Fig. 4: CS downregulates miR-1 through VEGF pathway.

A NSCLC patients were grouped into non-smokers and smokers based on their smoking status and VEGF/reference gene levels were measured in the tumor samples and expressed as 2^−∆Ct. (n = 9 for non-smokers and n = 57 for smokers, p = 0.0067). B Human lung tissues were cultured ex-vivo and exposed to various concentrations of CSE. VEGF/ reference gene levels were measured, normalized to the mean value of the control group(0), and expressed as 2^−∆∆Ct (n = 3 subjects, 4 replicates in each. *p < 0.0001). C Human ex-vivo cultured lungs were treated with 1% CSE or vehicle (PBS) for 24 h. Endothelial (CD45− CD31+), immune (CD45+), and double-negative (CD45−, CD31−) cells were isolated using magnetic sorting. The graph represents VEGF/reference gene levels in CSE-treated cellular fractions normalized to the levels in PBS groups and expressed as 2^−∆∆Ct (n = 3 patients, *p = 0.0366). D, E Endothelial cells were treated with various concentrations of CSE for 24 h and VEGF/reference gene levels were measured, normalized to the mean value of the control group(0), and expressed as 2^−∆∆Ct. D HUVECs (n = 8 in each group, from 2 experiments, *p = 0.0002). E EAhy926 (n = 10 or more from 2 experiments, *p = 0.0048, **p = 0.0062). F HUVECs were exposed to 10% CSE, collected at the time points shown on the X axis, and VEGF protein levels measured by ELISA (n = 3/time point, *p < 0.0) (G) EAhy926 were starved overnight, incubated with and without a VEGFR2 blocker (sUs, and treated with 2% CSE for 24 hours. MiR-1/reference gene levels were measured, normalized to the control group, (vehicle, DMSO) and expressed as 2^−∆∆Ct (n = 6 or more from 2 experiments, *p = 0.0221). H Murine lung endothelial cells (MLECs) were treated with blockers, exposed to 5% CSE, and miR-1 was measured and expressed as described in (G). (n = 4 per group, *p < 0.03).