Fig 2: WWOX modulation of ATR checkpoint is ATM-dependentA. WWOX associates with p-ATM following induction of DNA SSBs. HEK293 cells were transfected GST-WWOX. At 24 hours, cells were treated with IR (10Gy for an additional hour) or UV ((70 J/m2) or HU (2mM) for an additional 2 hours). Cells were then subjected to GST-pulldown and Lysates were blotted against ATM, p-ATM (Ser1981), KAP1, p-KAP1 (pThr824), CHK1, p-CHK1 (Ser296), GST (WWOX) and GAPDH. Pulled-down complexes were blotted with anti p-ATM (Ser1981) and anti-GST (WWOX). B. MCF7 cells were cultured in duplicates, first duplicate were left untreated ore treated with IR or UVC or HU, the second duplicate were treated first with ATM inhibitor KU-55933 for 48h then were treated as first duplicate as indicated in the figure. Whole cell lysates were analysed by western blot using antibodies against ATM, p-ATM, KAP1, p-KAP1, ITCH, CHK1, p-CHK1, WWOX, and GAPDH as loading control.

Fig 3: Knockdown of KAP1 resulted in a reduction in influenza virus replication. A549 cells were transfected twice with negative control siRNA (Allstars NC) or two siRNAs targeting human Kap1 (siKAP1 1#; siKAP1 2#). (A) qPCR to confirm the relatively low level of mRNA expression in siKAP1-treated A549 cells. (B) Western blotting with anti-KAP1 or anti-β-actin antibody to confirm the knockdown efficiency. (C) Cell viability of siRNA-treated cells was determined by using CellTiter-Glo reagent. (D) Virus growth in siRNA-treated A549 cells infected with WSN virus at an MOI of 0.001 and the supernatant was collected at 48h post-infection. (h.p.i: hours post-infection). Means ± SD of triplicate experiments is shown in (A,C,D). ** p < 0.01 (two-way ANOVA followed by Dunnett’s test).

Fig 4: Disulfiram and acetaldehyde induce DSBs, chromosome aberrations, and checkpoint activation in BRCA2‐deficient human cells Quantification of the frequency of cells with 10 or more γH2AX foci in BRCA2‐proficient (+BRCA2) or BRCA2‐deficient (−BRCA2) human DLD1 cells incubated with 10 μM disulfiram or 4 mM acetaldehyde for 4 days. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test.Cells treated as in (A) were incubated overnight with colcemid and metaphase spreads were stained with Giemsa. To quantify chromosome aberrations, approximately 50 metaphases were analyzed for each sample. Middle line represents median, and the box extends from the 25th to 75th percentiles. The whiskers mark the 10th and 90th percentiles. P‐values were calculated using an unpaired two‐tailed t‐test. ‐, DMSO; Di, disulfiram; Ac, acetaldehyde.Cells treated as in (A) were processed for FACS analyses of DNA content. Quantification of the percentage of cells in G2/M is shown. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test.Whole‐cell extracts prepared from BRCA2‐proficient or BRCA2‐deficient human DLD1 cells treated as in (A) were immunoblotted as indicated. SMC1, GAPDH, and H2AX were used as loading controls. ‐, DMSO; Di, disulfiram; Ac, acetaldehyde.BRCA2‐proficient (+BRCA2) and BRCA2‐deficient (−BRCA2) human DLD1 cells were incubated with 10 μM disulfiram or 4 mM acetaldehyde. Whole‐cell extracts prepared at the indicated time points during treatment were immunoblotted as shown. KAP1, SMC1, and GAPDH were used as loading controls. Di, disulfiram; Ac, acetaldehyde. Source data are available online for this figure.

Fig 5: Chloroquine induces phosphorylation of KAP1 at S824 and activates EBV lytic cycle in lymphoblastoid cells.A. Lymphoblastoid cells were treated with chloroquine (CQ) and harvested at different times post-treatment for immunoblotting with antibodies as indicated. B. Lymphoblastoid cells were untreated (open bar), treated with 10μM CQ (black bar) or 100μM CQ (horizontal striped bar) every 3 days, released virus particles were pelleted from supernatant 9 days later, treated with DNase and quantified using q-PCR. ND: not detectable; error bars represent 3 technical replicates from 2 experiments.