HepG2 and Bel‐7402HCC cell lines were purchased from the American Type Culture Collection and all cultured in high‐glucose DMEM containing 10% fetal bovine serum at 37°C in a humidified incubator (SANYO, Osaka, Japan) with 5% CO2. Epirubicin (EPI) and 5‐FU were purchased from Merck Millipore (Billerica, MA). MMC was purchased from Kyowa Hakko Kirin (Tokyo, Japan). Cisplatin was purchased from Sigma (St Louis, MO). The primary antibody against Mus81, CHK1, CDC25A, CDK2, CHK2, CDC25C, CDC2, p53, Bax, and Bcl‐2 were purchased from Abcam (Cambridge, UK). The primary antibody against phosphorylated CHK1 (Ser317), phosphorylated CHK2 (Thr68), phosphorylated CDC25C (Ser216), phosphorylated CDK2 (Tyr15), phosphorylated CDC2 (Tyr15), and phosphorylated p53 (Ser15) were purchased from Cell Signaling Technology (Danvers, MA). CHK1 inhibitor (CAS: 405168‐58‐3) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). CHK2 inhibitor (CAS: 516480‐79‐8) was purchased from Merck Millipore. Caspase‐3 activity assay kit was purchased from Chemicon International (Temecula, CA).
Cdc25c
Manufactured by Abcam
Sourced in United States, United Kingdom
CDC25C is a phosphatase enzyme involved in cell cycle regulation. It plays a key role in the G2/M transition by dephosphorylating and activating cyclin-dependent kinases.
Automatically generated - may contain errors
Lab products found in correlation
Cyclin b1, by Abcam (6 mentions)
Bcl 2, by Abcam (5 mentions)
Bcl2 associated x (bax), by Abcam (5 mentions)
Cdc25a, by Abcam (2 mentions)
P stat3, by Cell Signaling Technology (2 mentions)
Stat3, by Cell Signaling Technology (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Gapdh, by Abcam (2 mentions)
Bcl xl, by Abcam (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Phosphorylated p53 ser 15, by Cell Signaling Technology (1 mentions)
Chk2 inhibitor, by Merck Group (1 mentions)
Cisplatin, by Merck Group (1 mentions)
Humidified incubator, by Sanyo (1 mentions)
Chemidoc touch, by Bio-Rad (1 mentions)
Gapdh, by Keygen Biotech (1 mentions)
Phospho perk1 2, by Cell Signaling Technology (1 mentions)
12 protocols using cdc25c
1
Cytotoxic Regulation in Liver Cancer
2
Protein Expression Analysis in K562 Cells
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After K562 cells were treated with or without different concentrations of tested compound for 48 h, cells were harvested and washed with PBS for two times. The collected cells were lysed by the cold Lysis Buffer containing 5 µL of 100 mM phenylmethylsulfonyl fluoride (PMSF), following by centrifugation with 14,000 rpm for 15 min. Protein concentrations of supernatant were determined by using BCA assay, and then equal amounts of protein were separated by SDS-PAGE and transferred to poly(vinylidene fluoride) (PDVF) membranes. After blocked with 5% skim milk for 2 h, the membranes were incubated with primary antibodies against Cdc-2, Cyclin B1, Cdc25c, Bad, Bax, Bcl-2, Bcl-xl (Abcam, UK) and GAPDH (KeyGEN BioTECH, China) overnight at 4 °C. The membranes were then washed with TBST three times for 10 min, incubated with secondary antibody for 1–2 h, and washed with TBST again. Finally, the proteins were stained with ECL chemiluminescence immunoassay kit, and the images were captured by bio-rad ChemiDoc Touch (Bio-rad, US). The data was analysed by software Gel-Pro32.
