Figure 5

miR-23b directly binds to Blimp1 and regulates its expression. (A) miR-23b levels were evaluated using qRT-PCR in MCF10A.B2 Mock and Cas cells 48 hours after transfection with either the miR-23b precursor (pre-miR-23b) or negative controls (pre-control). Results were calculated as fold changes (mean ± s.e.m.) relative to controls, normalized to U44 (***p < 0.001). (B) Total cell extracts from Mock and Cas cells, that had been treated with 1 µM AP1510 or left untreated, 48 hours after transfection with either the miR-23b precursor (pre-miR-23b) or negative controls (pre-control) were probed for Blimp1 in a western blot analysis. Tubulin was used as the loading control. Blimp1 protein modulation was calculated relative to Mock level and normalized to tubulin as the loading control. (C) Densitometric analysis of protein levels in at least three independent experiments is shown (mean ± s.e.m.). Statistical analysis was performed using the Student’s t-test (**p < 0.01). (D) Total cell extracts from Mock and Cas cells, that had either been treated with 1 µM AP1510 or left untreated, 24 hours after transfection with either the miR-23b inhibitor (anti-miR-23b) or negative controls (anti-control) were probed for Blimp1 in a western blot analysis. Tubulin was used as the loading control. Blimp1 protein modulation was calculated relative to Mock level and normalized to tubulin as the loading control. (E) Densitometric analyses of protein levels in at least three independent experiments are shown (mean ± s.e.m.). Statistical analyses were performed using the Student’s t-test (*p < 0.05, **p < 0.01). (F) Seed matches and mutated binding sites (3′UTRmut PRDM1) of miR-23b in the 3′UTR of PRDM. Black bars indicate the seed matches, while bold letters indicate mutation sites. (G) Luciferase assays in HEK293 cells cotransfected with either wild-type or mutant PRDM1 3′UTR reporter constructs, together with miR-23b precursors (pre-miR-23b) and negative controls (pre-control).