Normocin

The MDA-MB-231 cell line is well established as a model for cancer research related to gene regulation. The MDA-MB-231 cell line is also being used for lipid research. Human MDA-MB-231 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), and then grown in DMEM culture medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 2 mM glutamine (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 1 mM sodium pyruvate, 10 mM HEPES, 100 μg/mL Normocin, 50 U/mL penicillin and 50 μg/mL streptomycin (P/S; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and incubated at 37 °C (with humidity) in 5% CO₂.

Peripheral blood mononuclear cells (PBMCs) were isolated from non-diabetic and diabetic volunteers in EDTA vacutainer tubes. PBMCs were isolated using the Histo-Paque density gradient method and Sepmate Tube (StemCell Technologies, Vancouver, CN, Canada) [10 (link)]. To generate monocytic cells, PBMCs were plated in either 6-well or 24-well plates (Costar, Corning Incorporated, Corning, NY, USA) at 3 × 106 cells/well and cultured in starvation media of RPMI-1640 culture medium (Gibco, Life Technologies, Grand Island, NY, USA) supplemented with 2 mM glutamine (Gibco), 1 mM sodium pyruvate, 10 mM HEPES, 100 ug/mL Normocin, 50 U/ml penicillin, and 50 μg/mL streptomycin (P/S; (Gibco). Cells were incubated at 37 °C (with humidity) in 5% CO₂ for 3 h. Non-adhered cells were removed, and monocytes adhered to the plate were washed with serum-free culture medium and later incubated for 24 h in RPMI with 2% fetal bovine serum.

THP-1 cells were obtained from the American Type Culture Collection (ATCC) and cultured according to their recommendation^{17 (link)–19 (link)}. In brief, cells were maintained in RPMI-1640 culture medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 100 ug/ml Normocin, 50 U/ml penicillin, and 50 μg/ml streptomycin (Gibco, Life Technologies, Grand Island, NY, USA). For experimentation, cells were plated in 12-well plates (Costar, Corning Incorporated, Corning, NY, USA) at 1 × 10⁶ cells/well (unless indicated otherwise). Cells were then stimulated for 24 h with 10 ng/ml TNFα (R&D Systems, Minneapolis, MN, USA) or 0.1% BSA as vehicle control. All cultures were incubated under recommended cell culture conditions at 37 °C (with humidity) in 5% CO₂. At the endpoint of the experiment, cells were harvested for RNA isolation, and the conditioned medium was used for the determination of MMP-9 secreted protein. For NF-kB/AP-1 reporter cells, cells were cultured in complete RPMI medium with the addition of zeocin (200 µg/ml) as a selective factor (InvivoGen, San Diego, CA, USA).

Human monocytic THP-1 cells were purchased from American Type Culture Collection (ATCC) and grown in RPMI-1640 culture medium (Gibco, Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco), 2 mM glutamine (Gibco), 1 mM sodium pyruvate, 10 mM HEPES, 100 ug/mL Normocin, 50 U/mL penicillin, and 50 μg/mL streptomycin (P/S; Gibco) [14 (link)]. Cells were incubated at 37 °C with humidity in 5% CO₂.

Adult human HSCs isolated from human liver tissue samples were obtained from ScienCell Research Laboratories. AICAR (2840, Tocris) was dissolved in water while GSK621 (S7898, Selleckchem) was dissolved in DMSO. 2DG (D8375, Sigma) was dissolved in water. Cells were grown in a humidified 5% CO₂ atmosphere at 37 °C in Dulbecco’s Modified Eagle Medium (DMEM with 4.5 g/L glucose) with 10% fetal bovine serum (FBS), 1% Penicillin/Streptomycin (P/S), and 1% Normocin (ant-nr-1, InvivoGen). For glucose starvation experiments, glucose-free DMEM (11966025, Gibco) with 10% FBS, 1% P/S, and 1% Normocin was used.