Journal: Nature

Article Title: MRE11 liberates cGAS from nucleosome sequestration during tumorigenesis

doi: 10.1038/s41586-023-06889-6

Figure Lengend Snippet: a , b , Images ( a ) and quantification ( b ) of cGAS and ISD90, or cGAS and human nucleosomal core particles (hNCP) colocalization in control cells and sg MRE11 MDA-MB-231 cells. Left to right, n = 344, 312, 491, 405, 366 and 243 independent cells. White arrowheads indicate colocalization of cGAS and ISD90 foci. WT versus sg MRE11 , ISD90, P = 0.007; hNCP, P = 0.0038. c , 2′3′-cGAMP ELISA after ISD90 transfection in sgControl versus sg MRE11 cells. Two independent biological replicates for WT, sg MRE11 , WT + ISD90 and sg MRE11 + ISD90. CDK1i refers to the CDK1 inhibitor (Ro-3306). Representative of two independent biological experiments. d , Top, schematic of the experiment. Bottom, western blot for the STING pathway in WT versus sg MRE11 cells after Lipofectamine (Lipo) only, ISD90 or hNCP transfection. e , cGAS + micronuclei after 24 h of irradiation in WT or sg MRE11 mitotic cells. n = 404 (WT), 441 (sg MRE11 ), 419 (WT + 4 Gy) and 438 (sg MRE11 + 4 Gy) independent cells. Representative of two independent biological experiments. f , g , cGAS–ISD90 colocalization. f , In the presence of MRE11 nuclease inhibitors, n = 500 independent cells per cell line per condition, **** P < 0.0001, representative of two independent biological experiments. g , sg MRE11 cells expressing different MRE11 constructs. n = 1,200 independent cells per cell line; WT versus sg MRE11 , P = 0.0001; sg MRE11 versus sg MRE11 + WT, P = 0.0002; sg MRE11 versus sg MRE11 + H129N, P = 0.0004; sg MRE11 versus sg MRE11 + DB1/DB2Δ, P = NS. Representative of two independent biological experiments. h , i , Schematic (top) and quantification (bottom) of TR-FRET fluorescence assays using europium–streptavidin (Eu–SA)-labelled hNCP and an anti-His antibody conjugated to the FRET acceptor ULight to measure cGAS–NCP interaction ( h ) and FRET acceptor AT647N conjugated hNCP to measure nucleosome stacking ( i ). Results representative of three independent experiments. j , Top, cGAMP accumulation in the presence of 1 µM cGAS, 5 µM dsDNA, 0.5 µM NCP and a 2-fold gradient of MRN concentrations (0.0156–2 µM). Bottom, quantified cGAS activity from triplicate experiments. Left to right, including comparisons to +NCP/–MRN: P < 0.0001, P = 0.0693 (NS), P = 0.0042, P < 0.0001, P = 0.0002, P < 0.0001, P < 0.0001, P < 0.0001 and P < 0.0001, by two-tailed one-way ANOVA. Representative of three independent experiments. k , l , Images ( k ) and quantification ( l ) FRAP assays in sgControl versus si MRE11 cells stably expressing GFP–cGAS. Images taken at 1-min intervals before and after photobleaching an arbitrary nuclear region of interest (ROI) for 60 min. Neocarzinostatin (NCS; 0.1 mg ml –1 ) was added immediately after photobleaching as indicated. n = 3 independent cells for each condition. Two-tailed two-way ANOVA: siControl + NCS versus si MRE11 + NCS, P = 0.041; siControl versus siControl + NCS, P = 0.0003; siControl + NCS versus si MRE11 , P < 0.0001. m , n , Images ( m ) and quantification ( n ) of colocalization of cGAS-ISD90 in cGAS-deficient control or sg MRE11 cells reconstituted with cGAS WT or cGAS(R255A). White arrowheads indicate colocalization of cGAS and ISD90 foci. Data are mean ± s.e.m., n = 1,200 independent cells per condition. sg cGAS + cGAS WT + doxycycline versus sg cGAS /sg MRE11 + cGAS WT + doxycycline, P < 0.0001; sg cGAS + cGAS(R255A) + doxycycline versus sg cGAS /sg MRE11 + cGAS(R255A) + doxycycline, P > 0.99 (NS). Unless otherwise specified, grouped analyses performed with a two-tailed t -test. Scale bars, 10 µm ( k , m ), 20 µm ( a ).

