Fig. 5
TAK1 activation engages p38 but its inhibition does not influence DDR signaling. a PSC27 cells were treated with bleomycin with or without increasing concentrations of the TAK1 inhibitor 5Z-7. Cell lysates were IPed with anti-p-TAK1, and subject to in vitro kinase assay with MKK6 as a substrate. Activation of kinase p38 was analyzed, with GAPDH as a loading control. Alternatively, IL-1α (20 ng/ml) was used to stimulate cells with or without 5Z-7, with the lysates subsequently analyzed. b IL-1α was eliminated from PSC27 cells by shRNAs. TAK1/MKK6 interaction and p38 activation in the cytosol were assessed by IP in the same conditions of a. c PSC27 cells were treated by bleomycin, or 5Z-7, or both, and subject to immunofluorescence (IF) staining of γ-H2AX. DNA damage extent in stromal cells was depicted as DDR statistics by counting the number of DDR foci per cell. Right, representative images of IF staining. Green, γ-H2AX; Blue, DAPI. Scale bars, 10 μm. Data analyzed by two-way ANOVA. d Top, representative images for clonogenic assay. PSC27 cells were treated by bleomycin alone for 6 h then released until 7d later, or treated by 5Z-7 alone continuously for 7d, or treated by both agents for individual time length in culture, after which clonogenic staining was performed. Bottom, statistics of stromal cell clonogenic growth. Data analyzed by Student’s t-test. e PSC27 cells were treated with bleomycin and/or 5Z-7, and cell lysates were collected 7d after drug treatment for immunoblot analysis. JNK1 phosphorylation and IL-8 expression were used to probe TAK1 activation and SASP development, respectively. Activation of mTOR and Akt were assayed with the same set of lysates, as well. Data in all bar plots are shown as mean ± SD. All data are representative of 3 biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001
