Anti cathepsin l

JAWS II and GM-BM cells were seeded on coverslips placed in a 24-well plate at a density of 1.5 × 10⁵ cells per well. Cells were left uninfected or were treated with ECTV for 60 min. at 37 °C. After 4, 12 and 24 h, cells were fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich, St Louis, MO, USA) and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) in PBS. Next, JAWS II and GM-BM cells were blocked with 3% bovine serum albumin (BSA, Sigma-Aldrich) in 0.1% Triton X-100 and incubated for 45 min. with anti-cathepsin B, anti-cathepsin L (both from Abcam, Cambridge, MA, USA) and anti-cystatin B (Thermo Fisher Scientific) primary antibodies. After washing with 0.1% Triton X-100 in PBS, cells were incubated with secondary anti-mouse or anti-rabbit antibodies conjugated with rhodamine Red-X (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) diluted in blocking solution for 1 h. ECTV antigens were stained with FITC-conjugated polyclonal antibodies for 1 h. Viral and nuclear DNA was stained with Hoechst 33342 (Sigma-Aldrich) solution for 10 min. in the dark. Slides were mounted in ProLong Gold Antifade Reagent (Life Technologies). Images were captured using Olympus BX60 fluorescence microscope and analyzed with Cell^F software (Soft Imaging System, Olympus, Tokyo, Japan) and ImageJ (NIH, Bathesda, MD, USA).

Cells were seeded in 96-well plates at a density of 0.5–5x10⁴/ml and formalin-fixed for 20 min following treatment. Cultures were washed three times in PBS and blocked (3% bovine serum albumin, 0.1% Triton) for 1 h before overnight incubation (4 °C) with primary antibodies and 1 h incubations with secondary antibodies. For LC3 and LAMP-I immunostaining, cultures were postfixed in 100% ice-cold methanol for 15 min. Antibodies were purchased from Cell Signalling Technologies (Danvers, MA, USA) except: anti-galectin-3 (BD Biosciences, San Jose, CA, USA), anti-cathepsin L (Abcam). Secondary antibodies from Molecular Probes were used at a 1:500 and visualised by ArrayScan. LAMP-I puncta were quantified using ImageJ (NIH, Bethesda, MD, USA) by measuring the area of the three largest and clearly defined immunopositive structures per cell (three fields; three independent experiments). Galectin-3-positive cells were defined by the presence of three or more cytosolic aggregates. For filipin staining, fixed cultures were quenched in glycine (1.5 mg/ml PBS; 10 min), rinsed and incubated with 25 μg/ml filipin-PBS (60 min) washed and visualised by ArrayScan.

Cells were lysed in Western-IP lysis buffer (Sigma, USA) at 4 °C. Totally 30 μg of proteins from cell lysates were separated by 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto a polyvinylidene fluoride (PVDF) membrane (Amersham Pharmacia Biotech., Little Chalfont, UK). The membranes were incubated at 4 °C overnight with following primary antibodies: rabbit anti-light chain 3B (LC3) (1:1000, Abcam, Cambridge, UK), anti-p62 (1:1000, Abcam, Cambridge, UK), anti-uncoupling protein 1 (UCP1) (1:1000, Cell Signaling Technology, Beverly, MA, USA) and anti-MitoProfile total OXPHOS rodent antibody cocktail (1:1000, Abcam, Cambridge, UK), anti-Cathepsin B (1:1000, CST, Beverly, MA, USA), anti-Cathepsin D (1:1000, CST, Beverly, MA, USA), anti-Cathepsin L (1:1000, Abcam, Cambridge, UK) and mouse anti-β-actin (1:1000, Sigma, St. Louis, MO, USA). Subsequently, the membranes were subjected to corresponding secondary antibodies [36 (link)].