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p-plk1 t210 antibody (Cell Signaling Technology Inc)

Name: p-plk1 t210 antibody
Brand: Cell Signaling Technology Inc
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Cell Signaling Technology Inc p-plk1 t210 antibody
P Plk1 T210 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/p-plk1 t210 antibody/product/Cell Signaling Technology Inc
Average 90 stars, based on 1 article reviews

p-plk1 t210 antibody - by Bioz Stars, 2026-03

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A Representative western blot (left) and quantification (right) of U2OS cells treated with MG132 (20 μM) in combination with PLK1i (1 μM BI2536), Chk2i (10 μM BML-277), CDK1i (5 μM RO-3306), CDK1i Chk2i combination, or a DMSO vehicle control. Each point represents one experimental replicate * p < 0.05, paired 1-way ANOVA with Bonferroni multiple comparisons correction. ns not significant ( p > 0.05). Error bars SD. B Representative western blot (left) and quantification (right) of U2OS cells treated with PLK1i (1 μM BI2536), Chk2i (10 μM BML-277), AurAi (50 nM MLN-8237), AurAi Chk2i combination, or a DMSO vehicle control. * p < 0.05, one-way ANOVA with Bonferroni multiple comparisons correction. ns, not significant ( p > 0.05). Error bars SD. C Left, immunoblot of mitotic HeLa cells treated with a vehicle control or Chk2 inhibitor for 1 h. Right, immunoblot of the same samples on a PhosTag gel. The putative phosphorylated specifies of PLK1 is indicated. *, uncharacterized bands recognized by PLK1 antibody. Right, quantification of phosphorylated PLK1 (slower-migrating PLK1 band on PhosTag gel) normalized to the total PLK1 protein abundance (68 kDa band on SDS-Page gel). Each point represents one biological replicate. * p < 0.05, two-tailed t -test. Error bars SD. D Experimental setup and representative western blot of PLK1 immunoprecipitated from mitotic HeLa cells treated with DMSO or Chk2i. E Quantification of phospho-antibody signal normalized to total PLK1 abundance from ( D ). Each point represents the value from one experimental replicate. * p < 0.05, two-tailed t -test. Error bars SD. F In vitro kinase assay with recombinant HIS-PLK1, MBP-Chk2 incubated with DMSO, PLK1i, or Chk2i as marked. Recombinant proteins were incubated in kinase assay buffer in a 1:1 molar ratio with 500 μM ATP for 30 minutes at 30 °C with gentle agitation. The reaction was quenched with 5 mM EDTA before being combined with denaturing sample buffer and analyzed via western blotting. p-PLK1 T210 antibody shown is Cell Signaling Technology D5H7. *, nonspecific recognition of MBP-Chk2. This experiment has been replicated 5 times with similar results. All panels, data are presented as mean values ± SD. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Chk2 sustains PLK1 activity in mitosis to ensure proper chromosome segregation

