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Fig. 5. Expression and distribution of {alpha}3-integrin and laminin-5 {alpha}3-chain in HMECs that express altered levels of CBP. (A) Immunofluorescence characterization of {alpha}3-integrin and laminin-5 {alpha}3-chain expression in cells sensitive and resistant to rECM-mediated growth arrest and apoptosis. Frozen section of early and late passage HMEC-LXSN controls (LXSN; passage 10 and 16) and HMEC-E6 cells (E6; passage 10 and 18) were grown in rECM for 6 days, cryosectioned, and immunostained for localization of {alpha}3-integrin and laminin-5 {alpha}3-chain expression. {alpha}3-integrin and laminin-5 {alpha}3-chain expression was primarily localized at the basal surface of early and late passage HMEC-LXSN and early passage HMEC-E6 cells (arrowheads). By contrast, CBP-poor late passage HMEC-E6 cells showed dispersed membrane and intracellular staining of {alpha}3-integrin (arrow) and qualitatively decreased laminin-5 {alpha}3-chain expression. (B) Immunofluorescent characterization of {alpha}3ß1-integrin and laminin-5 {alpha}3-chain expression in HMECs treated with CBP antisense ODNs. Frozen section of early passage HMEC-LXSN vector controls (LXSN) (passage 11) and HMEC-E6 cells (E6) (passage 11) treated either with CBP antisense ODN (A3342V; CBP-as) or inactive CBP ODN (scrA3342V; CBP-scr). Cells were grown in rECM for 6 days, cryosectioned, and immunostained for {alpha}3-integrin or laminin-5 {alpha}3-chain. {alpha}3-integrin and laminin-5 {alpha}3-chain expression was primarily localized at the basolateral surface in HMEC-LXSN and HMEC-E6 cells treated with inactive CBP ODNs (arrowheads). By contrast, HMEC-LXSN and HMEC-E6 cells treated with antisense CBP ODNs demonstrated disorganized membrane and cytosolic staining of {alpha}3-integrin (arrows) and markedly reduced laminin-5 {alpha}3-chain expression. (C) Immunofluorescent characterization of {alpha}3ß1-integrin and laminin-5 {alpha}3-chain expression in late passage HMEC-E6 cells expressing exogenous CBP. Frozen sections of immunostained late passage HMEC-E6-CBP– vector controls (CBP(–); passage 17) and late passage HMEC-E6-CBP+ cells (CBP(+); passage 17). Cells were grown in rECM for 6 days, cryosectioned, and immunostained for {alpha}3-integrin or laminin-5 {alpha}3-chain. HMEC-E6-CBP– cells demonstrated disorganized membrane and cytosolic staining of {alpha}3-integrin and markedly reduced levels of laminin-5 {alpha}3-chain expression. By contrast, HMEC-E6-CBP+ cells exhibited a qualitative increase in laminin-5 {alpha}3-chain expression. Although there was persistent membrane and cytosolic staining of {alpha}3-integrin and laminin-5 {alpha}3-chain, there was also increased localization at the basolateral surface (arrowheads).





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