Fig 1: Inhibition of RAC2 or PAK1 reverses the tumor-promoting effects of GADD45g deficiency in human MPN cells.a, b HEL and SET-2 cells were transfected with GADD45g-specific shRNA or shCtrl for 48 h, followed by transfection with RAC2-specific shRNA (shRAC2) or scrambled control (Scr) for another 48 h. Effects of RAC2 knockdown on colony formation (a) and apoptosis (b) of these cells. (c) HEL and SET-2 cells were transfected with GADD45g-specific shRNA or shCtrl for 48 h. Cells were then plated in methylcellulose containing IPA-3 (10 μM). Colonies were counted and analyzed on day 14. d HEL and SET-2 cells were transfected with GADD45g-specific shRNA or shCtrl for 48 h, followed by treatment with vehicle or IPA-3 (10 μM) for 48 h, and the percentage of apoptosis cells was examined. e Effects of EHT 1864 treatment (5 μM for 10–14 days) on colony formation of primary BM CD34+ cells from GADD45ghigh and GADD45glow patients with MPN (n = 3 per group). f Effects of IPA-3 treatment (10 μM for 10–14 days) on colony formation of primary BM CD34+ cells from GADD45ghigh and GADD45glow patients with MPN (n = 3 per group). For (a–d): Figures shown are representative of three independent experiments with similar results. Data are shown as mean ± SD (n = 3 technical replicates). Comparisons were evaluated by two-tailed Student’s t test, and multiple groups were analyzed with one-way ANOVA.
Fig 2: RAC2-PAK1 pathway mediates the Gadd45g insufficiency-induced activation of PI3K-AKT.a c-kit+ BM cells from diseased Gadd45g+/− and Ctrl mice were lysed, precipitated with anti-GADD45g antibody, and detected by Western blot with anti-RAC2 and -GADD45g antibodies. b Representative immunofluorescence micrographs showing colocalization of GADD45g with RAC2 in c-kit+ BM cells of Ctrl mice. Panels represent nucleus (blue), GADD45g (green), RAC2 (yellow), and merged images, respectively. Arrows in merged image indicate colocalization of GADD45g with RAC2. Bar represents 10 μm. c Representative immunofluorescence micrographs showing cellular distribution of GADD45g, RAC2 and RAC1 in cord blood CD34+ cells from healthy human donors. Panels represent nucleus (blue), GADD45g (green), RAC2 (orange), RAC1 (pink), and merged images, respectively. Arrows in merged image indicate colocalization of GADD45g with RAC2. Bar represents 5 μm. d Western blot analysis of RAC2-GTP and total RAC2 protein levels in c-kit+ BM cells from diseased Gadd45g+/− and Ctrl mice. e HEL and SET-2 cells transfected with GADD45g-specific shRNA or shCtrl were lysed, precipitated with anti-GADD45g antibody, and detected by Western blot with anti-RAC2 and -GADD45g antibodies. f Western blot analysis of RAC2-GTP and total RAC2 protein levels in HEL and SET-2 cells transfected with GADD45g-specific shRNA or shCtrl. g Western blot analysis of p-PAK1 and total PAK1 protein levels in c-kit+ BM cells from diseased Gadd45g+/− and Ctrl mice. h Western blot analysis of p-PAK1 and total PAK1 protein levels in HEL and SET-2 cells transfected with GADD45g-specific shRNA or shCtrl. i HEL and SET-2 cells were transfected with GADD45g-specific shRNA or shCtrl for 48 h, followed by transfection with RAC2-specific shRNA (shRAC2) or scrambled control (Scr) for another 48 h. The protein levels of p-PAK1, total PAK1, p-PI3K, total PI3K, pAKT-Ser473 and total AKT were examined by Western blot. j HEL and SET-2 cells were transfected with GADD45g-specific shRNA or shCtrl for 48 h, followed by treatment with vehicle or IPA-3 (10 μM for 48 h). The protein levels of p-PI3K, total PI3K, pAKT-Ser473 and total AKT were examined by Western blot. For (a–j): At least three independent experiments with similar results were performed.
Supplier Page from Abcam for Anti-RAC2 antibody