Journal: Genes

Article Title: WDR13 : A Novel Gene Implicated in Non-Syndromic Intellectual Disability

doi: 10.3390/genes12121911

Figure Lengend Snippet: List of probes used for qPCR.

Article Snippet: , THOC2 , NM_001081550.1 , Hs00396154_m1 , 33–34.

Techniques:

Journal: PLOS Biology

Article Title: CUL5 E3 ubiquitin ligase regulates the evasion of bladder cancer cells to CD8 + T cell-mediated killing by inhibiting autophagy

doi: 10.1371/journal.pbio.3003647

Figure Lengend Snippet: (A) Silver staining showed the proteins pulled down by CUL5 from the lysates of T24 cells. (B) Analysis pipeline was performed to identify potential proteins that interact with CUL5: (i) The proteins with unique peptides ≥5 and fold change ≥20 in T24-MS were selected; (ii) The RNA splicing biological process exhibited the most significant enrichment within the protein-protein interaction (PPI) network predicted by the STRING database ( https://cn.string-db.org/ ). (C) Co-IP assay was performed using an antibody specific for CUL5 to immunoprecipitate proteins from lysates of T24 and UMUC3 cells. The precipitate was subjected to western blotting with the antibodies against CUL5, PRMT5, THOC2, THRAP3, SNRNP200, PTBP1, SF3B1, PRPF8, SF3B2, and HNRNPC. (D) Western blotting with the indicated antibodies in T24 and UMUC3 cells following CUL5 knockout. (E) Co-IP assay using antibody specific for PTBP1 showed the interaction between CUL5 and PTBP1 in T24 and UMUC3 cells. The precipitate was subjected to western blotting with the antibodies against PTBP1 and CUL5. (F) Co-IP assay using antibody specific for CUL5 showed that the interaction between CUL5 and PTBP1 was not affected when bladder cancer cells were co-cultured with CD8 + T cells. The precipitate was subjected to western blotting with the antibodies against CUL5 and PTBP1. (G) Schematic diagram revealed the domains of PTBP1 full-length or truncations. (H) Co-IP assay using antibody specific for Flag showed the interaction between CUL5 and full-length or truncations of Flag-tagged recombinant PTBP1 in T24 cells. The precipitate was subjected to western blotting with the antibodies against Flag, CUL5, and β-actin. Original blots can be found in .

Article Snippet: Antibodies used included primary antibodies against CUL5 (Abclonal, A5369), β-Actin (Proteintech, 66009-1-Ig), PRMT5 (Proteintech, 18436-1-AP), THOC2 (Proteintech, 55178-1-AP), THRAP3 (Proteintech, 19744-1-AP), SNRNP200 (Proteintech, 23875-1-AP), PTBP1 (Proteintech, 12582-1-AP), SF3B1(Proteintech, 27684-1-AP), PRPF8(Proteintech, 11171-1-AP), SF3B2 (Proteintech, 10919-1-AP), HNRNPC (Proteintech, 11760-1-AP), LC3B (Abclonal, A5618), P62 (Proteintech, 18420-1-AP), Rubicon (Proteintech, 21444-1-AP), UVRAG (Proteintech, 29190-1-AP), Beclin1 (Proteintech, 11306-1-AP), HLA-class I (Proteintech, 15240-1-AP), Rabbit control IgG (Abclonal, AC005), Mouse control IgG (Abclonal, AC011), Mouse anti-HA tag (Abclonal, AE008), Rabbit anti-HA tag (Abclonal, AE036), Mouse anti-Flag tag (Abclonal, AE005), and Rabbit anti-Flag tag (Abclonal, AE004); HRP-conjugated secondary goat anti-mouse (Proteintech, SA00001-1), or goat anti-rabbit (Proteintech, SA00001-2) antibodies.

