Minimum essential medium (mem)

Human fetal lung fibroblasts, MRC5 (ATCC, Manassas, VA), grown in minimal essential medium (MEM) (Wisent, Montreal, QC, Canada) with 10% (v/v) FBS and 1% penicillin/streptomycin, were used between passages 5–15, such that studies were completed before the MRC‐5 cells enter senescence. CRISPLD2 shRNA constructs, and a scrambled shRNA used as a negative control, were cloned into a pTRIPZ lentiviral doxycycline (DOX)‐inducible vector (Thermo, Ottawa, ON, Canada). Lentivirus was produced by transfecting 293T cells with shRNA constructs pVSV‐G and psPAX2 using lipofectamine 2000 (Life technology, Burlington, ON, Canada). MRC‐5 cells were infected with lentivirus and puromycin (0.5 μg/mL) (Wisent) was added after 48 h to select stable clones. The characterization of MRC5^CRISPLD2KD has been described in detail previously by Zhang et al. (2015). Suppression of CRISPLD2 protein levels in MRC5^CRISPLD2KD cells after 4 days DOX induction was confirmed by western blot analysis. CRISPLD2 was suppressed by DOX induction only in MRC5^CRISPLD2KD and not in MRC5^control cells. All further experiments were carried out using 2 μg/mL DOX induction for 4 days in MRC5^CRISPLD2KD cells. The suppression of CRISPLD2 mRNA in MRC5^CRISPLD2KD cultures was repeatedly confirmed prior to each set of experiments.

MLO-Y4 osteocytes (courtesy of Dr. Bonewald, Indiana University School of Medicine) are cultured in growth media composed of 2.5% calf serum (CS, Gibco, USA), 2.5% fetal bovine serum (FBS, Gibco, USA), 1% penicillin-streptomycin (PS, Gibco, USA), and 94% Alpha Minimum Essential Medium (MEM) (WISENT, Canada). Cells are seeded during passage 29 at 10⁵ cells per 100 mm diameter collagen-coated (0.15 mg/ml Type I collagen (Corning, USA)) culture dishes and expanded until they achieve 80% confluency. The cells are then transferred onto collagen-coated experimental slides (75x25 mm) at a density of 500k cells per slide for overnight incubation till they reach 80% confluence again before imaging. MLO-Y4 cells are passaged between P29 and P35 while maintained in an incubator at 37 ˚C and 5% CO₂. Cell death was quantified using Trypan Blue Stain (Sigma-Aldrich, USA) and counted under a standard light microscope.

The glucose content of the medium was measured by the glucose oxidase-peroxidase method using a commercial kit (Rsbio, Shanghai, China). HepG2 and LO2 cells were seeded as described above and cultured in the presence of insulin (Sigma-Aldrich, St. Louis, MO, USA) or DEX (Jiangsu Hengrui Medicine Co., Lianyungang, China). The medium was changed to serum- and Phenol Red-free DMEM or MEM (Wisent) for 12 h, and the mixture of R1 and R2 in the kit was added to the supernatant at a ratio of 1:200 according to the instructions, followed by incubation at 37 °C for 15 min. OD₅₀₅ was measured and a standard curve of glucose content was generated. Glucose consumption was calculated as the difference in the glucose contents of experimental and blank wells [17 (link), 18 (link)].