Figure 3. Gsα and its effector cAMP inhibit Shh signaling and tumor growth in Gnas mutants.

(a) Bar graph (left) depicts expression changes in GNPs treated NF449 over control. Right: cAMP levels measured by ELISA.

(b) GNPs were treated with vehicle and SAG (200 nM) and/or transfected with pcDNA3 control and GsαQ227L (pGsCA) for 48 h. Bar graphs depict Gli1 and Ptch1 expression changes in treated cells over control.

(c) Average cAMP levels of control and mutant GNPs treated with vehicle and forskolin.

(d) GNPs were treated with vehicle or Rolipram (50 μM), forskolin (10 μM) and db-cAMP (100 μM) for 3 h. Gli1 and Ptch1 or Gli3FL and Gli3R were analyzed by qRT-PCR (left) and western blotting (right) as indicated. GAPDH: loading control.

(e) Quantification of Gli1, Ptch1 and Ccnd1 in GNPs treated with H89 or KT5720 over control.

(f) Bar graphs depict the relative fold change of Gli1 and Ptch1 expression in GNPs transfected with pGsCA with or without H89 treatment over control.

(g) Control and Gnas mice (n = eight for each group) were randomized to receive Rolipram or vehicle from P35 to P65. Representative images of H/E staining of cerebellar sections and brain morphology were shown. Arrows: the cerebellum.

(h) Scatter dot-plot depicts average tumor volumes estimated for vehicle- and Rolipram-treated Gnas mutants. Lines: mean values ± SEM.

(i) Images show immunostaining of Zic1 and BrdU in vehicle and Rolipram-treated GFAP:Gnas tumors at P65. Insets: high magnification in boxed areas. Bar graph (right) depicts the percentage of BrdU⁺/Zic1⁺ cells (n = eight animals each group).

(j) Kaplan-Meier survival curves for GFAP:Gnas mice (n = 12 per group) were randomized to receive Rolipram or vehicle at P30 for six weeks. P value for the log-rank test =0.0071.

Scale bars in g, 300 μm; i, 20 μm (insets 4 μm). Data shown in a-f are the mean ± SEM representing at least three independent experiments. *P < 0.05, ** P < 0.01; Student's t-test.