## 3 Results  
### Activation of the Akt1/Src/Cav1 Axis Promotes Cell Survival in Post-Irradiated RD and RH30 Lines  
The Akt and Src pathways are abnormally activated in RMS [8; 10]. By performing in silico analysis of RNA-seq data (GEO: GSE108022), we found that both AKT1 and SRC are significantly more expressed in human RMS samples, regardless of subtype, than in skeletal muscle samples (Figure 1A, top panels) [4]. Moreover, their expression is significantly correlated, as calculated by [PERSON]'s coefficient (Figure 1A, bottom panel). In light of the important role of these kinases in regulating anti-apoptotic and oxidative stress resistance mechanisms, we wondered whether their inhibition could affect intracellular ROS levels. For this purpose, proliferating human RD and RH30 cells were treated with LY294002, MK2206, and PP2, which are inhibitors of the phosphatidylinositol 3-kinase (PIJK), Akt, and Src kinases, respectively. All these treatments were effective in reducing cell proliferation without affecting cell viability and produced a significant increase in intracellular ROS levels measured after 24 h by ROS probe (CM-H2 DCFDA probe) (Figure 1B). Considering these data, we hypothesized that activation of the Akt1/Src pathway may play a role in the detoxification of intracellular ROS. Therefore, we sought to test whether the Akt/Src pathway can sense oxidative stress in response to IR therapy, which is known to cause high ROS accumulation. For this purpose, human RD and RH30 cells were subjected to an IR dose of 4 Gy and then collected at different time points for IB analysis. We found that phosphorylation of Akt1 on Ser473 (pAkt1) and phosphorylation of Src on Tyr416 were significantly increased between 4 and 8 h in the post-irradiated lines (Figure 1C). Between 8 and 24 h after IR, we found increased phosphorylation of Cav1 on Tyr14 (pCav1) (Figure 1C), a scaffolding protein that is a known target of Src kinases. In contrast, Erk1/2 phosphorylation (pErk) was not affected by IR treatment (Figure 1C). These data suggest that IR-induced oxidative stress could activate the Akt/Src pathway to promote cell survival by ROS detoxification. To test this, we evaluated the cell survival of RMS lines treated for 2 h with inhibitors of the PI3K/Akt, Src, and Erk pathways before receiving IR. As assessed by clonogenic assay, the inhibitors LY294002, MK2206, and PP2 were effective in reducing cell survival of RMS lines, whereas in contrast the ERK1/2 phosphorylation inhibitor PD098059 had no radiosensitizing effect (Figure 1D). Taken together, these results suggest that Akt1/Src activation in collaboration with Cav1 may be implicated in a pro-survival program through modulation of oxidative stress. Figure 1: Involvement of the Akt1, Src, and Cav1 proteins in oxidative stress and radiation response in RMS lines. (**A**) Quantification of AKT1 and SRC gene expression was performed using an RNA-seq dataset (GSE108022) relative to human FNRMS (\(n=66\)), FPRMS (\(n=35\)), and normal skeletal muscle (\(n=5\)) samples (top panels). Each point represents an individual sample. The mean expression is highlighted by a black horizontal bar. ***\(p\)-value \(<0.0001\); one-way ANOVA test vs. normal skeletal muscle. Correlation between AKT1 and SRC expression was calculated by Pearson’s coefficient (bottom panel). (**B**) RD and RH30 cells (\(3\times 10^{3}\)) were plated in black 96-well plates in triplicates 24 h prior to treatment with the indicated compounds. Quantification of ROS production with the CM-H2 DCFDA  
probe was performed after 24 h (\(n=2\)). Data are mean \(\pm\) SEM, * \(p\)-value \(<\) 0.05; ** \(p\)-value \(<\) 0.001; one-way ANOVA test vs. vehicle-treated cells. (**C**) RD and RH30 cells (1.5 \(\times\) 10\({}^{5}\)) were seeded into 60 mm dishes. After 24 h, cells were irradiated and collected at the indicated time points for IB (\(n=2\)). Densiometric quantifications are reported in the top graph after tubulin normalization.