FIGURE 5.
siRNA knockdown of α6 integrin in primary lens cell cultures led to suppression of survival proteins in the Bcl-2 and IAP families and cell death by apoptosis. A–F, α6 integrin was knocked down using a siRNA approach (siα6 integrin) in primary lens cell cultures. Controls were treated with non-targeting siRNA (siC). A, following α6 integrin siRNA knockdown cultures were immunoblotted for activated p-NFκB (p65 Ser-276) and total NFκB p65; Bcl-2 (B), Bcl-XL (C), and IAP family proteins (D–F): ch-IAP-1 (D), X-IAP (E), and survivin (F). G, induction of apoptosis following α6 integrin knockdown was examined by TUNEL assay. Confocal images show that α6 integrin knockdown induced cell death by apoptosis, TUNEL (red), nuclei (blue), F-actin (green); H, quantification for TUNEL positive cells showed a significant increase in the number of TUNEL positive cells in the absence of α6 integrin. Results show that α6 integrin is an essential upstream signal of NFκB-mediated expression of survival proteins in the Bcl-2 and IAP families in lens epithelial cells. Densitometric analyses of immunoblots for Bcl-2, Bcl-XL, ch-IAP-1, X-IAP, and survivin were plotted as a ratio to GAPDH; and p-NFκB p65 against total NFκB p65. For confocal imaging, z-stacks were collected and analyzed, and the data are presented as a single optical plane of 0.5 μm. Results are representative of at least three studies. Error bars represent S.E. *, p < 0.05, t test. Scale bar, 20 μm.