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Fig. 2 | Molecular Cancer

Fig. 2

From: Long non-coding RNA ANRIL promotes homologous recombination-mediated DNA repair by maintaining ATR protein stability to enhance cancer resistance

Fig. 2

ANRIL directly binds with ATR to maintain the stability of the ATR protein. A: upper, Representative images of RNA immunoprecipitation (RIP) with antibodies against ATR, RPA2 and RAD51; lower: RIP-qPCR assay of the relative expression of ANRIL in ATR-, RPA2- and RAD51-precipitated extracts. Error bars represent the SD of the mean of n = 3 experiments. RIP with IgG was used as a negative control. **P < 0.01 versus the IgG group as determined by two-tailed Student’s t test. B: RIP-qPCR assay of ANRIL expression in the presence of ATR antibodies with/without irradiation. The error bars represent the SD of the mean of n = 3 experiments. ns: non significance between control and IR group when normalized to ATR protein level. C: RIP-qPCR assay of ANRIL expression in the presence of Flag primary antibody in cells transfected with Flag-ATR, Flag-ATR-N and Flag-ATR-C. RIP with IgG was used as a negative control. ***P < 0.001 versus the IgG group as determined by two-tailed Student’s t test. D: Immunoblot assay of ATR, RPA2 and tubulin in the RNA pulldown extract with biotin-labeled full-length ANRIL. Biotin and Biotin-NC sequences were used as negative controls. E: Predicted structure of the lncRNA ANRIL determined by RNA fold software. F: Immunoblot of ATR in RNA pulldown extracts with different ANRIL fragments and their antisense (AS) sequences (1–880, 881–1640, 1641–2480, 2481–3857). G, H: Representative images (F) and quantitative analysis (G) of the Western blotting results of the ATR protein in H1299-NC and ANRIL-knockdown cells after 0, 4, 8, and 12 Gy irradiation. Phosphorylated ATR was also detected. The data are shown as the mean ± SEM. Significance was determined with Student’s t test. **P < 0.01. I: Real-time PCR assay of ATR mRNA expression in ANRIL NC and ANRIL-KD cells after irradiation. The data are shown as the mean ± SEM. NS, non-significance was observed with Student’s t test. J: Western blot analysis of pATR and ATR protein in ANRIL-knockdown cells pretreated with the proteasome inhibitor MG132. NC was used as positive control. K: Quantitative analysis was performed with ImageJ software. The data are shown as the mean ± SEM. Significance was determined with Student’s t test. **P < 0.01. L: Immunoprecipitation analysis of ubiquitinated ATR-irradiated ANRIL NC and ANRIL-KD cells. M: Quantitative analysis of ubiquitinated ATR was performed with ImageJ software. Error bars represent the SD of the mean of n = 3 experiments, *P < 0.05. N: Schematic diagram of how the lncRNA ANRIL regulates HR repair and radiosensitivity

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