doi: 10.1038/s41467-024-54922-7

Figure Lengend Snippet: A Representative western blot (left) and quantification (right) of U2OS cells treated with MG132 (20 μM) in combination with PLK1i (1 μM BI2536), Chk2i (10 μM BML-277), CDK1i (5 μM RO-3306), CDK1i Chk2i combination, or a DMSO vehicle control. Each point represents one experimental replicate * p < 0.05, paired 1-way ANOVA with Bonferroni multiple comparisons correction. ns not significant ( p > 0.05). Error bars SD. B Representative western blot (left) and quantification (right) of U2OS cells treated with PLK1i (1 μM BI2536), Chk2i (10 μM BML-277), AurAi (50 nM MLN-8237), AurAi Chk2i combination, or a DMSO vehicle control. * p < 0.05, one-way ANOVA with Bonferroni multiple comparisons correction. ns, not significant ( p > 0.05). Error bars SD. C Left, immunoblot of mitotic HeLa cells treated with a vehicle control or Chk2 inhibitor for 1 h. Right, immunoblot of the same samples on a PhosTag gel. The putative phosphorylated specifies of PLK1 is indicated. *, uncharacterized bands recognized by PLK1 antibody. Right, quantification of phosphorylated PLK1 (slower-migrating PLK1 band on PhosTag gel) normalized to the total PLK1 protein abundance (68 kDa band on SDS-Page gel). Each point represents one biological replicate. * p < 0.05, two-tailed t -test. Error bars SD. D Experimental setup and representative western blot of PLK1 immunoprecipitated from mitotic HeLa cells treated with DMSO or Chk2i. E Quantification of phospho-antibody signal normalized to total PLK1 abundance from ( D ). Each point represents the value from one experimental replicate. * p < 0.05, two-tailed t -test. Error bars SD. F In vitro kinase assay with recombinant HIS-PLK1, MBP-Chk2 incubated with DMSO, PLK1i, or Chk2i as marked. Recombinant proteins were incubated in kinase assay buffer in a 1:1 molar ratio with 500 μM ATP for 30 minutes at 30 °C with gentle agitation. The reaction was quenched with 5 mM EDTA before being combined with denaturing sample buffer and analyzed via western blotting. p-PLK1 T210 antibody shown is Cell Signaling Technology D5H7. *, nonspecific recognition of MBP-Chk2. This experiment has been replicated 5 times with similar results. All panels, data are presented as mean values ± SD. Source data are provided as a Source Data file.

Article Snippet: The reaction was quenched with 5 mM EDTA before being combined with denaturing sample buffer and analyzed via western blotting. p-PLK1 T210 antibody shown is Cell Signaling Technology D5H7.

Techniques: Western Blot, Control, Quantitative Proteomics, SDS Page, Two Tailed Test, Immunoprecipitation, In Vitro, Kinase Assay, Recombinant, Incubation, Gentle

Fig. 1. The expression of MEK1 and PLK1 are highly upregulated in colorectal cancer. (a) The overall survival rates of colon cancer patients depending on the status of KRAS mutation plotted using KM Plotter based on the GSE39582, GSE143985, and TCGA datasets. (b) The overall survival rates of colon cancer patients depending on the status of TP53 mutation plotted using KM Plotter based on the GSE39582, GSE143985, and TCGA datasets. (c) The overall survival rates of colon cancer patients based on KRAS and TP53 status were plotted using KM Plotter based on the GSE39582, GSE143985, and TCGA datasets. (d) The expression levels of PLK1 were analyzed in normal adjunctive tissues and COAD tumor tissues using the TCGA dataset. (e) Relapse-free survival (RFS) rates depending on the expression of PLK1 in colorectal cancer patients. (f) Relapse-free survival (RFS) rates depending on the expression of wild-type and KRAS mutant (n = 1125). (g) The relative expression levels of PLK1, TP53, KRAS, MAP2K1, and MYC were illustrated with a heatmap using the GSE227550, GSE156355, and GSE206800 datasets.

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Synergistic two-step inhibition approach using a combination of trametinib and onvansertib in KRAS and TP53-mutated colorectal adenocarcinoma.

doi: 10.1016/j.biopha.2024.117796

Figure Lengend Snippet: Fig. 1. The expression of MEK1 and PLK1 are highly upregulated in colorectal cancer. (a) The overall survival rates of colon cancer patients depending on the status of KRAS mutation plotted using KM Plotter based on the GSE39582, GSE143985, and TCGA datasets. (b) The overall survival rates of colon cancer patients depending on the status of TP53 mutation plotted using KM Plotter based on the GSE39582, GSE143985, and TCGA datasets. (c) The overall survival rates of colon cancer patients based on KRAS and TP53 status were plotted using KM Plotter based on the GSE39582, GSE143985, and TCGA datasets. (d) The expression levels of PLK1 were analyzed in normal adjunctive tissues and COAD tumor tissues using the TCGA dataset. (e) Relapse-free survival (RFS) rates depending on the expression of PLK1 in colorectal cancer patients. (f) Relapse-free survival (RFS) rates depending on the expression of wild-type and KRAS mutant (n = 1125). (g) The relative expression levels of PLK1, TP53, KRAS, MAP2K1, and MYC were illustrated with a heatmap using the GSE227550, GSE156355, and GSE206800 datasets.