Techniques: Silver Staining, Co-Immunoprecipitation Assay, Western Blot, Knock-Out, Cell Culture, Recombinant

Journal: American Journal of Cancer Research

Article Title: THOC2 expression and its impact on 5-fluorouracil resistance in glioblastoma multiforme

doi:

Figure Lengend Snippet: Primer list for RT-qPCR

Article Snippet: Lentivirus transduction The green fluorescence protein (GFP) expressing THOC2 shRNA lentiviral vector TL301111 (Origene, Rockville, MD, USA) and control scrambled vector (TR30021, Origene) were transduced to T98FR with 8 µg/mL polybrene (Sigma-Aldrich).

Techniques: Sequencing

Journal: American Journal of Cancer Research

Article Title: THOC2 expression and its impact on 5-fluorouracil resistance in glioblastoma multiforme

doi:

Figure Lengend Snippet: Correlation between 5-FU sensitivity, cell doubling time, and mRNA expression levels of 5-FU metabolism genes. A, B. Various GBM cells were obtained, and their 5-FU IC50 values and cell doubling times were determined. C. Linear regression was performed to correlate cell doubling time with 5-FU IC50 values, and R^2 values were calculated using Pearson’s correlation coefficient formula. D-F. Relative mRNA expression levels of 5-FU metabolism genes and THOC2 were measured by quantitative RT-qPCR. Linear regression was performed to correlate mRNA expression levels of 5-FU metabolism genes with 5-FU IC50 values.

Article Snippet: Lentivirus transduction The green fluorescence protein (GFP) expressing THOC2 shRNA lentiviral vector TL301111 (Origene, Rockville, MD, USA) and control scrambled vector (TR30021, Origene) were transduced to T98FR with 8 µg/mL polybrene (Sigma-Aldrich).

Techniques: Expressing, Quantitative RT-PCR

Journal: American Journal of Cancer Research

Article Title: THOC2 expression and its impact on 5-fluorouracil resistance in glioblastoma multiforme

doi:

Figure Lengend Snippet: Characteristics of 5-FU resistant acquired T98FR cells. A, B. GBM cells were exposed to 5-FU for 12 months to develop resistance. Notably, T98FR cells showed a significant increase in 5-FU resistance and upregulated THOC2 expression. C. Linear regression analysis showing the correlation between 5-FU IC50 values and THOC2 mRNA expression levels in GBM cells both before and after 5-FU challenge. Open circles and black regression line represent original GBM cells, while filled circles and red regression line represent 5-FU challenged GBM cells. R^2 values and P-values are also shown on the graph in matching colors. D. T98G, T98FR/sc, and T98FR/shTHOC2 were cultured in the presence of the indicated concentrations of 5-FU for 72 hr. Surviving cells were quantified by MTT assays and presented as relative survival with respect to the value in the absence of 5-FU (student’s t-test; **P < 0.01, ***P < 0.001). E. T98G, T98FG/sc, and T98FR/shTHOC2 growth rate comparison. Error bars indicate the SD. F, G. Relative mRNA expression level and protein level of THOC2 were quantified by RT-qPCR and western blot.

Article Snippet: Lentivirus transduction The green fluorescence protein (GFP) expressing THOC2 shRNA lentiviral vector TL301111 (Origene, Rockville, MD, USA) and control scrambled vector (TR30021, Origene) were transduced to T98FR with 8 µg/mL polybrene (Sigma-Aldrich).

Techniques: Expressing, Cell Culture, Comparison, Quantitative RT-PCR, Western Blot

Journal: American Journal of Cancer Research

Article Title: THOC2 expression and its impact on 5-fluorouracil resistance in glioblastoma multiforme

doi:

Figure Lengend Snippet: Effect of THOC2 knockdown in T98FR subcutaneous xenograft model. A. T98FR/sc and T98FR/shTHOC2 (1.5 × 10^6 cells, 20% matrigel) were inoculated into the right flank of NSG mice. After tumor formation, 80 mg/kg 5-FU was administered peritumorally, and tumor volume was measured once a week, for 3 weeks (student’s t-test; ***P < 0.001). B. Overall survival of at least seven animals/group represented in Kaplan-Meier graph (Log-rank test; *P < 0.05). C. Tumor images of T98FR/sc and T98FR/shTHOC2 at 21 days after initial 5-FU treatment.

