Incellis cell imager

PSCs or tumor cells were seeded in eight-well slide dishes (Ibidi, Gräfelfing, Germany) at 5 × 10⁴/well and incubated for 24 h. Then cells were fixed with 4% formaldehyde and blocked with normal horse serum for 30 min. Slides were incubated with primary antibody (anti-αSMA, 1:200, Abcam, Cambridge, UK; anti-IL-17RB, 1:200, ProteoTech, Attendorn, Germany; anti-vimentin, 1:200, Cell Signaling Technology, Danvers, MA, USA; anti-ATP5β, 1:500, Abcam, Cambridge, UK; anti-Tom20, 1:400, Abcam, Cambridge, UK) overnight at 4 °C. Subsequently, cells were incubated with Alexa 546-conjugated anti-mouse IgG (Thermo Fisher Scientific, Waltham, MA, USA) or Alexa 546-conjugated anti-rabbit IgG (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature. Finally, nuclear DNA was counterstained with DAPI (4′,6-diamidino-2-phenylindole, 1 mg/mL) for 15 min. After washing with PBS, images were acquired using a fluorescence microscope (to generate Figure 1C, InCellis Cell Imager, Bertin Instruments, Montigny-le-Bretonneux, France) or a confocal microscope (to generate Figure 6A, Zeiss Meta 710, Zeiss, Jena, Germany).

A total of 30,000–50,000 Huh-7 cells, 90,000 HepG2 cells, and 50,000 SNU-449 cells were seeded per a well in a Culture-Insert 2 well (80209, ibidi Technologies, Gräfelfing, Germany) in a 24-well plate; 24 h after, the culture insert was removed and cells were washed 2 times with PBS. Complete DMEM medium was added to each well and the wound area was imaged at indicated time points in the same areas. Cells were imaged on InCellis Cell Imager (Bertin Technologies SAS, Montigny-le-Bretonneux, France). The percentage of free wound area was calculated using the MRI Wound Healing Tool in ImageJ software.

To investigate cell proliferation and percentage of cell population in the S phase of the cell cycle we used EdU-Click 555 (BCK-EdU555, baseclick GmbH, Munchen, Germany) following the manufacturer’s protocol. Cells were incubated with the reagent for 2 h. Cells were imaged on InCellis Cell Imager (Bertin Technologies SAS, Montigny-le-Bretonneux, France).