Fig. 5: The Wild-type p53/oncogenic Ras nonautonomous STAT signal relay promotes the radioresistance of Drosophila Ras tumor tissues.

(a–d′) Upregulated p53 and dap/p21 within Ras^V12 clones after irradiation. GFP-labelled Ras^V12 clones were stained with anti-p53 antibody (a, a′, c, c′) or anti-p21 antibody (b, b′, d, d′) before irradiation (0 h) and 24 h after irradiation. Time was counted from the start of first faction of IR treatment. Scale bar is 20 µm. (e, f) Quantification by qPCR of upd, upd2, and upd3 expression in eye-antennal discs containing wild-type or Ras^V12 clones after 36 h of first fraction of IR treatment (IR+) or without IR treatment (IR−). Column bars represent the mean of fold changes for the expression level of indicated genes (e). Relative expression of upd2 and upd3 in irradiated eye-antennal discs containing wild type, Ras^V12 and Ras^V12p53^R155H clones (f). Three independent experiments were carried out. Error bars denote SD. P values are derived from Student’s t test analyses. (g) Diagram of setting Drosophila irradiation models. Larvae after egg laying (48 h) were irradiated with three fractions of 10 Gy and allowed to recover to late third-instar larval stage. All eye-antennal discs were dissected at the late third-instar larval stage to evaluate the irradiation results by measuring the relative size between GFP-labeled clones and whole eye-antennal discs. (h–l′) GFP-labeled clones homozygous for Ras^V12 (i, i′), sav³ (j, j′), Tsc1^Q600X (k, k′), or expressing dMyc (l, l′) as well as wild-type controls (h, h′) were induced in the eye-antennal discs of larvae, irradiated at 48 h, and then collected discs on day 5. (h–l) the eye-antennal discs without irradiation treatment (IR−). (h′–l′) show irradiated discs (IR+). (m) Quantification of relative eye disc size (blue) and GFP-clone size (green) treated with IR (IR+) or without IR (IR−). For each genotype, eye-antennal disc and GFP-clone were normalized to age-matched eye discs without IR. Column bars represent the mean size of samples (N = 5–10). Scale bar is 50 µm. (n–t′) GFP-labeled Ras^V12, p53^−/−, Ras^V12p53^−/−, upd^RNAiupd2^∆, Ras^V12Upd^RNAiupd2^∆, Dome^DN, and Ras^V12Dome^DN clones were induced in the eye-antennal discs and half were then irradiated at the second-instar larval stage. After 3 days of recovery, all eye discs at the late third-instar larval stage were dissected to evaluate the differences in response to irradiation. (n–t) Eye-antennal discs without irradiation treatment (IR−) and (n′–t′) eye discs treated with irradiation (IR+). Scale bar is 50 µm. (u) Quantification of clones and eye discs treated with or without irradiation. Eye-antennal disc and GFP-clone areas were measured by ImageJ and normalized to the eye-antennal discs with the same genotype at the same age without IR. Column bars represent the mean size of samples (N = 5–9). Blue columns represent the mean size of the entire eye-antennal tissue for the indicated genotypes; green columns represent the size of GFP-labeled tumors. Error bars denote SEM. P values are derived from Student’s t test analyses. Effect size (d) values for e, f, m, and u are greater than 0.8.