Article Snippet: Proteins were resolved by SDS-PAGE and subjected to immunoblot analysis with the following antibodies: pMEK1/2 (Santa Cruz Biotechnology, sc-81503; Santa Cruz, CA, USA), ERK1/2 (Cell Signaling, 4370; Danvers, MA, USA), p-ERK1/2 (Santa Cruz Biotechnology, sc-136521), PLK1 (Millipore, 05–844; Billerica, MA, USA), p-PLK1 (Cell Signaling, 5472S), c-Myc (Sigma-Aldrich, C3956), GAPDH (Santa Cruz, sc-32233), caspase-3 (Santa Cruz Biotechnology, sc-136521), cleaved caspase-3 (Cell Signaling, 9661S).

Techniques: Expressing, Mutagenesis

Fig. 2. The activities of MEK1 and PLK1 are highly upregulated in colorectal cancer patient. (a-b) Human colorectal tissues from patients with well- differentiated COAD and normal individuals, immunohistochemistry analysis displays anti-p-PLK1 (a) and anti-p-ERK1/2 (b) antibodies. The relative intensity of cells that exhibited positive p-PLK1 and p-ERK1/2 was analyzed and plotted. n > 5000. *p < 0.05; **p < 0.01; ***p < 0.001. Data are presented as mean ± SD.

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Synergistic two-step inhibition approach using a combination of trametinib and onvansertib in KRAS and TP53-mutated colorectal adenocarcinoma.

doi: 10.1016/j.biopha.2024.117796

Figure Lengend Snippet: Fig. 2. The activities of MEK1 and PLK1 are highly upregulated in colorectal cancer patient. (a-b) Human colorectal tissues from patients with well- differentiated COAD and normal individuals, immunohistochemistry analysis displays anti-p-PLK1 (a) and anti-p-ERK1/2 (b) antibodies. The relative intensity of cells that exhibited positive p-PLK1 and p-ERK1/2 was analyzed and plotted. n > 5000. *p < 0.05; **p < 0.01; ***p < 0.001. Data are presented as mean ± SD.

Techniques: Immunohistochemistry

Fig. 3. Cell viability assessment of KRAS, PLK1, and MEK1/2 inhibitors in COAD cells with different KRAS and TP53 status. (a) A heatmap analysis was performed with GSE59857 and KRAS, TP53, and BRAF status for four colon adenocarcinoma cell lines (SW48, HT29, HCT116, and HCT15). (b) Immunoblot analysis was performed using anti-KRAS, anti-ERK1/2, anti-p-ERK1/2, anti-PLK1, anti-p-PLK1, anti-MEK1/2, and anti-p-MEK1/2 antibodies in normal cell line hTERT-RPE1 and four colon adenocarcinoma cell lines with different KRAS and TP53 statuses. (c) qRT-PCR analysis was performed to observe the PLK1, KRAS, and ERK1/2 mRNA levels. (d-g) Salirasib (d), volasertib (e), onvansertib (f), and trametinib (g) were added in a concentration-dependent manner to SW48 (KRASwt/TP53wt), HT29 (KRASwt/TP53mt), HCT116 (KRASmt/TP53wt), and HCT15(KRASmt/TP53mt) COAD cells with different KRAS and TP53 mutation status. The percentage of cell viability was plotted after 48 hours of treatment. GI20, GI40, and GI50 values of each inhibitor in each cell line were calculated based on the viability assay. *p < 0.05; **p < 0.01; ***p < 0.001. Data are presented as mean ± SD.

