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Fig. 4 | Genome Biology

Fig. 4

From: A post-transcriptional program of chemoresistance by AU-rich elements and TTP in quiescent leukemic cells

Fig. 4

Phosphorylation of TTP by p38 MAPK-MK2 promotes chemoresistance. a The p38 MAPK (p38)-MK2 pathway enables stabilization and translation of ARE-bearing mRNAs via TTP phosphorylation and inactivation of its RNA decay function, in chemoresistant G0 cells. LY2228820 (LY) and BIRB396 (BB or BIRB) are p38 inhibitors. b Western analysis of in lysates from THP1 cells at indicated time points after AraC treatment. c Western analysis in S+ and AraCS cells treated with vehicle, 5 μM LY or 5 μM BB for 3 days. d Firefly luciferase activity of a reporter bearing TNFα ARE in its 3′UTR normalized to activity of co-transfected Renilla luciferase in S+ and AraCS cells treated with either vehicle, or 5 μM LY. e Sequential treatment with p38 inhibitors and AraC in leukemic cells. f, g Effect of p38 inhibition on survival of AraC-resistant cells after indicated treatments normalized to DMSO treatment (represented as a white bar); THP1 cells were treated with 5 μM BB, 5 μM LY, and vehicle in the absence (S+, top panels) or presence (AraC, bottom panels) of 5 μM AraC treatment for 3 days. Bar graphs show relative cell viability and death assessed by cell counting, MTS, and caspase 3/7 assays. In the presence of AraC, THP1 cells were treated with p38 inhibitors prior to AraC treatment (BB → AraC, LY → AraC), at the same time with AraC (AraC + BB) and 1 day after AraC (AraC → BB, AraC → LY). 4H and 1D indicate 4 h and 1 days, respectively. RU = relative units. h, i Effect of TTP-AA mutant on survival of AraC-resistant cells. TTP-AA mutant expression prior to 5 μM AraC treatment, which decreased TNFα in THP1 or K562 cells in Fig. 3f. Cell viability was assessed by cell count (H). TTP-AA, TTP wild-type, and vector were expressed in TTP-deficient BMDM cells prior to 1 μM AraC treatment. Bar graphs show relative cell viability and death (i). j Effect of p38 inhibition on resistant cells from five AML cell lines (M5 FAB subtype) after indicated treatments normalized to DMSO treatment for each cell line (represented as a white bar and set to 1). Cells were treated with 5 μM LY or vehicle 4 h prior to AraC treatment (top panel, AraC) or in the absence of AraC (bottom panel, S+). Human CD34+ cells from healthy donors were tested as a control. k Effect of p38 inhibition on survival of chemoresistant cells induced with various concentrations of AraC. MV4:11 leukemic cells were treated with 5 μM LY or vehicle prior to 0 μM, 0.2 μM, 0.5 μM, or 1 μM AraC for 3 days. *p ≤ 0.05. Data are represented as average ± SEM. See also Additional file 1: Figure S4

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