Ab21696

Mock or SINV WT- infected cells were plated on 8-well LabTek slide (Merck Millipore), were fixed with 4% formaldehyde (Merck) diluted in PBS 1X for 10 min at room temperature and then washed 3 times with PBS 1X. Cells were blocked in blocking buffer (0.1% Triton X-100; PBS 1 X; 5% normal goat serum) for 1 h. The following primary antibodies were diluted 1:400 in blocking buffer and incubated over night at 4 °C: mouse anti-dsRNA J2 (RNT-SCI-10010200; Jena bioscience), mouse anti-DDX5 (67025 Proteintech) or rabbit anti-DDX5 (ab21696; Abcam), anti-DDX17 (mouse, sc-27112; Santa Cruz Biotechnology), anti-capsid (rabbit, kind gift of Diane Griffin). Cells were washed with PBS 1X-Triton 0.1%. and incubated for 1 h at room temperature with goat anti-mouse Alexa 594 (A11032, Invitrogen) or goat anti-rabbit Alexa 488 (A11008, Invitrogen) secondary antibodiesdiluted at 1:1000 in PBS 1X-Triton X-100 0.1%. DAPI staining was performed for 5 min in PBS 1X to reveal the nuclei (D1306, Invitrogen, Thermo Fisher Scientific). Slides were mounted on coverslips with Fluoromount-G mounting media (Invitrogen, Thermo Fisher Scientific) and observed by confocal microscopy (LSM780, Zeiss) with a 40X or 63X objective. Images were analysed using Image J software and fluorescence intensity profiles were obtained.

Mock or SINV-WT infected (MOI 1, 24hpi) HCT116 cells were grown on 18 mm round cover glass in 12-well cell culture plates.
Cells were fixed with 3.7% formaldehyde diluted in PBS 1X for 10 min at room temperature. Cells were then permeabilized in 1 mL of 0.1% Triton X-100 in 1X PBS for 5 min at room temperature and incubated with anti-DDX5 primary antibody (rabbit, ab21696; Abcam) diluted to 1:400 in PBS 1X for 1 h. Goat anti-rabbit Alexa 488 (A11008, Invitrogen) secondary antibody diluted to 1:1000 was added on the cells for 1 h at room temperature.
Cells were fixed again with 3.7% formaldehyde (Biosearch technologies) diluted in PBS 1X for 10 min at room temperature and incubated over night at room temperature with the SINV genome specific LGC Biosearch Technologies’ Stellaris® RNA FISH Probe diluted in RNA FISH hybridization buffer (Stellaris, Biosearch technologies). DAPI staining was performed for 30 min to reveal the nuclei (D1306, Invitrogen, Thermo Fisher Scientific). Slides were mounted on coverslips with Fluoromount-G mounting anti-fading media (Invitrogen, Thermo Fisher Scientific) and observed by confocal microscopy (LSM780, Zeiss). Images were analysed using Image J software.

Cells were plated and treated with DOX at a concentration of 0.2 µg/mL or equivalent volume of vehicle for 24 h unless stated. After washing with cold PBS, cells were scraped following lysing with RIPA buffer. Ten percent total lysate was removed and kept as the input samples, and the remainder was used for immunoprecipitation following ImmunoCruz Optima Immunoprecipitation protocol, provided with Optima B and C (Santa Cruz Biotechnology). Preclearing Matrix B and C were used for optimal preclearing of the lysate (Santa Cruz Biotechnology). The following antibodies were used during the immunoprecipitation and immunoblotting: anti-TP53 (DO-1, sc-126; Santa Cruz); anti-AGO2 (ab32381, Abcam); anti-DDX5 (ab21696, Abcam); IgG (Santa Cruz).
To check for RNA dependence in TP53–AGO2 interaction, matrix-bound complexes were incubated with RNase A (Sigma) for 30 min at 37°C. Following incubation, samples were spun down and separated into supernatant- and bead-containing fractions and subjected to immunoblot analysis.