Figure 2.

Knocking Down ITGB4 Attenuates Proliferation and Migration and Increases Sensitivity of Cisplatin-Resistant Cells

H2009 and H1993 stable cell lines expressing mKate2 were transfected with control (Si Scramble) or ITGB4-specific (Si ITGB4) siRNA.

(A and B) (A) ITGB4 knockdown cells (red) had a significantly reduced proliferation rate than control cells (black). (∗∗∗∗p < 0.0001 two-way ANOVA). (B) Immunoblotting and qPCR data confirming the knockdown.

(C and D) (C) A scratch wound assay demonstrating the effect of knocking down ITGB4. ITGB4 knockdown (red) significantly halted the migration and did not close the wound completely after 96 h in both resistant cell lines. (∗∗∗∗p < 0.0001 two-way ANOVA). (D) Rate at which the wound closed was also significantly decreased in ITGB4 knockdown cells (red). (∗∗∗∗p < 0.0001 and ∗p < 0.0156 two-way ANOVA).

(E) Cisplatin (10 μM) treatment for 72 h reduced expression of phosphorylated PXN, PXN, and total FAK, but not ITGB4. (LE = low exposure, HE = high exposure).

(F) ITGB4 knockdown in H1993 cells inhibited proliferation and addition of cisplatin had a cytotoxic effect (∗∗∗∗p < 0.0001 two-way ANOVA).

(G) ITGB4 knockdown in H1993 cells increased caspase-3/7 activity. Treating ITGB4 knockdown cells with cisplatin had an added effect of inducing caspase activity, but drug treatment alone did not have a cytotoxic effect (∗∗∗∗p < 0.0001 two-way ANOVA).

(H) ITGB4 knockdown in H2009 cells inhibited proliferation by 72 h and addition of cisplatin had a cytostatic effect (∗∗∗∗p < 0.0001 two-way ANOVA).

(I) ITGB4 knockdown in H2009 cells did not induce caspase activity. Cisplatin treatment to ITGB4 knockdown cells for 72 h had an additive effect to induce caspase activity (∗∗∗∗p < 0.0001 two-way ANOVA).

(J) Immunoblot showing that MET protein expression in H1993 cells was reduced 4 days after knocking down ITGB4. Data are represented as mean ± SD.