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Synergistic two-step inhibition approach using a combination of trametinib and onvansertib in KRAS and TP53-mutated colorectal adenocarcinoma.

doi: 10.1016/j.biopha.2024.117796

Figure Lengend Snippet: Fig. 3. Cell viability assessment of KRAS, PLK1, and MEK1/2 inhibitors in COAD cells with different KRAS and TP53 status. (a) A heatmap analysis was performed with GSE59857 and KRAS, TP53, and BRAF status for four colon adenocarcinoma cell lines (SW48, HT29, HCT116, and HCT15). (b) Immunoblot analysis was performed using anti-KRAS, anti-ERK1/2, anti-p-ERK1/2, anti-PLK1, anti-p-PLK1, anti-MEK1/2, and anti-p-MEK1/2 antibodies in normal cell line hTERT-RPE1 and four colon adenocarcinoma cell lines with different KRAS and TP53 statuses. (c) qRT-PCR analysis was performed to observe the PLK1, KRAS, and ERK1/2 mRNA levels. (d-g) Salirasib (d), volasertib (e), onvansertib (f), and trametinib (g) were added in a concentration-dependent manner to SW48 (KRASwt/TP53wt), HT29 (KRASwt/TP53mt), HCT116 (KRASmt/TP53wt), and HCT15(KRASmt/TP53mt) COAD cells with different KRAS and TP53 mutation status. The percentage of cell viability was plotted after 48 hours of treatment. GI20, GI40, and GI50 values of each inhibitor in each cell line were calculated based on the viability assay. *p < 0.05; **p < 0.01; ***p < 0.001. Data are presented as mean ± SD.

Techniques: Western Blot, Quantitative RT-PCR, Concentration Assay, Mutagenesis, Viability Assay

Fig. 6. Cotreatment with trametinib and onvansertib synergistically induced apoptotic cell death through cell cycle arrest in KRAS and TP53-mutated HCT15 cells. (a) Flow cytometry analysis was performed on HCT15 cells treated with trametinib and/or onvansertib at their GI50 concentration. (b) The percentage of cells in each cell cycle phase were measured by flow cytometry. The population of cells was plotted. SubG1, gray; G1, orange; S, yellow; G2/M, skyblue. (c-d) HCT15 cells were treated with onvansertib (GI20, 0.340 μM) and trametinib (0.598 μM) at the concentration of the lower CI value in Table 3. (c) Immunoblot analysis was performed with anti-PLK1, anti-p-PLK1, anti-ERK1/2, anti-p-ERK1/2, anti-caspase-3, anti-cleaved caspase-3, and anti-GAPDH. The band intensity of cleaved caspase-3 was plotted (right panel). (d) The relative caspase-3 activity was measured with Ac-DEVD-AMC substrate and plotted. (e) A heatmap analysis was performed with GSE209767 in a trametinib-treated mouse model. The relative expression is displayed. (f-g) Immunofluorescence staining was performed in cells after single or combinational treatments with onvansertib and trametinib. (f) Cleaved caspase-3 (green), α-tubulin (red), and DNA (DAPI, blue) are displayed (n = 3039). Images of cells were collected and evaluated with a confocal microscope FW3000 (Olympus; Tokyo, Japan). Scale bar, 5 μm. (g) Quantification of the population of the cleaved caspase-3 (green fluorescence)-positive cells was plotted. n > 3000. *p < 0.05; **p < 0.01; ***p < 0.001. Data are presented as the mean ± SD.

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Synergistic two-step inhibition approach using a combination of trametinib and onvansertib in KRAS and TP53-mutated colorectal adenocarcinoma.

