Vitamin d3

Kasumi-1 cells (ATCC, Manassas, VA) were cultured in RPMI-1640 (Lonza, Walkersville, MD) supplemented with 10% FBS (Sigma, St Louis, MO) and 1% Penicillin/Streptomycin (Invitrogen, Carlsbad, CA). HL60 cells (ATCC) were cultured in MEMα (Invitrogen) supplemented with 20% FBS and 1% Penicillin/Streptomycin. All cells were maintained at 37 °C and 5% CO₂ at concentrations of 10⁶ cells/mL. When indicated, cells were also cultured with the following: 10 uM all-trans retinoic acid (Sigma), 50 uM indomethacin (Sigma), 100 uM cytarabine (Abcam, Cambridge, MA), 0.1 uM vitamin D3 (Sigma), 10 nM recombinant human Wnt3a (R&D Systems, Minneapolis, MN), or 10 nM ICG-001 (Selleck Chemical, Houston, TX). Non-targeting scramble control and TLE4-specific shRNA constructs were developed and delivered to cells via lentiviral delivery as previously described [10 (link)]. The shRNA used and their target sequences were: shTLE4 1 (AGTGATGACAACTTGGTGG) and shTLE4 2 (GGCATTATGTCATGTATTA). Data in figures were obtained using shTLE4 2 unless otherwise indicated. Infected cells were identified by GFP fluorescence detected using FACS LSRII or selected for via cell sorting with FACS Aria (BD, San Jose, CA). Full-length TLE4 (a.k.a.KIAA1261 kind gift of Dr. Ohara [18 (link)]) cDNAs were cloned into the MSCV-IRES-GFP retroviral vector.

Human monocytic cell line THP-1 (ATCC TIB-202), and dual THP-1 (Invivogen) cell lines were used for our experiments. The latter expresses two inducible reporter constructs for interferon regulatory factors (IRFs) as well as for NF-κB. The activities of these reporters were measured through a colorimetric assay for the secretory embryonic alkaline phosphatase (SEAP) reporter gene that is linked to NF-κB activation using the Quanti-blue substrate (InvivoGen), and a Luciferase reporter gene linked to IRFs using the QuantiLuc substrate (Invivogen), according to manufacturer’s instructions. Measurement of NF-kB and IRF activation were expressed as a response ratio for each stimulus relative to the reporter activity in unstimulated cells.
THP-1 cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 0.05 mM 2-mercaptoethanol, 100 U/ml Penicillin and 100 μg/ml Streptomycin at 37 °C and 5% CO₂. Differentiation and pre-treatment of THP-1 cells were done with 100 ng/mL phorbol ester 12-O-tetradecanoylphorbol-13-acetate (PMA, Sigma-Aldrich) or 0.5μM 1,25-dihydroxyVitamin D3 (Vitamin D3, Sigma-Aldrich) for 72 hours. For some experiments, cells were also activated with 100 ng/mL LPS plus 20 ng/mL IFN-γ for 8 hrs. Reagents were used in working dilutions (1/100) in PBS from stock solutions in DMSO.