Journal: Molecular Medicine Reports

Article Title: GINS2 regulates the proliferation and apoptosis of colon cancer cells through PTP4A1

doi: 10.3892/mmr.2022.12633

Figure Lengend Snippet: GINS2 knockdown activates the p53 pathway through PTP4A1. (A) RT-qPCR was performed to detect the PTP4A1 mRNA expression level in colon cancer cells. ***P<0.001 vs. HIEC-6. (B) The binding of GINS2 and PTP4A1 was detected using a co-immunoprecipitation assay. (C) mRNA and protein expression levels of PTP4A1 and p53 were detected using RT-qPCR and western blotting, respectively. ***P<0.001 vs. shRNA-NC. (D) PTP4A1 mRNA and protein expression levels were detected using RT-qPCR and western blotting, respectively. ***P<0.001 vs. Ov-NC. (E) p53 protein expression levels were detected using western blotting analysis. ***P<0.001 vs. Control; ### P<0.001 vs. shRNA-GINS2 + Ov-NC. GINS2, GINS complex subunit 2; PTP4A1, protein tyrosine phosphatase 4A1; RT-qPCR, reverse transcription-quantitative PCR; shRNA, short hairpin RNA; NC, negative control; Ov, overexpression.

Article Snippet: Briefly, the harvested cells were fully lysed using 1 ml Co-RIPA buffer (Applygen Technologies, Inc.) and then lysates were pre-cleared with 30 μl Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, Inc.) at 4°C for 1 h. After centrifuging the lysate, taking the supernatant and removing the beads in the supernatant, immunoprecipitation with antibodies against GINS2 (1:50; cat. no. sc-376595; Santa Cruz Biotechnology, Inc.), PTP4A1 (1:30; cat. no. sc-365659; Santa Cruz Biotechnology, Inc.) and IgG (1:50; cat. no. sc-69786; Santa Cruz Biotechnology, Inc.) was performed with shaking at 4°C overnight.

Techniques: Knockdown, Quantitative RT-PCR, Expressing, Binding Assay, Co-Immunoprecipitation Assay, Western Blot, shRNA, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control, Over Expression

Journal: Molecular Medicine Reports

Article Title: GINS2 regulates the proliferation and apoptosis of colon cancer cells through PTP4A1

doi: 10.3892/mmr.2022.12633

Figure Lengend Snippet: GINS2 knockdown regulates the proliferation, cycle arrest and apoptosis through the PTP4A1/p53 pathway. (A) Cell viability was detected using a Cell Counting Kit-8 assay. (B) Cell proliferation was detected using EdU staining. (C) Flow cytometry was performed to examine cell cycle distribution. (D) Cell apoptosis was detected using a TUNEL assay. (E) Western blotting analyses were performed and (F) quantified to detect the protein expression levels of cyclin D1, p21, Bcl2, Bax, cleaved PARP and PARP. (G) Schematic depiction of regulatory mechanism underlying colon cancer proliferation, cycle arrest and apoptosis via GINS2/PTP4A1/p53 pathway. ***P<0.001 vs. Control; ## P<0.01, ### P<0.001 vs. shRNA-GINS2 + Ov-NC. GINS2, GINS complex subunit 2; PTP4A1, protein tyrosine phosphatase 4A1; shRNA, short hairpin RNA; NC, negative control; Ov, overexpression; PARP, poly (ADP-ribose) polymerase.

Article Snippet: Briefly, the harvested cells were fully lysed using 1 ml Co-RIPA buffer (Applygen Technologies, Inc.) and then lysates were pre-cleared with 30 μl Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, Inc.) at 4°C for 1 h. After centrifuging the lysate, taking the supernatant and removing the beads in the supernatant, immunoprecipitation with antibodies against GINS2 (1:50; cat. no. sc-376595; Santa Cruz Biotechnology, Inc.), PTP4A1 (1:30; cat. no. sc-365659; Santa Cruz Biotechnology, Inc.) and IgG (1:50; cat. no. sc-69786; Santa Cruz Biotechnology, Inc.) was performed with shaking at 4°C overnight.

Techniques: Knockdown, Cell Counting, Staining, Flow Cytometry, TUNEL Assay, Western Blot, Expressing, Control, shRNA, Negative Control, Over Expression