Article Snippet: To generate cGAS knockout MDA-MB-231 cell lines, synthetic gRNAs targeting human cGAS were purchased from Synthego (CRISPR gene knockout kit V2). gRNAs were diluted to 30 µM (pmol µl –1 ) in 1× TE buffer (Synthego).

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Western Blot, Irradiation, Expressing, Construct, Fluorescence, Activity Assay, Two Tailed Test, Stable Transfection

Journal: Nature

Article Title: MRE11 liberates cGAS from nucleosome sequestration during tumorigenesis

doi: 10.1038/s41586-023-06889-6

Figure Lengend Snippet: a , b , ICC of cGAS localization to cytoplasmic interferon stimulating dsDNA 90 bp (ISD90) a , 1 h after transfection in siControl vs siMRE11 BJ-5ta cells (72 h after siRNA transfection). b , Percentage of cells with cGAS foci. Scale bar, 10 µm. n = 900 cells for each cell lines. siControl+ISD90 vs siMRE11 + ISD90; p = 0.0001. n = 3 independent biological experiments; c , Western blot was performed on innate immune signaling pathways in siControl, siMRE11 , and sicGAS BJ-5ta cells, 3 h post transfection with 4 μg/mL dsDNA 90 bp (ISD90), demonstrating consistent findings across three independent replicates. d , e , and f , Quantitative RT-PCR was used to analyze normalized gene expression for human interferon-stimulated genes (ISGs), namely CXCL10 , CCL5 , and IFIT1 in BJ-5ta cells. n = 2 independent biological experiments; 3 samples for each cell lines. ( d ) siControl (Lipo vs ISD90); p = 0.0027. siControl+ISD90 vs sicGAS +ISD90; p = 0.0004 and siMre11 + ISD90; p = 0.0009. ( e )siControl (Lipo vs ISD90); p = 0.0014. siControl+ISD90 vs sicGAS +ISD90; p = 0.0013 and siMRE11 + ISD90; p = 0.0076. ( f ) siControl (Lipo vs ISD90); p < 0.0001. siControl+ISD90 vs sicGAS +ISD90; p < 0.0001 and siMRE11 + ISD90; p = 0.0003. g , Representative immunocytochemistry analysis of cGAS (Red) localization to cytoplasmic Oregon Green conjugated human NCP (OG-hNCP) (Green), 60 min after transfection with 72 nM OG-hNCP in siControl or siMRE11 human MDA-MB-231 cells. Cell nuclei stained by 4′,6-diamidino-2-phenylindole (DAPI; Blue). Scale bar, 10 µm. Image quantification is shown in Fig. , right panel. h , i , qRT-PCR normalized gene expression for human interferon-stimulated genes ( ISG ) IFIT1 and CCL5 . Cells were transfected with Lipo only or 4 μg/mL ISD90 for 6 h. mRNA levels were normalized to β-actin mRNA levels. n = 3 independent biological experiments; 3 samples for each cell lines. ( h ) ISD90 (WT vs sgMRE11 ); p < 0.0001. ( i ) p = 0.0008. j , WT or two different Mre11 ATLD/ATLD MEF cell lines were transfected with 4 μg/mL Alexa488-ISD90 or 72 nM OG-hNCP for 60 min. Quantification of cells with colocalization for cGAS foci and cytosol DNA (ISD90 or OG-hNCP) is shown. Alexa488-ISD90 (WT vs ATLD #1; p = 0.0021, ATLD#2; p = 0.004). OG-hNCP (WT vs ATLD #1; p < 0.0001, ATLD#2; p < 0.0001). n = 3 independent biological experiments; 900 cells for each cell lines. k , l , qRT-PCR normalized gene expression for mouse ISG IFIT1 and CCL5 . ( k ) WT vs WT + ISD90; p = 0.0007, WT + ISD90 vs ATLD + ISD90; p = 0.0022. ( l ) WT vs WT + ISD90; p = 0. 0006, WT + ISD90 vs ATLD + ISD90; p = 0.0009. n = 3 independent biological experiments; 3 samples for each cell lines. Data are mean ± SEM. Unless otherwise specified, P values estimated using a two-tailed t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: To generate cGAS knockout MDA-MB-231 cell lines, synthetic gRNAs targeting human cGAS were purchased from Synthego (CRISPR gene knockout kit V2). gRNAs were diluted to 30 µM (pmol µl –1 ) in 1× TE buffer (Synthego).

