Fig. 4 |. 25HC restricts L. monocytogenes cell-to-cell dissemination.

a,b, Representative images (a) and quantification (b) of membrane protrusions induced by L. monocytogenes infection of Caco-2 cells expressing Neuro^RFP. Alexa Fluor 647 phalloidin staining was used to visualize F-actin. a, Scale bars, 20 μm (left) and 1 μm (right; magnifications of the region in the white box on the left). b, Left, frequency of host membrane protrusions per field of view collected across three independent experiments (vehicle treatment (−), n = 10 and 25HC, n = 13). Right, the total number of bacteria were similar between the two indicated treatments. The bars represent the mean values. The error bars show the s.d. and statistical significance was determined using a Student’s unpaired t-test (two-tailed). c, Schematic depicting the donor-to-recipient experiment. d, Representative images of recipient HEK293A^ΔMETΔCDH1 cells seeded with donor cells (WT HEK293A) infected with GFP-expressing L. monocytogenes as in c. The white boxes show the region of the merged inset (right). Scale bars, 100 μm (left and middle) and 50 μm (right). The cells were seeded at a donor:recipient ratio of 1:200. Images were acquired 24 h after donor-cell seeding. Images are representative of three independent experiments. e, Representative flow cytometry plots (n = 3) of donor (RFP-positive) and recipient (RFP-negative) cells infected with L. monocytogenes (GFP-positive). The percentage of L. monocytogenes infection in recipient cells (rectangular gate) is shown. f, Percentage of recipient cells infected, determined using the gating strategy in e. The bars represent the mean values. For the WT L. monocytogenes comparison (bars 1 and 2), the error bars show the s.d. from three independent experiments and statistical significance was determined using a Student’s unpaired t-test (two-tailed). Spread-deficient L. monocytogenes ΔactA served as a negative control and uninfected (UI) recipient cells are shown.