Acti stain 555

For staining HSV-1-infected and -uninfected cells, cells were seeded in 8-well chamber slides the day before infection and inoculated with HSV-1 at an moi of 5. At 12 h after the inoculation, cells were fixed with 4% paraformaldehyde at 37 °C for 10 min. After being washed twice with PBS, the fixed cells were incubated with blocking buffer consisting of 2.5% BSA, 50% Blocking One solution (Nacali Tesque), 0.01% sodium azide, and 0.1% saponin in PBS for 1 h at room temperature and then a combination of antibodies diluted in the blocking buffer overnight at 4 °C. After three washes with PBS containing 0.1% saponin, the cells were incubated with 2 µg/ml Alexa Fluor 488 anti-rabbit IgG, Alexa Fluor 555 anti-mouse IgG2a, Alexa Fluor 647 anti-mouse IgG1 (Invitrogen), and/or Acti-stain 555 (Cytoskeleton) diluted in the blocking buffer for 1 h at 37 °C. After three further washes with PBS containing 0.1% saponin, the slides were mounted with coverslips using Vectashield with DAPI (Vector Laboratory). Images were acquired with a confocal microscope (Nikon A1) and/or a super-resolution microscope (Nikon N-SIM). To evaluate colocalization, the acquired images were analyzed using the ImageJ plug-in Colocalization Threshold.

The following primary antibodies were used: anti-HA (chicken, A190-106A; Bethyl Laboratories), anti-actin (rabbit, A2066; Sigma), anti-myc (mouse, 9E10 clone; Juan Bonifacino, NIH), anti-myc (rabbit, 2278; Cell Signaling), anti-RhoA (mouse, ARH04; Cytoskeleton), anti-RhoA (mouse, SAB1400017; Sigma), anti-FLAG (mouse, F1804; Sigma), anti-FLAG (rabbit, 600-401-383; Rockland Immunochemical), anti-ARF1 (mouse, sc-53168; SCBT), anti-α-tubulin (mouse, T5168; Sigma), anti-acetylated α-tubulin (mouse, T6793; Sigma), anti-detyrosinated α-tubulin (rabbit, 48389; Abcam), ActiStain-488 (PHDG1; Cytoskeleton), Actistain-555 (PHDH1-A; Cytoskeleton), anti-heat shock protein 70 (HSP70) (chicken, SPC-178D; StressMarq), anti-InaC (mouse; T. Hackstadt), anti-IncA (rabbit; T. Hackstadt), and anti-MOMP (goat, 1621; ViroStat). The following secondary reagents were used: Hoechst dye (H1399) and goat and donkey anti-mouse, anti-goat, anti-rabbit, and anti-chicken (IgY) IgG Alexa Fluor 488-, 555-, or 647-conjugated secondary antibodies (Invitrogen). Donkey anti-chicken, anti-mouse, or anti-rabbit IgG and IgY horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Invitrogen.

Endothelial angiogenesis was studied by spheroid sprouting assay. HUVECs were first transfected with the indicated siRNAs for 24 h. Then, cells were trypsinized and added to a mixture of culture medium and methylcellulose (80%:20%) and transferred to a 96-well plate to allow the formation of spheroids for 24 h at 37 °C. The spheroids were collected, added to methylcellulose with FBS (80%:20%) and embedded in a collagen type I (BD Biosciences) gel. Following incubation for 24 h at 37 °C with 50 ng/ml VEGF the gels were fixed with formaldehyde and microscope images were taken at 10x magnification (Axio Observer Z1.0 microscope, Zeiss). The cumulative length of sprouts was quantified using image analysis software (AxioVision 4.8, Zeiss). For immunofluorescence (IF), 48 h after transfection cells were fixed with PFA 4% and incubated with anti-CD31 (1:40, BD Biosciences 550389) or anti-VE-Cadherin (1:400, CST 2500XP), and actin fibers were stained with Acti-stain 555 (Cytoskeleton Inc). Images were obtained in a Nikon A1R confocal microscope. Endothelial gaps were measured by quantifying the intercellular area versus the total area in 4 fields per image, 3 images per experiment.