Fig. 2.

miR-958 as a negative regulator of Toll signaling pathway. A and B: time course analysis of miR-958 expression (A) and Drosomycin protein (B) in wild-type (w¹¹¹⁸) adults after infection with M. luteus or water. C and D: miR-958 overexpression (C) or knockout (D) flies and controls were infected with M. luteus for 12, 24, and 48 h, and RNA was harvested for quantitative RT-PCR analysis to measure mRNA levels of Drosomycin. E and F: simultaneously, Drosomycin protein of miR-958 overexpression (E) or knockout (F) flies and controls was measured at 24 h and 48 h, respectively. The Toll pathway was activated by challenging flies with M. luteus; 24 h later, the flies were challenged with Enterococcus faecalis. G: the survival of the flies was monitored for 24 h (G). Gal80^ts;Tub-gal4/+ flies were used as a control. Gal80^ts;Tub-gal4/+ flies (n = 106); Gal80^ts;Tub>miR-958 flies (n = 95); Gal80^ts;Tub>miR-958 SP flies (n = 111). H: the Drosomycin-green fluorescent protein (GFP) reporter gene is expressed in adult flies during the immune response (H). Less fluorescence is observed in the representative miR-958 overexpression fly (right) than in the control fly (left) following infection with M. luteus for 6, 12, and 24 h. Data (A, C, and D) are presented as means ± SD from at least three independent experiments. Statistical significance was assessed by two-tailed Student’s t-test; *P < 0.05, **P < 0.01, ***P < 0.001.