Techniques: Transfection, Western Blot, Quantitative RT-PCR, Expressing, Immunocytochemistry, Staining, Two Tailed Test

Journal: Nature

Article Title: MRE11 liberates cGAS from nucleosome sequestration during tumorigenesis

doi: 10.1038/s41586-023-06889-6

Figure Lengend Snippet: a , Time-lapse microscopy of WT and sgMRE11 MDA-MB-231 cells expressing RFP-cGAS after transfection with 4 μg/mL Alexa 488-ISD90. Images were captured with a Nikon fluorescence microscope, and the time stamp is relative to transfection. White arrows indicate co-localization of RFP-cGAS foci and Alexa 488-ISD90 foci. Scale bar, 20 μm.

Article Snippet: To generate cGAS knockout MDA-MB-231 cell lines, synthetic gRNAs targeting human cGAS were purchased from Synthego (CRISPR gene knockout kit V2). gRNAs were diluted to 30 µM (pmol µl –1 ) in 1× TE buffer (Synthego).

Techniques: Time-lapse Microscopy, Expressing, Transfection, Fluorescence, Microscopy

Journal: Nature

Article Title: MRE11 liberates cGAS from nucleosome sequestration during tumorigenesis

doi: 10.1038/s41586-023-06889-6

Figure Lengend Snippet: a , b WT or sgMRE11 MDA-MB-231 cells transfected with control siRNA (siControl) or Mre11-targeting siRNA ( siMRE11 ) and analyzed 24 h after 15 Gy IR. Cells with micronuclei (a) or cGAS foci (b) were counted by ICC analysis. n = 2 independent experiments; 615 (WT + siControl), 387 ( sgMre11 + siControl), 467 (WT + siMre11 ), 620 ( sgMre11 + siMre11 ) samples for each cell lines. ( a ) WT+siControl vs sgMre11 +siControl; p = 0.029, WT+ siMRE11 ; p = 0.0022, sgMRE11+siMRE11 ; p = 0.0014. ( b ) WT+siControl vs sgMRE11 +siControl; p = 0.008, WT+ siMRE11 ; p = 0.0058, sgMRE11+siMRE11 ; p = 0.0074. c , d , human ISG expression IFIT1 (c) and CCL5 (d) were observed in WT and sgMRE11 MDA-MB-231 cells 48 h after IR (20 Gy) by RT-qPCR. mRNA levels were normalized to β-actin mRNA levels. ( c ) WT vs WT + 20 Gy; p = 0.0121, WT + 20 Gy vs sgMRE11 + 20 Gy; p = 0.0099. ( d ) WT vs WT + 20 Gy; p = 0.0048, WT + 20 Gy vs sgMRE11 + 20 Gy; p = 0.0025. n = 2 independent experiments; 3 samples for each cell lines. e , Mitotic cells of the indicated MDA-MB-231 cells were irradiated and then fixed 24 h after irradiation. Scale bar, 20 μm. Image quantification is shown in Fig. . Data are mean ± SEM. Unless otherwise specified, statistical analyses were determined by one-way ANOVA followed by Sidak’s multiple comparison post-test using a two-tailed test: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: To generate cGAS knockout MDA-MB-231 cell lines, synthetic gRNAs targeting human cGAS were purchased from Synthego (CRISPR gene knockout kit V2). gRNAs were diluted to 30 µM (pmol µl –1 ) in 1× TE buffer (Synthego).

