Panobinostat

Total bone marrow cells from WT and GPR109A^−/− mice were isolated by flushing the femora and tibiae. The cells were plated overnight at 37 °C with 5.5% CO₂ in alpha minimum essential medium (αMEM), supplemented with 10% FBS and 1% penicillin/streptomycin (Gibco, Carlsbad, CA, USA), and 40 ng/ml macrophage colony stimulating factor (M-CSF; R&D Systems). Next day, nonadherent cells were collected and cultured in osteoclast medium with 40 ng/ml M-CSF and 50 ng/ml receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL; R&D Systems) in 24-well plates (for tartrate-resistant acid phosphatase (TRAP) staining, immunofluorescence), 12-well plates (for RNA isolation), or 6-well plates (for Western blotting) at a density of 1 × 10⁶ cells/ml at 37 °C and 5.5% CO₂. The medium was changed every 3 d. In SCFA experiments, preosteoclasts in C3 and C4 groups were further stimulated on day 1 of culture with the following stimulants: 5 mmol/L propionate, 1 mmol/L butyrate (Sigma-Aldrich), 40 ng/ml IL-1β (MedChemExpress), 50 nmol/L TSA (MedChemExpress), 25 nmol/L Panobinostat (Sigma-Aldrich), 100 μmol/L 4-CMTB (Sigma-Aldrich), 1 mmol/L niacin (MedChemExpress), or 25 μmol/L AR42062 (Sigma-Aldrich). Then, fully differentiated osteoclasts (days 5–7) were collected for PCR, Western blotting, TRAP staining, and immunofluorescence staining.

Human lung cancer cell lines A549 (ATCC Cat#: CCL-185), NCI-H23 (ATCC Cat#: CRL-5800), NCI-H1299 (ATCC Cat#: CRL-5803), NCI-H441 (ATCC Cat#: HTB-174), HCC827 (ATCC Cat#: CRL-2868), NCI-H1975 (ATCC Cat#: CRL-5908) and NCI-H2172 (ATCC Cat#: CRL-5930) were obtained from American Type Culture Collection (ATCC) and cultured as instructed. The human lung cancer PC14 cell line was kindly provided by Mr. Yu (The Shanghai Cancer Institute, China). The cancer cells were maintained in Roswell Park Memorial Institute (RPMI)1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (all from Invitrogen). Cells were maintained under a standard gas atmosphere of humidified air/5% CO₂.
Cell growth was measured using CellTiter-Glo (Promega). The cells were seeded in 96-well plates in 100 μl of medium with different drug doses. Cisplatin (C2210000, Sigma Aldrich) and panobinostat (EPI009, Sigma Aldrich) were dissolved in dimethyl sulfoxide (DMSO). After the cells were cultured for 48 hours, 100 μl of CellTiter-Glo reagent was added to each well to measure cell growth according to the manufacturer's instructions.

Indicated chemicals were obtained from companies (in parentheses): brefeldin A (Sigma-Aldrich), golgicide A (Sigma-Aldrich), AG1478/tyrphostin (Sigma-Aldrich), Erastin (MedChem Express), ferrostatin-1 (Sigma-Aldrich), liproxstatin-1 (Sigma-Aldrich), NOX1/4 inhibitor (GKT137831, Cayman Chemical), Sulfasalazine (Sigma-Aldrich), Sorafenib (Sigma-Aldrich), RSL3 (MedChem Express), Glutathione (Sigma-Aldrich), N-acetyl-cysteine (Sigma-Aldrich), Ciclopirox olamine (CPX, Sigma-Aldrich), LOXi (PD-146176, Santa Cruz Biotechnology), Trolox (Sigma-Aldrich), Prankulast (Biomol), tunicamycin (Sigma-Aldrich), Erastin (MedChemExpress), BSO (Santa Cruz Biotechnology), cisplatin (Santa Cruz Biotechnology), doxorubicin (Sigma-Aldrich), 2-ME (Sigma-Aldrich), PPG (Sigma-Aldrich),
Nutlin-3 (Sigma-Aldrich), vildagliptin (Sigma-Aldrich), Q-VD-OPh hydrate (Sigma-Aldrich), bafilomycin A (Sigma-Aldrich), nocodazole (Sigma-Aldrich), panobinostat (Sigma-Aldrich), doxorubicin (Sigma-Aldrich). AMF-26 was synthesized by Merck KGaA^{23 (link)}.

The HDAC inhibitors Panobinostat (Sigma) and Vorinostat (Sigma) and the DOT1L inhibitors Pinometostat (EPZ-5676) (Selleck Chemicals) and SGC-0946 (Selleck Chemicals), and DMSO (Sigma) were diluted in culture medium. First, the inhibitors were diluted in culture medium to a stock concentration that was twice as high as the highest concentration tested. The stock concentration was further diluted to obtain to the desired range of concentrations. Twenty-thousand cells were plated per well in a flat-bottom culture-treated 96 wells plate, in 50 µl culture medium and 50 µl culture medium with inhibitor was added. Four hours before measuring cell viability, Cell Titer Blue (Promega) was added to the wells and cells were incubated in the dark at 37°C under 5% CO₂ conditions. After 4 hours, fluorescence was measured on the EnVision Multilabel Reader (Perkin Elmer). In order to calculate cell viability, values for fluorescence intensity of each condition were normalized against those of untreated cells. Each biological replicate represents an average of three technical replicates of an independent experiment.

Jurkat T cells (Clone E6-1) were obtained from the American Tissue Culture Collection. J-Lat 10.6 cells were obtained from the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH (contributed by Dr. Eric Verdin; Jordan et al., 2003 (link)). Cells were cultured in R10+ media [RPMI 1640 with HEPES and L-Glutamine, 10% fetal bovine serum, 100 U of penicillin/ml, and 100 µg of streptomycin/ml (Sigma)] and incubated at 37°C and 5% CO₂.
HDAC-Glo I/II Assay kits were obtained from Promega. Panobinostat was obtained from Sigma-Aldrich. Compounds 15, 16 and 17, and 18 were purchased from Sellick Chemical, Enamine, and Molport, respectively. Serpulanine A was gratefully obtained as a gift from Dr. Raymond J. Andersen (University of British Columbia). Compounds stocks were diluted in DMSO and stored at minus 20°C until use.