Journal: BMC Cancer

Article Title: Increased TCP11 gene expression can inhibit the proliferation, migration and promote apoptosis of cervical cancer cells

doi: 10.1186/s12885-023-11129-1

Figure Lengend Snippet: TCP11 overexpression induces the apoptosis of HeLa and SiHa cells. A and B : Apoptosis of HeLa and SiHa cells were detected by flow cytometry. C : Western blot was used to detect the protein expression of caspase-3, cleaved-caspase-3 and cleaved-PARP in HeLa and SiHa cells infected with overexpressing TCP11 lentivirus. Full-length blots/gels are presented in Supplementary Figure . Data are presented as mean ± SD of at least 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Anti-cleaved-PARP (BSM-33,138 M), anti-CDK1 (BS-0542R), and anti-CyclinB1 (BS-0572R) were purchased from Bioss (China).

Techniques: Over Expression, Flow Cytometry, Western Blot, Expressing, Infection

Journal: Endocrinology

Article Title: Silencing Med12 Gene Reduces Proliferation of Human Leiomyoma Cells Mediated via Wnt/ β -Catenin Signaling Pathway

doi: 10.1210/en.2016-1097

Figure Lengend Snippet: Antibody Table

Article Snippet: Cdk1/Cdk2 , Anti-Cdk1 antibody , Santa Cruz Biotechnology (Catalog # sc-53219) , Mouse monoclonal , 500.

Techniques:

Journal: Endocrinology

Article Title: Silencing Med12 Gene Reduces Proliferation of Human Leiomyoma Cells Mediated via Wnt/ β -Catenin Signaling Pathway

doi: 10.1210/en.2016-1097

Figure Lengend Snippet: Effect of Med12 knockdown on cell cycle regulatory and growth-promoting pathways in hUF cells. (A) Equal amounts of cell lysates from Med12-shRNA and control-shRNA cells were analyzed by Western blotting using anti-cyclin D1, Cdk1, Cdk2, Cdk4, and Cdk6 antibodies. β-actin Western blot was used as the loading control. (B) The intensity of each protein band was quantified and normalized with corresponding β-actin. *P < 0.05 when compared with control-shRNA. (C) Cell lysates were analyzed by Western blot using antibodies against p-ERK, total ERK, p-AKT, and total AKT. (D) The intensity of each protein band was quantified and normalized with corresponding β-actin. *P < 0.05 when compared with control shRNA.

Article Snippet: Cdk1/Cdk2 , Anti-Cdk1 antibody , Santa Cruz Biotechnology (Catalog # sc-53219) , Mouse monoclonal , 500.

Techniques: Knockdown, shRNA, Control, Western Blot

Journal:

Article Title: Chk1, but not Chk2, inhibits Cdc25 phosphatases by a novel common mechanism

doi: 10.1038/sj.emboj.7600328

Figure Lengend Snippet: Inhibition of Cdc25A/Cdk–cyclin interactions by Thr504 phosphorylation. (A) GST-Cdc25A fusion proteins (wild type (WT), T504A, or C428S) were first incubated with GST-Δ60-Chk1 (Δ60) or its kinase-dead form (DA) and then with Tyr15-phosphorylated Cdk1–cyclin A1 complexes. The reaction mixture was then analyzed by immunoblotting using anti-Cdc25A, anti-phospho-Thr504, anti-cyclin A1, or anti-Cdk1 phospho-Tyr15 antibodies. (For exact experimental conditions, see Supplementary data.) (B) Activated eggs were injected with 2 ng of mRNA encoding GST (Cont.) or GST-Cdc25A (WT or T504A), incubated for 2.5 h, reinjected or not with 2 ng of Δ60-Chk1 mRNA, and then incubated further for 1.5 h. Egg extracts (Input; equivalent to half an egg) and GST-pulled down proteins (GST-PD; equivalent to 10 eggs) were then immunoblotted for GST-Cdc25A or endogenous cyclins A1, B1, or E1 (left panel). (The blot of GST-Cdc25A in GST-PD is short-exposed.) The levels of cyclins (in both Input and GST-PD) were quantified using the NIH Image program from four independent experiments, and the levels of cyclins bound to GST-Cdc25A proteins were normalized to the input of cyclins; values obtained for WT Cdc25A (−Chk1) were set at 1.0 (right panel). (C) Extracts from the eggs expressing GST-Cdc25A proteins (together with or without Δ60-Chk1) as in (B) were mixed with extracts from the eggs expressing Myc-tagged cyclins A1 or B1 (together with Xe-Wee1B (Okamoto et al, 2002) to induce Cdk1 Tyr15 phosphorylation), incubated for 1 h at 4°C, and analyzed for GST-Cdc25A or ectopic cyclins A1 or B1 as in (B, left). (D, E) Activated eggs were injected with 2 ng of mRNA encoding wild-type GST-Cdc25A, GST-Cdc25A-ΔC23 (D) or GST-Cdc25A-3A (E), incubated for 2.5 h, and analyzed as in (B). In (B–E), all the GST-Cdc25A constructs had both S73A and C428S mutations (see text).

