Axiovert 200m apotome 2

HepG2 and HAT22/VGH were seeded on slides (4 × 10⁵ cells/slide) and cells were fixed with 4 % paraformaldehyde, permeabilized with 0.4 % Tryton X-100, and blocked with 5 % goat serum (Invitrogen Life Technologies, Waltham, MA, USA) in PBS containing 1 % BSA. Slides were incubated with rabbit monoclonal anti-C/EBP-β and anti-PAR2 (Abcam-Cambridge UK) antibodies for 1 h at room temperature, followed by incubation with the anti-rabbit Alexa Fluor 546 (Invitrogen Life Technologies, Waltham, MA, USA) secondary antibody. Cellular nuclei were counterstained with Dapi (Merck KGaA, Darmstadt, Germany). Slides were mounted with ELVANOL (Merck KGaA, Darmstadt, Germany) and observed under a fluorescence microscope (Axiovert 200M-Apotome.2, Carl Zeiss MicroImaging GmbH, Göttingen, Germany).

Adherent primary human monocytes and THP-1 cells (3 × 10⁵ cells/slide) were seeded on slides and cultured for 24 h. In some experiments, the Wnt inhibitor ICG-001 (5 uM for 24 h, kindly provided by Dr. M. Cadamuro, University of Padua, Padua, Italy), was used to confirm the activation of the Wnt pathway [19 (link)]. Cells were fixed in 4% paraformaldehyde and permeabilized with 0,4% Triton X-100 and blocked with 5% goat serum (Invitrogen Life Technologies, Waltham, MA, USA) in PBS containing 1% BSA. Slides were incubated with the primary antibodies (SerpinB3, LRP1, LRP6, Wnt1, Wnt7a, β-catenin, axin, cMyc) for one hour at room temperature and then incubated for 1 h at room temperature with a mouse or a rabbit secondary antibody, based on the origin of the primary antibody. The cellular nuclei were counterstained by DAPI (Merck KGaA, Darmstadt, Germany), mounted with ELVANOL Merck KGaA, Darmstadt, Germany), and observed under a fluorescence microscope (Axiovert 200M-Apotome.2, Carl Zeiss MicroImaging GmbH, Göttingen, Germany). The characteristics of the primary and secondary antibodies used in the study are reported in Supplementary Table S1.

HepG2/control cells were seeded on slides (4 × 10⁵ cells/slide) and cultured for 24 h. Cells were treated as follows: (a) overnight incubation with PBS as a negative control, (b) overnight incubation with 100 ng/mL of SerpinB3 recombinant protein as a positive control, (c) pre-treatment with 5 g/mL of the anti-LRP-1 85 kDa monoclonal antibody or with a generic mouse Ig for one hour followed by overnight incubation with 100 ng/mL of SerpinB3 recombinant protein, and (d) overnight incubation with 5 g/mL of RAP and 100 ng/mL of SerpinB3 recombinant protein. Cells were then fixed with 4% paraformaldehyde, permeabilized with 0.4% Tryton X-100, and blocked with 5% goat serum (Invitrogen Life Technologies, Waltham, MA, USA) in PBS containing 1% BSA. Slides were incubated with polyclonal anti-vimentin and anti-snail antibodies and with monoclonal anti-E-cadherin antibodies for 1 h at room temperature, followed by incubation with the Alexa-Goat 546 and 488 secondary antibodies, respectively. Cellular nuclei were counterstained with Dapi (Merck KGaA, Darmstadt, Germany). Slides were mounted with ELVANOL (Merck KGaA, Darmstadt, Germany) and observed under a fluorescence microscope (Axiovert 200M-Apotome.2, Carl Zeiss MicroImaging GmbH, Göttingen, Germany).