Enh001

Cells were fixed in 4% PFA/PBS followed by permeabilization with 0.4% Triton-X100 and blocking with 3% BSA/PBS. Cells were stained with S9.6 (Kerafast:ENH001; RRID:AB_2687463), and anti-nucleolin (Abcam:ab50279; RRID:AB_881762) primary antibodies and AlexaFluor 488/568 (Thermo Fisher Scientific) secondary antibodies. Cells were counterstained with DAPI before being imaged using a Nikon Eclipse Ti-S fluorescence microscope with 60× objective with quantification of nuclear S9.6 staining carried out by measuring mean fluorescence intensity within DAPI stained regions using imageJ (RRID:SCR_003070). Regions of nucleolin staining were subtracted from final quantification. Alternatively, GFP-RNH1^D210 was induced for 24 hours with 1 μg/mL doxycycline, after which, nonbound GFP-RNH1^D210 was extracted with extraction buffer (20 mmol/L Hepes, 20 mmol/L NaCl, 5 mmol/L MgCl₂, 1 mmol/L ATP, 0.5% NP40) at 4°C for 2 minutes before fixation with 4% PFA. Fixed cells were then stained with DAPI and nuclear GFP intensity quantified via high-content imaging using a Thermo ArrayScan XTI. Significant differences between quantified data were assessed using multiple two-tailed t tests, with post hoc Holm–Šidák multiple comparisons tests. Significant differences are indicated by *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Thin sections placed on nickel grids were blocked with 10% BSA in PBS for 20 min, and incubated with primary antibodies in incubation buffer (1% BSA in PBS) for 2 hr. Grids were subsequently washed with incubation buffer three times (10 min each). Secondary antibodies, goat anti-mouse IgG (EM.GMHL15, BB International) and protein A (EM.PAG15, BB International) conjugated to 15 nm gold particles, were used against the primary antibodies from mouse and rabbit respectively. Secondary antibodies at 20-fold dilution were applied and samples were incubated for 1 hr. After washing with PBS, the immune-complexes were fixed with 1% glutaraldehyde in PBS and washed three times with distilled water. The specimens were inspected by TEM operating at 120 kV (FEI Tecnai G2 TF20 Super TWIN).
The primary antibodies and applied dilution factors are listed as follows: mouse anti-dsDNA (500x, abcam ab27156), mouse anti-ATP5A (500x, abcam ab14748), mouse anti-Cytochrome C (8000x, abcam ab13575), mouse anti-PDHA1 (500x, abcam ab110334), mouse anti-Ubiquitin (1000x, abcam ab7254), mouse anti-DNA-RNA hybrid (500x, kerafast ENH001), and rabbit anti-SOD2 (500x, abcam ab13534).