Fig. 5
AWD regulates MAD signaling in ISCs. a, e pMAD level in wildtype (a and e arrowheads), awd over-expressing (a arrowheads) and awdRNAi (e arrowheads) ISCs (GFP+) at 18 h after Ecc15 challenge determined by immunohistochemistry. Average intensity levels of pMAD in ISCs per posterior midgut (PM) were quantified and normalized to the mean value of control samples expressing mCherryRNAi. c Expression of pMAD in highwireRNAi ISCs (arrowheads, GFP+) with and without awdOE was revealed by immunohistochemistry. b, d, f Histogram overlay of pMAD fluorescence in GFP + ISCs under conditions as noted, measured by intracellular flow cytometry assay. Median fluorescence intensity of pMAD in GFP + ISCs under these conditions, was computed by FlowJo software and normalized to the median value of control samples expressing mCherryRNAi collected on the same day of measurement. g Differential expression of pMAD between awd mutant ISCs (DELTA+, GFP+, indicated by yellow arrowheads) and wildtype ISCs (DELTA+, GFP−, indicated by red arrowheads) after 18 h of Ecc15 challenge. Lines connect data for wildtype and mutant ISCs from same guts in quantification. Error bars indicate SEM (a n = 7 flies, b n = 2529, 2919, 2815, 2294 cells for mCherryRNAi samples; n = 1923, 3162, 3359, 3551 cells for UAS::awdS1 samples; n = 16,325, 16,655, 15,328, 16,586 cells for UAS::awdT7 samples, c n = 6–7 flies, d n = 713, 743, 782, 1521, 2106, 2056 cells for mCherryRNAi samples; n = 973, 1256, 1516, 1325, 3765, 3793 cells for hiwRNAi samples; n = 233, 425, 473, 442, 2087 cells for UAS::awdS1;hiwRNAi samples, e n = 7 flies, f n = 10416, 6072, 6729, 10756, 9809, 10,267 cells for mCherryRNAi samples; n = 20866, 16933, 19561, 8922, 9318 cells for awdRNAi-1 samples; n = 9880, 15,935, 10,604, 8867, 9808, 6798 cells for awdRNAi-2 samples, g n = 6–7 flies). P values were calculated from Student’s t-test in a, c, g, and e, except that the ratio paired t-test was performed to examine the significant differences of pMAD expression between wildtype ISCs and awd mutant ISCs within the same gut in g. One-tailed Wilcoxon rank-sum test was performed in b, d and f. ****P < 0.0001; **P < 0.01; *P < 0.05; NS, not significant. Experiments were repeated 3 times (a, c, and e), or twice (g)
