Figure 3. CDC20-APC regulation of SOX2 protein and transcription controls GSC invasion and self-renewal.

(A) GSCs (B18, A1) were maintained in GSC or differentiating medium (Diff) (containing fetal bovine serum and no growth factors) for 14 days. Cell lysates were subjected to immunoblotting using indicated antibodies.

(B) B18 GSCs were transduced with CDC20 RNAi (CDC20i.1 and CDC20i.2) or control LacZ RNAi (C) lentivirus. 7 days later, cell lysates were subjected to immunoblotting using indicated antibodies.

(C) GSCs were transduced with the indicated lentiviruses. 7 days later, cell lysates were subjected to immunoblotting using indicated antibodies. Expression of CDC20-Res rescued the CDC20 RNAi-triggered decrease in SOX2 protein. C = SHC002 virus. Vec = control vector virus.

(D) GSCs transduced with CDC20-expressing lentivirus or control vector virus (Vec) were maintained in RHB-A media for 5 days. Cell lysates were subjected to immunoblotting using indicated antibodies.

(E) GSCs were transduced with ANAPC2 RNAi or control LacZ RNAi (C) lentivirus. 7 days later, cell lysates were subjected to immunoblotting using indicated antibodies.

(F) GSCs were treated with ProTAME or DMSO (Veh) for 12 hours. Cell lysates were subjected to immunoblotting using indicated antibodies.

(G) GSCs were treated with 20 μM of ProTAME or DMSO (Veh) as indicated. Cell lysates were subjected to immunoblotting using indicated antibodies.

(H) GSCs were treated with 20 μM of ProTAME, 5 μM of proteasome inhibitor MG132, or a combination of both for 8 hours. Cell lysates were subjected to immunoblotting using indicated antibodies. Veh = DMSO.

(I) GSCs were transduced with CDC20 RNAi (CDC20i.2) or control SHC002 (C) lentivirus for 7 days and treated with 10 μM of MG132 or DMSO (Veh) for 6 hours. Cell lysates were subjected to immunoblotting using indicated antibodies.

(J) GSCs stably infected with the SOX2 transcriptional reporter (hSRR2) or control reporter (mCMV) were transduced with CDC20 RNAi (CDC20i.2) or control scrambled (Scr) lentivirus. 7 days later, luciferase assays were performed. Luciferase values were normalized by total protein, and fold-change calculated by scaling to Scr + mCMV values (=1). Data represent mean+SEM. CDC20 RNAi decreased SOX2 reporter activity compared to control (ANOVA, P < 0.0001, n = 3).

(K) GSCs were infected with CDC20 RNAi (CDC20i.1 and CDC20i.2) or control scrambled (Scr) lentivirus. RNA was harvested 7 days later and reverse transcribed into cDNA. qPCR was performed on samples using specific primers for human NES. GAPDH and ACTB were used as reference genes. Data represent mean+SEM. CDC20 RNAi decreased NES mRNA in GSCs compared to control. (ANOVA, P < 0.0001, n = 3).

(L) GSCs infected with the CDC20-expressing or control vector (Vec) lentivirus together with the SOX2 RNAi (SOX2i) or control SHC002 RNAi (Scr) virus were subjected to the in vitro Matrigel transwell assay 7 days later. Data represent mean+SEM. Expression of CDC20 increased invasion compared to control infection (ANOVA, P = 0.007, n = 6). Expression of CDC20 plus SOX2 RNAi reduced invasion compared to infection with Scr plus either the CDC20-expressing or control vector virus. (ANOVA; P < 0.0001 and P = 0.001, respectively).

(M) GSCs infected with SOX2-expressing or control vector (Vec) lentivirus together with CDC20 RNAi or control SHC002 RNAi (Scr) were treated as in (L). Data represent mean+SEM. CDC20 knockdown decreased invasiveness compared to control (ANOVA, P < 0.0001, n = 4). Expression of SOX2 plus CDC20 RNAi increased invasion compared to infection with Vec plus CDC20 RNAi viruses (ANOVA; P = 0.011).

(N) GSCs infected as in (L) were subjected to the extreme limiting dilution assay as in Figure 1L. Data represent mean+SEM. Expression of CDC20 plus Scr increased self-renewal compared to control (ANOVA, P = 0.004, n = 3). Expression of CDC20 plus SOX2 RNAi reduced self-renewal compared to infection with Scr plus either the CDC20-expressing or control virus. (ANOVA; P < 0.0001 and P = 0.001, respectively).

(O) GSCs were transduced with the indicated lentiviruses and subjected to the in vitro Matrigel transwell assay as in (L). Data represent mean+SEM. SOX2 RNAi plus control vector virus (Vec) decreased GSC invasiveness compared to control (ANOVA; P < 0.001, n = 6). Expression of full-length SOX2-Res (FL) rescued the SOX2 RNAi-triggered defect in invasiveness (ANOVA; P < 0.0001) whereas SOX2-ResΔ110–200 did not (ANOVA; P = 0.9).

See also Figure S4.