doi: 10.1016/j.biopha.2024.117796

Figure Lengend Snippet: Fig. 6. Cotreatment with trametinib and onvansertib synergistically induced apoptotic cell death through cell cycle arrest in KRAS and TP53-mutated HCT15 cells. (a) Flow cytometry analysis was performed on HCT15 cells treated with trametinib and/or onvansertib at their GI50 concentration. (b) The percentage of cells in each cell cycle phase were measured by flow cytometry. The population of cells was plotted. SubG1, gray; G1, orange; S, yellow; G2/M, skyblue. (c-d) HCT15 cells were treated with onvansertib (GI20, 0.340 μM) and trametinib (0.598 μM) at the concentration of the lower CI value in Table 3. (c) Immunoblot analysis was performed with anti-PLK1, anti-p-PLK1, anti-ERK1/2, anti-p-ERK1/2, anti-caspase-3, anti-cleaved caspase-3, and anti-GAPDH. The band intensity of cleaved caspase-3 was plotted (right panel). (d) The relative caspase-3 activity was measured with Ac-DEVD-AMC substrate and plotted. (e) A heatmap analysis was performed with GSE209767 in a trametinib-treated mouse model. The relative expression is displayed. (f-g) Immunofluorescence staining was performed in cells after single or combinational treatments with onvansertib and trametinib. (f) Cleaved caspase-3 (green), α-tubulin (red), and DNA (DAPI, blue) are displayed (n = 3039). Images of cells were collected and evaluated with a confocal microscope FW3000 (Olympus; Tokyo, Japan). Scale bar, 5 μm. (g) Quantification of the population of the cleaved caspase-3 (green fluorescence)-positive cells was plotted. n > 3000. *p < 0.05; **p < 0.01; ***p < 0.001. Data are presented as the mean ± SD.

Techniques: Flow Cytometry, Concentration Assay, Western Blot, Activity Assay, Expressing, Immunofluorescence, Staining, Microscopy, Fluorescence

PLK1 expression is increased in CCA cell lines. (A) PLK1 mRNA levels were measured by reverse transcription-quantitative PCR. The relative mRNA expression was normalized to the reference gene GAPDH. The results are presented as the mean ± SD of three independent experiments. (B) A representative western blot of PLK1 protein expression in CCA cell lines. PLK1, polo-like kinase 1; CCA, cholangiocarcinoma.

Journal: Oncology Letters

Article Title: PLK1 inhibition leads to mitotic arrest and triggers apoptosis in cholangiocarcinoma cells

doi: 10.3892/ol.2024.14449

Figure Lengend Snippet: PLK1 expression is increased in CCA cell lines. (A) PLK1 mRNA levels were measured by reverse transcription-quantitative PCR. The relative mRNA expression was normalized to the reference gene GAPDH. The results are presented as the mean ± SD of three independent experiments. (B) A representative western blot of PLK1 protein expression in CCA cell lines. PLK1, polo-like kinase 1; CCA, cholangiocarcinoma.

Article Snippet: The primary antibodies for various proteins were obtained from Cell Signaling Technology, Inc., unless specified otherwise: Phosphorylated (p)-PLK1 (Thr210; cat. no. 9062; RRID: AB_11127447), aurora kinase A (aurora A/AIK; cat. no. 3092; RRID: AB_2061342), cyclin B1 (cat. no. 4138; RRID: AB_2072132), poly (ADP-ribose) polymerase (PARP; cat. no. 9542; RRID: AB_2160739), p-Histone H2AX (Ser139; γH2AX; cat. no. 9718; RRID: AB_2118009), Histone H2A.X (cat. no. 2595; RRID: AB_10694556), GAPDH (cat. no. 8884; RRID: AB_11129865), α-tubulin (cat. no. 2125; RRID: AB_2619646) and PLK1 (anti-PLK1; clone 35–206; cat. no. 05-844; RRID: AB_310836; Millipore).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot

PLK1 inhibitors reduce the proliferation of CCA cell lines. CCA cell lines were treated with 0–500 nM (A) BI2536 and (B) BI6727 compared with the VC DMSO for 24, 48 and 72 h. Cell viability was measured by MTT assay. The bar graphs show normalization to the VC-treated cells and the results are presented as the mean ± SD of three independent experiments. (C) Colony formation assay of CCA cells treated with BI2536 and BI6727. Representative images of the colony formation assay are shown. PLK1, polo-like kinase 1; CCA, cholangiocarcinoma; VC, vehicle control.

