Fig 1: Sequence-dependent inhibition of cGAS function. (A, B) THP-1 pre-treated 6 h with indicated doses (500, 250, 125, 62.5, 31.25 nM) (A) or 125 nM (B) of C2-Mut1 or A151 oligonucleotides, were transfected with 2.5 µg/ml of ISD70 overnight, and IP-10 (A) or IFN-ß (B) levels in supernatants were determined by ELISA. IP-10 (A) and IFN-ß (B) levels were normalised to the ‘ISD70 only’ condition, after background correction with the NT condition. (A, B) Data shown are averaged from three independent experiments in biological triplicate (± s.e.m. and ordinary two-way ANOVA with Sidak's multiple comparison tests relative to A151 are shown [A] or ordinary one-way ANOVA with Tukey's multiple comparison tests relative to condition ‘NT’, or otherwise indicated pairs of conditions are shown [B]). MG-63 (C) or mouse immortalised BMDMs (D) were pre-treated or not with 500 nM of C2-Mut1 for 6 h prior to stimulation with 2.5 µg/ml ISD (ISD70 for MG-63, ISD45 for BMDMs), LPS (1 µg/ml), GSK (100 nM), PAM3C (100 ng/ml), ODN1826 (500 nM), or DMXAA (50 µg/ml) overnight. IP-10 (C and D) and TNF-a (D) levels in supernatants were determined by ELISA. (C) Data were normalised to the ‘GSK’ condition, after background correction to the NT condition. Data shown are averaged from two independent experiments in biological triplicate (± s.e.m and Mann-Whitney U tests are shown). (D) IP-10 levels were normalised to the DMXAA condition, while TNF-a levels were normalised to the LPS condition. Data shown are averaged from three independent experiments in biological triplicate (± s.e.m and Mann-Whitney U tests are shown). (E) Recombinant cGAS protein was incubated in vitro with 2.3 µM ISD70 with or without (NT) increasing concentrations of ASOs (0.5, 2 and 10 µM) for 40 min. The reaction was stopped with EDTA and cGAMP levels were analysed by ELISA. Data were normalised to the condition A151 2 µM. Data shown are averaged from three independent experiments run in technical duplicate on the ELISA (± s.e.m and a Mann-Whitney U test is shown). (F) BJ hTERT SV40T cells were transfected overnight with an increasing amount of indicated ASO (20, 50, 100 nM), or 2 mM aspirin (ASP), prior to RNA purification. Expression of the panel of 3 human IFN-driven genes was analysed by RT-qPCR. Expression of the indicated genes was reported against to 18S expression and further normalised to the average of the ‘Mock’ condition. Data shown represent the average of three independent experiments conducted in biological duplicate (± s.e.m and Mann-Whitney U tests are shown). (G) Trex1-mutant primary BMDMs from three individual homozygous mice were transfected with 50 nM ASOs (or lipofectamine only, ‘Mock’) for 20 h, prior to RNA purification. Expression of the panel of three mouse IFN-driven genes was analysed by RT-qPCR. Expression of the indicated genes was reported against to 18s expression and further normalised to the average of the ‘Mock’ condition. Data shown represent the average of three mice conducted in biological duplicate (± s.e.m. and Mann–Whitney U tests are shown). *P = 0.05, **P = 0.01, ***P = 0.001, ****P = 0.0001, ns: non-significant.
Fig 2: 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.
Fig 3: HMGB2 is required for cGAS’ localization into CCF during senescence.a OVCAR3 cells expressing GFP-tagged cGAS were induced to senesce by cisplatin, and imaged under a confocal microscope. cGAS-GFP, ?H2AX, and HMGB2 co-localized CCF are indicated by arrows. b Expression of HMGB2, cGAS, and a loading control ß-actin in the indicated parental and two independent HMGB2 knockout OVCAR3 clones determined by immunoblot. c, d Representative images (c) and quantification (d) of endogenous cGAS localization into ?H2AX-positive CCF in senescent parental and HMGB2 knockout OVCAR3 cells with the indicated treatment. Arrows point to CCF. e, f Representative images (e) and quantification (f) of cGAS-GFP localization into ?H2AX-positive CCF in senescent parental and HMGB2 knockout OVCAR3 cells with the indicated treatment. Arrows point to CCF. g, h Quantification of endogenous cGAS (g) or cGAS-GFP (h) localization into CCF in senescent primary IMR90 cells induced by oncogenic RAS, with or without HMGB2 knockdown. i The secretion of soluble factors under the indicated conditions were detected by antibody arrays. The heatmap indicates the fold change (FC) in comparison with the control (Ctrl) or RAS-induced senescent IMR90 cells. Relative expression levels per replicate and average fold change differences are shown. Data represent mean ± s.e.m. n = 3 biologically independent experiments unless otherwise stated. Scale bar = 10 µm. P-values were calculated using a two-tailed t test. Source data are provided as a Source Data file.
