Anti dsdna antibody

Immunostaining for protein expression of cGAS or cdsDNA and image acquisition by fluorescence microscopy was performed as described previously [43 (link)]. Briefly, HNSCC cells were seeded on glass coverslips in 6-well plates and treated according to the CBMN assay. At 24 h after exposure, cells were fixed in 3.7% formaldehyde/PBS for 10 min, permeabilized in 5% bovine serum albumin/0.5% Triton-X-100/PBS for 30 min at room temperature, and incubated with an anti-cGAS antibody (Cell Signaling Technology, Danvers, MA, USA). For the immunostaining of cdsDNA with an anti-dsDNA antibody (Abcam, Cambridge, UK), a mild permeabilization protocol was applied using 0.1% Tween-20 and 0.01% Triton-X-100 in PBS for 3 × 5 min at room temperature not affecting the nuclear envelope. Incubation with the primary antibody was performed overnight at 4 °C. Cells were washed in PBS and incubated with a secondary antibody conjugated with Alexa Fluor 488^® or Cy5 (Thermo Fisher Scientific, Dreieich, Germany) for 1 h at room temperature followed by mounting in an HOECHST33258-containing antifade mountant. Fluorescence microscopic image acquisition was performed with an Axio Imager.A1 microscope (Zeiss, Jena, Germany).

Dot blot analysis was performed with minor changes according to the protocol reported elsewhere (Morales et al., 2016 (link)). Genomic DNA was isolated by standard extraction with Phenol/Clorophorm/Isoamylic Alcohol (pH 8.0) followed by precipitation with 3 M NaOAc and 70% Ethanol. The isolated genomic DNA was randomly fragmented for 4 hr at 37 °C with a cocktail restriction enzymes (BsrgI, EcoRI, HindIII, XbaI, Ssp1) supplemented with 1 M spermidine. After incubation, the digested DNA was cleaned up with Phenol/Clorophorm extraction and standard Ethanol precipitation. After sample quantification, 350 ng of digested DNA was incubated with RNase H overnight at 37 °C as a negative control. The same quantity of each sample was spotted onto a nitrocellulose membrane, blocked in 5% non-fat dry milk, and incubated with the anti-DNA-RNA hybrid [S9.6] antibody (Kerafast; 1:1000) or anti-dsDNA antibody (Abcam; 1:2000) overnight at 4 °C. A horseradish peroxidase-conjugated goat specie-specific secondary antibody (Santa Cruz Biotechnology) was used. Quantification on the scanned image of the blot was performed using Image Lab software with dsDNA as loading control.

DPCs were isolated using a modified Rapid Approach to DNA Adduct Recovery (RADAR) assay^{65 (link),25 (link)}. Briefly, 1–2 × 10⁶ cells were lysed in 1 mL of buffer containing 6 M guanidine thiocyanate (GTC); 10 mM Tris–HCl, pH 6.8; 20 mM EDTA; 4% Triton-X100; 1% Sarkosyl and 1% DTT. DNA was precipitated by adding 100% ethanol, washed three times in wash buffer (20 mM Tris–HCl, pH 6.8; 150 mM NaCl and 50% ethanol). DNA was then solubilized in 1 mL of 8 mM NaOH. The DNA concentration was quantified by treating a small aliquot of DNA with proteinase K (Invitrogen) for 3 h at 50 °C, followed by detection with PicoGreen dye (Invitrogen) according to manufacturer’s instructions. Equal dsDNA loading was confirmed by slot-blot immunodetection with an anti-dsDNA antibody (Abcam). Total DPCs after electrophoretic separation on polyacrylamide gels were visualised by silver staining using the ProteoSilver Plus Silver Stain Kit (Sigma-Aldrich) and specific proteins were detected by Western-blot.

Immunostaining for protein expression of cGAS or cdsDNA and image acquisition by fluorescence microscopy was performed as described previously [43 (link)]. Briefly, HNSCC cells were seeded on glass coverslips in 6-well plates and treated according to the CBMN assay. At 24 h after exposure, cells were fixed in 3.7% formaldehyde/PBS for 10 min, permeabilized in 5% bovine serum albumin/0.5% Triton-X-100/PBS for 30 min at room temperature, and incubated with an anti-cGAS antibody (Cell Signaling Technology, Danvers, MA, USA). For the immunostaining of cdsDNA with an anti-dsDNA antibody (Abcam, Cambridge, UK), a mild permeabilization protocol was applied using 0.1% Tween-20 and 0.01% Triton-X-100 in PBS for 3 × 5 min at room temperature not affecting the nuclear envelope. Incubation with the primary antibody was performed overnight at 4 °C. Cells were washed in PBS and incubated with a secondary antibody conjugated with Alexa Fluor 488^® or Cy5 (Thermo Fisher Scientific, Dreieich, Germany) for 1 h at room temperature followed by mounting in an HOECHST33258-containing antifade mountant. Fluorescence microscopic image acquisition was performed with an Axio Imager.A1 microscope (Zeiss, Jena, Germany).