Journal: International Journal of Molecular Medicine

Article Title: Tribbles pseudokinase 3 drives cancer stemness in oral squamous cell carcinoma cells by supporting the expression levels of SOX2 and EGFR

doi: 10.3892/ijmm.2025.5485

Figure Lengend Snippet: TRIB3 depletion decreases cell proliferation and affects cell cycle distribution. (A) SAS and HSC3 oral squamous cell carcinoma cells were transduced with lentiviruses carrying shRNA sequences for LacZ (control), sh-TRIB3#1 or sh-TRIB3#2. Western blotting results confirmed TRIB3 protein knockdown. Cell proliferation was assessed by clonogenic assay, with colonies stained using crystal violet and visualized on day 14. (B) SAS and (C) HSC3 cells were transfected with siRNA NC or siTRIB3 for 48 h. Cells were then harvested for western blotting analysis to verify TRIB3 protein depletion and for cell cycle analysis using BrdU. Anti-BrdU-FITC and PI fluorescence signals were detected using flow cytometry, analyzed using FlowJo software (version 10; FlowJo LLC; BD Biosciences) and quantified. Data represented the percentage of cells at each cycle phase and were presented as mean ± SD. (D) Western blotting analysis was used to determine the protein expression levels of cell cycle regulators Cyclin A2, Cyclin B1 and Cyclin D1 following TRIB3 shRNA transfection for 48 h in SAS and HSC3 cells. For quantification, CDK1 and CDK6 levels were normalized to Tubulin using the upper TRIB3 blot, while Cyclin A2, Cyclin B1 and Cyclin D1 levels were normalized to GAPDH using the lower TRIB3 blot. Blot images were representative of one of three independent experiments. Statistical analyses were conducted using one-way ANOVA with post-hoc Tukey's Honestly Significant Difference test. * P<0.05; ** P<0.01; *** P<0.001. TRIB3, tribbles pseudokinase 3; shRNA, short hairpin RNA; siRNA, small interfering RNA; BrdU, 5-bromo-2-deoxyuridine; NC, negative control; si, siRNA.

Article Snippet: In the present study, the decreased expression of the CDK6 protein in SAS and HSC3 cells was observed following knockdown of TRIB3.

Techniques: Transduction, shRNA, Control, Western Blot, Knockdown, Clonogenic Assay, Staining, Transfection, Cell Cycle Assay, Fluorescence, Flow Cytometry, Software, Expressing, Small Interfering RNA, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: Tribbles pseudokinase 3 drives cancer stemness in oral squamous cell carcinoma cells by supporting the expression levels of SOX2 and EGFR

doi: 10.3892/ijmm.2025.5485

Figure Lengend Snippet: Knockdown of TRIB3 downregulates cell cycle and stemness markers in xenograft tumors. Tumors derived from SAS cells transduced with shLacZ or shTRIB3 were analyzed by immunohistochemistry. Sections were stained for (A) TRIB3, (B) E2F1, (C) SOX2, (D) CDK1, (E) CDK6 and (F) EGFR. Representative immunohistochemical images are presented for each protein. Quantification of positive cells for each marker are presented. Staining intensities were quantitatively assessed from two random objective fields across three independent tumor sections per group. Data are presented as mean ± SD. Statistical analysis was performed using the unpaired Student's t-test. Scale bar, 50 μ m. TRIB3, tribbles pseudokinase 3; E2F1, E2F transcription factor 1; sh, short hairpin RNA.

Article Snippet: In the present study, the decreased expression of the CDK6 protein in SAS and HSC3 cells was observed following knockdown of TRIB3.

Techniques: Knockdown, Derivative Assay, Transduction, Immunohistochemistry, Staining, Immunohistochemical staining, Marker, shRNA

Journal: iScience

Article Title: RPRM negatively regulates ATM levels through its nuclear translocation on irradiation mediated by CDK4/6 and IPO11

