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. Author manuscript; available in PMC: 2019 Jul 21.
Published in final edited form as: Nat Cell Biol. 2019 Jan 21;21(2):203–213. doi: 10.1038/s41556-018-0260-7

Fig. 3. Targeting irak1 overcomes R-RT in p53MK/MK zebrafish.

Fig. 3.

(a-c) Morpholino (MO) depletion of Irak1 phenocopies oxfendazole and IRAK1/4 inhibitor (IRAK1/4i) in DTC (a), AO (b, top) and TUNEL (b, bottom) assays (oxfendazole shown in 1d). std, standard control. (c) MO dose response in p53MK/MK embryos in DTC assay. Data are means +/− SD of n = 3 independent experiments. (d) RT-PCR of pooled embryonic extracts showing reduced WT mRNA levels (upper band) and skipping of exon 4 (lower band). rppo, ribosomal protein P0. (e-f) Representative images (e) and quantification (f) of irak1-depleted p53MK/MK embryos reconstituted with WT vs. KD (left), WT vs. K2R (middle) or WT vs. E3A (right) human IRAK1 (hIRAK1) mRNA scored in AO assay. KD, kinase dead; K2R; K134R/K180R; E3A, E544A/E706A/E587A. Number of quantified, independent images (as boxed in e): Left: std MO+mock: n of non-irradiated = 14, n of irradiated = 14; irak1 MO+mock: n of non-irradiated = 13, n of irradiated = 13; irak1 MO+WT: n of non-irradiated = 12, n of irradiated = 13; irak1 MO+KD: n of non-irradiated = 15, n of irradiated = 13; Middle: std MO+mock: n of non-irradiated = 9, n of irradiated = 9; irak1 MO+mock: n of non-irradiated = 9, n of irradiated = 9; irak1 MO+WT: n of non-irradiated = 8, n of irradiated = 7; irak1 MO+KD: n of non-irradiated = 10, n of irradiated = 11; Right: std MO+mock: n of non-irradiated = 8, n of irradiated = 10; irak1 MO+mock: n of non-irradiated = 7, n of irradiated = 12; irak1 MO+WT: n of non-irradiated = 7, n of irradiated = 12; irak1 MO+KD: n of non-irradiated = 8, n of irradiated = 12. Raw images of all embryos and cropped spinal chord areas available at 10.6084/m9.figshare.7427942. (g) Western blot of embryonic extracts showing hIRAK1 levels and diagram of hIRAK1, with disrupted residues and interactions shown. All data represented as means +/− SD, *P < 0.05, **P < 0.005, ***P < 0.0005, ns, not significant, two-tailed Student’s t-test. See Supplementary Table 4 and Supplementary Fig. 8 for statistics source data including precise P values and unprocessed immunoblots, respectively. Scale bars, 0.5 (a) and 0.2 (b,e) mm.