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. 2016 Jun 25;7(30):47875–47890. doi: 10.18632/oncotarget.10293

Figure 2. Validation of RPL27A as a target for miR-595 by qRT-PCR and western blot analysis.

Figure 2

Endogenous RPL27A mRNA and protein levels were determined in untransfected, pBabePuro empty vector (pBabe), and pBabePuro-miR-595 (miR-595) transfected cells. miR-595 expression levels were determined in untransfected and miR-595 transfected KG-1. K562, HeLa and HepG2 cells 48 hrs after the miRNA transfection (A). To investigate if miR-595 overexpression downregulates RPL27A expression, RPL27A mRNA was quantified by qRT-PCR in untransfected cells, cells transfected with empty vector and cells transfected with miR-595. Downregulated expression of RPL27A occurred in all cell lines overexpressing miR-595, in contrast to cells transfected with pBabe empty vector and untransfected cells. Results represent three independent experiments; bars display the mean ± standard error of the mean (SEM) (B). HeLa and HepG2 cells were transfected with empty vector or miR-595. RPL27A protein expression is downregulated in HeLa and HepG2 cells expressing miR-595 compared to control. Tubulin was used as a loading control (C). HeLa cells which did not express miR-595 were transfected with either hairpin control inhibitor (HeLa+C) or miR-595 inhibitor (HeLa+ miR-595Inh). HeLa cells expressing miR-595 were also transfected with hairpin control or miR-595 inhibitor. Upregulated RPL27A mRNA and protein expression was evident in miR-595 expressing HeLa cells which underwent transfection with the miR-595 inhibitor compared to controls (D, E).