(A) Isogenic mouse embryonic fibroblasts expressing both Nck1 and 2 (WT), or lacking Nck1 and 2 expression (Nck^−/−) were transfected with Shank3-HA and infected with EPEC for six hours as described. Cells were then fixed and stained using anti-HA antibodies. (B) Untransfected MEFs were infected with EPEC for six hours and stained for endogenous Shank3 with specific antibodies. Following washing and staining, cells were examined by confocal microscopy. Single plane maximum projections of infected cells stained with anti-HA or shank3 antibodies (green in merge), DAPI (blue in merge) and phalloidin (red in merge) are shown. Scale bars represent 10 μm. (C) Fluorescent intensity profiles of actin (dotted line) and anti-HA (grey line) staining are shown. Pedestal profiles were drawn as straight lines between the arrowheads shown in the micrographs. Note the clear actin co-localisation present in both HA-tagged and endogenous Shank3 staining.

(A) HeLa cells were transfected with two levels of control shRNA vector (black bars) or one of two shank3-targeting shRNA vectors (grey and white bars). After 48 hours knockdown was assessed using real-time quantitative RT-PCR with Shank3 specific primers and normalized to GAPDH. Shown are means of two experiments, each using two independent cDNA samples, with standard deviations. A representative western blot following high-level (1000 ng) transfection of shRNA vectors is shown. Levels of endogenous Shank3 (as determined with anti-Shank3 antibody) are reduced in cells receiving Shank3-directed shRNAs 1 and 2, compared to control-transfected cells. Anti-tubulin is shown as a protein-level control.
(B) HeLa cells, WT MEFs or Nck−/− MEFs were transfected with shRNA constructs for 48 hours, then infected with EPEC for 6 hours as described. Pedestal formation was assessed by F-actin staining and is expressed as a percentage of total adherent bacteria. Means of data pooled from three independent experiments are shown, at least 40 cells per condition were counted in each experiment. Error bars indicate 95% confidence intervals, * signifies statistical significance p<0.05. Significance was measured using a two-sample T-test, comparing to the relevant shRNA control samples. Data is representative of three independent experiments.

HeLa cells were transfected with either Shank3-HA (A), HA vector alone (B) or Shank1-HA (C). After 24 hours cells were infected with EPEC for four hours, washed, fixed and stained using anti-HA antibodies, propidium iodide and phalloidin. Anti-HA (green in merge), DNA (red in merge) and actin (blue in merge) are shown, as single plane maximum projections, with orthogonal projections in the case of Shank3-HA, of confocal z-stacks. Scale bars represent 10 μm. (D) Fluorescent intensity profiles of actin (dotted line) and anti-HA (grey line) staining are shown. Pedestal profiles were drawn as straight lines between the arrowheads shown in the micrographs. In contrast to the colocalisation seen with Shank3 (A), HA or Shank1 (B and C) staining are both largely absent staining beneath attached bacteria.

(A) HeLa cells were transfected with Shank3-HA and stained with anti-HA antibodies (top panels). Identically transfected cells were infected with S. Typhimurium and fixed and stained 20 minutes following bacterial exposure to capture invasion events (bottom panels). Anti-HA (green in merge), actin (blue in merge) and bacteria (red in merge) are shown as maximum confocal projections. Arrows indicate sites of bacteria-induced ruffling. Scale bars represent 10 μm.
(B) HeLa, WT or Nck^−/− MEFs were infected for 20 minutes with S. Typhimurium and stained with DAPI (blue in merge), anti-Shank3 (green in merge) and anti-actin (red in merge) antibodies. Arrows mark pronounced dorsal actin- and Shank3-rich ruffles. Images shown are single confocal optical sections. Scale bars represent 10 μm.