indicates p < 0.05. ( B ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to WISP-1 + mouse non-immune IgG 1 control (mIgG) group. HCFs were pre-incubated with integrin β1-blocking antibodies (mouse IgG 1 clone) (β1 mAb, 10 μg/mL), integrin αVβ5-blocking antibodies (mouse IgG 1 clone) (αVβ5 mAb, 10 μg/mL), and mIgG control antibodies (10 μg/mL), respectively, for 30 min prior to WISP-1 protein treatment. Data shown as mean ± SEM (n = 5). Statistical analysis was performed using Kruskal–Wallis H test. * indicates p < 0.05. ( C ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to WISP-1 group. HCFs were pre-incubated with defactinib (5 μM) or CPD22 (2.5 μM) for 30 min prior to WISP-1 protein treatment. Data shown as mean ± SEM (n = 4). Statistical analysis was performed using Mann–Whitney U test. * indicates p < 0.05. Approximate molecular weights in kDa are indicated adjacent to representative immunoblots.
Article Snippet: In some experiments, HCFs were pre-incubated with a mouse IgG 1 clone antibody (mAb) (10 μg/mL integrin β1 mAb [Biotechne, MAB177781], 10 μg/mL integrin αVβ5 mAb [Biotechne, MAB2528], or 10 μg/mL mouse IgG 1 control antibody [Biotechne, MAB002]), or an inhibitor (5μM defactinib [Abcam, Cambridge, UK, ab254452], 2.5 μM CPD22 [Merck, Darmstadt, Germany, 407331], or 25 μM GM6001 [Tocris, Bristol, UK, 2983]) for 30 min prior to WISP-1 protein treatment.
Techniques: Cell Culture, Recombinant, Lysis, Western Blot, Expressing, MANN-WHITNEY, Incubation, Blocking Assay
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