Fig 1: TRABID regulates 53BP1 foci formation at DSB sites.U2OS (a–c) and PC-3 (d–f) cells infected with lentivirus expressing control shRNAs (shControl) or shRNAs specific for indicated genes were treated with IR and subjected to 53BP1 IFC 1 h post-treatment (a, d). Scale bar, 10 µm. The average 53BP1 foci number (b, e) and foci density (c, f) in each cell were quantified. Data were presented as means ± SD of more than 300 cells from three biological replicates. n.s., not significant. Two-tailed unpaired Student’s t-test. P values based on the order of appearance: b (0.882, 0.8655, and 0.583,1E-63); c (0.066, 0.9537, 0.2032, and 5.6E-47); e (0.0558, 0.6792, 0.9667, and 5E-27); f (0.8091, 0.8564, 0.7913, and 9.9E-12).
Fig 2: TRABID antagonizes SPOP-mediated removal of 53BP1 from DSB via deubiquitination of 53BP1.a PC-3 and U2OS cells infected with lentivirus expressing indicated shRNAs were treated with IR for 1 h and harvested for WB analysis. b Ubiquitination assay using Ni-NTA pull-down in 293T cells transfected with His-K29-single ubiquitin together with other indicated plasmids and treated with IR (5 Gy) for 1 h. c Ubiquitination assay using Ni-NTA pull-down in 293T control and TRABID knockdown cells transfected with indicated plasmids. Cells were harvested at different time points after IR. d–f U2OS cells infected with lentivirus expressing indicated shRNA were treated with IR followed by IFC of 53BP1 at the indicated time points after IR (d). Scale bar, 10 µm. The average 53BP1 foci number (e) and foci density (f) in each cell were quantified. Data were presented as means ± SD of more than 300 cells from three biological replicates. Two-tailed unpaired Student’s t-test. n.s., not significant. P values based on the order of appearance: e (0.154, 0.08, 0.054, 3E-35, 0.9716, 0.3365, 1.9E-44, 1.3E-19, 5.1E-6, 5.3E-20, 1.9E-50, and 2.3E-34); f (0.424, 0.8355, 0.9635, 5.8E-16, 0.2608, 0.34, 1.6E-10, 4.4E-8, 2.6E-13, 7.2E-29, 1.1E-62, and 3.5E-35). Source data are provided in this paper or the Mendeley database (https://data.mendeley.com/datasets/n9txt6y5cj/1). Similar results for (a–c) panels were obtained in two independent experiments.
Fig 3: TRABID is transcriptionally repressed by the NuRD complex after IR.a PC-3 and U2OS cells were treated with IR and harvested at the indicated time points (T) after IR for WB analysis. b PC-3 and U2OS cells were treated with or without IR and harvested at the indicated time points after IR for RT-qPCR analysis. Data were shown as the mean ± SD of three independent experiments (n = 3). Two-tailed unpaired Student’s t-test. P values based on the order of appearance: 0.0008, 3.3E-5, 0.0012, and 9.5E-5. c Heatmaps show LSD1 and MBD3 CUT&Tag profiling sequencing read intensity throughout the genome in PC-3 cells with or without IR treatment. d CUT&Tag signal tracks showing LSD1 and MBD3 occupancy at ZRANB1 gene locus in PC-3 cells with or without IR treatment. e, f ChIP-qPCR analysis of LSD1 (e) and MBD3 (f) occupancy at the ZRANB1 gene locus in both PC-3 and U2OS cells with or without IR treatment. Data were shown as the mean ± SD of three independent experiments (n = 3). Two-tailed unpaired Student’s t-test. P values based on the order of appearance: in e (0.0001, 0.0002, 0.0046, and 7.3E-5) and f (0.0026, 0.0007, 0.0044, and 0.0021). g–j PC-3 and U2OS cells were infected with lentivirus expressing indicated shRNAs and harvested at the indicated time points (T) after IR for WB analysis (g, h) and RT-qPCR analysis (i, j). Data were shown as the mean ± SD of three independent experiments (n = 3). Two-tailed unpaired Student’s t-test. P values based on the order of appearance: in i (0.0002, 2.7E-6, 0.91, 0.4781, 0.0072, 0.001, 0.7017, and 0.7798) and in j (0.0094, 0.0005, 0.7659, 0.8925, 0.047, 0.0109, 0.5247, and 0.9567). k, l PC-3 and U2OS cells were synchronized with nocodazole (100 ng/ml) for 12 h and released into the cell cycle. At the indicated time points (T), cells were harvested for cell cycle analysis by FACS (k) and WB analysis (l). m–p PC-3 cells infected with lentivirus expressing indicated shRNAs were synchronized with nocodazole (100 ng/ml) for 12 h and released into the cell cycle. At the indicated time points, cells were harvested for WB analysis (m, o) and RT-qPCR analysis (n, p). Data were shown as the mean ± SD of three independent experiments (n = 3). n.s. not significant. P values based on the order of appearance: in n (0.0004, 1E-5, 0.0026, 0.8436, 0.9526, and 0.7412) and in p (0.0001, 3.6E-5, 0.0062, 0.4242, 0.4398, and 0.2789). q PC-3 cells were infected with lentivirus expressing indicated shRNAs transfected with His-Ub-K29-single construct and released from nocodazole treatment. Cells were harvested for ubiquitination assay using Ni-NTA pull-down at indicated time points. The sequencing data for CUT&Tag have been deposited to the GEO database with the accession code GSE222267. Source data are provided in this paper or the Mendeley database (https://data.mendeley.com/datasets/n9txt6y5cj/1). Similar results for (a, g, h, l, m, o, q) panels were obtained in two independent experiments.
