Parafolmaldehyde

Transfected HeLa^DL-KO cells were stressed or not with 5 μg/ml actinomycin D for 3 h. Cells were then fixed with 4% parafolmaldehyde (Electron Microscopy Sciences, #15713-S), permeabilized with 0,2% Triton X-100 and blocked with 1% bovine serum albumin (BSA). Primary antibodies used were against G3BP (611127; BD Biosciences), nucleolin (sc-8031; Santa Cruz) and hnRNPDL (ab183136; Abcam). For visualization, the appropriate host-specific Alexa Fluor 488 or 555 (Molecular Probes) secondary antibodies were used. Slides were mounted using ProLong Gold antifade reagent with DAPI (Invitrogen). Images were captured using a Leica TCS SP8 STED 3x confocal microscope (Leica Biosystems) with a 63x oil objective.

iBMDM (3*10⁴ cells/well) were seeded in a 96-well plastic-bottom imaging plate (PerkinElmer) overnight and then infected with C. albicans (MOI 1) for 30 minutes. Wells were then fixed in 4% parafolmaldehyde (Electron Microscopy Sciences) for 15 minutes, washed with PBS (ThermoFisher), and blocked with PBS containing 3% bovine serum albumin (ThermoFisher) and 5% normal goat serum (Invitrogen) for 30 minutes. FITC-conjugated anti-Candida antibody (LSBio LS-C103355, 1:2000) was diluted in blocking buffer and added for 1 hour with agitation to label extracellular C. albicans, followed by 3 5 minute washes with PBS. Wells were then permeabilized in 0.1% Triton-X 100 (Sigma-Aldrich) for 15 minutes, followed by 3 washes in PBS. Calcofluor white (Sigma-Aldrich, 1:100) was diluted in blocking buffer and added to wells for 30 minutes with agitation, followed by 3 5 minute PBS washes, and images were captured on a BioTek Lionheart FX automated microscope. A CellProfiler pipeline was developed to segment extracellular (FITC+) and total (FITC+ CFW+) C. albicans, and the percent phagocytosed by macrophages was calculated as 100 * (1 – (FITC+ / FITC+ CFW+)).

Cells infected with GFP virus were collected 24 hpi using Hanks’-based, enzyme-free cell dissociation buffer (Gibco, 13150). Cells were washed once with PBS before they were fixed using 4% parafolmaldehyde (Electron Microscopy Sciences, USA) and suspended in fluorescence-activated cell sorting (FACS) buffer (PBS and 5% FBS). Cell suspensions were filtered through a 70-μm mesh, and cells were resuspended in FACS buffer before analyzing them using an in-house flow cytometer (BD Accuri C6 Plus). A total of 4 × 10⁴ cells were collected for each sample, and the analysis was performed using FlowJo (version 10).