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. 2011 Mar 11;30(8):1536–1548. doi: 10.1038/emboj.2011.69

Figure 4.

Figure 4

p38MAPK induces an SASP independent of the DNA damage response. (A) p38MAPK inhibition does not prevent the DDR. Whole cell lysates were collected at specified intervals after irradiation and analysed by western blot. Where indicated, p38MAPK was continuously inhibited by SB203580(+) beginning 48 h before irradiation. ATM-P, Ser 1981 phosphorylated ATM; CHK2-P, Thr 68 phosphorylated CHK2. (B) Constitutive p38MAPK activation does not induce 53BP1 foci. Cells were fixed 8 days after MKK6EE expression (MKK6EE), 10 days after RAS expression (SEN(RAS)), 8 days after irradiation (SEN(XRA)), or after replicative senescence (69 population doublings) (SEN(REP)) and immunostained for 53BP1. Foci were quantified using CellProfiler to count cells with ⩾3 53BP1 foci per nucleus. Error bars indicate 95% confidence interval. (C) Constitutive p38MAPK activation does not induce a DDR. Whole cell lysate was collected 8 days after MKK6EE infection and analysed by western blot. Presenescent controls (PRE) were infected with insertless vector. ATM-P, Ser 1981 phosphorylated ATM; CHK2-P, Thr 68 phosphorylated CHK2. (D) Neither ATM nor CHK2 depletion prevents the SASP induced by constitutive p38MAPK activation. PRE cells were irradiated (SEN(XRA)), infected with RAS lentivirus (SEN(RAS)), or infected with MKK6EE lentivirus (MKK6EE). Simultaneously, cells were infected with lentivirus expressing shRNAs against ATM (shATM #12), CHK2 (shChk2 #2, shChk2 #12), or GFP (shGFP; control) and selected. CM from 8 days after infection/irradiation were analysed by ELISA. (E) ATM or CHK2 depletion does not prevent p38MAPK phosphorylation at senescence. Top: cells were irradiated; 6 days later cells were infected with lentivirus expressing shRNAs against ATM (shATM #12), CHK2 (shChk2 #2, shChk2 #12), or GFP (shGFP; control) and selected. Bottom: cells were simultaneously infected with RAS lentivirus, lentivirus expressing an shRNA against ATM (shATM #12), CHK2 (shChk2 #2, shChk2 #12), or GFP (shGFP; control) and selected. Whole cell lysates from 8 days after irradiation/infection were analysed by western blot. p38-P: phosphorylated p38.