Techniques: Transfection, Expressing, Quantitative RT-PCR, Irradiation, Comparison, Two Tailed Test

Journal: Nature

Article Title: MRE11 liberates cGAS from nucleosome sequestration during tumorigenesis

doi: 10.1038/s41586-023-06889-6

Figure Lengend Snippet: a , b , MDA-MB-231 cells were transfected with the indicated siRNA and incubated for 48 h. Subsequently, cells were harvested 6 h ( a ) or 1 h ( b ) after transfection with 4 μg/mL ISD90. a , Western blot analysis of each cell line with the indicated antibodies. Mre11, Nbs1, Rad50 and GAPDH (a loading control). b , Quantification of cells positive for cGAS foci. (a) and (b) were double-checked using a blinded experiment. n = 2 independent biological experiments; 400 cells for each cell lines. WT + ISD90 vs siMRE11 + ISD90; p < 0.0001, siRad50 , siNBS1 and siMRN ; p = 0.0002. c , d , e , MDA-MB-231 cells were transfected with dsDNA, following treatment with indicated nuclease inhibitors (Mirin, PFM01 and PFM39) for 30 min. c , Representative images showing the localization of cGAS and Biotin-ISD90 were obtained 2 h after transfection with Biotin-ISD90. Scale bar, 10 μm. Image quantification is shown in Fig. . d , Western blot analysis for cGAS-STING pathways. Cells were obtained 3 h after transfection of ISD90. e , Cells were obtained 6 h after transfection of ISD90. qRT-PCR normalized gene expression for human interferon-stimulated genes IFIT1 . mRNA levels were normalized to human β-actin RNA levels. n = 3 independent biological experiments; 3 samples for each cell lines. p < 0.0001. f , g , sgMRE11 MDA-MB-231 stable cell lines were generated via retroviral infection, with each line expressing human Mre11 WT, H129N (nuclease dead mutant), and DB1/DB2 deletion (DNA binding mutant) in sgMRE11 MDA-MB-231 cells. These cell lines were then transfected with dsDNA. f , Western blot analysis of Halo, human Mre11, pSTING, pIRF3 and α-tubulin in each MDA-MB-231 cell lines, demonstrating consistent findings across three independent replicates. g , Representative images showing the localization of cGAS and Biotin-ISD90 were obtained 2 h after transfection with Biotin-ISD90. Scale bar, 10 μm. Image quantification is shown in Fig. . h , ICC 30 min after transfection with Oregon Green (OG)-NCP in MDA-MB-231 cells expressing RFP-cGAS and HaloTag-Mre11. Right panel, quantification of Mre11 and cGAS colocalization at cytoplasmic NCP foci. Scale bar, 10 μm. n = 3 independent biological experiments; 474 cells were analyzed for Fraction of NCP foci. NCP+cGAS vs NCP + cGAS+Mre11; p = 0.0006, NCP+Mre11 vs NCP+cGAS+Mre11; p = 0.0055. Data are mean ± SEM. Unless otherwise specified, statistical analyses were determined by one-way ANOVA followed by Sidak’s multiple comparison post-test using a two-tailed test: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: To generate cGAS knockout MDA-MB-231 cell lines, synthetic gRNAs targeting human cGAS were purchased from Synthego (CRISPR gene knockout kit V2). gRNAs were diluted to 30 µM (pmol µl –1 ) in 1× TE buffer (Synthego).

Techniques: Transfection, Incubation, Western Blot, Quantitative RT-PCR, Expressing, Stable Transfection, Generated, Infection, Mutagenesis, Binding Assay, Comparison, Two Tailed Test

Journal: Nature

Article Title: MRE11 liberates cGAS from nucleosome sequestration during tumorigenesis

