Fig. 2

PDHX mRNA and protein expression is suppressed by miR-27b in vitro. a and b Time course RT-PCR of cells transiently transfected with the indicated oligonucleotide. c and d RNA was collected from cells as above for evaluation of PDHX expression by quantitative RT-PCR using SYBR Green to probe for PDHX expression. e Cell lysates from transiently-transfected MCF7 and 4175 cells were collected and probed for PDHX protein and Vinculin loading control. ZR75 cells stably expressing elevated miR-27b or scr control, as well as 4175 cells expressing either the 27b-ZIP construct or scr control were used to replicate the experiments in transiently transfected cells. RNA was also collected from the stable cell and reverse transcribed for quantitation of PDHX expression by standard PCR followed by gel electrophoresis (f) as well as qPCR (g and h). Confirmation of the miR-27b expression status of the ZR75 (i) and 4175 (j) stable cell lines (previously generated in our lab) was completed by TaqMan qRT-PCR. The same RNA samples collected for PDHX qPCR were again used to measure the miR-27b level of these cells. k IB of PDHX protein using stable cell lysates as in panel e