Repression of p19^Ink4d expression partially abolished PTB’s inhibitory effect on cell proliferation.

<p>H1299 cells (1×10⁶) were transfected with either 2 µg pCMV-SPORT6 (EV) or equal molar of pCMV-SPORT6-PTB (PTB), or pCMV-SPORT6-PTB plus 75 nM siRNA_p19^Ink4d (sip19), or pCMV-SPORT6-PTB plus 75 nM siRNA_GFP (siGFP), cells were harvested 24 and 48 h post-transfection. (A) RT-qPCR analyses. Cells harvested 24 and 48 h post-transfection were measured for the levels of PTB mRNA and normalized to β-actin signals. Relative expression levels were calculated by comparing all the normalized signals to that of EV control of 24 h (to which a value of 1 was assigned). Data represent the means ± S.D. from three independent experiments. (B) BrdU incorporation assays. Cells harvested 0 h post-transfection were seeded into 96-well microplate for BrdU incorporation assays. The relative DNA synthesis was calculated by comparing the BrdU signals of PTB-overexpressing cells to that of EV control (to which a value of 100 was assigned). Data represent the means ± S.D. from three independent analyses. (C) MTT assays. Cells harvested 0 h post-transfection were seeded into 96-well microplate (1200 cells/well), cultured in regular medium for 20 (left) and 44 h (right), and subjected to MTT assays in the next 4 h (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058227#s2" target="_blank">Materials and Methods</a>). Data represent the means ± S.D. from three independent analyses.