Figure 6. Sema3C Activates Rac1 To Promote Survival of GSCs.

(A) Detection of the GTP-bound form of active Rac1 in a pull-down assay from the lysates of GSCs and NPCs expressing shNT or shSema3C. Cell lysates from the indicated cells were analyzed for protein levels of total Rac1, Sema3C, p-ERK1/2, p-Akt1, Sox2 and Olig2.

(B) GSCs transduced with shNT, shPlexinA2 or shPlexinD1 were cultured with different doses of recombinant human Sema3C protein (r-Sema3C). IB analysis of active GTP-Rac1 by pull-down assay from the lysates of indicated GSCs.

(C) RFP-GSCs and matched GFP-NSTCs were mixed at a 1:1 ratio, plated on stem cell Matrigel-coated plates and treated with Rac1 inhibitor NSC23766 (50|JM). Representative images of mixed cells on day 0 and day 3 (left). Quantification of RFP-GSC and GFP-NSTC cell number 3 days after treatment (right). Data are means ± standard deviation (SD) (n = 3).

(D) Knockdown of Rac1 with two separate shRNA resulted in significantly decreased cell viability in GSCs but not in NSTCs. Data are means ± standard deviation (SD) (n = 3).

(E) Knockdown of Rac1 via two separate shRNAs resulted in decreased tumorsphere formation. Data are means ± standard deviation (SD) (n = 3).

(F) IB analysis of cleaved-PARP and -caspase3 in GSCs and matched NSTCs transduced with shNT or two separate shRac1. Active GTP-Rac1 by pull-down assay is shown.

(G and H) IB analysis of p-p65, p65, CyclinD1, Survivin, XIAP and BCL2 in two GSCs treated with Rac inhibitor (G) or transduced with shNT or two separate shRac1 (H). *p< 0.05, **p < 0.01, ***p < 0.001. See also Figure S6