Fig 1: RNASEH1‐R‐loop signaling mediates PVC NPs‐induced STING pathway activation in macrophages. A) Representative composite fluorescence images showing the levels of R‐loops after treatment with different concentrations of PVC NPs. The R‐loop and nuclei were labeled with an anti‐DNA: RNA hybrid antibody (S9.6) (green) and DAPI (blue), respectively (scale bar = 5 µm). B) Quantitative analysis of the relative fluorescence intensity of R‐loops in the nucleus of cells treated with different concentrations of PVC NPs (n > 50 cells). C) The protein levels of RNASEH1 and RNASEH2A in cells exposed to different concentrations of PVC NPs were determined by western blotting. D,E) The relative mRNA levels of RNASEH1 (D) and RNASEH2A (E) were measured in THP‐1‐derived macrophages treated with different concentrations of PVC NPs for 48 h (n = 3). F) Representative R‐loop PLA fluorescence images demonstrating the RNASEH1‐R‐loop interaction (scale bar = 5 µm). G) Quantitative analysis of PLA foci in the nuclear compartment (n > 50 cells). H) Representative composite fluorescence images showing the levels of R‐loops after RNASEH1 overexpression following PVC NPs treatment (scale bar = 3 µm). I) The protein levels of TBK1, p‐TBK1, IRF3, p‐IRF3, and Flag‐RNASEH1 in cells exposed to or without PVC NPs were determined by western blotting. J–L) The relative mRNA levels of TNF‐α (J), CXCL10 (K), and CCL5 (L) were measured in THP‐1‐derived macrophages treated with PVC NPs after RNASEH1 overexpression (n = 3). Statistical analysis was performed using one‐way (B,D,E,G) or two‐way (J–L) ANOVA followed by Tukey's multiple comparison test. The data are presented as the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, NS, not significant. All the data are representative of three independent experiments.