Fig 2: Effects of bleomycin on formation of DSBs and expression of DNA damage response genes in tobacco leaves. (A) H2AX phosphorylation. Total protein load detected by Coomassie blue staining. (B) H2AX phosphorylation. Western blot with anti-gamma-H2AX. M, molecular size markers. Lanes 1 and 2, two independent biological repeats; mock or bleomycin (Ble) indicate infiltration with buffer or bleomycin, respectively. Arrowheads on right indicate the expected position of tobacco gamma-H2AX. (C) Transcriptional activation of the indicated DNA damage response genes in response to bleomycin treatment. The expression levels were measured 24 h after bleomycin treatment and are expressed as fold change after bleomycin treatment relatively to mock infiltration. Error bars represent the SD from two independent biological replicates. Statistical significance is indicated by asterisks (** = p < 0.05; *** = p < 0.01).

Fig 3: FL BARD1 functions in DNA damage response. SKNSH and SHSY5Y cell lines were silenced for FL BARD1 expression upon transfection with lentiviral plasmids (shBARD1#A, shBARD1#B). Unsilenced control cells were transfected with plasmid shCTR. The efficiency of short harpin silencing was verified by western blotting, using an antibody against FL BARD1 isoform. The molecular weight of FL BARD1 isoform is reported. The higher band in the blot is an aspecific staining. ß-Actin levels were used as loading control (A). The detection of ? -H2AX protein was verified in nuclear extract of silenced (shBARD1) and unsilenced control (shCTR) cells, by western blotting. Antibody against histone H3 was used as loading control (B). SKNSH shBARD1 and shCTR cells (C) and SHSY5Y shBARD1 and shCTR cells (D) were treated with 5 Gy IR. The expression of ?- H2AX was measured by western blotting in a time-course (30 min, 1h, 3h, 6h, 12h, 24h, 36h, 48h) after IR. The integral optical density (IOD) of ?- H2AX protein bands were measured and normalized respect to loading control protein band H3. The arrows indicate the higher increment of ?- H2AX in each cell line. The experiments were repeated twice.

Fig 4: Salinity induced accumulation of DSBs. (A) Representative Comet images of 7-days old wild-type, OE-1, sog1-6 and sog1-1 mutant plants incubated without NaCl for 1 h (control) and incubated with 150 mM NaCl for 12 h and then transferred to medium without NaCl for 5 h (recovery) (left, middle and right panel respectively). (B) Box plot of percentages of tail DNA of 7-days old wild-type, OE-1, sog1-6 and sog1-1 mutant plants. DNA percentage in the comet tails were determined by using TriTek Comet Score software. Box plots are based on analysis of approximately 100 cells per experimental sample from random microscopic fields of three independent biological replicates. Each box represents the interquartile range (IQR) of DNA damage, the line across each box represents the median value and the whiskers represent 5–95 percentile values. Brackets connect box plots of sample groups with significant statistical difference (*p < 0.05, **p < 0.01). (C) Immunostaining of ?-H2AX foci in the root cells of wild-type, overexpressor line and mutant line plants after 12 h of treatment without and with 150 mM NaCl, respectively. (D) Counted numbers of ?-H2AX foci per cell detected after 12 h of treatment with 150 mM NaCl in plants of all the genotypes. According to their number of ?-H2AX foci, approximately 100 nuclei for each sample were grouped into six classes; cell with 0, 1–2, 3–5, 6–10, 11–20 and > 20 foci per nuclei. (E) Detection of ?-H2AX accumulation by immunoblot analysis using the total histone protein isolated from untreated and NaCl treated of 7-days old wild-type, OE-1, sog1-6 and sog1-1 mutant plants, respectively. The blots were cut prior to incubation with the primary antibody. Replicates of the protein gel blots have been presented in Supplementary Fig. S11.