Activation of p38 by active MKK3 or MKK6 induces premature senescence. (A) Western blot analysis of BJ cells transduced at PD 18 with a vector control (BP) or constitutively active MKK3 (MKK3E) or MKK6 (MKK6E). Levels of MKK3, MKK6, phospho-ERK (P-ERK1/2), ERK2, phospho-p38 (P-p38), p38, p16INK4A, and p53 were determined 10 days postinfection. (B) PD of BJ cells between day 7 and day 11 after infection at PD 18 with a vector control (BP), Ha-RasV12 (HaRasV12), a constitutively active MKK3 (MKK3E) or MKK6 (MKK6E), or wild-type p38α (p38αWT). Values are means ± standard deviations (SD) for triplicates. (C) Morphology of BJ cells (BJ) or BJ cells immortalized with hTERT (BJ-hTERT) that had been transduced with a vector control (BP) or constitutively active MKK3 (MKK3E) or MKK6 (MKK6E), after staining for SA-β-gal (pH 6.0) at day 12 postinfection. (D) Quantification of percentages of SA-β-gal-positive cells within cell populations shown in panel C. Values are means ± SD for two separate wells. At least 200 cells were counted for each sample.