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Fig. 2. Laminin-5 3-chain expression is decreased in rECM-resistant, CBP-poor, late passage HMEC-E6 cells. (A) Analysis of differential gene expression in early (E6E) and late (E6L) passage HMEC-E6 cells (passage 10 and 18) relative to early passage HMEC-LXSN controls (passage 10) (LXSN). Cells were grown in contact with rECM and harvested for differential gene expression. All RNA combinations used for array analysis were obtained from cells that were matched for passage number, cultured under the identical growth conditions, and harvested at identical confluency. Data were collected in triplicate using independent biological replicates. Color-coding: green, downregulation of gene expression; red, induction; black, no significant change; grey, no data available. (B,C) Semi-quantitative RT-PCR analysis of integrin and laminin-5 mRNA expression in early (E) and late (L) passage HMEC-E6 cells (E6) (passage 10 and 18) and HMEC-LXSN controls (LXSN) (passage 10 and 16). Expression was normalized to ß-actin. These data are representative of three separate experiments. ^*Significantly different from LXSN-E (P<0.05); ^#significantly different from E6-E (P<0.05). (D) Laminin-5 3-chain protein expression is decreased in rECM-resistant, late passage HMEC-E6 cells (E6) (passage 18) relative to early passage HMEC-E6 cells (E6) (passage 11) and early and late passage HMEC-LXSN controls (LX) (passage 10 and 18). Western analysis was performed as described in Materials and Methods. Equal amounts of protein lysate were loaded per lane. Actin serves as a loading control.

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