Fig 4: TRABID overexpression enables synthetic lethality to PARP inhibitor in prostate cancer cells.a, b Dose-response survival curves of EV, TRABID-WT, and TRABID-C443S-expressing cells combine with siControl or si53BP1 exposed to increasing concentrations of olaparib in PC-3 (a) and U2OS (b) cells. Data were shown as the mean ± SD of three independent experiments (n = 3). Statistical analysis was performed using two-way ANOVA. P value were provided in the figure. c–f Colony formation assays were performed in PC-3 (c, d) and U2OS (e, f) cell lines infected with lentivirus expressing EV, TRABID-WT, or TRABID-C443S. The number of colonies was counted. Representative colonies are shown in (c, e), with quantification data shown in (d, f). Data were presented as the mean ± SD of three independent experiments. Two-tailed unpaired Student’s t-test. P value were provided in the figure. g, h. PC-3 cells infected with lentivirus expressing EV, TRABID-WT, or TRABID-C443S were injected s.c. into the right flank of SCID mice and treated with vehicle or olaparib (50 mg/kg). Tumor growth was measured every five days for 30 days. Tumors in each group at day 30 were harvested, photographed and shown in (g). Data in (h) are shown as mean ± SD (n = 5). Two-tailed unpaired Student’s t-test. P value were provided in the figure. Comparing the size of tumors in different groups at day 30. Source data are provided in this paper.
Fig 5: TRABID overexpression inhibits HR activity and promotes chromosomal instability.a–c Representative images of IHC staining (a) of TRABID protein on paired prostate cancer patient specimens (n = 30). Scale bar in 10 X fields: 100 µm; Scale bar in 40 X fields: 20 µm. Heat map showing TRABID IHC score in normal and paired tumor tissues (see calculation details in Methods) (b). Quantification of IHC score (c). Data were shown as the mean ± SD of three independent experiments (n = 3). Two-tailed unpaired Student’s t-test. P = 5.3E-7. d–g U2OS cells infected with lentivirus expressing EV, TRABID-WT, or TRABID-C443S and treated with siControl or si53BP1 were exposed to IR followed by IFC of BRCA1 (d) and RAD51 (f) at 1 h after IR. Scale bar, 10 µm. The average foci number (e, g) in each cell were quantified. Data were presented as means ± SD of more than 300 cells from three biological replicates. Two-tailed unpaired Student’s t-test. n.s. not significant. P values based on the order of appearance: e (2.5E-54, 0.3363, 1.9E-22, 0.0659, and 0.4015), g (1.9E-16, 0.9275, 0.0098, 0.4221, and 0.3285). h, i PC-3 cells were transfected with HR or NHEJ reporter in combination with TRABID WT/C443S or small interfering RNA (siRNA) for CtIP or 53BP1 and HR (h) and NHEJ (i) activities were measured. Data were represented as means ± SD of three biological replicates. Two-tailed unpaired Student’s t-test. n.s. not significant. GFP green fluorescent protein. P values based on the order of appearance: h (0.0002, 0.9571, 0.0711, 0.003, and 0.0537), i (0.0004, 0.9819, 0.0018, 0.0089, and 0.2552). j, k PC-3 cells infected with lentivirus expressing EV, TRABID-WT, or TRABID-C443S were treated with CPT (1 µM) for 24 h. Cells were harvested for karyotyping, and chromosome breaks in more than 80 cells from three biological replicates in each group were counted and quantified. Representative images were shown in (j) and quantitative data are shown in (k). Data were shown as the mean ± SD of three independent experiments (n = 3). Two-tailed unpaired Student’s t-test. P values based on the order of appearance:1.3E-37, 0.9348. n.s. not significant. Source data are provided in this paper.
Supplier Page from Abcam for Anti-ZRANB1 antibody