Fig. 4.

Combination of WNT974 and ART induces KRAS protein degradation by increasing β-TrCP and GSK-3β expressions. The mRNA level of β-TrCP in A HCT116 and B SW620 cells after treating with 20 μM ART, 20 μM WNT974 or a combination of both for 48 h. The protein level of C β-TrCP and D GSK-3β in the CRC cells after treating with 20 μM ART, 20 μM WNT974 or a combination of both for 48 h. The E–F mRNA expression and G–H protein expression of β-TrCP after siRNA-mediated knockdown of β-TrCP in the CRC cells. I–J Protein expression of KRAS in β-TrCP-knockdown or ANAPC2-knockdown cells. K Ubiquitination of KRAS in β-TrCP-knockdown cells after treating with 20 μM ART, 20 μM WNT974 or a combination of both treatments for 48 h, in the presence of GSK-3β inhibitor. L Protein expression of KRAS in the β-TrCP-knockdown CRC cells after treating with 20 μM ART, 20 μM WNT974 or a combination of both for 48 h, in the presence of GSK-3β inhibitor. M HCT116 and SW620 were treated with 20 μM WNT974, 20 μM ART or the combination of both for 48 h. The p-Akt (Ser473), t-Akt, p-PI3K (Tyr458), t-PI3K, p-mTOR (Ser2448), t-mTOR protein level were examined. Shown is mean ± SE, n = 3 individual experiments, *p < 0.05, **p < 0.01, *** p < 0.001 compared to control; a < 0.05, aa < 0.01, aaa < 0.001compared to ART; b < 0.05, bb < 0.01, bbb < 0.001 compared to WNT974