Article Snippet: Routinely, proteins equivalent to one egg or embryo were analyzed by immunoblotting ( Shimuta et al , 2002 ), using the above-described anti-phospho-Thr504 antibody, anti- Xenopus Cdc25A antibody ( Shimuta et al , 2002 ), anti-Myc antibody (A-14, Santa Cruz), anti-GST antibody (Z-5 or B-14, Santa Cruz), anti- Xenopus cyclin A1 or B1 antibodies (a gift from J Maller), anti- Xenopus cyclin E1 antibody (a gift from T Kishimoto), anti-Cdk1 phospho-Tyr15 antibody (♯911, Cell Signaling), anti-human Chk1 phospho-Ser345 antibody (♯2341, Cell Signaling), or anti-human 14-3-3β antibody (H8, Santa Cruz).

Techniques: Inhibition, Incubation, Western Blot, Injection, Expressing, Construct

Journal:

Article Title: Chk1, but not Chk2, inhibits Cdc25 phosphatases by a novel common mechanism

doi: 10.1038/sj.emboj.7600328

Figure Lengend Snippet: Requirement of Cdc25A phosphorylation on Thr504 for the DNA replication checkpoint. (A, B) Activated eggs were injected with 1 ng of mRNA encoding Myc-tagged wild-type Cdc25A or indicated Myc-tagged Cdc25A mutants, reinjected 2.5 h later with 2 ng of Δ60-Chk1 mRNA, and then analyzed by immunoblotting using anti-Myc or anti-Cdk1 phospho-Tyr15 antibodies. (C–E) One-cell embryos were uninjected (Cont.) or injected with 1 ng of mRNA encoding either wild-type Cdc25A or T504A Cdc25A, cultured, and analyzed for immunoblotting (after stage 8 with anti-Cdc25A or anti-Cdk1 phospho-Tyr15 antibodies; C), for the external morphology (D) and for the percentage embryonic death at stage 11 (E).

Article Snippet: Routinely, proteins equivalent to one egg or embryo were analyzed by immunoblotting ( Shimuta et al , 2002 ), using the above-described anti-phospho-Thr504 antibody, anti- Xenopus Cdc25A antibody ( Shimuta et al , 2002 ), anti-Myc antibody (A-14, Santa Cruz), anti-GST antibody (Z-5 or B-14, Santa Cruz), anti- Xenopus cyclin A1 or B1 antibodies (a gift from J Maller), anti- Xenopus cyclin E1 antibody (a gift from T Kishimoto), anti-Cdk1 phospho-Tyr15 antibody (♯911, Cell Signaling), anti-human Chk1 phospho-Ser345 antibody (♯2341, Cell Signaling), or anti-human 14-3-3β antibody (H8, Santa Cruz).

Techniques: Injection, Western Blot, Cell Culture

Journal:

Article Title: Chk1, but not Chk2, inhibits Cdc25 phosphatases by a novel common mechanism

doi: 10.1038/sj.emboj.7600328

Figure Lengend Snippet: No requirement of 14-3-3 binding for the inhibitory effect of Thr504 phosphorylation. (A) Activated eggs expressing GST-Cdc25A proteins (together with or without Δ60-Chk1) as in Figure 4B were subjected to GST pulldown assays and analyzed by immunoblotting using anti-GST antibody or anti-human 14-3-3β antibody (which can recognize all 14-3-3 isoforms). (B) Chk1-phosphorylated GST-Cdc25A protein (wild type) was GST-pulled down from activated eggs, washed with or without 0.4% Empigen, and analyzed by immunoblotting as in (A) (left). Extracts from the eggs expressing GST-cyclin A1 (together with Xe-Wee1B; see Figure 4C legend) were treated with the indicated peptide-coupled beads and immunoblotted for cyclin A1 or 14-3-3 protein (right). See Materials and methods for details. (C) GST-Cdc25A proteins (phosphorylated or not by Chk1) and egg extracts (equivalent to 10 eggs) were both prepared as in (B) (GST-Cdc25A proteins being eluted from glutathione beads), mixed together, and incubated for 1 h at 4°C. GST-Cdc25A proteins were then immunoprecipitated with anti-Cdc25A antibody (IP) and analyzed by immunoblotting using anti-GST or anti-cyclin A1 antibodies. As control of GST-Cdc25A proteins (wild type or T504A), GST alone was used (Cont.). (D) GST-Cdc25A proteins prepared as in (C) were first incubated or not with 0.1 μg of recombinant Xenopus 14-3-3ɛ protein in 30 μl of an egg extraction buffer for 15 min at 4°C and then with (GST-)Cdk1–cyclin A1 complexes (purified from 10 eggs) for 1 h. (Xenopus 14-3-3ɛ protein can bind to phosphorylated Thr504 of Cdc25A; see Materials and methods.) The mixture was then analyzed as in (C). In (A–D), all the GST-Cdc25A constructs had both S73A and C428S mutations.

