Penicillin streptomycin stock solution

Human multiple myeloma cell lines LP‐1, OPM‐2, RPMI‐8226, and ANBL‐6 cells, and the human stromal cell line HS‐5, were obtained from the American Type Culture Collection (ATCC, Molsheim, France). The XG‐2 cell line was kindly provided by J Moreaux (University of Montpellier, Montpellier, France). Myeloma cell lines and stromal cells were cultured in RPMI‐1640 and DMEM medium (Lonza, Basel, Switzerland) respectively, supplemented with 2 mm l‐glutamine (Thermo Fisher Scientific, Waltham, MA, USA), 1% penicillin/streptomycin stock solution (Thermo Fisher Scientific), and 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) at 37 °C in 5% CO₂. ANBL‐6 and XG‐2 cells were supplemented with 2 ng/ml recombinant IL‐6 (R&D Systems, Abingdon, UK). The authenticity of the human multiple myeloma cell lines was regularly confirmed by short tandem repeat analysis. The cell lines were checked monthly for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza).

HDL-c isolation with sequential potassium bromide (KBr) density ultracentrifugation as described [50 (link)]. To the plasma was added 1% antibiotics (penicillin/streptomycin stock solution, Thermo Fisher Scientific, Waltham, MA, USA), 0.5 mM EDTA (Thermo Fisher Scientific, Waltham, MA, USA), protease inhibitor cocktail (cOmplete^TM, Roche, Basel, Switzerland) to avoid ex vivo oxidation and degradation. Purified HDL-C was dialyzed against a degassed solution of Buffer A (20 mM Tris-HCl plus 0.5 mM EDTA) on a shaker at 4 °C with five buffer changes (once/day).

A murine renal tubular epithelial cell line (TEC; Fuheng Cell Center, Shanghai, China) was cultured in Dulbecco’s modified Eagle’s medium–nutrient mixture F-12 (HyClone Laboratories, Logan, UT, USA) with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin–streptomycin stock solution (Thermo Fisher Scientific). To simulate I/R injury in vitro, we used a model of hypoxia/reoxygenation (H/R). TEC cells were exposed to a hypoxia incubator chamber (STEMCELL Technologies, Vancouver, BC, Canada) with an anoxic mixture gas (95% N₂ and 5% CO₂) for 12h at 37 °C. Then, cells were returned to complete growth medium and placed in a normoxic chamber (37 °C, 5% CO₂) for 2 h of reoxygenation.
Lats2 was overexpressed via transfecting with a lentivirus incorporating the Lats2 recombinant plasmid (Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin, GENECHEM, Shanghai, China), referred to as LV-Lats2. LV-NC, a lentivirus incorporating an empty plasmid, served as a negative control. Viral particles were removed 16 h later after transfection, and cells were replenished with fresh TEC medium. Puromycin selection commenced 24 h later with the addition of 2 μg/mL puromycin. Surviving cells were isolated into single cell clones. The cells after treatment were lysed in RIPA lysis buffer (Beyotime, Shanghai, China) for western blotting analysis.

Stage VI X. laevis oocytes were obtained as previously described [71 (link)]. Oocytes were maintained in L-15 medium solution (Sigma-Aldrich Cat# L4386) supplemented with HEPES (Sigma-Aldrich Cat# H4034), 0.1% (v/v) of penicillin/streptomycin stock solution (ThermoFisher Scientific Cat# 15140–122) and 0.1% (v/v) of gentamycin (EMD Millipore Cat# 345814-1GM) at pH 7.6.

Cells were plated in flat-bottomed 24-well plates at a density of 2.5 x 10⁶ cells (for RBC-lysed total bone marrow cells or total splenocytes) in 500 μl of complete cell medium, or in flat-bottomed 96-well plates at a density of 50,000 cells (for HSPC-derived macrophages) in 200 µl of complete cell culture medium (RPMI 1640 medium supplemented with 2 mM L-glutamine, 5% heat-inactivated fetal bovine serum, and 1% penicillin-streptomycin stock solution [Gibco]). Whole blood was diluted 1:2 in complete cell culture medium at a final volume of 200 μl and plated in flat-bottomed 96-well plates. To prevent potential fungal growth, 0.5 μg/ml amphotericin B was added to the cultures of total bone marrow cells, total splenocytes and blood from infected animals. Cells were challenged with 100 ng/ml of Pam₃CSK₄, 100 ng/ml of ultrapure Salmonella minnesota LPS (all from In vivogen) or 25 x 10⁶ inactivated C. albicans ATCC 26555 yeasts for 24 h, and cell-free supernatants were then harvested and tested for cytokine release using commercial enzyme-linked immunosorbent assay (ELISA) kits [TNF-α and IL-6 (eBioscience)]. Unstimulated cells served as negative controls. Triplicate samples were analyzed in each assay.

Splenocytes were obtained from control or Pam₃CSK₄-treated mice as described elsewhere (Murciano et al., 2007 (link); Yáñez et al., 2011 (link)). Briefly, total spleen cells were obtained by collagenase D treatment of the organ, erythrocytes were lysed using ammonium chloride buffer (BD FACS™ lysing solution), and cells were washed and counted.
Resident peritoneal macrophages were harvested by instilling and withdrawing 10 ml of complete cell culture medium: RPMI 1640 medium supplemented with 2 mM L-glutamine, 5% heat-inactivated fetal bovine serum, and 1% penicillin-streptomycin stock solution (Gibco, Barcelona, Spain). Cells were washed once with the same medium, and plated at a density of 1.5 x 10⁵ cells in 200 μl of medium per well, in a 96 well tissue culture plate. Peritoneal macrophages were allowed to adhere for 5 h at 37°C in a 5 % CO₂ atmosphere, the non-adherent cells were removed, and the adherent macrophages were cultured for 72 h prior to be challenged with the indicated stimuli.

Lipoproteins were isolated with sequential potassium bromide density ultracentrifugation as described [42 (link)]. The plasma was obtained from freshly collected whole blood samples, and 1% antibiotics (penicillin/streptomycin stock solution, Gibco), 0.5 mM EDTA (Thermo Fisher Scientific, Waltham, MA, USA), and a protease inhibitor cocktail (cOmplete, Roche, Sigma-Aldrich, St. Louis, MO, USA) were added to avoid ex vivo oxidation and degradation. LDL-C particles were isolated by using sequential potassium bromide density ultracentrifugation. Purified LDL-C was dialyzed against a degassed solution of 20 mM Tris-HCl and 0.5 mM EDTA at 4 °C with five buffer changes (once/day).