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Fig. 5 | Genome Biology

Fig. 5

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

Fig. 5

TNFα induced by phosphorylation of TTP promotes chemoresistance. a Phosphorylation of TTP by the p38-MK2 pathway inactivates its RNA decay function, which leads to stabilization of ARE-bearing TNFα mRNA, resulting in activation of NF-kB signaling in resistant G0 leukemic cells. TNFα expression is inhibited by TTP-AA mutant, pirfenidone (PFD) or shRNAs, and NF-kB signaling by NF-kB inhibitor, Bay11-7082. b Expression of TNFα and NF-kB target genes at the translatome level at indicated time points after SS or AraC treatment. c TNFα protein level in S+, SS, and AraCS cells. d Effect of TNFα on chemoresistance. THP1 cells were transduced with doxycycline inducible shRNA against TNFα or control shRNA. ShRNA against TNFα was induced prior to AraC (shTNFα → AraC) or after AraC (AraC → shTNFα) and recombinant TNFα protein was added 1 day prior to AraC (ReTNFα → AraC). Cell viability and western analysis of TNFα are shown. e Effect of 300 μg/ml of PFD treatment for 3 days on TNFα expression at the translatome (middle) and protein levels (right) in AraCS cells. f Effect of pharmacological inhibition of TNFα by PFD on AraC resistance. THP1 cells were treated with 300 μg/ml PFD or vehicle in the absence of AraC (S+, top panels), in the presence of AraC (AraC, middle panels), or on serum starvation (SS, bottom panels). Bar graphs show cell viability and death assessed by cell counting, MTS, and caspase 3/7 assays. In the middle or bottom panels, THP1 cells were treated with PFD 1 day prior to AraC or SS (PFD → AraC, PFD → SS), at the same time with AraC or SS (AraC + PFD, SS + PFD), and 1 day after AraC or SS (AraC → PFD, SS → PFD). g Effect of TNFα inhibition on AraC resistance from six different leukemic cell lines. Cells were treated with PFD or vehicle 1 day prior to AraC (AraC, top panels) or in the absence of AraC (bottom panels, S+). h Effect of NF-kB inhibition on AraC resistance. THP1 cells were treated with 10 μM Bay 11-7082 (Bay) or vehicle in the absence of AraC (S+, top panels), in the presence of AraC (AraC, middle panels) or under serum starvation (SS, bottom panels). In the middle or bottom panels, THP1 cells were treated with Bay11-7082, 1 day prior to AraC or SS (Bay → AraC, Bay → SS), at the same time with AraC or SS (AraC + Bay, SS + Bay), and 1 day after AraC or SS (AraC → Bay, SS → Bay). *p ≤ 0.05. Data are represented as average ± SEM. See also Additional file 1: Figure S5

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