Article Snippet: Lentivirus transduction The green fluorescence protein (GFP) expressing THOC2 shRNA lentiviral vector TL301111 (Origene, Rockville, MD, USA) and control scrambled vector (TR30021, Origene) were transduced to T98FR with 8 µg/mL polybrene (Sigma-Aldrich).

Techniques: Knockdown

Journal: American Journal of Cancer Research

Article Title: THOC2 expression and its impact on 5-fluorouracil resistance in glioblastoma multiforme

doi:

Figure Lengend Snippet: Knockdown of THOC2 in T98FR leads to a shift in alternative splicing variants of apoptosis-related gene, Bcl-x. Expression of the Bcl-x splicing variant was examined on day 2 following 5-FU treatment in three cell lines: T98G, T98FR/sc, and T98FR/shTHOC2. (A and B) show the RT-qPCR results of the Bcl-x splicing variant expressed in the three cell types under culture conditions, while (C and D) display the RT-qPCR results of the Bcl-x splicing variant expressed in T98FR/sc, and T98FR/shTHOC2 treated with 5-FU at the indicated concentrations (Student’s t-test; ***P < 0.001).

Article Snippet: Lentivirus transduction The green fluorescence protein (GFP) expressing THOC2 shRNA lentiviral vector TL301111 (Origene, Rockville, MD, USA) and control scrambled vector (TR30021, Origene) were transduced to T98FR with 8 µg/mL polybrene (Sigma-Aldrich).

Techniques: Knockdown, Alternative Splicing, Expressing, Variant Assay, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Targeting THOC2-Mediated mRNA Export Induces PARP Inhibitor Vulnerability in DNA Repair-Competent Hepatocellular Carcinoma

doi: 10.64898/2026.05.17.725613

Figure Lengend Snippet: (A) Gene set enrichment analysis (GSEA) of mRNA biosynthesis, TREX, and nuclear pore complex (NPC) pathways in high versus low HR score groups. ( B) The heatmap depicting the correlation between TREX proteins and DNA damage repair (DDR) proteins. ( C) The density plot illustrates the distribution of correlation coefficients between the DNA repair proteins and the two components of TREX, and values represent the median correlation coefficient. Rose red represents THO complex, and yellow-brown represents other parts of TREX. ( D) The violin plot displaying the expression of the THO complex in the two datasets. ( E) The forest plot of survival analysis (OS) of THO complex in two proteomics data sets and TCGA. HR > 1 indicates worse prognosis with higher expression. ( F) Western blot (WB) analysis of the knockdown effect of siRNA on THO complex proteins in Huh7 cells. (G) Densitometric quantification of THO complex protein expression levels shown in (F) . Protein expression levels were normalized to β-tubulin and presented relative to the control group (NC) (mean ± SEM; n = 3). ( H, I) Immunofluorescence staining (IF) assay shows γH2AX foci in the control and THO complex knockdown cells after ionizing radiation (IR; 0.6 Gy, H ). And histograms presented the average number of foci per cell in each group (mean ± SEM; n = 3, I ). Scale bar, 20 μm. ( J) CCK-8 assays were applied to evaluate proliferation abilities of Huh7 cells after transiently knockdown of the components of THO complex with their relative siRNA, respectively (mean ± SEM; n = 3). ( K) WB analysis of THO complex expression in control and THOC2 stable knockdown Huh7 cells. Cellular β-tubulin expression served as the loading control. shCtrl: non-targeting shRNA; sh#2: sh-THOC2-2; sh#3: sh-THOC2-3; NC: negative control. Fisher’s exact test for ( A) ; Spearman correlation coefficient for ( B) ; two-tailed unpaired Welch test for ( D, G and I) ; Log-rank test for ( E) ; two-way ANOVA for (J) ; ns no significance; * P < 0.05; ** P < 0.01; *** P < 0.001 and **** P < 0.0001.

