Fig. 2. Inhibition of the ITGB4/PXN axis with siRNA or CFZ sensitizes cells to sotorasib treatment.

(A and B) Effect of PXN and ITGB4 single knockdowns and double knockdown with sotorasib on caspase activity after 72 hours of drug treatment. Two-way ANOVA was used to calculate the statistical significance for each group (si Control, si PXN, si ITGB4, and si PXN + si ITGB4. n = 3 per group; ns, not significant; *P < 0.05, **P < 0.001, ***P = 0.0001, and ****P < 0.0001). (B) Immunoblot confirmed knockdown with ITGB4 and PXN siRNA, and effect of sotorasib on protein expression and signaling after 72 hours was determined. (C) H23 cells with ITGB4 overexpression (OE) were treated with an increasing concentration (0.3 to 5 μM) of sotorasib for 72 hours to determine the percentage change in cell growth. (D) H23 cells with ITGB4 overexpression were treated with an increasing concentration (3 to 9 μM) of sotorasib for 72 hours to determine the effect on protein expression and signaling with immunoblot. (E and F) SW1573 cells were treated with eight different concentrations of sotorasib and CFZ in the form of a matrix to determine the % inhibition of proliferation. Synergy scores were calculated and represented as a Bliss synergy matrix. (G) SW1573 cells were treated with an increasing concentration of sotorasib (1 to 20 μM) without or with the addition of CFZ (10 nM) for 72 hours to determine changes in protein expression and signaling by immunoblot. (H) Immunoblot showing changes in the component of proteasomal complex or autophagy-associated genes.