## 3 Results
### Akt1/Src/Cav1/Catalase Signaling Promotes Radioresistance by Buffering Oxidative Stress in RD Cells  
We wondered whether Akt1 might be involved in the regulation of Src and Cav1. To answer this question, we exploited myr Akt1 cells that exhibit radioresistance hallmarks due to enhanced DNA repair [9]. As demonstrated by IB analysis, in three different RD clones carrying the myr Akt1 form we found significantly increased protein levels of total and phosphorylated Src and Cav1 forms as compared with control RD cells (Figure 2A). The myr Akt1 cells showed elevated expression of catalase (Figure 2A), an enzyme involved in H\({}_{2}\)O\({}_{2}\) detoxification [27], which we have previously recognized as being highly expressed in RDCav1 cells [23]. After staining the cells with antibodies against Cav1 or catalase, we used IF analysis to confirm the increased expression of these markers in myrAkt1 cells compared with control cells (Figure 2B). We demonstrated by semiquantitative PCR that myr Akt1 increases Cav1 gene transcription (Figure 2C). Following treatment with LY294002 and MK2206, Cav1 protein levels were downregulated in both control and myr Akt1 cells (Figure 2D), suggesting that Cav1 is a downstream target of the PI3K/Akt pathway. In myr Akt1 cells, intracellular levels of ROS measured under basal conditions were lower than those in control RD cells (Figure 2E). Accordingly, treatment of myrAkt1 cells with LY294002, MK2206, and PP2 increased ROS levels as measured after 24 h (Figure 2F), confirming that activation of Akt1 signaling is involved in maintaining low endogenous levels of oxidative stress. Next, we analyzed Akt1/Src/Cav1 signaling in response to IR treatment in myrAkt1 and RDCav1 radioresistancel. As shown in Figure 3A,B, the post-irradiated cell lines had higher levels of Src and pSrc as well as of Cav1 and pCav1 compared to the controls. Akt1 and pAkt1 levels in myr Akt1 cells increased at baseline, and remained higher than in controls after IR (Figure 3A). In contrast, the total form of Akt1 in RDCav1 cells did not change compared with controls. However, phosphorylation of Akt1 increased more rapidly in RDCav1 cells than in controls after IR (Figure 3B), suggesting that Cav1 may facilitate phosphorylation of Akt1 in response to IR. As demonstrated by clonogenic assay (Figure 3C,D), cell survival of both radioresistancel lines was significantly abrogated by pretreatment with LY294002, MK2206, and PP2, whereas PD098059 had no considerable effect (Figure 3C,D). These data suggest that activation of the Akt/Src/Cav1/catalase axis appears to be an essential prerequisite for the adaptation process leading to the development of radioresistance. Figure 2: MyrAkt1 signaling drives increased levels of Akt1, Src, Cav1, and catalase, promoting intracellular ROS reduction. **(A–C)** Control and myrAkt1 cells (\(1.5\times 10^{5}\)) were seeded into 60 mm dishes. After 48 h of proliferation, cells were processed for IB (\(n=2\)) **(A)**, IF (\(n=3\)) **(B)**, and PCR analyses (\(n=2\)) **(C)**. Densiometric quantifications are reported in the graphs after tubulin or GAPDH normalization. Data are mean \(\pm\) SEM, **\(p\)**-value \(<0.001\); ***\(p\)-value \(<0.0001\); unpaired Student’s \(t\)-test vs. control RD line. Representative images for IF analysis were taken using a fluorescent microscope at 63 X magnification. The scale bar corresponds to 50 \(\upmu\)m. **(D)** After 24 h had elapsed from plating cells (\(1.0\times 10^{5}\)) into 60 mm dishes, LY294002 and MK2206 or vehicle were provided for up to 72 h before IB analysis (\(n=2\)). Densiometric quantifications are reported in the top graph after tubulin normalization. Data are mean \(\pm\) SEM, **\(p\)**-value \(<0.001\); ***\(p\)-value \(<0.0001\); one-way ANOVA test vs. vehicle-treated cells. **(E,F)** Control and myrAkt cells (\(3\times 10^{3}\)) were plated in black 96-well plates in triplicates 24 h prior to treatment or not with the indicated compounds. Quantification of ROS production with the CM-H2 DCFDA probe was performed after 24 h (\(n=2\)).