3
Synthesis and Characterization of DCZ3301
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DCZ3301 (purity > 98%) was synthesized by Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China. This compound has been patented and the relevant patent number is 2016102204055 recorded by State Intellectual Property Office Of The P.R.C. DCZ3301 was dissolved in dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO, USA) at a concentration of 16 mmol/L (16 mM) and stored at -20℃ until use. IL-6 and VEGF were purchased from R&D Systems (Minneapolis, MN, USA). Human CD138 MicroBead was obtained from Miltenyi (Miltenyi Biotec GmbH, Germany). Antibodies for caspase 3, 8, and 9, PARP, ERK1/2, AKT, STAT3, phospho(p)-ERK1/2, p-AKT, p-STAT3, β-actin were purchased from Cell Signaling Technology; Cdc25C, CDK1, Cyclin B1, IKBα, p-IKBα(Ser32), p-p65(S536) were from Abcam. Z-VAD-FMK was provided by Selleck Chemicals (Houston, TX, USA). Cell Counting Kit-8 (CCK-8) was obtained from Dojindo. Annexin V-FITC and PI detection kit was purchased from BD Pharmingen (San Diego, CA). Mitochondrial membrane potential assay kit with JC-1 was obtained from Beyotime Institute of Biotechnology.
4
Protein Expression Evaluation Protocol
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CARD (purity ≥ 95% by HPLC) was purchased from Merck-Millipore Co, and was dissolved with DMSO and stored at –20°C. The antibody of Bax, Bcl-2, Caspase-3, E-cadherin, α-SMA, Vimentin, p21, CDC25c, Cyclin B1, CDK1 was purchased from Abcam, and Snail, p-STAT3, STAT3, and GAPDH from Cell Signaling Technology.
5
Western Blot Analysis of Cell Signaling
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Proteins were extracted by RIPA lysis buffer (Beyotime) with 1% phosphatase and protease inhibitors and separated by 10% SDS-PAGE (EpiZyme, Cambridge, MA, USA). Then, we transferred the proteins onto PVDF membranes (Pierce Biotechnology, Waltham, MA, USA) and blocked the membranes with 5% milk at 37 °C for 1 h. The membranes were incubated with primary antibody overnight at 4 °C. A subsequent 2 h incubation in secondary antibody was performed, and the membranes were then exposed to ECL reagents (Abcam, Cambridge, UK). Antibodies and dilutions were as follows: RAB20 antibody (Abcam, ab197209, 1:1000); α-tubulin (CST, #2144, 1:1000); β-actin (CST, #3700, 1:1000); CDK2 (CST, #2546, 1:1000); CyclinE1 (CST, #20808, 1:1000); CyclinD1 (CST, #55506, 1:1000); CyclinB1 (CST, #12231, 1:1000); cdc2 (CST, #9116, 1:1000); Chk1 (CST, #2360, 1:1000); cdc25C (CST, #4688, 1:1000); Phospho-cdc25C (CST, #4901, 1:1000); p53 (CST, #2527, 1:1000); Phospho-p53 (CST, #9286, 1:1000); and p21Waf1/Cip1 (CST, #2947, 1:1000).
6
Protein Expression Analysis in Lung Cell Lines
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In order to create protein lysates from MRC-5, A549, Calu-3, H1975, and H2228, protease and phosphatase inhibitors were added to radioimmunoprecipitation assay (RIPA) lysis solution (Thermo Fisher Scientific). The bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific) was used to measure the amounts of proteins. After sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for electrophoresis, polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA) were transferred. Immunoblotting was carried out using primary antibodies against CDC25C, Bax, Bcl-2, LC3 I, LC3 II, p62, cyclin B, CDK1-Y15, CDK1, c-PARP, and GAPDH (all at 1:1,000; Abcam, Cambridge, MA, USA). Post-washing thrice with tris-buffered saline with Tween 20 (TBST), membranes were processed for band visualization using an enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific). Using the ChemiDoc system from Bio-Rad (Hercules, CA, USA), the intensities of the protein bands were recorded and subsequently analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).
7
Immunoblotting Antibodies and Targets
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Immunoblotting was performed as described previously [18 (link)]. The following antibodies were used: α-tubulin and β-actin (Sigma-Aldrich), p21Cip1/WAF1 and p27KIP1 (BD-Pharmingen, San Diego, CA); p53 (DO-7; Dako, Carpinteria, CA); BRCA1(Santa Cruz Biotechnology, Dallas, TX); Cdc2 (MBL, Nagoya, Japan); PIM-1 and XPO1 (marketed as anti-CRM1) and p-HSF1Ser326 (Abcam, Cambridge, MA); CDC25C, c-Myc, cyclin D1, Hsp70, 4E-BP1, phosphorylated- (p-) 4E-BP1Thr37/Thr46, p-RbSer780, S6 (S6K), p-S6 ribosomal protein (S6K)Ser235/Ser236, PUMA, HSF1, Hsp70 and horseradish peroxidase–linked anti-mouse and anti-rabbit IgG (all from Cell Signaling Technology).
