Extended Data Fig. 10: cGAS promotes tumorigenesis. | Nature

Extended Data Fig. 10: cGAS promotes tumorigenesis.

From: Nuclear cGAS suppresses DNA repair and promotes tumorigenesis

Extended Data Fig. 10

a, Results of the MTT assay showing the proliferation of PC-9 cells that had been stably transfected with pcDNA3.1-HA (HA) or HA–cGAS (HA–cGAS). Data are expressed as mean ± s.e.m. of n = 3 independent experiments. Student’s t tests (unpaired and two-tailed) were used for statistical analysis. *P < 0.05; ***P < 0.001. b, Colonogenic assay results depicting the colony-forming ability of PC-9 cells that were stably transfected with pcDNA3.1-HA (HA) or HA–cGAS (HA–cGAS). Data represent the mean ± s.e.m. of n = 6 independent experiments. The Mann–Whitney U-test was used for statistical analysis. c, The survival fraction of control shRNA LLC and Cgas shRNA LLC cells was determined by the MTT assay 6 days after exposure to etoposide (100 μg ml−1) or camptothecin (1 μM) for 4 h or H2O2 (10 mM) for 30 min. Data are expressed as mean ± s.e.m. of n = 3 independent experiments. Student’s t tests (unpaired and two-tailed) were used for statistical analysis. d, The survival fraction of PC-9 cells that had been stably transfected with pcDNA3.1-HA (HA) or HA–cGAS was determined by the MTT assay 6 days after exposure to etoposide (100 μg ml−1) or camptothecin (1 μM) for 4 h or H2O2 (10 mM) for 30 min. Data are expressed as mean ± s.e.m. of n = 3 independent experiments. Student’s t tests (unpaired and two-tailed) were used for statistical analysis. ef, Soft agar assays of anchorage-independent colony formation of human skin fibroblast from two different donors that were transfected with vectors encoding SV40 large tumour antigen (LT), HRAS V12 (Ras) and TERT together with pcDNA3.1-HA (HA) or HA–cGAS followed by plating in soft agar. Colonies were photographed after 5 weeks of growth at 200× original magnification (e). To assess anchorage-independent growth, 105 cells were plated in 0.4% Noble agar and colonies were counted 5 weeks after seeding. Quantification data are shown in f and represent the mean ± s.e.m. of n = 3 independent experiments. The Mann–Whitney U-test was used for statistical analysis. g, Representative results of the comet assay depicting the tail moment of PC-9 cells that had been stably transfected with pcDNA3.1-HA (HA) or HA–cGAS under alkaline conditions. h, Quantification of the tail moment of PC-9 cells that had been stably transfected with pcDNA3.1-HA (HA) or HA–cGAS under alkaline conditions. Data represent mean ± s.d. of the tail moment of n = 143 (HA) and n = 127 (HA–cGAS) cells from 3 independent experiments. The Mann–Whitney U-test was used for statistical analysis. i, Schematic of the role of cGAS in control of the DNA-damage response. In response to DNA damage, cGAS translocates to the nucleus and is recruited to DSBs where it interacts with PARP1 via PAR and prevents the formation of the PARP1–Timeless complex. This interferes with the process of homologous recombination. Nuclear cGAS suppresses DNA repair and promotes tumorigenesis. j, The relative expression of cGAS in normal and tumour (T) stages from specimens of patients with lung adenocarcinoma (LUAD). Data were obtained from the TCGA database. log2(fold-changes) and P values of cGAS expression are 0.47 (P = 0.12, T1 versus normal), 0.73 (P = 0.01, T2 versus normal), 0.76 (P = 0.005, T3 versus normal) and 0.93 (P = 0.003, T4 versus normal). NS, not significant. k, The relative expression of cGAS in normal and T stages from specimens of patients with lung squamous carcinoma (LUSC). Data were obtained from the TCGA database. log2(fold-changes) and P values of cGAS expression are 0.56 (P = 0.06, T1 versus normal), 0.78 (P = 0.006, T2 versus normal), 0.94 (P = 0.0003, T3 versus normal) and 0.79 (P = 0.004, T4 versus normal). NS, not significant. l, m, Kaplan–Meier curve analysis of the overall survival probabilities of patients with lung adenocarcinoma or lung squamous carcinoma based on the expression levels of BLK and KPNA2. Patients with lung adenocarcinoma were assigned to 1 of 4 groups based on low or high levels of gene expression.