2.10. Immunocytochemistry (ICC)

SVG-A cells were plated onto 60-mm tissue culture plates (Falcon, catalog no. 353002), grown to ~80% confluency, and transfected with the following expression plasmid combinations: Either with pcDNA 3.1(+) T7-2xStrep-JCV LT-Ag plus pcDNA 3.1 (+)-Smc6-flag or pCGT7-JCV Sm t-Ag plus pcDNA 3.1 (+)-Smarca5 combinations using lipofectamine 3000 reagent (Invitrogen, catalog no. L3000008). At 16 h post-transfection, cells were washed with 1× PBS, transferred onto glass-slide chambers (Nunc, catalog no. 154461), and incubated for an additional 24 h. Next, the cells were washed twice with 1× PBS, fixed in cold acetone for 2 min, washed twice with 1× PBS, and incubated with 5% bovine serum albumin prepared in 1× PBS for 2 h. Chamber slides were then incubated with a combination of α-T7 polyclonal (Genescript, Piscataway, NJ, USA, catalog no. A00622) (1:200 dilution) and α-FLAG monoclonal (Invitrogen, catalog no. MA1-91878) antibodies overnight. Cells were then washed three times with TBST buffer for 10-min intervals and subsequently incubated either with a fluorescein isothiocyanate (FITC)-conjugated goat α-rabbit (Abcam, Cambridge, MA, USA, catalog no. Ab6717) or Rhodamine-conjugated goat α-mouse (MilliporeSigma, Burlington, MA, USA, catalog no. AP124R) secondary antibodies for 45 min. Cells were then washed with TBST buffer three times for 10 min each and incubated with DAPI (4′,6-Diamidino-2-Phenylindole, Dihydrochloride) (ThermoFisher, Waltham, MA, USA, catalog no. D1306) (300 ng/mL prepared in 1× PBS) to stain the nucleus. The cover glass was mounted onto the slide chambers using “ProLong^® Gold Antifade” mounting medium (ThermoFisher, Waltham, MA, USA, catalog no. {"type":"entrez-protein","attrs":{"text":"P36934","term_id":"549428","term_text":"P36934"}}P36934) and dried overnight. Slides were then examined under a fluorescence microscope (Leica, Morrisville, NC, USA, DMI-6000B, objective: HCX PL APD 60×/1.25 oil, employing LAS AF operating software) to examine the proteins of interest. In addition, the cellular distribution profiles of both LT-Ag and Sm t-Ag were also analyzed by ICC in SVG-A cells, as described under their respective figure legends.