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. 2014 Jan;5(1-2):41–55. doi: 10.18632/genesandcancer.5

Fig 3. CmpdA reduced growth of KRAS positive lung cancer cells depends on loss or mutation of p53.

Fig 3

A) Human and mouse KRAS positive lung cancer cell lines harboring wildtype (WT) p53 (A549, H460, KE67), mutant p53 (H1792) or lacking (null) p53 (H358, KPF54) were treated with either 0.1% DMSO or 5μM CmpdA as indicated. 2μg/mL Doxorubicin (Doxo) was used as a positive control. Cell growth was measured at the indicated timepoints using a colorimetric MTS tetrazolium assay (CellTiter 96® AQueous One Solution Cell Proliferation Assay from Promega, Madison, WI). B) p53 WT KE67 and p53 null KPF54 cell lines were transfected with 100ηg of an NF-κB-responsive firefly luciferase reporter vector (3x-κB-Luc) and 5ηg of Renilla luciferase vector (pRL-TK) and NF-κB activity was analyzed by dual luciferase reporter assays. –LUC) negative control (cells transfected with pcDNA3 instead of 3x-κB-Luc and pRL-TK); RLUs) Relative luciferase units. C) H358 and A549 cells were analyzed for NF-κB activity by Western Blotting. Antibodies used are indicated. D) p53 null H358 cells were transfected with 100ηg of an NF-κB-responsive firefly luciferase reporter vector (3x-κB-Luc), 5ηg of Renilla luciferase vector (pRL-TK) and 200ηg of either empty pcDNA3 or pcDNA3-p53. After transfection, cells were treated with either 0.1% DMSO or 5μM CmpdA for 16h. NF-κB activity was analyzed by dual luciferase reporter assays. Expression of recombinant p53 was evaluated by Western Blotting. Antibodies used are indicated. RLUs) Relative luciferase units. E) p53 WT murine KE67 cells were transfected as described in methods with a siRNA targeting murine p53 (sip53) or non-targeting siRNA (NTctrl). At 48h these cells were transfected with 100ηg of an NF-κB-responsive firefly luciferase reporter vector (3x-κB-Luc) and 5ηg of Renilla luciferase vector (pRL-TK) and analyzed at 72h after siRNA transfection for knockdown efficiency by Western Blotting. Antibodies used are indicated. F) KE67 cells were transfected as described in (E) and NF-κB activity was analyzed by dual luciferase reporter assays. –LUC) negative control (cells transfected with pcDNA3 instead of 3x-κB-Luc and pRL-TK); RLUs) Relative luciferase units. G) KE67 cells were transfected as described in methods with a siRNA targeting murine p53 (sip53) or non-targeting siRNA (NTctrl). Subsequently cell growth was measured at 72h post-transfection using a colorimetric MTS tetrazolium assay (CellTiter 96® AQueous One Solution Cell Proliferation Assay from Promega, Madison, WI). H) A549 cells were transfected as described in methods with a siRNA smartpool targeting human p53 (sip53) or a non-targeting siRNA (NTctrl). Subsequently, cells transfected with sip53 were transfected either with pcDNA3 (empty vector control) or with pcDNA3-p53 vector. Analysis of p53 knockdown and p53 re-expression was performed by Western Blotting. Antibodies used are indicated. I) A549 cells were transfected as described in H and cell growth was evaluated at 72h post-transfection using a colorimetric MTS tetrazolium assay (CellTiter 96® AQueous One Solution Cell Proliferation Assay from Promega, Madison, WI). Statistical significance in all cases was measured by Student's t-test (*p<0.05) when compared to experimental control samples (siCtrl). Error bars represent average ± 1s.d.