Extended Data Fig. 3: rDNA fragments accumulate in the cytoplasm of Abro1-deficient cells.
From: Stalled replication fork protection limits cGAS–STING and P-body-dependent innate immune signalling
(a) Co-staining of ssDNA and nucleolin in Abro1 KO cells treated with HU (4 mM, 4 h). Scale bars, 10 μm. (b) Leptomycin B (LMB) blocks accumulation of cytosolic ssDNA. Cells incubated with LMB for 8 h followed by 4 h continuous incubation with or without addition of 4 mM HU were stained with antibody to ssDNA. Scale bars, 10 μm. (c) pSTAT1 levels were reduced when cells were treated with LMB. (d) Detection of rDNA in the cytoplasm of Abro1 KO cells by slot blot with biotin-labelled rDNA probe. ‘Nuclear DNA’ (from 2 × 105 cells) and ‘Cytosolic DNA’ (‘1x’ from 5 × 105 cells, ‘2x’ from 1 × 106 cells) were loaded. Band intensity (a.u.) is quantified using ImageJ. (e) Knockdown DNA2 reduces cytosolic rDNA in the cytoplasm of Abro1 KO cells by slot blot with biotin-labelled rDNA 18S probe. ‘Nuclear DNA’ (from 2 × 105 cells) and ‘Cytosolic DNA’ (from 5 × 105 cells) were loaded. Band intensity (a.u.) is quantified using ImageJ. (f) Detection of rDNA by FISH with biotin-labelled rDNA 18S probe. Scale bars, 10 μm. (g) Treatment with RNAPI inhibitor, CX5461, reduces cytosolic ssDNA accumulation in Abro1 KO cells treated with HU (4 mM, 4 h). CX-5461 (1 μM) was added for 1 h before the end of the HU treatment. IF staining was carried out with ssDNA antibody. Scale bars, 10 μm. Data shown represent three independent experiments in a, d-g and two independent experiments in b, c. (h) Treatment with CX5461 reduces the upregulation of IL6 and CXCL10 in Abro1 KO cells treated with HU (4 mM, 16 h). CX-5461 (1 μM) was added for 1 h before the end of the HU treatment. Relative fold change was quantified and shown with mean ± SD (n = 3 independent experiments). Two way Anova was used for statistics.
