spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

Right arrow Help viewing high resolution images
Right arrow Return to article
(Downloading may take up to 30 seconds.
If the slide opens in your browser, select File -> Save As to save it.)
Click on image to view larger version.



Fig. 3. Loss or suppression of CBP expression inhibits CBP occupancy of the LAMA3A promoter and suppresses LAMA3A promoter activity and laminin-5 expression. (A) Late passage HMEC-E6 cells do not exhibit CBP occupancy of the 277 bp AP-1-rich region of the LAMA3A promoter. ChIP was performed in HMEC-LXSN controls (LXSN) (passage 11 and 16), and early passage HMEC-E6 cells (E6) (passage 11) and compared with CBP-poor, late passage HMEC-E6 cells (E6) (passage 18). Input controls test the integrity of the DNA samples. (B) LAMA3A promoter activity was measured in early and late passage HMEC-LXSN controls (LXSN) (passage 11 and 16), and early passage HMEC-E6 cells (E6) (passage 10) and compared with CBP-poor, late passage HMEC-E6 cells (E6) (passage 18). Data represent two independent experiments performed in triplicate. (C) CBP protein expression is suppressed by antisense ODNs. HMEC-LXSN vector controls (LXSN) (passage 12) and early passage HMEC-E6 cells (E6) (passage 12) were cultured in the presence of (1) no treatment, (2) active CBP-specific ODN (A3342V), and (3) inactive CBP ODN (scrA3342V). Resultant cells were analyzed for CBP protein expression as described in Materials and Methods. Equal amounts of protein lysate were added per lane. Actin was used as a loading control. (D) CBP protein expression is suppressed in rECM culture on days 1-11. Early passage HMEC-E6 cells (passage 12) were cultured in rECM and treated daily with active CBP-specific ODN (A3342V) (AS) or inactive CBP ODN (scrA3342) (S). Resultant cells were tested for CBP protein expression as described in Materials and Methods. Equal amounts of protein lysate were added per lane and actin was used as a loading control. (E) Early passage HMEC-E6 cells (passage 10) were treated with CBP-specific ODN (A3342A; As) and inactive CBP ODN (scrA3342V; Scr), grown in contact with rECM, and tested by ChIP to determine whether suppression of CBP expression resulted in a loss of CBP binding to the AP-1-rich site of the LAMA3A promoter. Input controls test the integrity of the DNA samples. (F) Suppression of CBP expression results in decreased LAMA3A promoter activity. Early passage HMEC-LXSN controls (passage 11) and HMEC-E6 cells (passage 11) grown in rECM and treated with CBP-specific antisense ODNs (A3342V) (AS-CBP) exhibit decreased LAMA3A promoter activity relative to cells treated with inactive ODNs (scrA3342V) (scr-CBP). Data represent two independent experiments performed in triplicate. Error bars show standard error. (G) Laminin-5 {alpha}3-chain mRNA expression is suppressed by antisense ODNs. Early passage HMEC-E6 cells (passage 12) were cultured in the presence of no treatment (C), inactive CBP ODN (scrA3342V; Scr), and active CBP-specific ODN (A3342V; As). Resultant cells were analyzed for laminin-5 {alpha}3-chain mRNA expression by semi-quantitative RT-PCR as described in Materials and Methods. Actin was used as a loading control.





Right arrow Return to article