Flavopiridol

E2 was obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany), Flavopiridol was supplied by Cayman Chemical Company (Ann Arbor, MI, USA) and 2-ME was purchased from Selleck Chemicals (Houston, TX, USA).

Human CCA cell lines, including H1 (OCUCh-LM1-H1)[9 (link)], HuCCT1[10 (link)], SSP-25[11 (link)], and ETK1[12 (link)] cell lines were kindly provided by Dr. Jack Wands. TFK-1 and RBE were purchased from RIKEN Cell Bank (Tsukuba, Japan). The non-malignant human bile duct cells hBD was purchased from Celprogen Inc (Torrance, CA, United States, Catalog Number: 36755-12). CCA cell lines were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS; Gibco^TM, Thermo Scientific, Waltham, MA, United States; Cat# 10099141C), 100 μg/mL streptomycin, and 100 U/mL penicillin (HyClone™, Logan, UT, United States). The hBD cell line was grown in DMEM containing 10% FBS, 100 μg/mL strep. All cell lines were authenticated by STR and were negative for contamination of mycoplasma recently. Arcyriaflavin A, Flavopiridol, Roscovitine, and γ-secretase inhibitor, DAPT (N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-s-phenylglycinet-butyl ester) were all purchased from Cayman Chemical (Ann Arbor, MI, United States).

In vitro differentiated neutrophils from ECOMG cells were used as a model system to study nuclear architecture in response to calcium influx in neutrophils. ECOMG cells were cultured and differentiated as previously described (Zhu et al. 2017 (link)). For inhibitors, neutrophils were treated with 10 µM Triptolide (Cayman), 100 µM 5,6-dichloro-1-β-D-ribofuranosyl-1H-benzimidazole (Cayman) or 10 µM flavopiridol (Cayman) for 30 min, or 2 µM FK506 for 2 h before activation. Primary B cells were treated with 2 µM FK506 (Cayman) for 2 h before activation. Inhibitors were kept in culture during activation time course. Acute degradation was induced by treating the cells with 0.5 µM dTAG-13 (Tocris) before activation. dTAG-13 was kept in culture during activation time course. The time series degradation experiments were performed by inducing protein degradation at the beginning of the time course and harvesting the samples at different time points. RPMI-1640 (Gibco) contains 0.42 mM calcium. Additional CaCl₂ was supplied to the medium before activation to a final concentration of 1 mM. A23187 was purchased from Sigma and Cayman. Neutrophils were activated either using fast activation conditions (20 µM A23187 for 15 min), or using slow activation conditions (5 µM A23187 for 4 h). These two activation conditions were used throughout the manuscript unless otherwise mentioned.