8
Examining Cellular Signaling Pathways
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A 16 mM DCZ3301 stock solution was dissolved in DMSO (Sigma, St. Louis, MO, USA) and stored at −20 °C. Antibodies for ERK1/2, phospho-ERK1/2, Akt, phospho-Akt, STAT3, phospho-STAT3, Lyn, phospho-Lyn, Syk, phospho-Syk, JAK2, phospho-JAK2, cleaved Caspase-8, Bax, B cell lymphoma-2 (Bcl-2), Bcl-xl, PARP, c-Myc, p21 and Actin (for western blotting) were purchased from Cell Signaling Technology (Danvers, MA, USA). Caspase-9, cleaved caspase-3, p-CHK2, cdc25A, cdc25C and cyclinB1 antibodies were purchased from Abcam (Cambridge, MA, USA). IL-6 and IGF-1 (R&D Systems, Minneapolis, MN, USA) dissolved in phosphate-buffered saline (PBS) containing 0.1% albumin from bovine serum albumin (BSA) were prepared as 10 μg/ml stock solution and stored at −20 °C. Panobinostat and vorinostat were provided by Selleck Chemicals (Houston, TX, USA). CCK-8 was obtained from Yeasen (Shanghai, China), the Annexin-V/PI apoptosis detection kit from BD Pharmingen (Franklin Lakes, NJ, USA) and the JC-1 Mitochondrial Membrane Potential Detection Kit from Beyotime Institute of Biotechnology (Shanghai, China).
9
Tissue Microarray Analysis of HCC
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Tissue microarrays (TMAs) were made using pathological specimens collected from 52 patients with HCC collected from Sun Yat-sen Memorial Hospital, including tumor tissue and adjacent tissues. AURKA, CDC25C, KPNA2, MCM3 and TUBG1 antibodies were purchased from Abcam (ab52973, ab32444, ab289858, ab128923, ab179503, CN, Shanghai) and diluted into 1:100, 1:1,250, 1:2,500, 1:100, 1:100, and NEK2 antibodies were purchased from Pygcn (NEK2 Polyclonal antibody, CN, Wuhan), and diluted into 1:400. The average optical density value calculated by IamgeJ was used as the observation index of IHC. Three fields of view for each tissue were selected, the average optical density value of its score was calculated, and the average of the three was taken as the result (mean). The top 26 patients with high expression scores were defined as high expression groups (available online https://cdn.amegroups.cn/static/public/tcr-23-1237-2.xlsx ; https://cdn.amegroups.cn/static/public/tcr-23-1237-3.xlsx ).
10
Protein Expression Analysis in U251 Cells
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Total protein of transfected U251 cells was extracted using RIPA buffer with protease inhibitor. Separated by 10% SDS-PAGE gel electrophoresis, transferred to polypropylene fluoride membrane (PVDF) treated with methanol for 2 h, transferred to a shaker, sealed with TBS-T blocking solution containing 5% skimmed milk powder for 1 h. Then, the primary antibodies GAPDH (1:5000, Abcam, UK), CCNB1 (1:500, Abcam, UK), CDK1 (1:500, Abcam, UK) and Cdc25C (1:500, Abcam, UK) were added to incubate overnight at 4 °C. Washed by TBS-T 5 times for 5 min each time, secondary antibody (1:5000) was added to incubate for 1 h at room temperature, Washed by TBS-T 5 times for 5 min each time, and finally ECL color developing solution was added for color development. The developed bands were subjected to gray level analysis by ImageJ Software (v1.8.0), The difference in protein expression levels between the HOXD11-NC group and the HOXD11-siRNA group is represented by the gray value of the corresponding protein bands.
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