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. 2021 May 31;49(11):6082–6099. doi: 10.1093/nar/gkab451

Figure 1.

Figure 1.

Sequence-dependent inhibition of cGAS sensing by ASOs. (A) HeLa and HT-29 cells were transfected for 24 h with 20 nM of indicated ASOs targeted to cGAS (Supplementary Table S1), prior to RNA purification and RT-qPCR analyses. cGAS levels were reported relative to 18S, and normalised to Mock condition. Data shown represent the median of two independent experiments for each cell line. (B) THP-1 pre-treated overnight with 100 nM of the indicated ASO, were transfected or not (non-treated [NT]) with 2.5 μg/ml ISD70 for 8.5 h and IP-10 levels in supernatants were determined by ELISA. Data shown are averaged from two independent experiments in biological triplicate (± s.e.m and ordinary one-way ANOVA with Tukey's multiple comparison tests to the ‘ISD70 only’ condition, or otherwise indicated pairs of conditions are shown). There was no basal effect of the ASOs on NT cells (21). (C) HT-29 cells pre-treated overnight with 125, 250 or 500 nM of indicated ASOs, were transfected or not (non-treated [NT]) with 2.5 μg/ml of ISD70 for 24h, and IP-10 levels in supernatants were determined by ELISA. IP-10 levels were normalised to the ‘ISD70 only’ condition, after background correction with the NT condition. Data shown are averaged from two independent experiments in biological triplicate (± s.e.m. and Mann–Whitney U tests to the ‘ISD70 only’ condition are shown). (D, E) THP-1 pre-treated overnight with 100 nM of the indicated ASOs, were transfected with 2.5 μg/ml of ISD70 for 7–8 h, and IP-10 levels in supernatants were determined by ELISA. (D) Stimulations and ELISAs were carried out in two independent plates and the results presented on each axis (with a correlation r = 0.7716, P < 0.0001). Averaged values from both plates are given in Supplementary Table S2. ISD70 only condition is shown in blue. (E) IP-10 levels were normalised to the ‘ISD70 only’ condition, after background correction with NT condition. Data shown are averaged from three independent experiments in biological triplicate (± s.e.m. and ordinary one-way ANOVA with Tukey's multiple comparison tests to the condition ‘ISD70 only’, or otherwise indicated pairs of conditions, which are shown). (F) HT-29 cells pre-treated overnight with 187.5 nM of indicated ASOs were transfected or not with 2.5 μg/ml of ISD70 for 24 h, and IP-10 levels in supernatants determined by ELISA. IP-10 levels were normalised to the ‘ISD70 only’ condition, after background correction with NT condition. Data shown are averaged from three independent experiments in biological triplicate (± s.e.m. and ordinary one-way ANOVA with Dunnett's multiple comparison tests to the condition ‘ISD70 only’ condition are shown). (G) cGAS–/–, UNC93B1–/– and matched controls with rescued UNC93B1 expression (UNC93B1 WT) THP-1 were pre-treated 6 h with 100 or 250 nM ASOs, and transfected with 2.5 μg/ml of ISD70 overnight. GSK (100 nM) and ODN2006 (500 nM) were used as human STING and TLR9 agonists, respectively. IP-10 levels in supernatants were determined by ELISA and normalised to the ‘GSK’ condition, after background correction with NT condition. Data shown are averaged from three independent experiments in biological triplicate (± s.e.m and ordinary two-way ANOVA with Tukey's multiple comparison tests relative to ‘ISD70 only’ condition are shown). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns: non-significant.