Fig 2: Effect of CYLD on RAD18, DTL, POLH and PCNA monoubiquitination. (A) MCF-7 cells were transfected with CYLD-targeted siRNA (siCYLD) or siControl. At 65 h post-transfection, the cells were irradiated at 20 J/m2 UV and harvested 1 or 6 h post-irradiation. (B) MCF-7 cells were transfected with siRNA against CYLD, RAD18 and DTL or their double or triple combinations. At 65 h post-transfection, the cells were UV-irradiated at 20 J/m2 and harvested 6 h post-irradiation. Whole-cell extracts were fractionated by SDS-PAGE and analyzed by Western blot with indicated antibodies. Protein levels were normalized to GAPDH and presented each relative to its level in unirradiated or UV-irradiated siControl-treated cells.

Fig 3: Effect of TENT4A on binding and stability of TLS-related mRNAs. (A) FLAG-tagged TENT4A expressed in HEK293T cells was immunoprecipitated with an anti-FLAG antibody and RNA bound to it was analyzed by qPCR. The results are shown as fold-change relative to empty vector. The error bars indicate SEM (two independent experiments). (B–D) Representative gel images of RAD18, CYLD and POLH poly(A) tail are shown, each with the poly(A) tail densitometric trace underneath, using Image J. The position of TVN is indicated by an asterisk that predominantly recognizes A12, serving as an internal control. (E–H), TENT4A and/or TENT4B expression was knocked down in MCF-7 cells for 48 h, after which the cells were treated with 5 µg/mL Actinomycin D for up to 8 h. The expression levels of RAD18, CYLD, POLH and POLD3 genes were each determined by qPCR relative to the expression level at time 0. Half-life was calculated by using non-linear fit one-phase decay curve equation. The significance of the differences was calculated using Student’s t-test (one-tailed); Data are presented as mean ± SEM from three independent experiments. ns, non-significant; p > 0.05.

Fig 4: Effect of TENT4A on levels and translation of TLS proteins. (A) MCF-7 cells were transfected with TENT4A-targeted siRNA (siTENT4A) or non-targeting control siRNA (siControl). At 65 h post-transfection, the cells were UV-irradiated at 20 J/m2 and harvested 1 or 6 h post-irradiation. Whole-cell extracts were fractionated by SDS-PAGE followed by Western blot with indicated antibodies. Protein levels were normalized to GAPDH and presented each relative to its level in unirradiated siControl-treated cells. (B) Effect of cell treatment (72 h) with siTENT4A or siControl on the levels of the CYLD and POLH proteins in MCF-7 cells. Whole-cell extracts were fractionated by SDS-PAGE and analyzed by Western blot with indicated antibodies. Protein levels were normalized to GAPDH and presented each relative to its level in siControl-treated cells. (C) Polysome profile analysis in MCF-7 cells in which TENT4A expression was knocked down. Absorbance trace of polysome fractionation on a sucrose gradient. (D) qPCR analysis of mRNA levels of TLS-related genes in each fraction was expressed as a percentage of total levels summed over all fractions. The results are presented as the mean ± SEM of two independent experiments. (E–G) Fraction of polysome-associated mRNA of the genes CYLD, POLH and GAPDH, respectively. The values were taken from (D). (H) Fraction of polysome-associated mRNA of the POLD3 control gene. The results are presented as the mean ± SEM of two independent experiments. Statistical significance was determined using Student’s t-test (one-tailed); * p < 0.05, ns, non-significant; p > 0.05.