Figure 5

Down-regulation of retinoic acid receptor (RAR) isotypes in immortalized normal human mammary MCF-10A cells using shRNA affects morphology and response to All-trans retinoic acid (ATRA). A) Phase-contrast micrographs of MCF-10A cells transfected with shRNA against RARα, RARβ or RARγ show distinct effects on cell morphology compared to nontransfected control cells. Equal numbers of transfected and control cells were seeded and then photographed after 48 h; bar = 20 μm. B) Confocal micrographs showing effects on MCF-10A cell proliferation as determined by staining for cell-cycle arrest marker p27 (red) and proliferation marker pRB (green) after transfection with shRARα, shRARβ, shRARγ or scrambled control (SCR) in the presence or absence of 1 uM ATRA; bar = 100 μm. C) Immunoblotting with isotype-selective antibodies illustrates silencing achieved with individual shRNAs. Tubulin was used as the loading control. Results shown are representative of three independent experiments.