Supplementary Figure 4: Extended data for main Figs. 4 and 5.
From: An IRAK1–PIN1 signalling axis drives intrinsic tumour resistance to radiation therapy
(a-b) AlamarBlue survival curves of SAS, MDA-MB-231 and CAL27 shTRIPZ (control) and shIRAK1 cells (a) and IRAK1/4i-treated HeLa cells (b) at indicated IR doses. Western blots showing shRNA knockdown efficacy also shown in (a). All data represented as means ± SD, n = 3 independent experiments performed in triplicate, *P < 0.05, **P < 0.005, ***P < 0.0005, two-tailed Student’s t-test. (c-f) Clonogenic assays of HeLa (c-d), CAL27 (e) and SAS (f) cells either stably expressing IRAK1 shRNA (c) or treated with R406 (1 μg/ml, d-f) and irradiated with indicated γIR doses (Gy). Representative images shown to the left, surviving fractions to the right. All data represented as means ± SD, n = 3 independent experiments performed in triplicate, *P < 0.05, **P < 0.005, ***P < 0.0005, two-tailed Student’s t-test. (g) HeLa cells transfected with control LACZ or MyD88 siRNA and treated with IL-1β (100 ng/ml) analyzed by immunofluorescence with indicated antibodies 5 minutes after stimulation. siMyD88 abrogates recruitment of IRAK1 to cell surface, and is thus sufficient to eliminate MyD88 function. Scale bar, 20 μm. (h) Control and IRAK1 shRNA HeLa cells treated with IL-1β (100 ng/ml) or IR (10 Gy), harvested at indicated time points after stimulus and analyzed by western blot. (i) Extended data for western blot shown in Fig. 4k to show RAIDD, Chk2 phosphorylation (marker of ATM activity), total Chk2 and GAPDH levels. (j) Extended data for western blot shown in Fig. 4d to show caspase-2 cleavage levels. (k-l) Quantification and representative images of p53MK/MK embryos injected with indicated MOs, irradiated (15 Gy) at 18 hpf and scored for DTCs at 5 dpf. Data in (k) are means ± SD of n = 3 independent experiments (bars 3,4,7,8) or n = 4 independent experiments (bars 1,2,5,6). *P < 0.05; ***P < 0.0005; ns, not significant, two-tailed Student’s t-test. Scale bar, 0.5 mm. See Supplementary Table 4 and Supplementary Figure 8 for statistics source data including precise P values and unprocessed immunoblots, respectively.
