tdt in situ apoptosis detection kit fluorescein  (R&D Systems)

Journal: Cancer Cell International

Article Title: Novel function of GTPBP2 in promoting hepatocellular carcinoma progression through inhibition of BTRC-mediated KRAS degradation

doi: 10.1186/s12935-025-04029-w

Figure Lengend Snippet: GTPBP2 promotes KRAS and its associated signaling cascade in HCC. (A) Gene Set Enrichment Analysis (GSEA) was performed to investigate the co-expression network and calculate Pearson’s correlation coefficient between GTPBP2 and various hallmarks of cancer in the Roessler HCC cohort ( GSE14520 ). The lower panel presents an enrichment plot depicting genes that are correlated with GTPBP2 within the KRAS signaling pathway. (B) Knockdown of GTPBP2 in HA22T, Mahlavu, and J7 cells was performed to investigate KRAS expression and KRAS-mediated signaling, including phosphorylation of AKT (p-AKT) and MEK (p-MEK), using western blot analysis. Quantitative results are presented in bar plots on the right panel of the immunoblots. (C) The impact of GTPBP2 overexpression on KRAS and its associated signaling cascade was assessed by western blotting and quantitatively analyzed in HA22T, Mahlavu, and J7 cells. β-Actin served as the loading control. Quantitative results are displayed below each signal. Data are presented as means ± SD from three independent experiments. (D) Migration assays were performed to evaluate the effect of KRAS on GTPBP2-overexpressing Mahlavu and J7 cells, using the pcDNA3 empty plasmid as the transfection control. The quantitative results are presented in the right panel (**p < 0.01)

Article Snippet: Coverslips were initially blocked for 45 min at 37 °C with a blocking solution and then incubated with a 1:200 dilution of primary antibodies, including KRAS (sc-30, Santa Cruz Biotechnology)/GTPBP2 (ab222195, Abcam) or BTRC (sc-390629, Santa Cruz Biotechnology)/KRAS (A1190, ABclonal), overnight at 4 °C.

Techniques: Expressing, Knockdown, Phospho-proteomics, Western Blot, Over Expression, Control, Migration, Plasmid Preparation, Transfection

Journal: Cancer Cell International

Article Title: Novel function of GTPBP2 in promoting hepatocellular carcinoma progression through inhibition of BTRC-mediated KRAS degradation

doi: 10.1186/s12935-025-04029-w

Figure Lengend Snippet: Knockdown of GTPBP2 decreases KRAS protein stability in HCC cells. (A ) J7 cells, both with GTPBP2 knockdown and control, were treated with 20 μM cycloheximide (CHX) for time points ranging from 0 to 12 hours. The collected cell lysates were subsequently analyzed by western blot to evaluate KRAS protein stability, with β-actin serving as the loading control. (B) Control or GTPBP2-knockdown J7 cells were treated with or without MG-132 (10 μM) for 4 hours to evaluate the impact of GTPBP2 expression on KRAS protein stability mediated by proteasomal degradation. (C) GTPBP2-knockdown J7 cells transfected with the HA-Ub plasmid were treated with MG-132 for 4 hours prior to KRAS immunoprecipitation and subsequent immunoblotting against Ubiquitin. (D) The RBD pulldown assay was utilized to determine the effect of GTPBP2 overexpression on KRAS activation and to investigate the protein-protein interaction between GTPBP2 and active KRAS in J7 cells. (E) Protein-protein interactions between KRAS and GTPBP2 were detected using the Duolink PLA technique. The close proximity of the two proteins (less than 40 nm apart) is visualized by red fluorescent dots. Nuclei are counterstained with DAPI. Scale bars: 20 μm. (F) Immunohistochemistry (IHC) was performed to detect the expressions of GTPBP2 and KRAS in human HCC tissues. (G) Immunofluorescence (IF) was used to examine cellular location of GTPBP2 and BTRC in J7 cells using antibodies specifically against GTPBP2 and BTRC. Scale bars: 20 μm. (H) Analysis on the amino acid sequence of GTPBP2 for the presence of a BTRC recognition site, DSGKS motif. Two key serine (Ser) residues to binding capacity were mutated to alanine (Ala) to create GTPBP2 mutant (Mt). (I) Co-immunoprecipitation (Co-IP) experiments were conducted using J7 cells transfected with wild-type (Wt) or mutant GTPBP2 containing mutated BTRC to assess potential protein-protein interaction between GTPBP2 and BTRC

Article Snippet: Coverslips were initially blocked for 45 min at 37 °C with a blocking solution and then incubated with a 1:200 dilution of primary antibodies, including KRAS (sc-30, Santa Cruz Biotechnology)/GTPBP2 (ab222195, Abcam) or BTRC (sc-390629, Santa Cruz Biotechnology)/KRAS (A1190, ABclonal), overnight at 4 °C.