Journal: Oncology Letters

Article Title: PLK1 inhibition leads to mitotic arrest and triggers apoptosis in cholangiocarcinoma cells

doi: 10.3892/ol.2024.14449

Figure Lengend Snippet: PLK1 inhibitors reduce the proliferation of CCA cell lines. CCA cell lines were treated with 0–500 nM (A) BI2536 and (B) BI6727 compared with the VC DMSO for 24, 48 and 72 h. Cell viability was measured by MTT assay. The bar graphs show normalization to the VC-treated cells and the results are presented as the mean ± SD of three independent experiments. (C) Colony formation assay of CCA cells treated with BI2536 and BI6727. Representative images of the colony formation assay are shown. PLK1, polo-like kinase 1; CCA, cholangiocarcinoma; VC, vehicle control.

Techniques: MTT Assay, Colony Assay, Control

PLK1 inhibitors induce G 2 /M-phase arrest in CCA cell lines. CCA cells were treated with VC, 10 and 100 nM (A) BI2536 and (B) BI6727 for 24 h. Cell cycle distribution was analyzed by flow cytometry using the Muse cell cycle assay kit. The percentages of cells in each phase of the cell cycle are shown and the results are presented as the mean ± SD of three independent experiments. A significant difference was observed when comparing treated cells with control cells (*P<0.05). Representative cell cycle distribution of CCA cells treated with VC, BI2536, and BI6727 are shown below the graphs. PLK1, polo-like kinase 1; CCA, cholangiocarcinoma; VC, vehicle control.

Journal: Oncology Letters

Article Title: PLK1 inhibition leads to mitotic arrest and triggers apoptosis in cholangiocarcinoma cells

doi: 10.3892/ol.2024.14449

Figure Lengend Snippet: PLK1 inhibitors induce G 2 /M-phase arrest in CCA cell lines. CCA cells were treated with VC, 10 and 100 nM (A) BI2536 and (B) BI6727 for 24 h. Cell cycle distribution was analyzed by flow cytometry using the Muse cell cycle assay kit. The percentages of cells in each phase of the cell cycle are shown and the results are presented as the mean ± SD of three independent experiments. A significant difference was observed when comparing treated cells with control cells (*P<0.05). Representative cell cycle distribution of CCA cells treated with VC, BI2536, and BI6727 are shown below the graphs. PLK1, polo-like kinase 1; CCA, cholangiocarcinoma; VC, vehicle control.

Techniques: Flow Cytometry, Cell Cycle Assay, Control

BI2536 and BI6727 upregulate the expression of (A) mitotic proteins and (B) induce DNA damage, as well as cleavage of PARP and caspase-9. CCA cells were treated with VC, or 10 and 100 nM BI2536 and BI6727 for 24 h. The expression of AURKA, p-PLK1 (T210), PLK1, cyclin B1, gH2AX, H2AX, PARP, caspase-9 and GAPDH proteins were detected by western blot analysis. Representative western blot images are shown. The bar graphs demonstrate the relative expression of the proteins normalized to GAPDH and VC-treated cells. The relative expression of p-PLK1 (T210) was normalized to total PLK1 and VC-treated cells. The relative expression of gH2AX was normalized to total H2AX and VC-treated cells. The relative protein expression was calculated from three independent experiments. PARP, poly (ADP-ribose) polymerase; CCA, cholangiocarcinoma; VC, vehicle control; AURKA, aurora kinase A; p-, phosphorylated; PLK1, polo-like kinase 1; H2AX, histone H2AX.

