1. Volume 27, Issue 23, Pages 3616-3625.e5 (December 2017)
Fbxl4 Serves as a Clock Output Molecule that Regulates Sleep through Promotion of Rhythmic Degradation of the GABAA Receptor 
Qian Li, Yi Li, Xiao Wang, Junxia Qi, Xi Jin, Huawei Tong, Zikai Zhou, Zi Chao Zhang, Junhai Han 
Current Biology 
Volume 27, Issue 23, Pages e5 (December 2017)
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2. Figure 1 RDL Undergoes Rhythmic Degradation in lLNvs
(A) Images of immunostained lLNvs showing endogenous RDL levels at ZT1–ZT3 and ZT13–ZT15. Adult heads were stained with anti-RDL (green) and anti-PDF (red) antibodies. The scale bar represents 5 μm. Relative RDL levels in lLNvs at indicated time points are shown as boxplots with whiskers with maximum 1.5 interquartile ranges (IQRs) in the lower panel; n = 10.
(B) The response of WT lLNvs at ZT1–3 (blue) and at ZT13–15 (red) to puffer applications of 1 mM GABA in the presence (black) and absence of the GABAA receptor inhibitor picrotoxin (200 μM). The membrane potentials of lLNvs were held at −90 mV, and the time point of puffing GABA is indicated by the black arrow. The saturated GABA currents at indicated time points are shown in boxplots as whiskers with maximum 1.5 IQRs in the right panel. ZT1–3, n = 8; ZT13–15, n = 12.
(C) Images of immunostained lLNvs showing RDL-HA levels after pulse expression. Whole heads were stained with anti-HA (green) and anti-PDF (red) antibodies. The scale bar represents 10 μm. Relative RDL-HA levels are shown as boxplots with whiskers with 1.5 IQRs in the right panel; n = 6.
(D) Images of immunostained lLNvs showing RDL-HA degradation rate in early morning (left panel) and in early night (middle panel), respectively. Schematics for heat shock treatment and fly collection are presented in each top panel. The time points for the start of heat shock and fly collections are indicted with the red arrow and black arrows, respectively. Relative RDL-HA reductions are shown as boxplots with whiskers with maximum 1.5 IQRs in the right panel; n = 9.
All statistical comparisons are made by two-tailed Student’s t tests. ∗∗p < 0.01; ∗∗∗p < See also Figures S1 and S2 and Table S1.
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3. Figure 2 Fbxl4 Promotes RDL Ubiquitination and Degradation
(A) Yeast two-hybrid screening identified RDL interacting with Fbxl4. Schematic representation of RDL is shown in the upper panel, and its large intracellular loop is indicated in red. The encoded region of P4 is shown in the middle panel. Yeast two-hybrid screening shows one cDNA of RDL (P4) interacting with Fbxl4. Bait, pGBKT7-fbxl4; V1, pGADT7-RecAB; V2, pGBKT7.
(B) Distribution of Fbxl4 in lLNv somata (left) and terminals of the accessory medulla (right). Whole heads were stained with anti-Fbxl4 (green) and anti-PDF (red) antibodies. The scale bar represents 5 μm.
(C) Coimmunoprecipitation of Fbxl4 and RDL in vivo. Fly head extracts were immunoprecipitated with anti-HA antibodies. The precipitate and 5% of the input were subjected to western blotting with anti-Fbxl4, as well as anti-Nrv3 (ATPase β subunit, for the negative control) antibodies.
(D) Pull-down assay shows that the large intracellular loop of RDL (RDLil) bound to Fbxl4. The pull-down samples, as well as a portion (5% of the input for pull-down) of the head extracts, were loaded for western blotting and detected with anti-Fbxl4 and anti-PLC (as negative control).
