Alexa fluor 488 or 555

Following infection, cells were washed with 1× phosphate-buffered saline (PBS; Corning), fixed with 100 µL 4% paraformaldehyde (ThermoFisher, Waltham, MA, USA) for 20 min at room temperature and permeabilized for 15 min with 0.05% Triton X-100 (Sigma Aldrich, St. Louis, MO, USA). Viral capsid was detected by immunofluorescent microscopy as previously described [35 (link)]. Briefly, fixed monolayers were blocked for 1 h at room temperature with PBS containing 5% normal goat serum (NGS; Gibco). Cells were then incubated with anti-dsRNA (J2; Scicons, Szirák, Hungary), anti-HAstV-1 mouse monoclonal antibody 8e7 (Invitrogen ThermoFisher, Waltham, MA, USA) or anti-VA1 rabbit monoclonal antibody (generously provided by Dr. David Wang, Washington University School of Medicine in St. Louis, St. Louis, MO, USA) diluted in 1% NGS for 1 h at room temperature. Cell monolayers were then washed 3 times with 1× PBS and incubated with anti-mouse or anti-rabbit IgG labeled with either Alexa Fluor 488 or 555 (Invitrogen) secondary antibody and with Hoescht stain (ThermoFisher) at room temperature for 30 min. Imaging was performed on the EVOS FL cell imaging system followed by analysis with ImageJ 1.50i software.

Immunohistochemistry was performed according to previous publications [53 (link), 54 (link)]. Retinal sections were mounted onto coated glass slides. Sections were rehydrated in PBS for 20 min then immersed in a blocking buffer containing 2 % BSA, 0.5 % Tween-20 and 0.05 % Triton-X 100 for 1 h. Primary antibody for PK2 (Hamster monoclonal, 1:200, Roche Inc.) or OPN4 (Affinity purified rabbit polyclonal, 1:200, Millipore Inc.) was added to the sections overnight at 4 °C. Slides were washed with PBS containing 0.5 % Tween-20 five times for 5 min each. Anti-rabbit or anti-hamster secondary antibodies (Alexa Fluor 488 or 555 1:2000; Invitrogen Inc.) were then applied, followed by incubation with 10 μg/ml Hoechst 33342 (Invitrogen Inc) for 5 min at room temperature to stain the nucleus. Sections were viewed under a Nikon inverted fluorescence microscope (Model TE-2000U; Nikon Inc, Tokyo, Japan). Images were captured with a SPOT digital camera (Diagnostic Instruments, Inc, Sterling Heights, MI). Immunofluorescence intensity was quantified with Image J. For DAB (3,3′-diaminobenzidine) immunostaining, sections were incubated with anti-PK2 antibody (Hamster monoclonal, 1:500 dilution) antibody, followed by incubation with biotinylated anti-hamster secondary antibody. Color development of DAB immunostaining was carried out with the standard ABC method [52 (link)].

Immunofluorescent staining was conducted using standard protocols.¹⁸ Briefly, cells were incubated on no. 1.5 coverslips in tissue culture plates to adhere overnight. PHEMO buffer, which consisted of 3.7% formaldehyde, 60 mM piperazine‐N,N′‐bis(2‐ethanesulfonic acid), 25 mM HEPES, 0.05% glutaraldehyde, 10 mM EGTA, and 4 mM MgSO₄, was used to fix cells for 10 min. After washing in PBS, cells were blocked with 2.5% normal goat serum for 15 min, then incubated overnight with primary antibody (1:1000) at 4°C. After washing with PBS, cells were incubated with 1:500 Alexa Fluor‐conjugated secondary antibody at room temperature for 2 h. For each pair of primary–secondary antibodies, the same steps were sequentially repeated.
Primary antibodies used include Rabbit polyclonal antipericentrin (Abcam® Cat. no. ab 4448) and mouse monoclonal anti‐α‐Tubulin (Sigma® Cat. no. T‐9026). Secondary antibodies used include Alexa Fluor 488 or 555 (Invitrogen®). Nuclei were stained by incubating cells with mounting media containing DAPI (4′,6‐diamidino‐2‐phenylindole; Vectashield®).

Cells were seeded on glass coverslips in a 12–well plate and transfected with the indicated plasmid for 24 h. Then, the cells were treated with or without DNA damage reagents according to the experimental setup. The cells were fixed in 4% paraformaldehyde (PFA) in PBS for 20 min at RT and then permeabilized in 0.5% Triton X–100 for 10 min. Slides were blocked in 5% normal goat serum (NGS) and incubated with primary antibodies diluted in 1% NGS overnight at 4 °C. Samples were then incubated with the indicated secondary antibodies labeled with Alexa Fluor 488 or 555 (Invitrogen) diluted in 1% NGS at RT for 1 h. Thereafter, they were stained with DAPI for 15 min at RT. Coverslips were mounted using Dako Fluorescence Mounting Medium (Agilent) and imaged using a Nikon confocal microscope (Eclipse C1 Plus). All scoring was performed under blinded conditions.

The following antibodies were used: previously described 8F3 anti-FANCC [15] (link), a gift from Dr. M. Hoatlin (OHSU), and Novus Biologicals (Littleton, CO); anti-FANCA (Santa Cruz Biotechnologies, Santa Cruz, CA), anti-UNC5A (Sigma-Aldrich, St. Louis, MO); anti-HA (12CA5, Roche Diagnostics, Indianapolis, IN); anti-cMyc (9E10, Santa Cruz Biotechnologies, Santa Cruz, CA); anti-GFP (clone B2; Santa Cruz Biotechnologies); anti-cleaved-caspase-3 (Cell Signaling Technologies, Danvers, MA), anti-mouse and anti-rabbit (Santa Cruz Biotechnologies); and donkey anti-rabbit Alexa Fluor 488 or 555 and anti-mouse Alexa Fluor 555 or 488 (Invitrogen, Burlington, ON). F-actin was labeled with Alexa Fluor 546 phalloidin (Life Technologies, Burlington, ON).

For plasmid transfection, 1 × 10⁴ of A549 cells were cultured in two-well chamber slides. ERGIC3-TurboGFP or ERGIC3-Myc plasmid vectors were transfected into these cells using Metafectene Pro (Biontex Laboratories, Martinsried, Germany) according to the manufacturer's protocol. CellLight^™ ER-RFP BacMan 2.0 (an ER marker) was purchased from Invitrogen (Carlsbad, CA, USA).
For immunostaining, slides were washed in phosphate buffered-saline (PBS), fixed in 3% paraformaldehyde (PFA) solution for 5 min, and post fixed in a 4% PFA solution for 10 min. Samples were incubated in 3% BSA and 0.1% saponin (Sigma) in PBS for 1h at RT, for the blocking of non-specific binding sites. Primary antibodies (diluted 1:250) were incubated overnight at 4°C. After washing, cells were incubated with secondary antibodies (diluted 1:500) conjugated to Alexa Fluor 488 or 555 (Invitrogen) for 1h at RT. Slides were washed in PBS and cover slipped using Fluoroshield^™ (Sigma) for DAPI staining. Slides were observed using a confocal laser scanning microscope (LSM710, Carl Zeiss GmbH, Jena, Germany).