Fig. 2

Validation of interaction between let-7a and ND4. A RT-PCR showing pulldown of ND4 with biotin labelled let-7a in cellular system of MCF-7 cells. M-marker, Lane 1: Biotin labelled let-7a and 300 µg of cellular lysate were incubated for 1 h and then magnetic streptavidin beads were added and incubated for another 1 h followed by RNA isolation and RT-PCR analysis, Lane 2: Biotin labelled let-7a and magnetic streptavidin beads were incubated for 1 h then lysate was added for another 1 h. Lane 3: Biotin labelled let-7a, magnetic streptavidin beads and lysate were added at the same time and incubated for 2 h followed by RNA isolation and RT-PCR analysis. B RT-PCR showing pulldown of ND4 in MCF-7 cells transfected with biotin labelled let-7a (20 nM). Lane 1: Biotin labelled let-7a and 300 µg of cellular lysate were incubated for 1 h and then magnetic streptavidin beads were added and incubated for another 1 h followed by RNA isolation and RT-PCR analysis, Lane 2: Biotin labelled let-7a and magnetic streptavidin beads were incubated for 1 h then lysate was added for another 1 h. C ND4 levels in RNA immunoprecipitation complex was analysed by RT-PCR and its negative control. D Relative activity of Luciferase in let-7a transfected MCF-7 cells using pMIR REPORT vector system. Let-7a significantly inhibit the translation of luciferase by directly binding to target site on ND4. E RT-PCR analysis showing half-life of ND4 transcript after combined treatment of EtBr and let-7a mimic in MCF-7 cells at different time points (12, 24, 36 and 48 h). F MCF-7 cells were treated with EtBr and transfected with let-7a mimic for 24 h. ND4 levels were analysed by Western blot analysis. G MDA-MB-231 cells were transfected with mimic and antisense of let-7a for 24 h. ND4 levels were determined by Western blot analysis (H) ND4 expression in MCF-7 cells. β-Actin is used as a loading control