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Fig. 2. Further analysis of SNX1-recruitment to plasma membrane ruffles and SCVs. (A-C) MDCK cells were infected with the SL1344 strain for 15 minutes, then fixed and immunolabeled. Ten optical z-slices were deconvolved and 3D-image volume rendered (see also supplementary material Movie 1). (Ai-iii) SNX1 (green) clearly accumulates at membrane ruffles (arrows) and around bacteria (boxed area in Ai, magnification in Aii). (B) Magnification of the membrane ruffle shown in (Aiii), with the green (SNX1) channel omitted in (Biii). (C) A single optical z-section of the maximum projections shown in (Aiii) and (B), respectively, is displayed with insets providing the respective YZ- and XZ-view. Note that SNX1 is localized as a ring around the bacterium. (D) HeLa cells infected with SL1344 for 15 minutes were fixed, immunolabeled for endogenous SNX1 (Alexa594, red) and EEA1 (Alexa488, green), and labeled with DAPI (blue) and TRITC-phalloidin (magenta). Whereas SNX1 appears as globular or tubular at sites of infection, EEA1 displays a punctate distribution (arrowheads). (E) Quantification of SNX1 and EEA1 acquisition on SCVs, scoring between 40-100 SCVs in individual z-sections, expressed as percentage of all SNX1- and/or EEA1-positive bacteria (n=3 for 15 minutes, n=2 for 60 minutes, error ± minimum or maximum). Scale bar: 10 µm.

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