Figure 2.

Overexpression of ∆40p53 downregulates YY1 expression by upregulating miR-186-5p

(A) Quantitative PCR of YY1 mRNA levels normalized to actin in H1299 cells transfected with C: GFP (vector control)/p53FL only/∆40p53 only/14A (expresses both p53FL and ∆40p53), 48 h post transfection (n = 3). (B) Western blot analysis of cell extracts from H1299 cells expressing C: GFP (vector control), p53FL only, ∆40p53 only, and 14A (expresses both p53FL and ∆40p53), probed with anti YY1 antibody, anti p53 antibody, CM1 and anti-actin antibody after 48 h. (C) Quantitative PCR of YY1 mRNA levels normalized to actin in H1299 cells cotransfected with C: GFP (vector control)/p53FL only/∆40p53 only/14A (expresses both p53FL and ∆40p53) and anti-miR-186-5p, 48 h post transfection (n = 3). (D) Western blot analysis of cell extracts from H1299 cells co-transfected with C: GFP (vector control)/p53FL only/∆40p53 only/14A (expresses both p53FL and ∆40p53) and anti-miR-186-5p, probed with CM1. (E) Quantitative PCR using Taqman probe for miR-186-5p in H1299 cells cotransfected with C: GFP (vector control)/p53FL only/∆40p53 only/14A (expresses both p53FL and ∆40p53) and anti-miR-186-5p. (F) Schematic of mutation generated in the YY1 3'UTR WT in the miR-186 binding site. (G)Western blot analysis of cell extracts from H1299 cells transfected with control and ∆40p53 followed by transfection of YY1 3'UTR WT and YY1 3'UTR Mutant, probed with CM1. (H) Effects of ∆40p53 mediated miR‑186 expression on the activity of wild‑type and mutant 3'‑UTRs of YY1 was analyzed by luciferase reporter assay 48 h post transfection [The luciferase values for each condition were normalized with protein concentration. The relative luciferase activity for each condition was calculated in comparison to the YY1-3'UTR WT control samples.] (I) Quantitative PCR using Taqman probe for miR-186 from RNA extracted from H1299 cells transfected with Control and ∆40p53 followed by transfection of YY1 3'UTR WT and YY1 3'UTR Mutant.