doi: 10.1038/s41586-023-06889-6

Figure Lengend Snippet: a , b , ICC of cGAS re-localization to cytoplasmic ISD90 in a subset of cells, a , 6 h after transfection in siControl versus siMRE11 BJ-5ta cells (72 h after siRNA transfection). b , Percentage of cells with cGAS localization in both cytosol and nucleus (C + N; Cytosol + Nucleus [0.4 1.25]), and predominantly nuclear (N: Nucleus [C/N ratio <0.4]) 0, 3, and 6 h after 4 µg/mL ISD90 transfection in a series of images containing at least 10 evaluable cells each. n = 2 independent experiments; n = 500 cells for each cell condition. siControl (C + N) 0 h vs 3 h: p = 0.0002, 0 h vs 6 h: p < 0.0001; (C) 0 h vs 3 h: p = 0.0001, 0 h vs 6 h: p = 0.0002; (N) 0 h vs 3 h: p < 0.0001, 0 h vs 6 h: p < 0.0001. siMRE11 (C) 0 h vs 3 h: p = 0.0234, 0 h vs 6 h: p = 0.0012; (N) 0 h vs 3 h: p = 0.0116. c , d , Indicated MDA-MB-231 cells were either transfected with dsDNA or treated with NCS for 6 h. c , The Log 2 -transformed cGAS localization ratio (Cytosol/Nucleus). N = 2 independent experiments; 10 (WT and sgMRE11 ) cells. WT + Lipo vs WT + ISD90; p = 0.034, WT + Lipo vs WT + NCS; p < 0.0001. d , Representative images of cGAS staining, demonstrating consistent findings across three independent replicates. Scale bar, 10μm. Data are mean ± SEM. Unless otherwise specified, statistical analyses were determined by one-way ANOVA followed by Sidak’s multiple comparison post-test using a two-tailed test: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: To generate cGAS knockout MDA-MB-231 cell lines, synthetic gRNAs targeting human cGAS were purchased from Synthego (CRISPR gene knockout kit V2). gRNAs were diluted to 30 µM (pmol µl –1 ) in 1× TE buffer (Synthego).

Techniques: Transfection, Transformation Assay, Staining, Comparison, Two Tailed Test

Journal: Nature

Article Title: MRE11 liberates cGAS from nucleosome sequestration during tumorigenesis

doi: 10.1038/s41586-023-06889-6

Figure Lengend Snippet: a , b , Human cGAS knock-out MDA-MB231 cell lines were made with CRISPR technology using multiguided sgRNA (Synthego). Then, MDA-MB-231 sgcGAS stable cell lines were transfected with mcGASt1, hNCP v52, or mcGASt1/hNCP v52. a , After 2 h, cells were harvested for Western blot analysis to confirm human Mre11, pTBK, pSTING, and Actin, demonstrating consistent findings across two independent replicates. b , Six hours after transfection, qRT-PCR was performed to normalize gene expression levels for interferon-stimulated genes CXCL10 , with mRNA levels normalized to β-actin mRNA levels. n = 3 independent experiments; 3 samples for each cell lines. sgcGAS + Lipo vs sgcGAS + cGAS-NCP; p < 0.0001, sgcGAS/sgMRE11 + Lipo vs sgcGAS/sgMRE11 + cGAS-NCP; p < 0.0001, sgcGAS + cGAS vs sgcGAS/sgMRE11 + cGAS; p = 0.0067. c , d , PiggyBac Transposon system was used to deliver PB-cGAS-WT or PB-cGAS-R255A (an AP site binding mutant) to each cell line, followed by selection with puromycin for two days. After selection, cells were incubated with doxycycline for 24 h, and then transfected with ISD90. c , Western blot analysis of human Mre11, cGAS and Actin 24 h after doxycycline treatment, demonstrating consistent findings across two independent replicates. d , The cGAS localization ratio (Nucleus/cytosol) 2 h after Biotin-ISD90 transfection. n = 2 independent experiments; 31 cells for each cell lines. sgcGAS + cGAS-WT + ISD90 vs sgcGAS/sgMRE11 + cGAS-WT + ISD90; p = 0.0394, sgcGAS/sgMRE11 + cGAS-WT + ISD90 vs s sgcGAS/sgMRE11 + cGAS-R255A + ISD90; p = 0.022. e , Representative images showing the localization of cGAS and Biotin-ISD90 were obtained 2 h after transfection with Biotin-ISD90, demonstrating consistent findings across two independent replicates. Scale bar, 10 μm. Data are mean ± SEM. Unless otherwise specified, statistical analyses were determined by one-way ANOVA followed by Sidak’s multiple comparison post-test using a two-tailed test: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. n.s., not significant.

Article Snippet: To generate cGAS knockout MDA-MB-231 cell lines, synthetic gRNAs targeting human cGAS were purchased from Synthego (CRISPR gene knockout kit V2). gRNAs were diluted to 30 µM (pmol µl –1 ) in 1× TE buffer (Synthego).

Techniques: Knock-Out, CRISPR, Stable Transfection, Transfection, Western Blot, Quantitative RT-PCR, Expressing, Binding Assay, Mutagenesis, Selection, Incubation, Comparison, Two Tailed Test