Fig. 3
AWD promotes internalization of Tkv. a AWD protein level in ISCs (GFP+) with or without Ecc15 infection determined by immunohistochemistry. b Median fluorescence of AWD in GFP + ISCs during the course of a 24 h Ecc15 infection, as measured by intracellular Flow Cytometry analysis. Fluorescence was normalized to the median value of control samples at 0 h collected on the same day of measurement. c Tkv-3xHA puncta (arrowheads) in wildtype or awd overexpressing ISCs (GFP+) after 18 h of Ecc15 infection were revealed by immunohistochemistry. Sizes of Tkv-3xHA puncta were quantified. d Co-localization between Tkv-3xHA puncta and lysosomes in wildtype or awd overexpressing ISCs after 18 h of Ecc15 infection was quantified. e Tkv-3xHA expression in wildtype or awdRNAi expressing ISCs upon 18 h of Ecc15 infection. Average number of Tkv-3xHA puncta in ISCs per posterior midgut (PM) was quantified. f Differential Tkv-3xHA expression patterns inside (GFP+, indicated by yellow arrowhead) and outside (GFP−, indicated by red arrowhead) of awd mutant clones after Ecc15 challenge. Numbers of Tkv-3xHA puncta was quantified. g Maximal numbers of Tkv-GFP puncta within 30 min of time-lapse imaging were quantified in ISCs in which awd was overexpressed in the absence of infection, or after 20 h of Ecc15 infection in ISCs in which awd was knocked down. h Quantification of the percentage of Tkv-GFP puncta co-localized with lysosomes in wildtype and awd overexpressing ISCs in the absence of infection, from 30 min time-lapse movies. Error bars indicate SEM (a n = 7 flies, b n = 3086, 3887, 3908 cells for 0 h samples; n = 1770,1710,1694 cells for 6 h samples; n = 3148, 2623, 109, 2534 cells for 12 h samples; n = 4403, 3535, 4519, 4387 cells for 18 h samples; n = 5871, 5033, 5394, 4688 cells for 24 h samples; n = 4053, 4141, 4186, 3888 cells for 3d post infection samples, c n = 7–9 flies; d n = 6–10 flies; e n = 9 flies; f n = 7–9 flies; g, h n = 5 flies). P values from Student’s t-test (a, c–h) or from one-tailed Wilcoxon rank-sum test (b): ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; NS,  = not significant. Experiments were repeated three times (a, c–f)
