Methanol acetone

HeLa cells were transfected with 2 µg of GFP-TRIM68 for 18–24 hr and were then treated with poly(I:C) (20 µg/ml) for 6 or 24 hr. Cells were fixed with methanol/acetone (Sigma) at a 1∶1 ratio, stained with an anti-TFG antibody and mounted in DAPI containing mounting media (Dako). Localisation of TFG was visualised using an Alexa fluor 546 conjugated anti-rabbit secondary antibody (Molecular Probes) and cells were imaged by confocal microscopy on a Zeiss LSM 510 META (Oberkochen, Germany).

Per well, 15,000 cells were seeded in glass 8-chamber slides (Milian, Boswil, Switzerland). For LC3B, Atg5 and Beclin-1 detection, cells were fixed in ice-cold 100% methanol for 10 min at −20 °C and permeabilized for 15 min with 0.1% TritonX-100 (Sigma-Aldrich). For α-smooth muscle actin (αSMA) detection, cells were fixed in ice-cold methanol:acetone in ratio 7:3 (both Sigma-Aldrich) for 10 min at −20 °C. Blocking was performed for 20 min with 10% FBS in PBS. Next, slides were incubated with the primary antibody (Table 3) diluted in PBS for 1 h at room temperature, followed by incubation with AlexaFluor 546 goat anti-rabbit IgG antibody (1:400, polyclonal, Invitrogen) or AlexaFluor 546 goat anti-mouse IgG antibody (1:400, polyclonal, Invitrogen) for 45 min at room temperature in the dark. Mounting medium (Dako, Baar, Switzerland) complemented with nuclear staining (4′,6-diamidino-2-phenylindole (DAPI 1 μg/mL, Roche)) were used to apply the cover slip to each slide. Images were acquired with an Olympus BX53 microscope equipped with a DP80 camera and edited with ImageJ 1.52 a.

Niclosamide (Sigma-Aldrich, St. Louis, MI, USA) working solutions were prepared prior to each experiment and used immediately after preparation to avoid precipitation. More specifically, Niclosamide was first weighed and diluted into a 1:1 solution of methanol:acetone (Sigma-Aldrich, St. Louis, MI, USA), followed by intense vortexing to yield a homogenous solution of 20 mM. The working solution was subsequently prepared through serial dilutions in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MI, USA) to a concentration of 10 μM. Adequate quantities of the working solution were then diluted in a serum-free culture medium to yield the 30 nM and 100 nM desired concentrations for SELM stimulation. The final concentration percentage of DMSO in our cell cultures was 0.003% and 0.01% for the 30 nM and 100 nM concentrations of Niclosamide, respectively. The same solvent used for Niclosamide dilution was used as blank in SELM stimulations with no effect on SELM mRNA transcription, collagen production, or migration (Supplementary Table S1).

Lungs were harvested at day 5 pi and homogenized in 1 mL DMEM using GentleMACS tissue homogenizer (Miltenyi Biotec, Gaithersburg, MD) as described (55 (link)). Homogenates were centrifuged at 500 xG at 4°C for 8 min, supernatant was 10-fold diluted in DMEM (Hyclone) and overlaid onto 90% confluent Vero E6 cells in 24-well plates. After 2h of absorption, cells were overlaid with 2% methylcellulose (Sigma Aldrich) and incubated at 37°C for 6 days. Following incubation, methylcellulose was aspirated, wells were washed with PBS, fixed with acetone: methanol (60:40, Sigma-Aldrich), and air-dried overnight. Wells were washed 3x with KPL wash buffer and blocked with blotto overnight at 4°C. The next day, Blotto was removed and a mAb cocktail against RSV F and G proteins (clones 131-2A, 131-2G) was diluted in blotto was added overnight at 4°C. Wells were washed 3x with KPL wash buffer and goat anti-mouse-AP (ThermoFisher) was added overnight at 4°C. Wells were washed 3x with KPL wash buffer and virus plaques were developed with 1-Step™ NBT/BCIP substrate solution (ThermoFisher) for 5 min, rinsed with diH2O, and enumerated using a dissection microscope.

PASMC proliferation was assessed using the Click-iT Edu Alexa Fluor 488 Imaging Kit (ThermoFisher Scientific, Waltham, Massachusetts, United States, Cat#10337). Isolated PASMCs (passage 0) cultured in 24-well plates were exposed to chronic hypoxia for 96 h (1% O₂, 5% CO₂, balanced with N₂), incubated with 5-Ethynyl-2′-Desoxyuridin (EdU, 10 µM) for further 24 h under chronic hypoxia and finally fixed with acetone-methanol (1:1; Sigma-Aldrich, St. Louis, Missouri, United States, Cat#32201 and Cat#32213) for 5 min at room temperature. Fixed PASMCs were blocked with 3% BSA (Sigma-Aldrich, St. Louis, Missouri, United States, Cat#A7030) and stained according to manufacturer’s protocol. Afterwards, PASMC nuclei were visualized with Hoechst^®33342 (4µM; ThermoFisher Scientific, Cat# 62249). Fluorescence emission was detected using Leica DMI6000 CS fluorescence microscope (Leica, Wetzlar, Germany). Hoechst-positive (Hoechst⁺; total cell number) and EdU-positive (EdU⁺; proliferation cell number) PASMCs were counted using the Leica Las X (Leica, Wetzlar, Germany) software. Finally, proliferation rate was calculated by assessing the ratio between EdU⁺ and Hoechst⁺ PASMCs as previously described (Malczyk et al., 2013 (link)).

After 4 and 24 h of infection, the chamber slides were washed two times with PBS 1× and were then fixed with acetone/methanol (1:1 v/v; Sigma-Aldrich, St. Louis, MO, USA) and were incubated at −20 °C for 24 h followed by Giemsa staining (Fast Panotic Kit, Laborclin, São Paulo, Brazil). At least 600 macrophages were counted per well to achieve the parameters of the Macrophage Infection Rate [(infected macrophage/total macrophage) ∗ 100], amastigotes per infected macrophage, and the infection index, which was calculated by multiplying the rate of infected macrophages by the average number of amastigotes per macrophage.