Spatially fractionated mini-GRID radiotherapy differentially modulates radiation-induced senescence in murine glioma and normal cells (IMAGE)
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Figure 2. Molecular and secretory profiling reveals a reduced senescence burden after mini-GRID irradiation in F98 and RG2 cells. (A, B) Relative mRNA expression of Cdkn1a (p21) and Cdkn2a (p16) in F98 (A) and RG2 (B) cells 7 days after irradiation with 20 Gy delivered as Conv-RT or mini-GRID RT, normalized to the reference gene Wdr55 and expressed as fold-change versus non-irradiated controls (2^–ΔΔCt). Conv-RT induces a robust upregulation of p21 and p16, whereas expression levels in mini-GRID–treated cells remain close to those of controls. (C) Representative Western blot analysis of p53, p21, p16 and γH2AX protein levels in F98 and RG2 cells 7 days after treatment. N.I., non-irradiated. Conv-RT at 20 Gy leads to accumulation of DNA damage and senescence markers, whereas mini-GRID RT reduces the induction of these proteins and brings their levels closer to those observed in non-irradiated cells. Vinculin is shown as a loading control. (D, E) Expression of selected senescence-associated secretory phenotype (SASP) factors Il1a, Il6, Serpine1 and Cxcl1 in F98 (D) and RG2 (E) cells 7 days after irradiation, quantified by RT-qPCR and expressed as fold-change versus non-irradiated controls. Conv-RT elicits strong induction of all four SASP genes, whereas mini-GRID RT markedly attenuates this response, with Il1a, Il6 and Serpine1 approaching baseline levels and Cxcl1 induction being strongly blunted or almost abolished. Data are mean ± SD of at least three independent experiments. One-way ANOVA was used to assess statistical significance (*p < 0.05; **p < 0.01; ***p < 0.001. ns, not significant).
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