Functional links between MK3 and PRC in proliferative life span.

<p>TIG3 cells were sequentially transduced with either <i>Bmi1</i>.<i>ires</i>.<i>GFP</i> (<i>Bmi1OE</i>) or <i>GFP</i> (con) virus, and <i>MK3</i>/<i>puromycin</i> (<i>MK3OE</i>) or control <i>puromycin</i> virus (con) at 48 hrs intervals. Retroviral vectors expressing murine <i>Bmi1/</i>GFP reporter were transduced first (or empty vector control), followed by a MK3/<i>puromycin</i> resistance marker (or empty vector control). Transduction of <i>Bmi1OE</i> and control transduced cells was simultaneously carried out with the same <i>MK3</i>^<i>WT</i><i>OE</i> viral preparation (or control virus) to minimize inter-experimental variation. Cells were grown on selection medium and proliferation capacity was tested ± 2–3 weeks post-transduction. (A) Proliferation curves of normal human TIG3 fibroblasts transduced with a retroviral <i>MK3</i>^<i>WT</i><i>overexpression</i> vector (<i>MK3</i>^<i>WT</i><i>OE</i>; black symbols) or <i>shcon</i> vector (white symbols), in conjunction with either an empty vector control (con; circles) or a murine <i>Bmi1</i> expression vector (<i>Bmi1OE</i>; triangles). (B) Proliferation curves of normal human TIG3 fibroblasts transduced with a retroviral <i>MK3</i> knock-down vector (<i>shMK3</i>; black symbols) or <i>shcon</i> vector (white symbols), in conjunction with either an empty vector control (con; circles) or a murine <i>Bmi1</i> expression vector (<i>Bmi1OE</i>; squares). Cell counts at t = 2 through t = 8 (A, B) were normalized to cell counts at t = 0 for each transduced cell culture individually (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118840#sec013" target="_blank">Methods</a> section for details); statistical significance was determined by two-tailed Student’s t-test and is presented relative to the empty vector control (* p < 0.05). (C) Comparative morphology of TIG3 cells expression <i>Bmi1</i> and/or <i>MK3</i> versus control cells as recorded by GFP fluorescent imaging ± 3 weeks after transduction (D) Immunoblot analysis of EZH2, CBX4, RNF2 and H3K27me3 in <i>MK3</i>^<i>WT</i><i>OE</i>, <i>Bmi1OE</i>, <i>Bmi1OE/MK3</i>^<i>WT</i><i>OE</i> and <i>control</i> TIG3 cell lysates. (E) Expression analysis of BMI1, MK3, and TP53 at the indicated time points in (corresponding to experiment in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118840#pone.0118840.g004" target="_blank">Fig 4C</a>). Cells were grown on selection medium and analysed at 1 or 4 weeks after serial transduction; expression vectors and antibodies are indicated in the figure. Early and late samples were loaded on the same gel for BMI1 analysis; corresponding sections are shown separately.