Fig 2: Suppression of RA-induced RARß2 activation by BER/SSBR inhibition. (A–D) Real-time RT-PCR analysis of RA-induced RARß2 expression, normalized to HPRT1 expression, in (A) PARP1-down-regulated, (B) PARP-inhibited, (C) XRCC1-down-regulated, and (D) PNKP-down-regulated HEK293 cells as a function of time of 1 µM RA treatment. Before RA treatment, down-regulation of PARP1, XRCC1, or PNKP was achieved by 80 nM siRNA via transient transfection for 48 hr, or PARP inhibition was achieved by 1 µM rucaparib treatment for 24 hr. Western blot or real-time RT-PCR analysis was performed to verify the down-regulation of each BER/SSBR protein and is shown in Inset. (E–G) RT-PCR analysis of RARß2 premRNA transcript. (E) A schematic of the experimental design showing primer locations and PCR products for amplifying a region of the RARß2 premRNA and mature mRNA. Ethidium bromide-stained 0.8% agarose gel and the associated ImageJ quantitation showing the PCR products from (F) untreated and 3-hr 1 µM RA-treated control cells and (G) XRCC1-down-regulated or PARP-inhibited cells, compared with control cells, after RA treatment. The data represent the average (with SD) of three or more independent experiments. Statistical analysis was performed in GraphPad Prism by using two-way ANOVA with Tukey’s multiple comparisons test, except in F where unpaired t test with Welch’s correction was used. *P value < 0.05; **P value < 0.01.

Fig 3: Involvement of hnRNP UL1 in the DDR in human nucleoli. (A) The DNA damage sensitivity of HEK UL1 KO cells compared to HEK WT cells was tested after 2.5 h of treatment with the genotoxic reagents ETO and CPT. After this time, the cells were harvested at the following time points: 18 h, 24 h, 30 h, 48 h, 72 h and 80 h. Cell viability was assessed by Trypan blue staining, and the percentage of survival was calculated. The results were normalized to those for the control cells treated with DMSO. (B) DNA damage was assessed by Comet Assay in HEK UL1 KO cells compared to HEK WT cells after DNA damage induced with ETO and CPT. Cells treated with DMSO were used as negative controls. For the assay, 50 cells per sample were analyzed. The error bars represent the SDs of three biological replicates. The p values were calculated using Student's t test, and the statistical significance is represented as follows: *P = 0.05; **P = 0.01; ***P = 0.001. (C) IP was performed using an anti-hnRNP UL1 antibody and protein extracts from the NO and CN fractions of HEK WT cells. After elution, the immunoprecipitated proteins were identified by western blotting followed by immunostaining. Four independent biological replicates from each fraction were used for the experiment. For the input, 5% of the total volume applied to the beads was used; for the negative controls, beads without antibody were used; for immunostaining, anti-hnRNP UL1, anti-pChk1, and anti-pRPA32 antibodies were used; and actin was used as a loading control. (D) IP was performed using magnetic beads conjugated with an anti-FLAG antibody and protein extracts from the NO and CN fractions of HEK UL1 OE cells. Three independent biological replicates from each fraction were used for the experiment. For the input, 5% of the total volume applied to the beads was used. For immunostaining, anti-53BP1, anti-RAD50, anti-FLAG, and anti-XRCC1 were used, and an anti-actin antibody was used as a control. * - nonspecific signal.

Fig 4: Constitutive association of BER/SSBR proteins in immunocomplexes of demethylases. (A and B) Co-IP analysis showing the presence of the indicated BER/SSBR proteins in western blots of chromatin-bound (A) LSD1 and (B) TET1 IP complexes from control and 1 µM RA-treated HEK293 cells. (C) Co-IP analysis showing the presence of LSD1, TET1, and APE1 in the XRCC1 IP from the total nuclear extracts of control and 2-hr 1 µM RA-treated cells. (D) Co-IP analysis could not detect non-BER/SSBR proteins, such as MLH1, PMS2, and XRCC4, in the TET1 IP from the total nuclear extracts of control and 2-hr 1 µM RA-treated cells.

Fig 5: The interaction of APTX with XRCC1 and XRCC4 and their roles in the recruitment of APTX to DNA damage sites. (A) The interaction of GFP-APTX with XRCC1 and XRCC4 examined by IP. GFP-Trap magnetic agarose beads were used for IP and GFP, GFP-APTX, XRCC1 and XRCC4 proteins in the precipitate were examined by western blotting; 5% of whole cell lysate that was used in IP experiment is represented as input 5%. (B) The efficacy of knocking down XRCC1 and XRCC4 by siRNA. GFP-APTX stably expressing U2OS cells following the transfection of the indicated siRNAs were subjected to western blotting analysis of XRCC1, XRCC4 and GFP-APTX. KAP1 was shown as the loading control. The expression level of XRCC1 and XRCC4 normalized by KAP1 expression after siRNA treatment was indicated of 53 and 18% against siCON, respectively. (C) Representative live-cell images of the GFP-APTX stably expressing U2OS cells under the depletion of XRCC1 or XRCC4 after photobleaching. Micro-laser was irradiated on the area inside the red square. (D) Relative green fluorescent intensity of C. Fifteen cells were scored at each point. The data represent the mean ± SEM.