Nucleofection kit 5

MCF10A cells were cultured in DMEM/F12 (Gibco, 11320-033) supplemented with 5% horse serum (Gibco, 16050-122), 20 ng/ml EGF (Peprotech, AF-100-15), 10 µg/ml insulin (Sigma I9278), 0.5 µg/ml hydrocortisone (Sigma, H0888), 100 ng/µl cholera toxin (Sigma, C8052) and 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco 15140-122). Cells were transfected with the GFP–PLCδ-PH containing plasmid (Addgene 21179) using a Nucleofector II instrument (Lonza) and Lonza nucleofection kit V (Lonza, VCA-1003) with programme T-024, following the manufacturer's guidelines, and cultured for 1 day. Transfected cells were re-seeded onto 35-mm gridded glass-bottom dishes (MatTek Corp., # P35G-2-14-CGRD) and used the following day.
Live-cell images were acquired with a Confocal Zeiss LSM 780 microscope (Carl Zeiss Ltd) at 37°C in a temperature- and CO₂-controlled chamber, using Zen software (Carl Zeiss Ltd) (see Table S1 for full parameters). Cells were fixed by adding 8% formaldehyde (Sigma F8775) in 0.2 M phosphate buffer pH 7.4, in a 1:1 ratio with culture medium in the dish. Samples were further fixed in 2.5% glutaraldehyde (Sigma G5882) and 4% formaldehyde in 0.1 M phosphate buffer pH 7.4 for 30 min at room temperature. Samples were then kept in 1% formaldehyde in 0.1 M phosphate buffer pH 7.4 and stored at 4°C until processing.

Transient transfection was performed by electroporation using a Nucleofector II instrument (Lonza) and Lonza nucleofection kit V (VCA-1003; Lonza) following the manufacturer’s guidelines. Briefly, 2 × 10⁶ RAW264.7 were electroporated with 2 μg mCherry-SopF or RFP-Rab7 using program D-032.

Transient transfection of GFP-VAPA and LAMP1-RFP was performed by electroporation using a Nucleofector II instrument (Lonza) and Lonza nucleofection kit V (VCA-1003; Lonza) following the manufacturer’s guidelines.

Control non-silencing siRNA was from Qiagen. Human-specific Notch1 siRNA pool was from Dharmacon and panMena and Mena^INV siRNA from Ambion1 (link). A total of 1 × 106 MDA-MB-231 cells were transfected with 2 μm siRNA using the Lonza Nucleofection Kit V 72 h before each experiment. Immunoblot analysis and/or qPCR were performed to confirm knockdown for each experiment.

Transfection of siRNA (30 pmoles) and cDNA (3 μg) was performed by nucleofection (kit V) as recommended (Lonza, Cologne, Germany). siRNA against EB1 (sense strand: UUAAAUACUCUUAAGGCAUTT), ch-TOG (sense strand: GAAAUACUCUUAAUUCUAATT) and LacZ (control siRNA; sense strand GCGGCUGCCGGAAUUUACCTT) were synthesized by Life technologies. Efficiency and specificity of siRNA are presented in figure S3A. cDNA coding for EGFP-α-tubulin was obtained from Clontech (Mountain View, CA); mCherry-α-tubulin was a gift from Gia Voeltz, University of Colorado, Boulder (Addgene plasmid # 49149).

Pre-designed siRNA products were synthesized from Genewiz (South Plainfield, NJ), including HCMV US11, human TREM129, and Ube2j1 or Ube2j2 (Supplementary Table 3). Transfection of siRNA oligonucleotides corresponding to US11, TREM129, or Ube2j genes was carried out using Lipofectamine 2000 transfection agent (Invitrogen) at a final concentration of 20 nM mixed siRNA oligomers per well. Mock control was transfected without adding the mixed siRNA oligomers. The US11 and TMEM129 genes were targeted with two non-overlapping siRNAs to enhance effectiveness. For US11 knockdown in primary HUVEC cells by siRNA, cells were transfected with 20 nM siRNA oligomers per well using Nucleofection kit V (Lonza) 24 h before HCMV infection. Knockdown efficiency was confirmed by western blot.