Keratinocyte sfm 1 medium

Human HK-2 cells was cultured in DMEM and Keratinocyte-SFM (1×) medium, (Life Technologies Green Island NY) medium respectively. When the cells on the adhesion reagent reached 70% confluence, 10 ng/ml recombinant human TGFβ1 for 48 h was placed in the serum diluted medium with or without DCA (100 nM) and 2-DG (100 nM) pre-incubation for 2 h.

HK-2 cells, which are human kidney proximal tubular cells, were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). HK-2 cells were maintained in Keratinocyte-SFM (1×) medium (Life Technologies Green Island NY).
Autophagic flux was determined by analyzing LC3-II turnover by preventing lysosomal degradation using CQ (Sigma-Aldrich, St. Louis, MO, USA). LC3-II expression was evaluated by western blotting using anti-LC3 antibodies (Cell Signaling Technology, Danvers, MA, USA) [37 (link), 46 (link)]. LC3-flux was calculated by subtracting the densitometry value of normalized LC3-II in the sample treated with CQ by the value in the sample treated without CQ [37 (link), 46 (link)]. LC3-II flux was expressed relative to the respective controls [37 (link), 46 (link)]. HK-2 cells were cultured in Hanks’ Balanced Salt Solution (HBSS), amino acid-free solution, with SAM (0.5 mM, 2 mM or 4 mM) or vehicle for 2 hours, and then 50 μM CQ or vehicle was administered for 1 hour before collection of samples.