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Fig. 9. Suppression of laminin-5 {alpha}3-chain in early passage HMEC-E6 cells blocks apoptosis in rECM. (A) Laminin-5 {alpha}3-chain mRNA expression is compared by semi-quantitative RT-PCR in three early passage HMEC-E6 cells (passage 10) clones expressing siRNA sequences directed against laminin-5 {alpha}3-chain (si#1, si#2, si#3) or control siRNA (si-cont), and in HMEC-E6 control cells (no tx). Actin was used as a loading control. (B,C) Suppression of laminin-5 {alpha}3-chain expression in early passage HMEC-E6 cells results in increased proliferation in rECM culture. (B) Ki-67 staining indices in early passage HMEC-E6 cells (passage 11) expressing siRNA sequence #2 directed against laminin-5 {alpha}3-chain or siRNA control. (C) DAPI staining of cell nuclei demonstrates the number of nuclei per cell cluster formed by early passage HMEC-E6 cells (passage 11) expressing either siRNA sequence #2 directed against laminin-5 {alpha}3-chain or siRNA control. Two hundred cells were surveyed per time point and indices were calculated from an average of three independent experiments. Error bars show standard error. (D) Electron micrographs of early passage HMEC-E6 cells (passage 11) expressing siRNA sequence #2 directed against laminin-5 {alpha}3-chain (si-laminin-5) or control siRNA (si-control). Early passage HMEC-E6 cells expressing control siRNA underwent apoptosis when grown in rECM for 7 days as shown by (1) nuclear condensation (n), (2) cell shrinkage and separation, and (3) margination of chromatin (mr). By contrast, HMEC-E6 cells expressing siRNA directed against laminin-5 {alpha}3-chain did not undergo apoptosis and instead formed large, dense, irregularly shaped multicellular colonies that have no central lumen. (E) Relative levels of TdT-positive cells in early passage HMEC-E6 cells (passage 10) treated either with siRNA sequence #2 directed against laminin-5 {alpha}3-chain (laminin-5 si-RNA) or control siRNA (si-control). Apoptosis was measured by TUNEL-staining. Data represents an average of two independent experiments in triplicate. Error bars show standard error. (F) Immunofluorescent characterization of laminin-5 {alpha}3-chain expression in early passage HMEC-E6 cells (passage 12) expressing si-RNA directed against laminin-5 {alpha}3-chain (si-laminin-5) or control siRNA (si-control). Cells were grown in rECM for 6 days, cryosectioned, and immunostained for laminin-5 {alpha}3-chain. Laminin-5 {alpha}3-chain expression was primarily localized at the basolateral surface in early passage HMEC-E6 cells expressing control siRNA. By contrast, early passage HMEC-E6 cells expressing si-RNA directed against laminin-5 {alpha}3-chain exhibited markedly reduced laminin-5 {alpha}3-chain expression.





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