Figure 2.

Cholesterol-conjugated chemically modified siRNA reverses RA-FLSs dysfunction

(A) The inhibiting effect on SMO protein level after si-S1A3-Chol treatment. Representative western blot of SMO protein in total protein after RA-FLSs were treated with si-Scr-Chol or si-S1A3-Chol (800 nM) for 72 h. GAPDH was used as a loading control. Relative expression was calculated as the ratio of SMO/GAPDH and is presented as fold change versus si-Scr-Chol (n = 6). (B) The inhibiting effect on GLI1 protein level after si-S1A3-Chol treatment. Representative western blot of GLI1 protein in nuclear protein after RA-FLSs were treated with si-Scr-Chol or si-S1A3-Chol (800 nM) for 72 h. Histone H3 was used as a loading control. Relative expression was calculated as the ratio of GLI1/Histone H3 and is presented as fold change versus si-Scr-Chol (n = 6). (C) ELISA assay determined the concentrations of cytokines IL-6, IL-8, MMP-1, MMP-3, MMP-9, and MMP-13 in cell-culture medium supernatants after RA-FLSs were treated with si-Scr-Chol or si-S1A3-Chol (800 nM) for 72 h (n = 4). (D) The impact on cell viability was evaluated by CCK-8 assay after RA-FLSs were treated with si-Scr-Chol or si-S1A3-Chol (800 nM) for 0, 24, 48, and 72 h, and the absorbance was detected at indicated time points (n = 6). (E) The cell proliferation was determined by flow cytometry. The percentages of EdU-positive cells were detected after RA-FLSs were treated with si-Scr-Chol or si-S1A3-Chol (800 nM) for 48 h, shown in the bar graph (n = 6). (F) The cell-cycle phase distribution was detected by flow cytometry after RA-FLSs were treated with si-Scr-Chol or si-S1A3-Chol (800 nM) for 48 h, and the percentages of cells in the S and G₂/M phases are shown in the bar graph (n = 6). (G) The migration capacity of RA-FLSs after si-S1A3-Chol treatment was examined by scratch wound healing assay. Representative images were captured after RA-FLSs were treated with si-Scr-Chol or si-S1A3-Chol (800 nM) for 24 h, and the number of migrated cells beyond the scratch line is shown in the bar graph (n = 6). Scale bars, 400 μm. (H) The migration ability was detected using chemotaxis assay for 12 h after RA-FLSs were treated with si-Scr-Chol or si-S1A3-Chol (800 nM) for 48 h. Representative images were captured, and the number of migrated cells is shown in the bar graph (n = 6). Scale bars, 200 μm. (I) The invasion ability was assessed using transwell chambers with Matrigel-coated membrane for 24 h after RA-FLSs were treated with si-Scr-Chol or si-S1A3-Chol (800 nM) for 48 h. Representative images were captured, and the number of invaded cells is shown in the bar graph (n = 6). Scale bars, 200 μm. Data are presented as mean ± SD. ns, p > 0.05; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001 versus si-Scr-Chol group by one-way ANOVA with Dunnett’s test for multiple comparisons.