Figure 2. Loss of IL1R1 or IL1RAP leads to radiation resistance.

(A) Western blots were performed to determine the efficacy of sgRNAs targeting IL1RAP. MCF10A cells were infected with lentiviruses expressing the indicated sgRNAs. After treatment with puromycin for 48 h, cells were collected and lysed by sodium dodecyl sulfate loading buffer. IL1RAP and vinculin were blotted with indicated antibodies. (B) Western blots to confirm the efficacy of sgRNAs targeting IL1R1 and IL1RAP. MCF10A cells were infected with lentivirus expressing indicated sgRNAs and selected with puromycin. Cells were treated with IL-1α (10 ng/ml) or IL-1β (10 ng/ml) for 0.5 h and then collected. Cell lysates were prepared and blotted with indicated antibodies. (C) Cell growth assays were performed using MCF10A cells infected with lentivirus expressing sgRNAs targeting IL1R1 or IL1RAP and under a single high dose of radiation treatment. 1 × 10⁵ cells were seeded in a 12-well plate and incubated 24 h. Cells were irradiated with a single high dose of 20 Gy and maintained for 14 d. Cells were then collected and counted with an automated cell counter (TC20, Bio-Rad). Experiments were performed in triplicate with three biological replicates. Representative images and results are shown. (C, D) Quantification of relative survival in cell growth assay presented in (C). t tests were performed to estimate differences between two groups, and the data are presented as means ± SD. **P < 0.01, ***P < 0.001. (E) Results of crystal violet staining and quantification assay. MCF10A cells infected with indicated sgRNAs were treated with a conventional radiation schedule, which is 2 Gy per day for 5 d, 2 d off, and then continued for another 5 d (scheme on top). 2 × 10⁵ cells were seeded in six-well plates and 24 h later treated with indicated conditions. After 14 d, cells were stained with crystal violet solution, and relative survival was quantified by the Synergy multimode microplate reader (histogram on the bottom). The data are presented as means ± SD. **P < 0.01; ***P < 0.001 (t test). (F) Cell growth assays were performed using MCF10A cells infected with lentivirus expressing indicated sgRNAs and under conventional radiation treatment. (E) 2 × 10⁵ cells were seeded in a six-well plate, and 24 h later, cells were irradiated with the same conditions as in (E). Cells were fixed and stained. Experiments were performed in triplicate with three or four biological replicates. Representative images and results are shown.