Techniques: Knockdown, Control, Western Blot, Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Ubiquitin Proteomics, Over Expression, Activation Assay, Protein-Protein interactions, Immunohistochemistry, Immunofluorescence, Sequencing, Binding Assay, Mutagenesis, Co-Immunoprecipitation Assay

Journal: Cancer Cell International

Article Title: Novel function of GTPBP2 in promoting hepatocellular carcinoma progression through inhibition of BTRC-mediated KRAS degradation

doi: 10.1186/s12935-025-04029-w

Figure Lengend Snippet: GTPBP2 inhibits KRAS ubiquitination by attenuating the interaction between BTRC and KRAS. (A) BTRC immunoprecipitation analysis was performed to evaluate the effect of GTPBP2 overexpression on the interaction between BTRC and KRAS in J7 and HA22T cells. (B) The interaction between KRAS and BTRC was assessed using Duolink PLA in control, GTPBP2-Wt, and GTPBP2-Mt overexpressing J7 cells. KRAS antibody alone was used as a negative control. The interaction between KRAS and BTRC was detected using both KRAS and BTRC antibodies. Close proximity (<40 nm) between the two proteins is represented by small, distinct red fluorescent puncta observed by fluorescence microscopy. Nuclei were counterstained with DAPI. Scale bars: 20 μm. (C) Quantification of PLA signals was determined by counting the number of puncta per cell. Histograms show the mean± SEM from 30 cells across three independent experiments (*p < 0.05; **p< 0.01). ( D ) Ubiquitination of KRAS was analyzed in HA-Ub plasmid transfected control or BTRC-knockdown J7 cells by immunoprecipitation with the KRAS-specific antibody, followed by immunoblotting with an HA-Ub antibody. ( E ) GTPBP2 protein levels were analyzed by western blotting in control and BTRC-knockdown HA22T and J7 cells treated with or without MG-132 for 4 hours. β-actin served as the loading control. (F) Ubiquitination of GTPBP2 was assessed using the same procedure as in (D), with immunoprecipitation performed using a GTPBP2-specific antibody. The asterisk (*) indicates the antibody heavy chain

Article Snippet: Coverslips were initially blocked for 45 min at 37 °C with a blocking solution and then incubated with a 1:200 dilution of primary antibodies, including KRAS (sc-30, Santa Cruz Biotechnology)/GTPBP2 (ab222195, Abcam) or BTRC (sc-390629, Santa Cruz Biotechnology)/KRAS (A1190, ABclonal), overnight at 4 °C.

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Over Expression, Control, Negative Control, Fluorescence, Microscopy, Plasmid Preparation, Transfection, Knockdown, Western Blot

Journal: Cancer Cell International

Article Title: Novel function of GTPBP2 in promoting hepatocellular carcinoma progression through inhibition of BTRC-mediated KRAS degradation

doi: 10.1186/s12935-025-04029-w

Figure Lengend Snippet: GTPBP2 expression is correlated to AFP content and is required for sorafenib resistance. (A, C) Representative images from the colony formation assay in control and GTPBP2-knocked down Mahlavu and J7 cells, with or without sorafenib (5 μM) treatment, stained with crystal violet. The right panels display the quantified results. (B, D) MTS assays were performed to assess the impact of GTPBP2 on cellular sensitivity to sorafenib in control and GTPBP2-knockdown Mahlavu and J7 cells over a 3-day period. ( E, F ) Cell viability assays in Mahlavu (E) and J7 (F) cells showing that overexpression of GTPBP2 increased resistance to sorafenib, and this effect was overcome by KRAS knockdown. The corresponding IC50 values are indicated. Data are presented as mean ± SEM from three independent experiments (**p<0.05, ** p < 0.01). Experiments were performed in triplicates. GTPBP2 expression was analyzed using the Roessier Liver array ( G) and Cohort 2 (H) in relation to AFP contents. Serum concentration higher than 300 ng/ml is presented as high, less than or equal to 300 ng/ml is presented as low. (* p< 0.05; ** p < 0.01; *** p < 0.001). (I) A schematic illustration of the proposed model where GTPBP2 attenuates BTRC-mediated KRAS degradation in liver cancer. Abnormal expression of GTPBP2 in liver cancer tissue competes with KRAS for BTRC binding, leading to enhanced KRAS protein stability and activation of its downstream signaling pathways. This, in turn, promotes increased metastasis, exacerbates malignancy, and contributes to sorafenib resistance

Article Snippet: Coverslips were initially blocked for 45 min at 37 °C with a blocking solution and then incubated with a 1:200 dilution of primary antibodies, including KRAS (sc-30, Santa Cruz Biotechnology)/GTPBP2 (ab222195, Abcam) or BTRC (sc-390629, Santa Cruz Biotechnology)/KRAS (A1190, ABclonal), overnight at 4 °C.

Techniques: Expressing, Colony Assay, Control, Staining, Knockdown, Over Expression, Concentration Assay, Binding Assay, Activation Assay, Protein-Protein interactions