α p21 f 5 mouse mab  (Santa Cruz Biotechnology)

Journal: The Journal of Biological Chemistry

Article Title: Targeting EphA2 under DNA damage causes mitotic bypass via p21 induction

doi: 10.1016/j.jbc.2026.111271

Figure Lengend Snippet: p21 is responsible for EphA2 knockdown–caused mitotic bypass. A and E , HeLa S3 cells were transfected with either siCtrl or siEphA2. After 12 h, the cells were treated with 0.5 μM ADR for 12 h, washed, and then further cultured for 48 h before Western blot analysis ( A ). Band intensities of cyclin B1 ( B ), p53 ( C ), and p21 ( D ) were quantified, and the ratios against the band intensity of α-tubulin are plotted. Three or four independent experimental results are shown as dots together with mean ± SD. p -values were determined using Welch's t test. E , HeLa S3 cells were transfected with either nontargeting or p21-targeting siRNA and further cultured for 48 h before Western blot analysis. At 24 h after transfection, the medium was changed with fresh medium. F and G , HeLa S3/tFucci(CA)5 cells were transfected with siEphA2, along with either siCtrl or sip21. After 12 h, the cells were treated with 0.5 μM ADR for 12 h, washed, and then further cultured for 24 h before time-lapse imaging for 24 h. G2 cells at the beginning of imaging were examined. In F , percentages of cells in G2 phase, cell death during G2 phase, and the cumulative percentage of mitotic bypass are shown as mean ± SD from three independent experiments. No cells entered M phase. G2, orange ; M/post-M, gray ; cell death, black ; mitotic bypass, blue . Percentages of mitotic bypass during imaging are shown in the graph ( G ) as mean ± SD calculated from three independent experimental results. p -values were determined using Student's t test. n = 40 cells per condition. ADR, adriamycin.

Article Snippet: Primary antibodies used for immunoblotting (IB), immunofluorescence (IF) or flow cytometry were as follows: mouse monoclonal anti-Chk1 (IB, 1:1000; G-4, sc-8408, Santa Cruz Biotechnology), anti-γ-tubulin (IF, 1:250; GTU-88, T6557, Merck), anti-p21 (IB, 1:1000, DCS60, 2946, Cell Signaling Technology), anti-p53 (IB, 1:1000; DO-1, sc-126, Santa Cruz Biotechnology), and anti-phospho-histone H3 (IF, 1:400; 6G3, 9706, Cell Signaling Technology) antibodies; rabbit monoclonal anti-EphA2 (IB, 1:1000; 6997S, Cell Signaling Technology), anti-phospho-Chk1 (Ser345, IB, 1:1000; 133D3, #2348, Cell Signaling Technology), and anti-phospho-EphA2 (Ser897, IB, 1:1000; D9A1, 6347, Cell Signaling Technology) antibodies; rabbit polyclonal anti-cyclin B1 (IB, 1:3000; IF and flow cytometry, 1:250; H-433, sc-752, Santa Cruz Biotechnology), anti-phospho-histone H2A.X (γH2AX, IB, 1:500; 2577S, Cell Signaling Technology), and anti-phospho KAP1 (Ser824, IB, 1:1000; A300–767A, Bethyl Laboratories, Montgomery) antibodies; and rat monoclonal anti-α-tubulin (IB, 1:4000; IF, 1:800; MCA78 G, Bio-Rad) antibody.

Techniques: Knockdown, Transfection, Cell Culture, Western Blot, Imaging

Journal: The Journal of Biological Chemistry

Article Title: Targeting EphA2 under DNA damage causes mitotic bypass via p21 induction

doi: 10.1016/j.jbc.2026.111271

Figure Lengend Snippet: p21 upregulation induced by EphA2 knockdown in human cervical cancer cells. Cells were transfected with either siCtrl or siEphA2. After 12 h, cells were treated with ADR for 12 h, washed with PBS (−), and further cultured for 48 h prior to Western blot analysis. Ca Ski cells ( A ), SKG-II cells ( C ), HCT116 cells ( D ), and MCF7 cells ( E ) were subjected to this using the ADR concentrations indicated in the figure. Ca Ski cells were additionally subjected to time-lapse imaging during the last 24 h of the 48 h culture period. The same procedure was applied to Ca Ski cells, and time-lapse imaging was performed during the last 24 h of a 48 h culture ( B ). Representative results were shown from two independent results. Percentages of cells in G2 phase, M/post-M, cell death in G2 phase and the cumulative percentage of mitotic bypass at the time points are shown. n = 40 cells per condition. ADR, adriamycin.

Article Snippet: Primary antibodies used for immunoblotting (IB), immunofluorescence (IF) or flow cytometry were as follows: mouse monoclonal anti-Chk1 (IB, 1:1000; G-4, sc-8408, Santa Cruz Biotechnology), anti-γ-tubulin (IF, 1:250; GTU-88, T6557, Merck), anti-p21 (IB, 1:1000, DCS60, 2946, Cell Signaling Technology), anti-p53 (IB, 1:1000; DO-1, sc-126, Santa Cruz Biotechnology), and anti-phospho-histone H3 (IF, 1:400; 6G3, 9706, Cell Signaling Technology) antibodies; rabbit monoclonal anti-EphA2 (IB, 1:1000; 6997S, Cell Signaling Technology), anti-phospho-Chk1 (Ser345, IB, 1:1000; 133D3, #2348, Cell Signaling Technology), and anti-phospho-EphA2 (Ser897, IB, 1:1000; D9A1, 6347, Cell Signaling Technology) antibodies; rabbit polyclonal anti-cyclin B1 (IB, 1:3000; IF and flow cytometry, 1:250; H-433, sc-752, Santa Cruz Biotechnology), anti-phospho-histone H2A.X (γH2AX, IB, 1:500; 2577S, Cell Signaling Technology), and anti-phospho KAP1 (Ser824, IB, 1:1000; A300–767A, Bethyl Laboratories, Montgomery) antibodies; and rat monoclonal anti-α-tubulin (IB, 1:4000; IF, 1:800; MCA78 G, Bio-Rad) antibody.