Article Snippet: The RIP sequencing data of THOC2 in Huh7 cells are available at the National Center for Biotechnology Information (PRJNA1119719).

Techniques: Expressing, Western Blot, Knockdown, Control, Immunofluorescence, Staining, CCK-8 Assay, shRNA, Negative Control, Two Tailed Test

Journal: bioRxiv

Article Title: Targeting THOC2-Mediated mRNA Export Induces PARP Inhibitor Vulnerability in DNA Repair-Competent Hepatocellular Carcinoma

doi: 10.64898/2026.05.17.725613

Figure Lengend Snippet: B) Kaplan-Meier overall survival (OS) curves stratified by THOC2 expression (optimal cut-off) in Jiang ( A) and Gao et al.’s cohort ( B . C) Representative immunohistochemistry (IHC) images of THOC2 in tumor and adjacent non-tumorous tissues (NAT). On the left: Samples with longer OS (57 months). On the right: Samples with shorter OS (6 months). Scale bars: 400 μm (top); 100μm (bottom). ( D, E) THOC2 IHC scores were significantly higher in tumor than NAT ( n = 79). ( F) Kaplan-Meier analysis of the OS based on THOC2 IHC scores. Patients were stratified into low (score = 0) and high (score > 0). ( G) Univariate and multivariate Cox regression analyses of THOC2 and clinical characteristics in the Jiang et al.’s cohort. ( H) The correlation between THOC2 expression and clinical indicators in Jiang et al.’s cohort. Patients were stratified into high- and low-THOC2 groups based on the cut-off value of THOC2 expression in ( A) . DT: diameter of tumor; TN: tumor number; MVI: microvascular invasion. ( I) Nomogram constructed by combining THOC2 and clinical risk factors. ( J) Calibration plots of the nomogram for predicting the probability of 5-year survival. Log-rank test for ( A, B and F) ; Two-tailed unpaired Wilcoxon t test for ( D) ; chi-square test for ( E, H) .

Article Snippet: The RIP sequencing data of THOC2 in Huh7 cells are available at the National Center for Biotechnology Information (PRJNA1119719).

Techniques: Expressing, Immunohistochemistry, Construct, Two Tailed Test

Journal: bioRxiv

Article Title: Targeting THOC2-Mediated mRNA Export Induces PARP Inhibitor Vulnerability in DNA Repair-Competent Hepatocellular Carcinoma

doi: 10.64898/2026.05.17.725613

Figure Lengend Snippet: CCK8 A, colony formation (B, C) and EdU assays (D, E) were applied to evaluate the proliferation abilities of control or THOC2-knockdown Huh7 and MHCC97H cells (mean ± SEM; n = 3). Scale bar, 50 μm. ( F-J) Tumor xenograft model was constructed with stable control and THOC2-knockdown Huh7 cells (mean ± SEM; n = 6). Tumors were collected from sacrificed mice and tumor weights were measured ( G) . Tumor sizes were recorded consecutively to establish tumor growth curves ( H) . IHC staining of Ki67 in mouse subcutaneous tumors ( I-J) . Scale bar, 50 μm. ( K, L) Apoptosis experiment in the control and THOC2-knockdown cells after irradiation (IR) treatment (dose = 6 Gy). And histograms presented the percentage of apoptosis cells in each group (mean ± SEM; n = 3). ( M, N) IF assay shows γH2AX foci in the control and THOC2-knockdown cells after IR treatment (dose = 6 Gy). And histograms presented the average number of foci per cell in each group (mean ± SEM; n = 3). Scale bar, 20 μm. ( O, P) The alkaline comet assays show that the DSB repair capability is reduced in THOC2-knockdown Huh7 and MHCC97H cells. HCC cells are harvested at the indicated time (NT, and 0.5 h and 8 h post-treatment) upon IR treatment (6 Gy). The tail moment was analyzed using the CometScore software (mean ± SEM; n = 3). Scale bar, 200 μm. shCtrl: nontargeting shRNA; sh#2: sh-THOC2-2; sh#3: sh-THOC2-3; two-tailed unpaired Wilcoxon t test for ( A, C, E, L, N and P) ; two-tailed paired Wilcoxon t test for ( G, H and J) ; * P < 0.05; ** P < 0.01; *** P < 0.001 and **** P < 0.0001.