Article Snippet: Routinely, proteins equivalent to one egg or embryo were analyzed by immunoblotting ( Shimuta et al , 2002 ), using the above-described anti-phospho-Thr504 antibody, anti- Xenopus Cdc25A antibody ( Shimuta et al , 2002 ), anti-Myc antibody (A-14, Santa Cruz), anti-GST antibody (Z-5 or B-14, Santa Cruz), anti- Xenopus cyclin A1 or B1 antibodies (a gift from J Maller), anti- Xenopus cyclin E1 antibody (a gift from T Kishimoto), anti-Cdk1 phospho-Tyr15 antibody (♯911, Cell Signaling), anti-human Chk1 phospho-Ser345 antibody (♯2341, Cell Signaling), or anti-human 14-3-3β antibody (H8, Santa Cruz).

Techniques: Binding Assay, Expressing, Western Blot, Incubation, Immunoprecipitation, Recombinant, Purification, Construct

Journal: International Journal of Oncology

Article Title: Knockdown of PGBD5 inhibits the malignant progression of glioma through upregulation of the PPAR pathway

doi: 10.3892/ijo.2024.5643

Figure Lengend Snippet: List of antibodies used.

Article Snippet: CDK1 Rabbit mAb , ABclonal Biotech Co., Ltd. (cat. no. A11420).

Techniques:

Journal: International Journal of Oncology

Article Title: Knockdown of PGBD5 inhibits the malignant progression of glioma through upregulation of the PPAR pathway

doi: 10.3892/ijo.2024.5643

Figure Lengend Snippet: Effects of PGBD5 knockdown on the apoptosis of glioma cells. (A and B) Effects of PGBD5 knockdown on the cell cycle progression of glioma cells. (D and E) Apoptosis and cell cycle distribution of glioma cells were detected using flow cytometry. Western blotting showed the expression levels of (C) apoptosis-related proteins (Bax, Bcl-2 and caspase-3) and (F) cell cycle-related proteins (CDK1 and cyclin B1) in glioma cell lines after PGBD5 knockdown. β-actin or GAPDH were used as the protein loading controls. The experiments were repeated three times. * P<0.05, ** P<0.01, *** P<0.001 vs. sh-NC or as indicated. NC, negative control; PGBD5, piggyBac transportable element derived; sh, short hairpin.

Article Snippet: CDK1 Rabbit mAb , ABclonal Biotech Co., Ltd. (cat. no. A11420).

Techniques: Knockdown, Flow Cytometry, Western Blot, Expressing, Negative Control, Derivative Assay

Journal: Gland Surgery

Article Title: Transcriptome analysis and functional identification of adipose-derived mesenchymal stem cells in secondary lymphedema