Journal: Oncology Letters

Article Title: PLK1 inhibition leads to mitotic arrest and triggers apoptosis in cholangiocarcinoma cells

doi: 10.3892/ol.2024.14449

Figure Lengend Snippet: BI2536 and BI6727 upregulate the expression of (A) mitotic proteins and (B) induce DNA damage, as well as cleavage of PARP and caspase-9. CCA cells were treated with VC, or 10 and 100 nM BI2536 and BI6727 for 24 h. The expression of AURKA, p-PLK1 (T210), PLK1, cyclin B1, gH2AX, H2AX, PARP, caspase-9 and GAPDH proteins were detected by western blot analysis. Representative western blot images are shown. The bar graphs demonstrate the relative expression of the proteins normalized to GAPDH and VC-treated cells. The relative expression of p-PLK1 (T210) was normalized to total PLK1 and VC-treated cells. The relative expression of gH2AX was normalized to total H2AX and VC-treated cells. The relative protein expression was calculated from three independent experiments. PARP, poly (ADP-ribose) polymerase; CCA, cholangiocarcinoma; VC, vehicle control; AURKA, aurora kinase A; p-, phosphorylated; PLK1, polo-like kinase 1; H2AX, histone H2AX.

Techniques: Expressing, Western Blot, Control

Silencing PLK1 shows antiproliferative effects on CCA cell lines. (A) CCA cells were transfected with 100 nM siSC or siPLK1 for 48 h and cell viability was measured by MTT assay. The bar graphs show the normalization to siSC-transfected cells. (B) Cell cycle analysis was performed by flow cytometry using the Muse cell cycle assay kit. The percentages of cells in each phase of the cell cycle are shown. Representative cell cycle distribution of CCA cells transfected with siSC or siPLK1 were shown in . (C) Total apoptotic cells were determined by flow cytometry using the Muse Annexin-V and Dead cell kit. The percentages of total apoptotic and dead necrotic cells are shown. The results are presented as the mean ± SD of at least three independent experiments. Representative plots showing Annexin V/7-AAD double staining of CCA cells transfected with siSC or siPLK1 are shown in . A significant difference was observed when comparing siPLK1-transfected cells with siSC-transfected cells (*P<0.05). (D) Protein levels of AURKA, PLK1, cyclin B1, gH2AX, H2AX, PARP and a-tubulin were examined by western blotting. Representative western blot images are shown. PLK1, polo-like kinase 1; CCA, cholangiocarcinoma; si, short interfering; SC, negative control; AURKA, aurora kinase A; PLK1, polo-like kinase 1; H2AX, histone H2AX; PARP, poly (ADP-ribose) polymerase.

Journal: Oncology Letters

Article Title: PLK1 inhibition leads to mitotic arrest and triggers apoptosis in cholangiocarcinoma cells

doi: 10.3892/ol.2024.14449

Figure Lengend Snippet: Silencing PLK1 shows antiproliferative effects on CCA cell lines. (A) CCA cells were transfected with 100 nM siSC or siPLK1 for 48 h and cell viability was measured by MTT assay. The bar graphs show the normalization to siSC-transfected cells. (B) Cell cycle analysis was performed by flow cytometry using the Muse cell cycle assay kit. The percentages of cells in each phase of the cell cycle are shown. Representative cell cycle distribution of CCA cells transfected with siSC or siPLK1 were shown in . (C) Total apoptotic cells were determined by flow cytometry using the Muse Annexin-V and Dead cell kit. The percentages of total apoptotic and dead necrotic cells are shown. The results are presented as the mean ± SD of at least three independent experiments. Representative plots showing Annexin V/7-AAD double staining of CCA cells transfected with siSC or siPLK1 are shown in . A significant difference was observed when comparing siPLK1-transfected cells with siSC-transfected cells (*P<0.05). (D) Protein levels of AURKA, PLK1, cyclin B1, gH2AX, H2AX, PARP and a-tubulin were examined by western blotting. Representative western blot images are shown. PLK1, polo-like kinase 1; CCA, cholangiocarcinoma; si, short interfering; SC, negative control; AURKA, aurora kinase A; PLK1, polo-like kinase 1; H2AX, histone H2AX; PARP, poly (ADP-ribose) polymerase.