Fig 4: cGAS activation requires TOP1cc during senescence.a Slot blot analysis of total TOP1 proteins in CCF purified from senescent IMR90 cells induced by the indicated treatment, with or without HMGB2 knockdown. b, c IMR90 cells induced to senesce by etoposide or oncogenic RAS were stained for TOP1cc and ?H2AX (b), and percentages of ?H2AX-positive CCF positive for TOP1cc were quantified (c). CCF are indicated by arrows. d Slot blot analysis of TOP1cc levels in CCF purified from senescent IMR90 cells induced by the indicated treatment with or without HMGB2 knockdown. e Slot blot analysis of TOP1cc levels in CCF purified from the indicated IMR90 cells treated with or without TOP1 inhibitor Camptothecin (CPT). f, g Examination of STING dimerization (f) or 2’3’-cGAMP levels (g) in the indicated cells. h Quantification of cGAS localization into CCF in the indicated IMR90 cells. i The secretion of soluble factors under the indicated conditions were detected by antibody arrays. The heatmap indicates the fold change (FC) in comparison with the control (Ctr) or RAS condition. Relative expression levels per replicate and average fold change differences are shown. j, k IMR90 cells were transfected with chromatin fragments isolated from IMR90 cells with or without CPT treatment. Benzonase was used to digest chromatin into fragments. Representative images (j) and quantification (k) of cGAS and TOP1cc co-localization in the transfected IMR90 cells. Arrows point to cGAS foci induced by the transfected chromatin fragments without or with TOP1cc. Data represent mean ± s.e.m. n = 3 biologically independent experiments unless otherwise stated. Scale bar = 10 µm. P-values were calculated using a two-tailed t test. Source data are provided as a Source Data file.
Fig 5: Identification of a highly potent inhibitor of cGAS sensing. (A) Top: sequence alignments of cGAS inhibitors and identification of important bases predicted by MEME analysis, highlighted with arrows (Supplementary Figure S1A). Bottom: design of C2 and ASO2 mutants incorporating point mutations at selected positions of the MEME motif (mutations are highlighted in yellow). (B, D) HT-29 cells pre-treated overnight with indicated doses of ASOs (500, 250, 125, 62.5 nM), were transfected or not with 2.5 µg/ml of ISD70 for 24 h and IP-10 levels in supernatants were determined by ELISA. IP-10 levels were normalised to the ‘ISD70 only’ condition, after background correction to the NT condition. Data shown are averaged from two (D) or three (B) independent experiments in biological triplicate (± s.e.m and ordinary two-way ANOVA with Tukey's multiple comparison tests relative to C2 [B] or ASO2 [D] conditions, are shown; comparisons were not significant between the ASOs for 250 and 500 nM). (C) Mouse LL171 cells were treated for 6 h with indicated amount of ASOs (200, 400, 600 nM) prior to overnight stimulation with 2.5 µg/ml ISD45. Cells were lysed and ISRE-Luc levels were analysed by luciferase assay the next day. ISRE-luciferase levels were normalised to the ‘ISD45 only’ condition, after background correction with NT condition. Data shown are averaged from two independent experiments in biological triplicate (± s.e.m and Mann-Whitney U tests are shown). (E) Top: sequence alignments of C2-Mut1 variants; C2-ASO2-A and C2-ASO2-B have the 3' ends from ASO2up and ASO2down underlined. Bottom: variants of the homopolymer dC20; 2'OMe bases are in pink and the cGAS inhibitory motif underlined. (F) HT-29 cells pre-treated overnight with 187.5 nM indicated ASOs were transfected or not with 2.5 µg/ml of ISD70 for 24 h and IP-10 levels in supernatants were determined by ELISA. (G) THP-1 pre-treated overnight with 100 nM indicated ASOs were transfected with 2.5 µg/ml of ISD70 for 24 h and IP-10 levels in supernatants determined by ELISA. (F, G) IP-10 levels were normalised to the ‘ISD70 only’ condition, after background correction with the 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 relative to condition ‘ISD70 only’, or otherwise indicated pairs of conditions are shown). (H) LL171 cells were treated for 6 h with 200 nM ASOs prior to overnight stimulation with 2.5 µg/ml ISD45. Cells were lysed and ISRE-Luc levels were analysed by luciferase assay the next day. ISRE-luciferase levels were normalised to the ‘ISD45 only’ condition, after background correction with the 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 ‘ISD70 only’ condition are shown). (I) THP-1 cells treated for 6 h with 100 nM C2-Mut1, or 100, 250, 500 nM of C2-Mut1-PS were transfected with 2.5 µg/ml of ISD70 overnight. 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). (J) Sequences of [LINC-PINT] ASOs 101–116, showing the location of the inhibitory GGUCCC motif (in pink) from ASO103 identified by MEME (see E and Supplementary Figure S1B). The yellow region highlights the DNA moiety of the gapmers. (K) THP-1 pre-treated overnight with 100 nM indicated [LINC-PINT] ASOs, were transfected with 2.5 µg/ml of ISD70 for 7.5 h 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 three independent experiments in biological triplicate (± s.e.m and one-way ANOVA with Dunnett's multiple comparison tests to the ‘ISD70 only’ condition are shown). (L, M) LL171 cells were treated for 6 h or 20 min with 200 nM ASOs prior to being ‘washed’ or not (L), or treated with 50, 100 or 200 nM ASOs for 20 min (M), and stimulated overnight with 2.5 µg/ml ISD45. Cells were lysed and ISRE-Luc levels were analysed by luciferase assay the next day. ISRE-luciferase levels were normalised to the ‘ISD45 only’ condition, after background correction with the NT condition. Data shown are averaged from two (L) or three (M) independent experiments in biological triplicate (± s.e.m. and one-way ANOVA with Tukey's multiple comparison tests to the ‘ISD45 only’ condition (L), or Mann-Whitney U tests to the ‘ISD45 only’ condition (M), or otherwise indicated pairs of conditions, are shown). (N) THP-1 pre-treated for 40 min with 250 nM indicated ASOs, were transfected overnight with 2.5 µg/ml of ISD70 and IP-10 levels in supernatants were determined by ELISA. 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 ‘ISD70 only’ condition, and a Mann-Whitney U test comparing Mut1-dC and dC20 are shown). *P = 0.05, **P = 0.01, ***P = 0.001, ****P = 0.0001, ns: non-significant.
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