Supplementary Figure 2: Extended data for Main Figs. 1 and 2.
From: An IRAK1–PIN1 signalling axis drives intrinsic tumour resistance to radiation therapy
(a) Representative images of anterior spinal cords (as depicted on top) of WT and p53MK/MK embryos injected with standard control MO (std MO) or p53 MO targeted to initiator ATG (p53 MO (atg)) and treated with 15 Gy IR with or without oxfendazole (20 µg/ml) at 18 hpf. Embryos stained with AO at 7.5 hpIR. (b) Quantification of spinal cord areas shown in (a). Data represented as means ± SD of n = 3 independent experiments with at least 8 spinal chord images analyzed per condition; ***, P < 0.0005, ns, non-significant, two-tailed student’s t test. See Supplementary Table 4 for statistics source data including precise P values. (c-d) Pooled embryonic lysates analyzed by western blot with indicated antibodies. (e) Confocal microscopy of p53MK/MK zebrafish tails stained with DAPI (blue) and anti-γH2AX (green) after 0 Gy or 15 Gy IR and DMSO vs oxfendazole (20 µg/ml). Arrowheads indicates positively staining cells. hpIR, hours post ionizing radiation. hpf, hours post fertilization. (f) HeLa cells treated with indicated IRAK1 inhibitors or PIN1 inhibitor (EGCG) were irradiated (10 Gy), harvested at indicated time points and analyzed by western blot with indicated antibodies. (g) Acridine orange (AO) staining of 48 hpf embryos after 0 or 15 Gy IR and increasing doses of oxfendazole, with magnified views of spinal cords shown below. (h) Magnified images of spinal cords stained with AO at 6 hpIR, 18 hpIR, and 30 hpIR. (i) Oxfendazole (20 µg/ml) suppression of R-RT requires a 4-hr post-IR window as shown through a temporal requirement experiment. Readouts of R-RT observed as penetrance (%, absolute numbers) after AO staining at 48 hpf and DTC at 120 hpf with drug incubation windows depicted in green. (j) Quantification of DTCs in >12 p53MK/MK embryos at 120 hpf treated with inhibitors to SEA-predicted oxfendazole targets and irradiated at 18 hpf. Highest doses tested here tested in Fig. 2b. Shown are means of n = 2 independent experiments. Scale bars, 0.2 (a,g-h) and 0.1 (e) mm. See Supplementary Fig. 8 for unprocessed immunoblots.