doi: 10.1016/j.isci.2022.105115

Figure Lengend Snippet: CDK4/6 phosphorylate RPRM at serine 98, but the unphosphorylation status of RPRM is critical for its stabilization and nuclear translocation (A and B) Mass spectrometry analysis of the phosphorylation site of RPRM corresponding to CDK4 (A) and CDK6 (B). (C) Time-course analysis of the changes in the RPRM levels of H460-RPRM cells after treated with different inhibitors. (D) Diagrams of full-length RPRM protein and different mutants. (E) Representative immunofluorescence images of the RPRM −/− MEFs exogenously expressing full-length RPRM, RPRM-S98A and ΔRPRM (79–109) at 30 min after 20 Gy X-irradiation. Scale bars, 10 μm. (F) Co-IP assay confirmed an interaction between ATM and both RPRM-S98A and ΔRPRM (79–109) in H460 cells. (G and H) Change in the ATM levels of H460-NC/RPRM/S98A cells at different times after 2 Gy X-irradiation. (I) Change in the ATM expression of H460-NC/RPRM/S998A cells after cisplatin (CDDP, 15 μM) treatment for 24 h. (J) Relative plating efficiency of H460-NC/RPRM/S98A cells irradiated with 4 Gy X-rays. (K) Exogenous expression of both RPRM and RPRM-S98A increased micronucleus formation in H460 cells upon 2 Gy X-rays. (L) Relative plating efficiency of H460-NC/RPRM/S98A cells treated with 15 μM CDDP. (M) Micronucleus formation of RPRM −/− MEFs expressing full-length RPRM, RPRM-S98A and ΔRPRM (79–109) after exposed to 5 Gy X-irradiation. Data shown represent the means (±SEM) of three biological replicates, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; Significance was determined by unpaired two-sample t test.

Article Snippet: CDK6 , Beyotime , Cat#AC256.

Techniques: Translocation Assay, Mass Spectrometry, Phospho-proteomics, Immunofluorescence, Expressing, Irradiation, Co-Immunoprecipitation Assay

Journal: iScience

Article Title: RPRM negatively regulates ATM levels through its nuclear translocation on irradiation mediated by CDK4/6 and IPO11

doi: 10.1016/j.isci.2022.105115

Figure Lengend Snippet:

Article Snippet: CDK6 , Beyotime , Cat#AC256.

Techniques: Control, Virus, shRNA, Recombinant, Purification, Sequencing, Dominant Negative Mutation, Mutagenesis, Software

Journal: iScience

Article Title: RPRM negatively regulates ATM levels through its nuclear translocation on irradiation mediated by CDK4/6 and IPO11

doi: 10.1016/j.isci.2022.105115

Figure Lengend Snippet: CDK4/6 phosphorylate RPRM at serine 98, but the unphosphorylation status of RPRM is critical for its stabilization and nuclear translocation (A and B) Mass spectrometry analysis of the phosphorylation site of RPRM corresponding to CDK4 (A) and CDK6 (B). (C) Time-course analysis of the changes in the RPRM levels of H460-RPRM cells after treated with different inhibitors. (D) Diagrams of full-length RPRM protein and different mutants. (E) Representative immunofluorescence images of the RPRM −/− MEFs exogenously expressing full-length RPRM, RPRM-S98A and ΔRPRM (79–109) at 30 min after 20 Gy X-irradiation. Scale bars, 10 μm. (F) Co-IP assay confirmed an interaction between ATM and both RPRM-S98A and ΔRPRM (79–109) in H460 cells. (G and H) Change in the ATM levels of H460-NC/RPRM/S98A cells at different times after 2 Gy X-irradiation. (I) Change in the ATM expression of H460-NC/RPRM/S998A cells after cisplatin (CDDP, 15 μM) treatment for 24 h. (J) Relative plating efficiency of H460-NC/RPRM/S98A cells irradiated with 4 Gy X-rays. (K) Exogenous expression of both RPRM and RPRM-S98A increased micronucleus formation in H460 cells upon 2 Gy X-rays. (L) Relative plating efficiency of H460-NC/RPRM/S98A cells treated with 15 μM CDDP. (M) Micronucleus formation of RPRM −/− MEFs expressing full-length RPRM, RPRM-S98A and ΔRPRM (79–109) after exposed to 5 Gy X-irradiation. Data shown represent the means (±SEM) of three biological replicates, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; Significance was determined by unpaired two-sample t test.

Article Snippet: Purified recombinant CDK6 , Origene , TP327978.

Techniques: Translocation Assay, Mass Spectrometry, Phospho-proteomics, Immunofluorescence, Expressing, Irradiation, Co-Immunoprecipitation Assay

Journal: iScience

Article Title: RPRM negatively regulates ATM levels through its nuclear translocation on irradiation mediated by CDK4/6 and IPO11

doi: 10.1016/j.isci.2022.105115

Figure Lengend Snippet:

Article Snippet: Purified recombinant CDK6 , Origene , TP327978.

Techniques: Control, Virus, shRNA, Recombinant, Purification, Sequencing, Dominant Negative Mutation, Mutagenesis, Software