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A549 KRAS mutant G12S LKB1 null M CDKN2A (p16) M CDKN2a (p14) SMARCA4 H1299 p53 null N-Ras Q16K SK-MES-1 p53 null M CDKN2A (p16) M CDKN2a (p14) PC3 p53.

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Presentation on theme: "A549 KRAS mutant G12S LKB1 null M CDKN2A (p16) M CDKN2a (p14) SMARCA4 H1299 p53 null N-Ras Q16K SK-MES-1 p53 null M CDKN2A (p16) M CDKN2a (p14) PC3 p53."— Presentation transcript:

1 A549 KRAS mutant G12S LKB1 null M CDKN2A (p16) M CDKN2a (p14) SMARCA4 H1299 p53 null N-Ras Q16K SK-MES-1 p53 null M CDKN2A (p16) M CDKN2a (p14) PC3 p53 null PTEN null MCF7 PIK3CA mutation Estrogen receptor positive M CDKN2A (p16) M CDKN2a (p14) MDAMB231 p53 null KRAS mutant Estrogen receptor negative B-Raf M CDKN2A (p16) M CDKN2a (p14) NF2 Figure s1. Metformin (MET) effects on proliferation of various human cancer cell lines. Lung carcinoma cells (A549, H1299, SK-MES1), prostate carcinoma cells (PC3) and breast cancer cells (MCF7 and MDA-MD231) were treated with an increasing concentrations of MET (0µmol-5mM) for a period of 48 hours. Cells were subsequently fixed with ethanol and DNA content was used as a marker for proliferation rate determined by crystal violet staining. Results of three independent experiments are shown. Values represented are Mean±SEM. Specific genotypic make-up of each cell line with oncogenic mutations is also provided.

2 Figure s2. Metformin (MET) effects on proliferation of human lung embryonic fibroblast cell line. Human lung embryonic fibroblast cell line was treated with an increasing concentrations of MET (0µmol-25µmol) for a period of 48 hours. Cells were subsequently fixed with ethanol and DNA content was used as a marker for proliferation rate determined by crystal violet staining. Results of three independent experiments are shown. Values represented are Mean±SEM.

3 Figure s3. Rapamycin and Ionizing Radiation (IR) inhibit proliferation of A549 human lung cancer cells. A549 human lung cancer cells were treated with increasing doses of rapamycin (5-500nM) for 24 hours before treatment with 0, 2 or 8 Gy IR. Cells were fixed 48h later with methanol. DNA content, as a marker of proliferation, was determined with crystal violet staining. Results of three independent experiments are shown as Mean ± SEM. Statistically significant differences compared to corresponding control cells (not treated with rapamycin) within the 0Gy 2Gy and 8Gy IR treatment groups are shown (×:P<0.05, ××:P<0.01 for 0Gy group, #:P<0.05, ##:P<0.01 for 2 Gy group, and *: P< 0.05, **: P<0.01 for 8Gy group, respectively).

4 Figure s4. Effects of Rapamycin and/or IR treatment on expression and phosphorylation of members of the Akt – mTOR pathway. A549 cells were treated with Rapamycin (0nM or 5nM) for 24 or 48 hours, IR (0, 2 or 8Gy) for 1 or 24 hours or combined Rapamycin + IR treatments as indicated. The cells were washed and lysed. Lysates were analyzed with immunoblotting using indicated antibodies. Representative images of 3 independent immunoblotting experiments are shown for total and phosphorylated Akt (S473 and T308), mTOR and phosphorylated 4EBP1.

5 Figure s5. Comparison of the anti-proliferative effects of MET with Rapamycin or Gefitinib in untreated and irradiated A549 lung cancer cells. A549 human lung cancer cells were treated with increasing MET (5µM-5mM), rapamycin (5nM-500nM), or Gefitinib (1µM- 4µM) doses for 24 hours before treatment with 0, 2 or 8 Gy IR. Cells were fixed 48h later with methanol. DNA content, as a marker of proliferation, was determined with crystal violet staining. Results of three independent experiments are shown as Mean ± SEM. Statistically significant differences compared to corresponding control cells (not treated with either agent) within the 0Gy 2Gy and 8Gy IR treatment groups are shown (*:P<0.05, **:P<0.01 for MET treated cells, #:P<0.05, ##:P<0.01 for rapamycin treatment, and ×:P< 0.05,××: P<0.01 for Gefitinib treatment respectively). Horizontal lines facilitate a visual comparison of the effects of MET + 2Gy IR and MET + 8 Gy IR with the rest of the treatment conditions.


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