Axiovert 40c phase contrast microscope

Signal transduction inhibitors, BAY 87‐2243 (HIF‐1α inhibitor, Selleck Chemicals, Houston, TX, USA), U0126 (ERK1/2 inhibitor, Cell Signaling Technology), and DMOG (PDH inhibitor, Sigma‐Aldrich) were resuspended in DMSO (Wako, Tokyo, Japan) and added to serum‐free medium at the indicated concentrations. BAY 87‐2243 was used at concentrations of 50 and 100 μm, U0126 at 0, 10, 20, and 40 μm concentration, and DMOG at 0 and 2000 μm concentration. The control medium contained the same volume of DMSO. SMGs were treated with BAY 87‐2243 and U0126 for 4 h and cultured for an additional 4 or 30 h under hypoxic conditions. SMGs that were treated with U0126 were cultured for a further 4 or 30 h under normoxic conditions as a control group. SMGs that were treated with DMOG were cultured for 10 h. For immunoblotting, SMGs were collected 4 h after hypoxia treatment. Photographs were taken at 0 and 30 h after hypoxia treatment using a digital SLR camera (Fuji FinePix, Fuji, Tokyo, Japan) fitted onto an Axiovert 40C phase‐contrast microscope (Carl Zeiss).

BC formed in HepaRG cell cultures appear as characteristic refractive structures, corresponding to bright/white objects on phase-contrast photographs, as previously reported^{21 (link)}, whereas cells appear as dense/black elements, containing nuclei recognizable by their round/delimited shape and differential density. After capturing images using an Axiovert 40 C phase contrast microscope (Carl Zeiss, Le Pecq, France) and adjusting brightness parameters to optimally distinguish between white and black densities, bright objects reflecting BC as well as round/delimited nuclei-related objects were segmented by adjusting the shape and area parameters to exclude non-corresponding objects, using a dedicated in-house macro-program based on ImageJ 1.48 software^{22 (link)}. BC numbers and areas as well as nuclei numbers were next determined from 3 zones per condition counting from at least 3 independent experiments. In some experiments, BC were additionally visualized through immuno-labelling of BC with anti-P-glycoprotein (P-gp) or anti-multidrug resistance-associated protein (MRP) 2 antibodies, as reported below, and their number was quantified using ImageJ and relatively to 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei.