MK3 Modulation Affects BMI1-Dependent and Independent Cell Cycle Check-Points
Fig 4
Functional links between MK3 and PRC in proliferative life span.
TIG3 cells were sequentially transduced with either Bmi1.ires.GFP (Bmi1OE) or GFP (con) virus, and MK3/puromycin (MK3OE) or control puromycin virus (con) at 48 hrs intervals. Retroviral vectors expressing murine Bmi1/GFP reporter were transduced first (or empty vector control), followed by a MK3/puromycin resistance marker (or empty vector control). Transduction of Bmi1OE and control transduced cells was simultaneously carried out with the same MK3WTOE 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 MK3WT overexpression vector (MK3WTOE; black symbols) or shcon vector (white symbols), in conjunction with either an empty vector control (con; circles) or a murine Bmi1 expression vector (Bmi1OE; triangles). (B) Proliferation curves of normal human TIG3 fibroblasts transduced with a retroviral MK3 knock-down vector (shMK3; black symbols) or shcon vector (white symbols), in conjunction with either an empty vector control (con; circles) or a murine Bmi1 expression vector (Bmi1OE; 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 Methods 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 Bmi1 and/or MK3 versus control cells as recorded by GFP fluorescent imaging ± 3 weeks after transduction (D) Immunoblot analysis of EZH2, CBX4, RNF2 and H3K27me3 in MK3WTOE, Bmi1OE, Bmi1OE/MK3WTOE and control TIG3 cell lysates. (E) Expression analysis of BMI1, MK3, and TP53 at the indicated time points in (corresponding to experiment in Fig 4C). 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.