Techniques: Transfection, MTT Assay, Cell Cycle Assay, Flow Cytometry, Double Staining, Western Blot, Negative Control

The proposed mechanism illustrates how PLK1 inhibition mediates apoptosis in CCA cells. Inhibiting PLK1 triggers mitotic arrest, activates the SAC, increases DNA damage and ultimately induces apoptosis. PLK1, polo-like kinase 1; CCA, cholangiocarcinoma; SAC, spindle assembly checkpoint; AURKA, aurora kinase A; c-, cleaved; PARP, poly (ADP-ribose) polymerase.

Journal: Oncology Letters

Article Title: PLK1 inhibition leads to mitotic arrest and triggers apoptosis in cholangiocarcinoma cells

doi: 10.3892/ol.2024.14449

Figure Lengend Snippet: The proposed mechanism illustrates how PLK1 inhibition mediates apoptosis in CCA cells. Inhibiting PLK1 triggers mitotic arrest, activates the SAC, increases DNA damage and ultimately induces apoptosis. PLK1, polo-like kinase 1; CCA, cholangiocarcinoma; SAC, spindle assembly checkpoint; AURKA, aurora kinase A; c-, cleaved; PARP, poly (ADP-ribose) polymerase.

Techniques: Inhibition

Gene tracks HSF1 and MYC at the PLK1 promoter analyzed from CUT&RUN in .

Journal: bioRxiv

Article Title: MYC and HSF1 Cooperate to Drive PLK1 inhibitor Sensitivity in High Grade Serous Ovarian Cancer

doi: 10.1101/2024.06.11.598486

Figure Lengend Snippet: Gene tracks HSF1 and MYC at the PLK1 promoter analyzed from CUT&RUN in .

Article Snippet: Antibodies used for IHC included MYC (Abcam #ab32072; RRID: AB_731658), HSF1 (CST #4356; RRID: AB_2120258), p-PLK1 (T210) (Abcam #ab155095).

Techniques:

A) Diagram of the relationship between PLK1 and MYC/HSF1 indicating PLK1 can directly regulate MYC and HSF1 through phosphorylation but also indirectly by regulating the PI3K-AKT-mTOR and FBXW7 pathways. B-C) PLK1 activity was assessed using a published gene signature in the TCGA-OV cohort and correlated with activity signatures for MYC (B) or HSF1 (C). D) A cohort of 58 high grade serous ovarian tumors were subjected to immunohistochemistry with antibodies for MYC, HSF1, and active PLK1 (pT210). E-F) Immunohistochemistry in D was analyzed with quPath to identify percent of cells that have positive nuclei for these markers to indicate active levels. Pearson correlation was used to correlate active HSF1 and MYC (E), HSF1 and PLK1 (F), and MYC and PLK1 (G).

Journal: bioRxiv

Article Title: MYC and HSF1 Cooperate to Drive PLK1 inhibitor Sensitivity in High Grade Serous Ovarian Cancer

doi: 10.1101/2024.06.11.598486

Figure Lengend Snippet: A) Diagram of the relationship between PLK1 and MYC/HSF1 indicating PLK1 can directly regulate MYC and HSF1 through phosphorylation but also indirectly by regulating the PI3K-AKT-mTOR and FBXW7 pathways. B-C) PLK1 activity was assessed using a published gene signature in the TCGA-OV cohort and correlated with activity signatures for MYC (B) or HSF1 (C). D) A cohort of 58 high grade serous ovarian tumors were subjected to immunohistochemistry with antibodies for MYC, HSF1, and active PLK1 (pT210). E-F) Immunohistochemistry in D was analyzed with quPath to identify percent of cells that have positive nuclei for these markers to indicate active levels. Pearson correlation was used to correlate active HSF1 and MYC (E), HSF1 and PLK1 (F), and MYC and PLK1 (G).

Article Snippet: Antibodies used for IHC included MYC (Abcam #ab32072; RRID: AB_731658), HSF1 (CST #4356; RRID: AB_2120258), p-PLK1 (T210) (Abcam #ab155095).

Techniques: Activity Assay, Immunohistochemistry

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