FIGURE 6.

PKCα is a novel target of miR-142-3p. (A) Venn diagram showing the distribution of unique and overlapping genes downregulated by miR-24, miR-30b and miR-142-3p mimics in DC and Mφ. (B) Sequence alignment of predicted miR-142-3p binding site in the 3′UTR of PKCα of various mammals. (C) HEK293 cells were cotransfected with PKCα 3′UTR construct and with either miR-142-3p or control mimic. Renilla activity was normalized to firefly activity and the ratios subsequently normalized to empty vector transfected with miR-142-3p mimic set as 1. Data are expressed as ± SEM of four independent transfections. Western blot analysis of PKCα levels in miR-142-3p overexpressing (D) Mφ and (E) DC. Day 7 differentiated cells were transfected with miR-142-3p mimic, inhibitor or control mimics. Cell lysates were prepared after 36 h and PKCα levels were detected by western blotting. GAPDH was used as internal control. Densitometric analysis of bands was performed with ImageJ. The normalized PKCα in control-transfected cells was set as 1. (F) Expression kinetics of PKCα in Mφ and DC. Expression levels of PKCα transcript were monitored at indicated time points during the monocyte to Mφ and monocyte to DC differentiation. (G) siRNA mediated knockdown of PKCα as observed by RT-PCR and immunoblotting in Mφ. GAPDH served as endogenous control. Phagocytosis assays were performed with labelled E. coli in Mφ transfected with PKCα or scramble siRNA and untreated cells. (H) Representative images were captured using a florescence microscope and (I) Geo. MFI values obtained from flow cytometry values. Scale bar, 200 μm. The data presented are representative of three independent experiments in each cell type. Student's t-test was conducted to calculate p values. *p<0.01 was considered significant.