doi: 10.21037/gs.2020.02.09

Figure Lengend Snippet: Cell cycle inhibitor influences the proliferative ability and differentiation potential of ASCs. (A) Immunoblot analysis of p-CDK1, CDK1, p-CDK2, CDK2, Cyclin B and Cyclin A expression levels after the cells were treated or not treated with CDK inhibitors JNJ, Ro-3306, or purvalanol A for 2 h; (B) representative photos of cell density and morphology of ASCs after treatment with CDK inhibitors JNJ, Ro-3306, or purvalanol A for 48 h. Bar =100 µm; (C) the division potential of ASCs was detected with CCK8 after the cells were treated or not treated with CDK inhibitors JNJ, Ro-3306, or purvalanol A for 48 h. Values were expressed as the mean ± SEM of three replicates. **, P<0.01, significantly different from L+DMSO; #, P<0.01, significantly different from N+DMSO; ns, no statistical difference (one-way ANOVA). (D) ASCs treated with or without CDK inhibitors, JNJ, Ro-3306, or purvalanol A were induced to differentiate into adipocytes and were stained with Oil Red O; 10 images of each condition were captured for statistical analysis; (E) the areas stained with Oil Red O was expressed as the mean ± SEM (n=3). Bar=100 µm. *, P<0.05, **, P<0.01, significantly different from L+DMSO; ##, P<0.01, significantly different from N+DMSO; ns, no statistical difference (one-way ANOVA); (F) RT-qPCR detected the expression of mature adipocyte markers adiponectin, LPL, or PPARG in induced ASCs, which were treated with CDK inhibitors before adipogenesis. ASCs derived from lymphedema group and normal group of 3 random donors in triplicates. *, P<0.05, **, P<0.01, significantly different from L+DMSO; #, P<0.05, ##, P<0.01, significantly different from N+DMSO; ns, no statistical difference (one-way ANOVA). N, normal; L, lymphedema; ASCs, adipose-derived mesenchymal stem cells; RT-qPCR, quantitative reverse transcriptase-PCR; CDK, cyclin-dependent kinase; CCK8, cell counting kit-8.

Article Snippet: After separation by NuPAGE 4–12% Bis-Tris precast gels (NP0322BOX, Invitrogen) with NuPAGE ® MOPS SDS Running Buffer (NP0001, Invitrogen), the proteins were blotted onto a PVDF transfer membrane at 300 mA for 90 min. After blocking with 5% skim milk powder in TBST (0.1 M Tris-HCl pH 8, 1.5 M NaCl and 0.1% Tween-20) for 1 h at room temperature, the membrane was incubated overnight at 4 °C with the primary antibodies CDK1, p-CDK1, CDK2, p-CDK2, Cyclin B, Cyclin A, or β-tubulin (9116, 4539, 2546, 2561, 12231, 4656, 2128, Cell Signaling Technology) followed by 1 h incubation with peroxidase-conjugated rabbit anti-goat IgG (H+L) (ZB2306, Zsbio) (p-CDK1, CDK2, p-CDK2, Cyclin B), peroxidase-conjugated mouse anti-goat IgG (ZB2305, Zsbio) (CDK1) or peroxidase-conjugated rabbit anti-goat IgE (1110-05, SouthernBiotech) (Cyclin A) at room temperature and was developed with the Chemiluminescent Substrate (34080&34095, Thermo Scientific).

Techniques: Western Blot, Expressing, Staining, Quantitative RT-PCR, Derivative Assay, Cell Counting

Journal: Gland Surgery

Article Title: Transcriptome analysis and functional identification of adipose-derived mesenchymal stem cells in secondary lymphedema

doi: 10.21037/gs.2020.02.09

Figure Lengend Snippet: Schematic difference of lymphedema-associated ASCs and ASCs from normal tissue of the same patients. Lymphedema-associated ASCs had more rapid proliferation and adipogenic differentiation capacity than ASC from normal tissue. CDK1 is a key driver that may prompt these cells toward proliferation and adipogenic differentiation could explain the adipose tissue accumulation widely observed in secondary lymphedema. ASCs from lymphedema adipose tissues showed immunomodulation dysfunction which may play an important role in the pathogenesis of lymphedema. ASCs, adipose-derived mesenchymal stem cells; CDK1, cyclin-dependent kinase 1.

Article Snippet: After separation by NuPAGE 4–12% Bis-Tris precast gels (NP0322BOX, Invitrogen) with NuPAGE ® MOPS SDS Running Buffer (NP0001, Invitrogen), the proteins were blotted onto a PVDF transfer membrane at 300 mA for 90 min. After blocking with 5% skim milk powder in TBST (0.1 M Tris-HCl pH 8, 1.5 M NaCl and 0.1% Tween-20) for 1 h at room temperature, the membrane was incubated overnight at 4 °C with the primary antibodies CDK1, p-CDK1, CDK2, p-CDK2, Cyclin B, Cyclin A, or β-tubulin (9116, 4539, 2546, 2561, 12231, 4656, 2128, Cell Signaling Technology) followed by 1 h incubation with peroxidase-conjugated rabbit anti-goat IgG (H+L) (ZB2306, Zsbio) (p-CDK1, CDK2, p-CDK2, Cyclin B), peroxidase-conjugated mouse anti-goat IgG (ZB2305, Zsbio) (CDK1) or peroxidase-conjugated rabbit anti-goat IgE (1110-05, SouthernBiotech) (Cyclin A) at room temperature and was developed with the Chemiluminescent Substrate (34080&34095, Thermo Scientific).

Techniques: Derivative Assay