Article Figures & Data
Figures
- Figure 1.
Sedimentation properties of ΨCx43 RNA. Hs578T and MDA 435 cell lysates were fractionated by sucrose gradient centrifugation and collected while continuously monitoring the absorbance at 254 nm. Analysis of Cx43/ΨCx43 mRNAs was done using semiquantitative RT-PCR followed by NcoI digestion.
- Figure 2.
ΨCx43 is preferentially translated and binds with higher affinity to the translational machinery than Cx43. Cx43V5 and ΨCx43Myc cRNAs were in vitro translated in a cell-free system. Translation products were analyzed by electrophoresis on a 12% SDS-PAGE, transferred, and immunoblotted with V5 and Myc antibodies, respectively. Band intensities were quantified using Scion Image and results were plotted in GraphPad Prism 3.0. A, ratios of concentrations of Cx43V5/ΨCx43Myc were varied from 100 to 0. B, cRNAs were added to the translation reaction with increasing amounts of KCl.
- Figure 3.
Polyribosome profile analysis of Cx43 mRNA following the addition of ΨCx43 mRNA. Cx43 and ΨCx43 mRNAs were extracted and analyzed as mentioned in Materials and Methods. A, polysome profile for Hs578T neo and Hs578T ΨCx43 cells. B, polysome profile for MDA 231 neo and MDA 231 ΨCx43 cells. Band intensities were quantified using Scion Image and results were plotted in GraphPad Prism 3.0. Cx43 mRNA levels in each fraction were expressed as a percentage of total levels summed over all fractions.
- Figure 4.
Effects on Cx43 RNA and protein expression in response to exogenous Cx43 and ΨCx43 siRNAs. A, RT-PCR products digested with NcoI from MDA 231 neo and ΨCx43 cells. Cx43 RNA decline was quantified using Scion Image and results were plotted in GraphPad Prism 3.0 as the decline in MDA 231 ΨCx43 cells relative to the control (neo cells). B, Cx43 protein levels were determined by Western blot detection of Cx43 as described in Materials and Methods. C, expression of Cx43 and ΨCx43 RNAs was determined by RT-PCR followed by NcoI digestion in MCF7 cells stably expressing Cx43 and ΨCx43 siRNAs. Band intensities were quantified using Scion Image and results were plotted in GraphPad Prism 3.0. Cx43 and ΨCx43 expression levels were analyzed relative to the control cells (MCF7 PSR). D, Cx43 expression was determined by Western blot of protein lysates from MCF7 cells expressing Cx43 and ΨCx43 siRNAs using Cx43 antibody.
- Figure 5.
ΨCx43 knockdown sensitizes cells to chemotherapeutics. A, Cx43 expression levels analyzed by Western blot in MDA 231 cells stably expressing ΨCx43 siRNA. Band intensities were quantified using Scion Image and results were plotted in GraphPad Prism 3.0. Cx43 and ΨCx43 expression levels were analyzed relative to the control cells (MDA 231 PSR). B, IC50 values were estimated from dose-response curves obtained after 48 h of exposure to different doxorubicin and paclitaxel concentrations as indicated in Materials and Methods. Points, mean of four independent experiments; bars, SE. ***, P < 0.0001.
Tables
- Table 1.
Expression of Cx43 and ΨCx43 in breast cancer cell lines
Cell lines ΨCx43 Cx43 RNA Cx43 protein Hs578T − ++++ ++++ MDA 231 + ++++ +++ MCF7 ++ + − MDA 435 +++ + −
Volume 8, Issue 4
