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Fig. 6. RNAi of CASK leads to increased cell proliferation in HaCaT keratinocyte monolayer cultures. (A) Efficiency of CASK RNAi. HaCaT cells were transfected with control siRNA or three different siRNAs targeting CASK for 96 hours, and then harvested for immunoblotting and probed with antibodies against CASK and -tubulin. (B) Growth of non-synchronised HaCaT cells transfected with control siRNA, CASK siRNA1 or CASK siRNA2. Cells were subjected to the MTT proliferation assay at 72 hours post siRNA transfection. Error bars indicate +s.d. (n=3). (C) Growth curves of control-siRNA- and CASK-siRNA-transfected HaCaT cells. Serum-starved and re-stimulated cells were transfected with control siRNA or CASK siRNA, and cells were subjected to MTT proliferation assay at 0, 24, 48, 72 and 96 hours post siRNA transfection. Error bars indicate ±s.d., (n=3). (D) Phase-contrast micrographs of control-siRNA and CASK-siRNA-transfected HaCaT cells at 24, 48, 72 or 96 hours post siRNA transfection. Scale bars, 100 µm. (E) Protein extracts from control-siRNA and CASK-siRNA-transfected HaCaT cells were immunoblotting and probed with antibodies against CASK, phosphorylated Rb (pRb), total Rb and actin. (F) FACS cell-cycle analysis of DNA content of control-siRNA and CASK-siRNA-transfected synchronised HaCaT cells at 0 and 60 hours post siRNA transfection. Percentages of cells in G1 and S phases, and at G2 or M phase are indicated. (G) FACS cell-cycle analysis of DNA content of control-siRNA and CASK-siRNA-transfected non-synchronised HaCaT cells at 60 hours post siRNA transfection. Percentages in G1 and S phases, and at G2 or M phase are indicated.

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