Figure 3

CALM knock-down inhibits infectious Ad2 uptake but not Ad2-ts1. HeLa cells were Lipofectamine 2000 (Invitrogen, Basel, Switzerland) transfected with 23 nM CALM siRNA (consisting of annealed sense strand GAAAUGGAACCACUAAGAA·(dTT) and antisense strand UUCUUAGUGGUUCCAUUUC·(dTT)) [52] (Microsynth, Balgach, Switzerland), or 23 nM non-silencing (n.s.) control siRNA (5'-AATTCTCCGAACGTGTCACGT-3', Qiagen, Hilden, Germany). A, B) Total projections of confocal fluorescence micrographs showing immunostainings of CALM in knock-down and control cells with goat anti-CALM (Santa Cruz Biotechnology, USA), and Alexa 488-rabbit anti-goat antibodies. Quantification with ImageJ http://rsbweb.nih.gov/ij/ as described [39]. C) Flow cytometric analyses of Ad5-eGFP in CALM or non-silencing (n.s.) siRNA-transfected cells 6, 9 or 12 h pi [39]. D, E) E1A expression of Ad2, Ad2-ts1 (grown at 40°C) and Ad2-BAC46 (40°C) infected HeLa-ATCC cells at different moi pretreated with siRNA against CALM or non-silencing siRNA 24 h pi at 37°C. Panel D depicts immunofluorescence images of M73 mouse monoclonal antibody-stained cells [53] and DAPI (4',6-diamidino-2-phenylindole) signals of the nuclei. Panel E is the quantification of immediate early protein E1A fluorescence expressed as % of non-silencing siRNA-treated cells infected with Ad2, Ad2-ts1 or Ad2-BAC46, respectively. Note that at moi 0.3, 3, 30 or 300, Ad2-ts1 and Ad2-BAC46 gave 15 and 13-fold less E1A-positive cells than Ad2, respectively. F, G) Mean values from TEM analyses of siRNA-transfected cells cold-incubated with Ad2 or Ad2-BAC46, washed and warmed at 37°C for 60 min. v/c denotes virus particles per cell; n number of cells, PM plasma membrane and P-values are from one-sided t-tests. H) Colocalization of incoming Ad2-texas red (Ad2-TR) or Ad2-ts1-TR in cold-synchronized infections at 37°C with mouse anti-Lamp1, followed by Alexa488 goat anti-mouse antibodies [39]. Images of 10-15 cells per time point were acquired by confocal microscopy, and colocalization of virus particles and lysosomes analyzed in single sections with ImageJ.