Article Snippet: The RIP sequencing data of THOC2 in Huh7 cells are available at the National Center for Biotechnology Information (PRJNA1119719).

Techniques: Control, Knockdown, Construct, Immunohistochemistry, Irradiation, Software, shRNA, Two Tailed Test

Journal: bioRxiv

Article Title: Targeting THOC2-Mediated mRNA Export Induces PARP Inhibitor Vulnerability in DNA Repair-Competent Hepatocellular Carcinoma

doi: 10.64898/2026.05.17.725613

Figure Lengend Snippet: (A) Venn diagram for screening THOC2-selective export of mRNAs, wherein Jiang et al.’s cohort and Gao et al.’s cohort represent co-expressed proteins of THOC2 ( P < 0.01; r > 0), RIP-Seq represents mRNA bound by THOC2 in the Huh7 cells ( P < 0.05; IP/Input > 2), and LC-MS/MS of mouse subcutaneous tumors represents proteins down-regulated in the THOC2 knockdown group compared to the control group (in at least four samples of paired shTHOC2 and shCtrl samples, shTHOC2/shCtrl is less than 1). ( B) Pathways enriched by intersection proteins in ( A) . Color represents the number of intersecting proteins in pathways. ( C, D) Heatmaps of proteins in cell cycle (C) and DNA repair (D) contained in intersection proteins. LC-MS/MS represents mass spectrometry data of mouse subcutaneous tumors, Cor represents correlation analysis with THOC2 and HR represents the relationship between protein and prognosis in the proteomic datasets. ( E) RIP-Seq results of THOC2 binding to the selected mRNA in Huh7 cells. ( F) Expression intensity of TOP2A, MDC1, MSH6, and PRKDC in proteomic data of mouse subcutaneous tumors (mean ± SEM; n = 5). ( G) Correlation between TOP2A, MDC1, MSH6, PRKDC and THOC2 expression in Gao et al.’s cohort and Jiang et al.’s cohort, respectively. shCtrl: nontargeting shRNA; sh#2: shTHOC2-2; two-tailed paired Wilcoxon t test for ( F) ; Pearson correlation coefficient for ( G) .

Article Snippet: The RIP sequencing data of THOC2 in Huh7 cells are available at the National Center for Biotechnology Information (PRJNA1119719).

Techniques: Liquid Chromatography with Mass Spectroscopy, Knockdown, Control, Mass Spectrometry, Binding Assay, Expressing, shRNA, Two Tailed Test

Journal: bioRxiv

Article Title: Targeting THOC2-Mediated mRNA Export Induces PARP Inhibitor Vulnerability in DNA Repair-Competent Hepatocellular Carcinoma

