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. Author manuscript; available in PMC: 2014 Jun 2.
Published in final edited form as: Oncogene. 2011 Jun 6;30(48):4765–4779. doi: 10.1038/onc.2011.196

Figure 2. c-Myc modifies sensitivity to HAMLET-induced cell death.

Figure 2

A Healthy kidney cells (HRPTEC), kidney carcinoma cells (A498) and lung carcinoma cells (A549) were treated with HAMLET (21 μM) and ATP levels were measured after 3, 6 and 24 hours. HAMLET caused a reduction in ATP levels in the carcinoma cells but not in the healthy cells. ATP levels are given in % of untreated cells at the same time point. Means of 3 experiments + SEMs. B qPCR quantification of c-Myc mRNA levels. Relative c-Myc mRNA levels are shown (c-Myc/GAPDH, in % of levels in healthy cells). Means of 2 experiments + SEMs. C c-Myc Western blot with Actin as a loading control. D, E Inhibition of c-Myc expression by transfection of A549 cells with c-Myc shRNAs (72 hours) was confirmed by qPCR (D) and c-Myc Western blot with Actin as loading control (E). Relative c-Myc mRNA levels are shown (c-Myc/GAPDH, in % of levels in CT shRNA-transfected cells). Means of 2 experiments + SEMs. ntf = non-transfected. F c-Myc shRNA-transfected A549 cells (72 hours) become more resistant to the lethal effect of HAMLET. The cytotoxic effect of HAMLET was quantified after 3 hours as a reduction in ATP levels or in the number of cells, compared to non-transfected cells (ntf) and cells transfected with CT shRNA. ATP levels in % of levels in respective untreated cells. Means of 2-3 experiments ± SEMs (ATP) or means of triplicates in 1 experiment + SDs (Cell survival). G A549 cells were cell cycle-arrested by serum starvation (48 hours) or double thymidine block and treated with HAMLET. Cancer cell viability was assessed after 3 hours after using Trypan blue exclusion or ATP measurements. Means ± SEMs of 3 experiments.