1 25 dihydroxyvitamin d3

The HL-60 human myeloblastic leukemia cells were originally an early pass of cells derived from the original patient samples, generously gifted by Dr. Robert Gallagher and continuously maintained in this laboratory as previously published [23 (link)]. The cells used were certified as mycoplasma free HL-60 by Bio-Synthesis, Lewisville, TX, in August 2017. The HL-60 cells were grown in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 5% fetal bovine serum (Hyclone, Logan, UT) and 1x antibiotic/antimycotic (Sigma, St. Louis, MO) in a 5% CO2 humidified atmosphere at 37°C. Cells were continuously cultured in the constant exponential growth phase, not exceeding 2.0 x10⁶, with cell viability exceeding 95% by 0.2% trypan blue (Invitrogen, Carlsbad, CA) exclusion dye via hemocytometry. Experimental cultures were initiated at a density of either 0.1 x 10⁶ for 24, 48, and 72 hour time points for flow cytometry, or 0.2 x 10⁶ for 8 hour flow cytometry time points and 48 hour lysate collection. All cells and lysate collected used the same treatment conditions of 0 μM or 10 μM RRD-251 Hydrochloride (Sigma) and 0 μM or 0.5 μM 1,25-dihydroxyvitamin D3 (Cayman, Ann Arbor, MI). Cells were harvested at 8, 24, 48, and 72 hours post-treatment. Three biological replicates of each experiment were performed unless stated otherwise.

Dulbecco’s modified Eagle’s medium (DMEM), α-Minimum Essential Medium (α-MEM), antibiotic-antimycotic (anti-anti), fetal bovine serum (FBS), and phosphate-buffered saline (PBS) were purchased from the Life Technologies (Grand Island, NY). OxPAPC, CLI-095, and monophosphoryl lipid A (MPLA) were purchased from the Invivogen (San Diego, CA). Fibroblast growth factor-2 (FGF-2) was obtained from the R&D System (Minneapolis, MN). Tryptic soy broth was purchased from the Acumedia (Lansing, MI). Agar was obtained from the Fisher Scientific (Pittsburgh, PA). Human LL-37 ELISA kit was purchased from the Hycult Biotech (Plymouth Meeting, PA). LL-37 peptide was purchased from the AnaSpec (Fremont, CA). GW0742, trypsin IIS, and ampicillin (Amp) were purchased from the Sigma-Aldrich (St. Louis, MO). Collagenase type I was purchased from the Worthington (Lakewood, NJ). 1,25-dihydroxy vitamin D₃ was purchased from the Cayman Chemical (Ann Arbor, MI).

All cells were cultured in RPMI1640 with 10 or 20% FBS. HL60 (acute myelogenous leukemia cell line), U937 (histiocytic lymphoma cell line), KG-1a (acute myeloid leukemia cell line), NB4 (acute promyelocytic leukemia cell line), Nalm-6 (acute B acute lymphoblastic leukemia cell line), Molt-4 (acute T acute lymphoblastic leukemia cell line), Kasumi-1 (acute myelogenous leukemia cell line), and K562 (chromic granulocytic leukemia cell line) were cultured at the Fujian Institution of Hematology. HL-60, U937, and KG-1a cell lines were cultured at the BIDMC and were gifts from Dr. Daniel G Tenen. Antibodies against various proteins were obtained from the following sources: Pin1 was previously described [36 (link)]; β-actin from Sigma; Cyclin D1 (DCS-6) from Biolegend; NF-κB/p65 (D14E12, 8242) from Cell Signaling Technology; β-catenin from BD biosciences; and Rab-2A (15420-1-AP) from Proteintech Group. All-trans retinoic acid (ATRA) were purchased from Sigma, 1,25-dihydroxyvitamin D3 were purchased from Cayman Chemicals, and ATRA-releasing pellets were from Innovative Research of America.