Epigenetic regulator Stuxnet modulates octopamine effect on sleep through a Stuxnet-Polycomb-OctB2R cascade (2020)
Figure 3

Figure 3. Stx regulates sleep through regulating Pc downstream targets Octβ2R.

(A-E) Statistical analysis of Pc and stx double mutant stxd77;; pcXT109 sleep profile compared with controls. (A) total sleep (From left to right mean±SEM: 378.6±12.25, n=77; 455.0±11.17, n=80; 286.6±13.68, n=41; 356.7±14.50, n=43; 468.7±10.22, n=77; 467.2±13.49, n=80; 498.5±11.91, n=41; 500.9±17.84, n=43). (B) sleep bout duration (From left to right mean±SEM: 22.55±1.219, n=77; 40.86±3.271, n=80; 14.33±0.889, n=41; 20.84±2.209, n=43; 53.72±4.560, n=77; 64.26±6.380, n=80; 66.39±9.010, n=41; 81.38±11.49, n=43). (C) number of sleep bouts (From left to right mean±SEM: 19.67±0.5610, n=77; 15.91±0.6843, n=80; 21.79±0.8670, n=41; 21.20±0.9624, n=43; 12.69±0.5957, n=77; 11.58±0.5415, n=80; 13.31±0.7201, n=41; 11.34±1.001, n=43). (D)sleep latency of stxd77;; pcXT109 (From left to right mean±SEM: 52.20±5.496, N=7; 22.76±2.621, N=9; 53.68±8.744, N=3; 31.55±7.770, N=3). (E) Quantitative RT-PCR of Octβ2R in adult head of stxd77 and w1118 control. (Data represent mean±SEM: w1118, 0.8853±0.1177; N=3; stxd77/Y, 0.3263±0.1575；N=3). (F) Rescue of stxd77 by over expressing Octβ2R in ellipsoid body. From left to right mean±SEM: 399.6±11.49, n=50; 400.3±10.48, n=60; 349.9±14.77, n=48; 495.8±16.43, n=28; 470.1±13.04, n=44; 423.6±14.32, n=47; 548.6±8.707, n=50; 499.0±8.225, n=60; 490.7±16.26, n=48; 494.7±11.99, n=28; 480.2±14.64, n=44; 437.4±14.85, n=47. (G) Rescue of stxd77 by Pc RNAi in ellipsoid body. From left to right mean±SEM: 399.9±11.88, n=48; 409.8±11.50, n=49; 348.7±20.19, n=32; 495.8±16.43, n=28; 459.3±14.78, n=61; 407.5±15.66, n=40; 550.7±8.881, n=48; 516.0±11.76, n=49; 502.3±15.08, n=32; 494.7±11.99, n=28; 546.3±11.35, n=61; 514.2±18.94, n=40. Note that the same data is used for genotype stxd77;; EB1-Gal4 in panel F and G. (H) Colocalization of stx-Gal4 with Octβ2R-Gal4. Scale bar showed was 10um. (I-J) Dam-ID experiment to identify the binding of Pc on Octβ2R genomic locus. Primers used were shown in (I). The fold enrichment of different sites were shown in (J), iab-7 was used as a positive control for Pc binding (From left to right mean±SEM: 8.9±0.3, 1.2±0.06, 2.2±0.3, 3.3±0.7, 1.7±0.3, 14±0.06, 1.2±0.3, 5.3±0.7, 1.9±0.3, 15.8±0.06, 19.2±0.3, 26.7±0.7, 15.2±0.7, N=3). (K) Octβ2R was upregulated in PcXT109 mutant (Data represent mean±SEM: w1118, 1.004±0.0609, N=3；pcXT109/+, 1.447±0.1300, N=3).

Data information: Bar graphs are presented as mean ± SEM. (A-D, F, G) Statistical differences were measured using one-way ANOVA, Tukey's multiple comparison test, n.s. indicates no significant difference, *P < 0.05, **p <0.01, and ***P < 0.001. Horizontal white and black boxes along the x axis indicate light and dark periods under LD, respectively. (E, K) Statistical differences were measured using unpaired Student's t-test, *P < 0.05. n indicates the number of tested flies; N indicates the number of biological repetitions (For E and K, each repeat with a sample size of 30 individual fly heads; for J, each repeat with a sample size of 50 individual fly heads). All the p-values are listed in supplementary table (Table EV3).