Figure 1.

Chemically modified siRNAs inhibit SMO expression with high efficiency

(A) Silencing efficiency screening of anti-SMO siRNAs. Relative SMO mRNA expression was assessed by qPCR after RA-FLSs were transfected with different siRNAs (50 nM) for 48 h, shown as fold change versus si-NC (50 nM) (n = 12). (B) Concentration dependence of siRNAs in the silence of SMO mRNA expression was determined by qPCR after RA-FLSs were transfected with si-h-5 (left) or si-h/m-2 (right) at indicated concentrations (0.001–50 nM) for 48 h. Relative expression is shown as fold change versus si-NC (50 nM) (n = 4). (C) Concentration dependence of si-h/m-2 in inhibiting SMO protein level was determined by western blot after RA-FLSs were transfected with si-h/m-2 at indicated concentrations (10, 20, and 50 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-NC (50 nM) (n = 12). (D) Representative schematic diagram of chemical modification pattern of si-S1A3-Chol. (E) Silencing efficiency screening of siRNAs with different chemical modification patterns. Relative SMO mRNA expression was assessed by qPCR after RA-FLSs were transfected with naked siRNA or chemically modified siRNAs (50 nM) for 48 h, shown as fold change versus si-NC (50 nM) (left) (n = 3). Heatmap (right) shows the percentages of the mean inhibition rate of siRNAs with different modifications in RA-FLSs. (F) Half-maximal inhibitory concentration of selected chemical modification anti-SMO siRNAs. Concentration dependence of siRNAs in the silence of SMO mRNA expression was assessed by qPCR after RA-FLSs were transfected with naked siRNA or chemically modified siRNAs at indicated concentrations (0.01, 0.1, 1, 2, 5, 10, 20, and 50 nM) for 48 h, shown as percentages of si-NC (50 nM) (n = 3). (G) Protein silencing efficiency of selected chemically modified siRNA. Representative western blot of SMO protein after RA-FLSs were transfected with si-h/m-2, si-S1A3, or si-S1A4 (50 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-NC (50 nM) (n = 6). (H) Concentration dependence of si-S1A3-Chol in the silence of SMO mRNA expression was determined by qPCR after RA-FLSs were transfected with si-S1A3-Chol at indicated concentrations without any transfection reagent for 48 h. si-S1A3 (50 nM) transfected with Lipofectamine 3000 served as the positive control, and relative expression is shown as fold change versus si-NC (50 nM) (n = 4). Data are presented as mean ± SD. ns, p > 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 versus si-NC group (in A, C, and H) or versus si-h/m-2 (naked siRNA with transfection reagent) group (E and G) by one-way ANOVA with Dunnett’s test for multiple comparisons.