(E) Fbxl4 ubiquitinates the large intracellular loop of RDL in vitro. Purified His6-RDLil and HA-Ub component were incubated with PBS buffer (blank), the head extracts from fbxl4 mutants, or the head extracts from WT flies. All fly heads were collected at ZT2. His6-RDLil was immunoprecipitated with anti-His, and ubiquitinated His6-RDLil was detected by anti-HA.
(F) In vivo ubiquitination of RDL. RDL-myc was expressed alone or co-expressed with Fbxl4 in S2R+ cells; the lysates were immunoprecipitated with anti-myc and the ubiquitinated RDL was detected by anti-ubiquitin antibody.
(G) Depletion of Fbxl4 led to accumulation of synthesized RDL-HA. Fly heads were collected at ZT10, and the extracts were immediately prepared for western blot analysis.
(H) Images of immunostained lLNvs show endogenous RDL levels in WT and fbxl4 mutant lLNvs at ZT1–ZT3. Dissected adult fly heads were stained with anti-RDL (green) and anti-PDF (red) antibodies. The scale bar represents 5 μm. Relative RDL levels in lLNvs in each genotype are shown as boxplots with whiskers with 1.5 IQRs in the right panel. WT, n = 6; fbxl4, n = 7. Two-tailed Student’s t tests were used to compare genotypes. ∗∗∗p <
See also Figure S3 and Table S1.
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4. Figure 3 The Expression Level of Fbxl4 Is Oscillated in lLNvs
(A) Western blot showing the protein levels of RDL-HA and Fbxl4 with the indicated treatments are presented in the top panel.
(B) Quantification of relative Fbxl4 and RDL-HA levels with the indicated treatment. Data averaged from three independent experiments are represented as mean ± SEM.
(C) Images of immunostained lLNvs showing Fbxl4 levels throughout 1 day. Adult heads were collected at indicated time points; dissected whole heads were stained with anti-Fbxl4 (green) and anti-PDF (red) antibodies. The scale bar represents 5 μm.
(D) Quantification of Fbxl4 levels in lLNvs at indicated time points. Data are represented as mean ± SEM; n = 8.
(E) Single cell qRT-PCR showing fbxl4 mRNA levels in lLNvs throughout 1 day. Data are represented as mean ± SEM; n = 5.
See also Figure S4 and Table S1.
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5. Figure 4 Fbxl4 Expression in lLNvs Is Dependent on CLOCK Activity
(A) In S2R+ cells, CLOCK stimulated luciferase reporter-gene expression through an fbxl4 promoter sequence (seq. 1, −627∼+183). A truncated promoter (seq. 2, −350∼+183) that lacks the E-box or a promoter (seq. 4) with mutations in the two E-box elements failed to drive luciferase-reported gene expression. Data averaged from three independent triplicate experiments are presented as mean ± SEM. Two-tailed Student’s t tests were used to compare conditions. ns, not significant; ∗∗∗p <
(B) Images of immunostained lLNvs showing Fbxl4 levels in Clkjrk mutant lLNvs throughout 1 day. The scale bar represents 5 μm. Quantification of relative Fbxl4 levels in WT and Clkjrk flies is shown in the lower panel. Data are presented as mean ± SEM; n = 8.
(C) Images of immunostained lLNvs showing Fbxl4 levels in WT (left upper panel) and per01 mutant lLNvs (left lower panel) throughout 1 day in constant dark condition. The scale bar represents 5 μm. Quantification of relative Fbxl4 levels in WT and per01 mutant flies is shown in the right panel. Data are presented as mean ± SEM; n = 6.
(D) Locomotor traces of WT (left) and fbxl4 mutant (right) flies. Quantification of circadian periods, power, and rhythmicity is presented in the top panel.
See also Table S1.
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6. Figure 5 fbxl4 Mutant lLNvs Exhibit Enhanced GABA Sensitivity
(A) Representative current-clamp traces from WT and fbxl4 mutant lLNvs at day and night, respectively.
(B) Percentage of spikes fired in bursts in WT and fbxl4 mutant flies.
