Fig 2: A mechanistic roadmap explaining how RT upregulates CD73 and how combination therapy exerts antitumor effects.A Schematic diagram of RT up-regulates CD73 in MC38 cells through ATR-Chk1-STAT3 pathway and the combination of αCD73 and RT triggers CD8 + T cell-mediated adaptive immune responses by potentiating activation of the cGAS-STING pathway and type I IFN production. B The schematic summary of synergistic mechanism between αCD73 + RT. The combination of αCD73 and RT (right) significantly reduces the infiltration of TAM, MDSCs, and Tregs, while augmenting the infiltration of mature DCs and CD8 + T cells. Furthermore, it stimulates the generation of TNF-α, IFN-γ, and GzmB and reduces T-cell exhaustion compared to the control group (left).

Fig 3: DNA replication stress and genome instability following LUC7L3 depletionA. DNA replication speed was analyzed through DNA fiber assay in LUC7L3-depleted A549 cells following indicated treatment. ImageJ software was used for analysis of DNA replication fork length, and statistical analysis was shown. n＞100.B. Immunofluorescence analysis of p-CHK1(S345) phosphorylation levels in LUC7L3-knockdown HeLa cells. Green fluorescence indicated p-CHK1(S345), and blue represented the nucleus stained with DAPI. Intranuclear fluorescence signals of p-CHK1(S345) were analyzed using ImageJ, and statistical analysis was shown. n＞70. a. u. means arbitrary units.C. Western blot analysis of p-CHK1(S345) and CHK1 protein phosphorylation levels in LUC7L3-and RBM25-depleted HeLa cells.D. Western blot analysis of p-CHK1(S345) phosphorylation levels in LUC7L3-depleted and GFP-RNase H1-overexpressing A549 and HeLa cells.E. Chromosome spreading assay to assess the effect of LUC7L3 depletion on chromosomes in A549 ​cells. n＞40. ∗p ​< ​0.05, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001, ∗∗∗∗p ​< ​0.0001, indicating statistical significance and ns indicates non-significance. Scale bar: 10 ​μm.

Fig 4: RT up-regulates CD73 in MC38 cells, which depends on the DNA damage repair pathway.Flow cytometric analysis of CD73 expression on MC38 cells at different time points after RT. Images (A), representative histograms of percentage and MFI (B) of CD73 on MC38 cells 48 h after 8 Gy or 12 Gy irradiation. C, D Immunofluorescence staining analysis of CD73 and γ-H2AX on MC38 cells 48 h after 8 Gy irradiation. Representative images (C) and MFI (D) of CD73 and γ-H2AX. Scale bars: 100 μm. Western blot analysis of the expression of CD73 and ATR-Chk1-STAT3 pathway in MC38 cells treated with or without 8 Gy RT (E), and ± Chk1i (50 nM, SCH900776) (F), ±Stat3i (500 nM, Angoline) (G), and harvested 24 h later. Flow cytometric analysis of CD73 expression on the surface of MC38 cells treated ± 8 Gy RT, ±Chk1i (H), ±Stat3i (J). Percentage and MFI (I, K) of CD73 on MC38 cells 48 h after RT. Statistical variations were analyzed utilizing the unpaired t-test. Data are expressed as mean ± SEM (n = 3 per group).*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 5: Age-related changes in histone levels. A H3.1/3.2, H3.3, H3.3S31ph, H3K4me3, H3K27me3, and total H3 were analyzed by western blotting using histone-rich extracts from the tibialis anterior muscle. Representative blot images from 8-wk-old and 75-wk-old mice are shown. H3.3 levels are presented as the ratio relative to H3.1/3.2. The levels of H3.3S31ph, H3K4me3, and H3K27me3 were normalized to total H3. B H3.3S31ph, CHK1 phosphorylated at serine 296 (pCHK1), total CHK1, H3T3ph, H3S10ph, H3S28ph, H3.3, and total H3 were re-analyzed using histone-rich extracts of tibialis anterior muscle obtained in our previous study [8]. C Correlation analysis between H3.3S31ph and both pCHK1 and total CHK1 was performed using all data points from the western blot results shown in panel B. H3.3 levels are presented as the ratio relative to H3.1/3.2 (panel A). H3.3S31ph levels were normalized to H3.3 (panel A). H3K4me3 (panel A), H3K27me3 (panel B), pCHK1 (panel B), total CHK1 (panel B), H3T3ph (panel B), H3S10ph (panel B), and H3S28ph (panel B) were normalized to total H3. Data are presented as mean ± SD (n = 3 biological replicates per time point). Statistical significance was assessed by unpaired t-test (panels A and B) and Pearson correlation analysis (panel C). p-values are shown when < 0.05