Techniques: Knockdown, Transfection, Cell Culture, Western Blot, Imaging

Journal: Nutrients

Article Title: Malva sylvestris Flower Extract Exhibits Antineoplastic Potential Against Human Colon Cancer Cell Lines and Induces CDK2 Transcript Instability via Plant miR160-5p

doi: 10.3390/nu18030495

Figure Lengend Snippet: Molecular mechanisms activated by MFE in the tumour cells. ( A – D ) Results of RT-qPCR analyses carried out on the RNA isolated from HCT-116 (panels ( A , C )) and Caco-2 (panels ( B , D )) cells treated for 48 h with the highest dose of MFE. The data relative to VIM , E-CAD , ITGB3 (panels ( A , B )), P53 , P27 and P21 (panels ( C , D )) transcript levels were expressed as fold change compared to the respective internal CNT taken as unit (i.e., 1), after normalization for β-ACTIN mRNA according to the 2 −∆∆Ct formula. ( E – G ) Representative immunoblots of P53, P27, P21 (panel ( E )), phospho c-MYC (Ser62), phospho CDK2 (Thr160), phospho AKT (Ser 473), AKT and β-ACTIN (panel ( F )) protein levels in HCT-116 and Caco-2 cells, exposed or not for 48 h to the highest dose of MFE. Considering the protein signal of the CNT as a unit (i.e., 1), the quantitation of each band is reported immediately below its representative image in the panel ( E ). Results of the immunoblots presented in panel ( F ) are reported in the form of graphs in panel ( G ) as percentage variation compared to the CNT taken as unit (100%). β-ACTIN was used as a loading control to normalise the results. ( H ) The graph shows the transfection efficiency of the synthetic miR160b-5p after transfection in HCT-116 and Caco-2 cells for 48 h. ( I ) CDK2 mRNA levels, measured by RT-qPCR, in HCT-116 and Caco-2 cells transfected for 48 h with synthetic miR160b-5p. Data were reported relative to CNT cells transfected with a scramble miRNA (SiR) and taken as unit (i.e., 1) after normalization for β-ACTIN mRNA, according to the 2 −∆∆Ct formula. ( J ) Representative immunoblots of phospho c-MYC (Ser62), phospho CDK2 (Thr160) and β-ACTIN protein levels in HCT-116 and Caco-2 cells transfected for 48 h with synthetic miR160b-5p. Considering the protein signal of the CNT as a unit (i.e., 1), the quantitation of each band was reported immediately below its representative image. β-ACTIN was used as a loading control for the normalisation. In all panels, data were indicated as mean ± s.d. of three independent experiments (* p < 0.05 vs. control; ** p < 0.01; *** p < 0.001).

Article Snippet: Rabbit polyclonal phoshpo-AKT (Ser473) (cs-4060), mouse monoclonal cleaved CASPASE-3 (cCASP-3) (cs-94530), rabbit monoclonal phospho-CDK2 (Thr160) (cs-2561), mouse monoclonal phosho-c-MYC (Ser62) (cs-13748) and mouse monoclonal P21 WAF1/Cip1 (cs-2946) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Quantitative RT-PCR, Isolation, Western Blot, Quantitation Assay, Control, Transfection

Journal: Nutrients

Article Title: Malva sylvestris Flower Extract Exhibits Antineoplastic Potential Against Human Colon Cancer Cell Lines and Induces CDK2 Transcript Instability via Plant miR160-5p

doi: 10.3390/nu18030495

Figure Lengend Snippet: Hypothetical model of MFE action in tumour cells. The possible signalling activated in HCT-116 (on the left) and Caco-2 (on the right) cells treated with MFE (indicated with light violet spots) is shown. In the HCT-116 cells, a block of senescence mediated by an upregulation of the CDK2/c-MYC/AKT axis and a hypothetic differentiation event modulated by cytoplasmatic P27 is outlined. On the other hand, in Caco-2 cells, the activation of senescence mediated by an increase in P21, a reduction in AKT and an accumulation of intracellular ROS levels is represented. Legend—green up arrows indicate an increase in the related components inside the cell; red down arrows indicate a decrease in the related components inside the cell; continuous black arrows indicate that the signal is active; dashed black arrows indicate that the signal is not active; black arrows with tips indicate a promoting activity; black arrows without tips indicate inhibitory activity. Abbreviations—Cyt: cytoplasm; CM: cell membrane; ES: extracellular space; Nuc: nucleus; P: phosphorylated protein; ROS: reactive oxygen species.

Article Snippet: Rabbit polyclonal phoshpo-AKT (Ser473) (cs-4060), mouse monoclonal cleaved CASPASE-3 (cCASP-3) (cs-94530), rabbit monoclonal phospho-CDK2 (Thr160) (cs-2561), mouse monoclonal phosho-c-MYC (Ser62) (cs-13748) and mouse monoclonal P21 WAF1/Cip1 (cs-2946) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Blocking Assay, Activation Assay, Activity Assay, Membrane