(C) Frequency of spontaneous action potential firing in each genotype and condition. The burst firing pattern was characterized by firing composed of several spikes of decreasing amplitude.
(D) Resting membrane potential in each genotype and condition.
(E) Spike amplitude for each genotype and condition. For (B)–(E), data are presented as boxplots with whiskers with maximum 1.5 IQRs. Dots indicate outliers. WT: daytime, n = 12; nighttime, n = 11. fbxl4: daytime, n = 11; nighttime, n = 9.
(F) The responses of WT (dark), fbxl4 mutant (blue), and pdf>fbxl4RNAi lLNvs (red) to puffer applications of 1 mM GABA at ZT1–ZT3. The membrane potentials of lLNvs were held at −90 mV, and the time point of puffing GABA is indicated with an arrow.
(G) GABA currents in WT, fbxl4 mutant, and pdf>fbxl4RNAi lLNvs are shown as boxplots with whiskers with maximum 1.5 IQRs. WT, n = 8. fbxl4Δ45, n = 8. pdf>fbxl4RNAi, n = 7.
Statistical comparisons are made by two-tailed Student’s t tests. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < See also Figure S5 and Table S1.
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7. Figure 6 Fbxl4 Regulates Sleep by Promoting RDL Degradation
(A) Average sleep traces for WT (black, n = 181) and fbxl4Δ45 mutant (blue, n = 173) female flies plotted as a 30 min sleep average.
(B) Total daytime sleep for each genotype.
(C) Total nighttime sleep for each genotype.
(D) Sleep latency after lights off for each genotype. For (B)–(D), data are shown as boxplots with whiskers with maximum 1.5 IQRs. Dots indicate outliers. WT, n = 181. fbxl4Δ45, n = 173. fbxl4e02322, n = 51.
(E) Average sleep traces for pdf-GAL4/+ (black, n = 172), UAS-Fbxl4/+ (red, n = 140), and pdf-GAL4/UAS-Fbxl4 (blue, n = 127) male flies plotted as a 30 min sleep average.
(F) Total nighttime sleep for each genotype.
(G) Sleep latency after lights off for each genotype. For (F)–(G), data are presented as boxplots with whiskers with maximum 1.5 IQRs. Dots indicate outliers. pdf-GAL4/+, n = 172. UAS-Fbxl4/+, n = 140. pdf-GAL4/UAS-Fbxl4, n = 127.
(H) Average sleep traces for UAS-RdlRNAi/+ (black, n = 52), pdf>RdlRNAi (blue, n = 74), fbxl4;UAS-RdlRNAi/+ (green, n = 162), and fbxl4;pdf>RdlRNAi (red, n = 94) female flies plotted as a 30 min sleep average.
(I) Total daytime sleep for each genotype.
(J) Total nighttime sleep for each genotype.
(K) Sleep latency after lights off for each genotype. For (I)–(K), data are shown as boxplots with whiskers with maximum 1.5 IQRs. Dots indicate outliers. UAS-RdlRNAi/+ (black), n = 52. pdf>RdlRNAi (blue), n = 74. fbxl4;UAS-RdlRNAi/+ (green), n = 162. fbxl4;pdf>RdlRNAi (red), n = 94.
(L) Average sleep traces for WT (black, n = 100), fbxl4 (blue, n = 116), wake (pink, n = 66), and fbxl4;wake (cyan, n = 76) male flies plotted as a 30 min sleep average. Please note that male flies were used for (L)–(O).
(M) Total daytime sleep for each genotype.
(N) Total nighttime sleep for each genotype.
(O) Sleep latency after lights off for each genotype. For (M)–(O), data are shown as boxplots with whiskers with maximum 1.5 IQRs. Dots indicate outliers. WT (black), n = 100. fbxl4 (blue), n = 116. wake (pink), n = 66. fbxl4;wake (cyan), n = 76.
Statistical comparisons are made by two-tailed Student’s t tests. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < See also Figure S6 and Table S1.
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