Regulator of G-Protein Signaling – 5 (RGS5) Is a Novel Repressor of Hedgehog Signaling
Figure 2
Direct effect of RGS5 on Shh-mediated reporter expression.
(A) RGS5 and RGS4 are inhibited by Shh stimulation. C3H10T1/2 cells were stimulated with Shh (1 µg/mL) for 2 hours, 6 hours, and 24 hours. Relative expression of (i) Smo, (ii) RGS5, (iii) RGS4, and (iv) RGS2 was determined by qPCR, and normalized to the expression of each gene in the absence of Shh (untreated cells). n = 3–7; error bar = SEM; *p<0.05 by t-test. (B) Knock-down of RGS5 activates Shh-mediated reporter expression. The expression of RGS5 was knocked-down following transfection with gene-specific siRNA in C3H10T1/2 cells (i), whereas RGS4 and RGS2 expression is not affected by the RGS5 siRNA. Cells were treated with Shh (1 µg/mL) for 24 hours, and RNA was isolated. The relative expression of (ii) RGS5, (iii) Ptc1, (iv) Ptc2, (v) Gli1, and (vi) Gli2 was assessed by qPCR. Expression is corrected for GAPDH and normalized to the expression of each gene in the absence of siRNA transfection and in the absence of Shh stimulation (-Shh, -RGS5 siRNA). n = 4–5; error bars = SEM; *p<0.05 by t-test. (C) Knock-down of RGS4 does not potentiate Shh-mediated expression of reporter genes. The expression of RGS4 was knocked-down following transfection with gene-specific siRNA in C3H10T1/2 cells. Cells were treated with Shh (1 µg/mL) for 24 hours, and RNA was isolated. The relative expression of RGS4 (i), Ptc1 (ii) and Gli1 (iii) was assessed by qPCR. Expression is corrected for GAPDH and normalized to the expression of each gene in the absence of Shh stimulation (untreated, -siRNA). n = 3–5; error bars = SD.