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. 2021 May 8;296:100761. doi: 10.1016/j.jbc.2021.100761

Figure 7.

Figure 7

Involvement of C3aR and protein kinase C in Peptide-2-induced activation of RhoA and vascular permeability.A, semiquantitative PCR to monitor the knockdown efficiency of C3a receptor (C3aR) in HUVECs by siRNA against C3aR (siC3aR). B, FITC-Peptide 2 attachment on HUVECs observed by fluorescent confocal microscopy. HUVECs were incubated with 200 μmol/l FITC-Peptide 2. C, summary graph of the intensity of FITC-Peptide 2 on HUVECs, which were incubated with the indicated concentrations of FITC-Peptide 2. D, inhibition of Peptide-2-induced RhoA activation by knockdown of C3aR in HUVECs. HUVECs were incubated with Peptide 2 for the indicated times. E, summary graph of RhoA activity examined in (D). F, inhibition of Peptide-2-induced U937 cell migration through a HUVEC monolayer coating in the Transwell chamber by knockdown of C3aR in HUVECs. G, protein kinase C (PKC) phosphorylation (activation) induced by Peptide 2 with or without siC3aR in HUVECs. H, summary graph of the P-PKC/GAPDH density ratio. I, RhoA activity induced by Peptide 2 in the presence and absence of PKC inhibitor in HUVECs. J, summary graph of RhoA activity examined in (I). K, Peptide 2-induced U937 cell migration in the Transwell chamber coated with HUVECs in the presence and absence of PKC inhibitor. L, Peptide-2-stimulated vascular permeability in the presence and absence of PKC inhibitor. Dashed oval indicates the extravasated Evans blue. M, schematic representation of the protective effect of DPPIII on the heart and kidney. Scale bar: 20 μm (B) and 1 mm (L). In A, the data was analyzed by one-way ANOVA; in C, H and J, two-way ANOVA was applied for comparing the data between groups, and one-way ANOVA was applied for comparing the results at 0 μmol/l or Time 0 with those at other concentrations or time points; in F and K, two-way ANOVA was used to compare the data between groups. ∗∗ p < 0.01 versus Control peptide; †p < 0.05 and ††p < 0.01 versus 0 min; §§p < 0.01 versus Scramble or DMSO. A.U., arbitrary units; P-PKC, phosphorylated PKC.