Cyclin b1

Proteins were extracted from human pulmonary artery tissues or PAECs and lysed in RIPA lysis buffer (RIPA, Beyotime, China) in accordance with the instructions of the manufacturer. Protein concentrations were evaluated using the BCA assay based on the albumin standard. The primary antibodies were used as follows: Dec1 and PPARγ (Abcam, United States, dilution: 1/1,000, for both), Cyclin B1 and Cyclin D1 (Sigma, United States, dilution 1/1,000, for both), Bax (Cell Signaling Technology, United States, dilution: 1/1,000), Bcl-2 (Sigma, United States, dilution 1/1,000), cleaved caspase 3, and β-actin (Santa Cruz, United States, dilution 1/1,000, for both). Detailed information of Western blot was described previously (Li et al., 2020 (link)). β-actin was used as a loading control. Fold change of protein expression was evaluated as average fold change for the ratio of targeting genes/β-actin in treated samples compared with that in the controls. Measurements were repeated three times.

MDA-MB-468 or HCC1937 cells were treated with PTX at their respective IC25 and IC50 doses, 0.75 µM SMI#9, or a combination of PTX and 0.75 µM SMI#9, and whole cell lysates were prepared at 48 h as previously described [36 (link)]. To analyze the effects of proteasome inhibitor on cyclin B1 regulation, PTX and SMI#9 treated HCC1937 cells were also treated with the 26S proteasome inhibitor MG132 (700 nM). 50–100 µg of protein were subjected to 4–20% gradient SDS-PAGE and western blot analysis of RAD6 [34 (link)], TAU (antibody recognizing nonphosphorylated and phosphorylated TAU isoforms, Abcam, Waltham, MA), cyclin B1 (Sigma-Aldrich Chemicals, St. Louis, MO), and β-actin (Sigma-Aldrich Chemicals). Although molecular and gene silencing analysis have established RAD6B as the predominant RAD6 gene overexpressed in breast cancer cell lines and tissues, the RAD6 protein detected by our antibody is referred as RAD6 rather than RAD6B as the peptide we used for RAD6B antibody generation is 91% conserved in human RAD6A and thus fails to distinguish RAD6A and RAD6B proteins [34 (link)]. Protein levels relative to the loading control β-actin were quantified by Image J.

Caco-2 cells were directly lysed in Laemmli buffer (3% (v/v) glycerol, 5% (v/v) β-mercaptoethanol, 2% (v/v) SDS, 0.1% (w/v) bromophenol blue in 100 mM Tris-HCl, pH 6.8). Samples were boiled in a hot block at 95ºC for 5 min, resolved by SDS-PAGE (10% polyacrylamide gel) and proteins transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were blocked in 5% skimmed milk in TBS-T (20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 0.1% Triton X-100), immunoblotted with primary antibodies recognizing cyclinB1 (Sigma-Aldrich, St Louis, MO, USA) and GAPDH (sc-32233, Santa Cruz Biotechnology, Dallas, TX, USA) and horseradish peroxidase-conjugated secondary antibodies (BI2413C and BI2407, PARIS Biotech, Compiègne, Paris, France). Chemiluminescence (Pierce ECL Western Blotting Substrate, Thermo Fischer Scientific, Waltham, MA, USA) was used for protein detection, and band intensities were quantified using Image J software.

Recombinant KIND2 was expressed and purified as previously described^{43 (link)}. In brief, KIND2 was cloned into pCoofy17 to add an amino-terminal His10-Sumo tag and expressed in Escherichia coli Rosetta at 18 °C overnight. After purification by IMAC in high-salt TBS buffer (20 mM Tris, pH 7.5, 500 mM NaCl and 1 mM TCEP), the Sumo tag was removed by SenP2 (obtained from the in-house facility) digestion overnight at 4 °C. The protein was further purified by SEC using TBS (20 mM Tris, pH 7.5, 200 mM NaCl and 1 mM TCEP) containing 5% glycerol as running and storage buffer. Recombinant CDK1 and cyclin B1 were purchased from Sigma (SRP5009).

MCF7 cells were washed once with sterile PBS and harvested using a rubber cell scraper as previously described [34 (link)], with the following changes: cells were pelleted via centrifugation at 1000 rpm and resuspended in ice cold RIPA buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1 mM EGTA, 1% NP-40) with protease and phosphatase inhibitors. The cell suspensions were then sonicated with a 70% duty pulse sonication cycle, and centrifuged to remove cell debris. The antibodies used in this study, typically at a 1:1000/2000 dilution, included APC1^tot (Abcam133397), APC1^S355phos (Abcam10923), CDC20 (PA5-34775), FZR1/CDH1 (Sigma), CDC27 (Abcam10538), Cyclin B1 (Sigma), HURP (Abcam70744, Proteintech), Securin (Abcam79546), MDR-1 (Sigma), BCRP (Santa Cruz Biotechnology, Dallas, TX, USA; SCBt), TFPI (Abcam, Cambridge, UK), PARP (Sigma), γH2AX (NovusBio, Centennial, CO, USA), histone H3^K9Ac (Millipore, Burlington, MA, USA), histone H3^S10phospho, histone H3^tot (Millipore), GAPDH (Millipore), and tubulin (Sigma). Following primary antibody incubation overnight at 4 °C, the blots were probed with a 1:10,000 dilution of a horseradish peroxidase (HRP) secondary antibody.

Commercially available antibodies obtained from Cell Signaling against the indicated proteins were: Cdc2/CDK1 (Cat # 9112S), CDK1-P-Y15 (Cat # 4539S), Histone H3-P-S28 (Cat # 9713S), p53-P-S15 (Cat # 9284), SMC1-P-957 (Cat # 4805S), SMC1 (Cat # 4802S), CDC25A (Cat # 3652S) and Chk2-P-T68 (Cat # 2661S). Antibodies obtained from Santa Cruz Biotechnology were CDC25A F-6 (Cat # sc -7389), CDC25C H-150 (Cat # sc-5620), Cdc2 (Cat # sc-54). Others were against actin (Cat. 109 # MA515739, Pierce), RPA32-P-S4/8 (Cat # A300-245A, Bethyl), cyclin A (Cat # 06-138, Upstate) γH2AX (Cat # 05-636, Millipore), Chk2 Clone 7 (Cat # 05-649, Millipore), cyclin B1 (Cat. # 05-373, Millipore), and CDK1 (Cat. #Ab18, Abcam). Secondary antibodies have previously been described. Additional antibodies used for immunofluorescence include: cyclin B1 (Cat # 4138S, Cell signaling), Alexa 488 (anti-mouse Cat # A11029, anti-rabbit Cat # A11034, Invitrogen) and Alexa 568 (anti-mouse Cat # A11031, anti-rabbit Cat # A11036, Invitrogen). NS1 CE10 and NS1 91W were described previously [7] (link).