Ctr 6000 microscope

For dominant-negative overexpression assays, 3 × 10⁴ hTERT RPE-1 cells (ATCC) were incubated overnight on 13 mm diameter cover slips (Knittel, Braunschweig, Germany) into 24-well plates (Corning incorporated Costar^®). The cells were transfected using Lipofectamine^® 3000 (Thermo Fisher Scientific, Carlsbad, CA). For overexpression, pEGFP-C1 plasmids encoding chicken brain MyoVa-mGTD (residues 1377-1830; CAA77782.1) or MyoVa-GTD (residues 1423–1830; CAA77782.1) fused to EGFP as well as the empty plasmid encoding only EGFP as control were used. MyoVa-mGTD was identical to the one described in19 (link)20 (link), except that the insert, previously in pS65T-C1, was transferred to pEGFP-C1. MyoVa-GTD (residues 1423–1830; CAA77782.1) was PCR amplified using as template a chicken brain MyoVa full tail cDNA clone53 (link), and the PCR product was inserted into pEGFP-C1 plasmid in fusion with EGFP. The transfections were carried out in a final volume of 500 μL medium following the manufactures’ instructions. After 72 h of incubation for overexpression of EGFP or EGFP-MyoVa-GTD, cells were washed with PBS, fixed with 4% (v/v) paraformaldehyde pH 7.4 for 15 min, washed with PBS and processed for immunofluorescence and data acquisition as described above. For these assays, cells were imaged using a Leica CTR 6000 microscope (Leica).

Photography of fluorescent cells were carried out in an inverted Leica CTR 6000 microscope equipped with a digital camera Leica DC500 or Leica DM IRB microscope equipped with a digital camera Leica DFC350FX (Leica Microsystems, Wetzar, Germany). In-gel fluorescence scanning was performed on a Typhoon FLA 9500 scanner (GE Healthcare, Little Chalfont, UK) using 432 nm excitation laser and 610 BP40 emmision filter.

Paraffin embedded liver sections at 4 μm thickness were prepared for Masson's trichrome, immunohistochemistry (IHC), and TUNEL labeling. Masson’s trichrome staining was performed following the manufacturer’s instructions (Abcam, MA, USA) and photomicrographs were acquired using a CTR6000 microscope with a DFC450 C camera (Leica, Wetzlar, Germany). The hepatic fibrosis score was evaluated from eight randomly selected fields, as described [32 (link)]. IHC was performed as previously described [34 (link)]. A list of antibodies used in this study is included in Additional file 1: Table S3. DAB-based TUNEL assay kit (#ab206386, Abcam, MA, USA) was used to measure apoptosis in the liver according to the manufacturer’s instructions. After mice were anesthetized by 0.02 g/ml tribromoethanol (18 µl/g BW), blood was collected from the abdominal aorta using coagulation tubes (BD, NJ, USA). Supernatants were collected for the measurement of cytokines with ELISA detection kits according to the manufacturer’s instructions. A list of ELISA kits used in this study is included in Additional file 1: Table S4.

For the analysis of CD74 cell surface expression, cells were incubated with monoclonal antibody to CD74 (sc-20062 or sc-6262; both from Santa Cruz), monoclonal antibody to MET (MAB3582; R&D Systems, Wiesbaden, Germany) or the respective isotype control (MAB002; R&D Systems), followed by incubation with a phytoerythrin (PE)-conjugated F(ab′)₂ fragment (115-116-071; Dianova). For the analysis of primary lymphoid cells, indirect staining for CD74 was performed in a first step as described above, followed by incubation with APC-labeled anti-CD19 (C7224; Dako, Hamburg, Germany) or anti-CD4 (IM2468; Beckman Coulter, Krefeld, Germany) antibodies. Immunofluorescence was analyzed using a FACSAria flow cytometer and CELLQuest software (Becton Dickinson). The percentage of viable and apoptotic cells was determined by Annexin V-FITC/propidium iodide (PI) double staining (Bender MedSystems) and flow cytometry using a FACSAria flow cytometer. Cells double negative for Annexin V-FITC and PI were considered as viable cells. For light microscopy, a Leica CTR6000 microscope equipped with a Leica DFC350FX camera was used.

For histological analysis, tissues were fixed in 4% paraformaldehyde, then ethanol dehydrated, embedded in paraffin, and sectioned as described previously (54 (link)). Hematoxylin-eosin (HE) staining was performed by standard methods. Histological scoring was performed by measuring inflammation and damage as previously described (54 (link)).
All the images were taken by using a Leica CTR6000 microscope. Brightness and contrast were adjusted linearly across the entire image for any particular image.