Figure 4. Rac proteins regulate the activation of STAT3 and ERK, and the expression of stemness markers in glioblastoma tumorspheres.

A. U87-MG cells transiently overexpressed HA-Rac proteins, or B. U251-MG cells stably expressing short-hairpin sequences to target specific Racs were starved for overnight and then treated with or without IL-6 (10 ng/mL) or oncostatin M (OSM, 10 ng/mL) for indicated time to induce the STAT3 and ERK activation. Protein lysates were prepared and subjected to immunoblot analysis with antibodies against phosphorylated STAT Tyr705, total STAT, phosphorylated ERK1/2 and total ERK1/2 antibodies. Ectopic expressed HA-Rac proteins were detected by anti-HA antibody. C. Lysates of U373-MG cells harboring control scramble sequences or shRacs were subjected to immunoblot analysis with antibodies against CD133 and Sox2 respectively. GAPDH detected by its specific antibody served as the loading control. D. The quantitative results for expression levels of CD133 and Sox2 from three independent Western blot analysis. E. U373 tumor spheroid cells stably expressing scramble shRNA or Rac1-3-targeting shRNA were treated with CoCl₂ for 16 hrs. The surface CD133 expression was then detected by staining with PE-conjugated anti-CD133 antibody, and analyzed by flow cytometry. The results were normalized with IgG control staining. (*: p<0.05, t-test.)