doi: 10.64898/2026.05.17.725613

Figure Lengend Snippet: B) Protein level of TOP2A, MDC1, MSH6 and PRKDC was measured in THOC2-knockdown Huh7 and MHCC97H cells or THOC2-overexpressing Huh7 cells. ( C, D) The total mRNA expression (mean ± SEM; n = 3) of TOP2A, PRKDC, MDC1, and MSH6 was detected by qRT-PCR in Huh7 (C) and MHCC97H cells (D) . ( E) FISH/IF assays conducted on control and THOC2 knockdown cells of Huh7 and MHCC97H. Scale bar, 10 μm. (F) Quantification of nuclear/cytoplasmic RNA fluorescence intensity ratio in control and THOC2-knockdown Huh7 and MHCC97H cells (mean ± SEM, n = 3) based on FISH/IF assays shown in (E). (G) Nucleocytoplasmic fractionation followed by qRT–PCR analysis of THOC2 target mRNAs. Data are presented as nuclear-to-cytoplasmic (N/C) ratios (mean ± SEM, n = 3). (H) Cell proliferation assays in THOC2-knockdown MHCC97H cells following TOP2A overexpression (mean ± SEM, n = 3). (I) Representative immunofluorescence images of γH2AX foci in THOC2-knockdown MHCC97H cells with or without MDC1 or MSH6 overexpression. Scale bar, 20 μm. (J) Quantification of γH2AX foci per cell in (I) (mean ± SEM, n = 3). shCtrl: nontargeting shRNA; sh#2: shTHOC2-2; sh#3: shTHOC2-3; Vector: empty vector; OE: THOC2 overexpression; two-tailed unpaired Wilcoxon t test for ( C, D, F, G and J) ; two-way ANOVA for (H) ; ns, no significance; * P < 0.05; ** P < 0.01; *** P < 0.001 and **** P < 0.0001.

Article Snippet: The RIP sequencing data of THOC2 in Huh7 cells are available at the National Center for Biotechnology Information (PRJNA1119719).

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Control, Fluorescence, Fractionation, Over Expression, Immunofluorescence, shRNA, Plasmid Preparation, Two Tailed Test

Journal: bioRxiv

Article Title: Targeting THOC2-Mediated mRNA Export Induces PARP Inhibitor Vulnerability in DNA Repair-Competent Hepatocellular Carcinoma

doi: 10.64898/2026.05.17.725613

Figure Lengend Snippet: The dose-response curves of PARP inhibitor Olaparib on Huh7 (A) , MHCC97H (B) and Hepa1-6 (C) cells, both control and THOC2 knockdown, were generated using CCK8 assays (mean ± SEM; n = 3). The half maximal inhibitory concentrations (IC50) were shown. ( D) Apoptosis experiment after THOC2 knockdown combined with PARPi (0.8 µM) in Huh7 and MHCC97H. PARPi: cells treated with Olaparib which is dissolved in DMSO; DMSO: cells treated with an equal volume of DMSO to Olaparib. (E, F) Immunofluorescence staining (IF) assay shows γH2AX foci in control and THOC2-knockdown MHCC97 cells treated with or without PARP inhibitor after ionizing radiation (IR; 0.6 Gy). And histograms presented the average number of foci per cell in each group (mean ± SEM; n = 3). Scale bar, 20 μm. ( G-I) Subcutaneous Hepa1-6 cells tumors, tumor growth curves and tumor weight in the indicated treatment groups (mean ± SEM; n = 6). The dose of Olaparib used is 50 mg/kg. ( J) Representative images of IHC Ki67 and γH2AX staining and the relative IHC scores (mean ± SEM; n = 6) in Hepa1-6 tumor tissues in ( G) . Scale bar, 50 μm. ( K) Representative images of TUNEL staining and the positive cells (%) (mean ± SEM; n = 6) in Hepa1-6 tumor tissues in e . Scale bar, 50 μm. The colors above the image represent the groups. shCtrl: nontargeting shRNA; sh#2: shTHOC2-2; sh#3: shTHOC2-3; sh#1: sh Thoc2 -1; PARPi: mouses treated with Olaparib (50 mg/kg); Control: mouses treated with an equal volume of DMSO to Olaparib; two-tailed unpaired Wilcoxon t test for ( D, F, I, J and K) ; two-way ANOVA for (H) ; ns, no significance; * P < 0.05; ** P < 0.01; *** P < 0.001 and **** P < 0.0001.

Article Snippet: The RIP sequencing data of THOC2 in Huh7 cells are available at the National Center for Biotechnology Information (PRJNA1119719).

Techniques: Control, Knockdown, Generated, Immunofluorescence, Staining, TUNEL Assay, shRNA, Two Tailed Test