EMBO/soda-nel huggingface dataset:
Identification of novel genes and networks governing hematopoietic stem cell development (2016)
Figure 5

Figure 5. Identification of genes required for HSPC specification and validation in zebrafish.

A: GO analysis of biological processes among Group IV target genes, showing enrichment of networks in the endothelial–hematopoietic transition.

B: Heat map showing bona fide HSC regulators in Group IV target genes.

C: Heat map showing the most depleted shRNAs in pair-wise comparisons between the D20LSK and D20LK populations.

D: Validation of the involvement of Group X target genes Lrsam1, Ap2a1, Mettl22, and Hal in zebrafish hematopoiesis by MO-mediated knockdown. Whole-mount in situ hybridization of Runx1 and c-Myb expression is shown at 26 hpf and 30hpf, respectively. Prdm16 knockdown serves as a positive control. Note that nonspecific staining for Runx1 and c-Myb was largely unaffected by targeted MO knockdown, whereas HSC markers Runx1 and c-Myb staining were notably reduced in discrete cells in the ventral region of the dorsal aorta (arrows). Scale bar = 100 µm.
Identification of novel genes and networks governing hematopoietic stem cell development (2016)
Figure 6

Figure 6. Novel genes from Group X are required for maintaining human HSCs.

A: Representative flow cytometric analysis showed that CD34+ cells were significantly decreased at 5dpi with knockdown of AP2A1, METTL22, LRSAM1, or HAL. The human cord blood CD34+ cells were transduced with pLKO-shRNAs targeting human AP2A1, METTL22, LRSAM1, or HAL and cultured for 5 days. Then, the flow cytometric analysis was performed to examine the percentage of CD34+ cells in culture.

B: Quantification of CD34+ cells at 5dpi with knockdown of AP2A1, METTL22, LRSAM1, and HAL showed significant decrease of CD34+ cells compared to control. Error bars represent standard deviation (SD) from duplicates, *p<0.05. C: Flow cytometric analysis showed CD34+/CD90+ cells were strikingly decreased at 5dpi with knockdown of AP2A1, METTL22, LRSAM1, and HAL.

D: Quantification of CD34+CD90+ cells at 5dpi with knockdown of AP2A1, METTL22, LRSAM1, and HAL showed significant decrease of CD34+CD90+ cells compared to control. Error bars represent standard deviation (SD) from duplicates, *p<0.05.
Molecular basis of accessible plasma membrane cholesterol recognition by the GRAM domain of GRAMD1b (2021)
Figure 2

Figure 2. A basic patch within the GRAM domain of GRAMD1b is critical for anionic lipid recognition

(A) The ribbon diagram of the modelled GRAM domain of GRAMD1b (GRAM1b) (see Materials and Methods). Amino acid side chains that contribute to the basic patch of GRAM1b are indicated. The bottom panel shows the diagram that was rotated 45° about the indicated axis. N, N-terminus; C, C-terminus.

(B) Electrostatic surface representations of the modelled wild-type GRAM1b (WT) and mutant versions of GRAM1b carrying mutations that are predicted to alter charge distribution of the basic patch (basic patch mutants: K161A, R191A, and K161A/R191A). Blue indicates positively charged surface, while red indicates negatively charged surface at the level of ±2 kT/e. The electrostatic surface representations were generated by APBS Electrostatics plugin using PyMoL. The dashed boxes indicate the positions of the mutated amino acid residues.

(C-E) Liposome sedimentation assays of wild-type GRAM1b proteins (WT) and mutant versions of GRAM1b proteins carrying indicated mutations (K161A, R191A, and K161A/R191A). Liposomes containing the indicated mole% lipids were incubated with purified GRAM1b proteins as shown. Bound proteins [pellet, (P)] were separated from the unbound proteins [supernatant, (S)], run on SDS-PAGE and visualized by colloidal blue staining (mean ± SEM, n = 3 independent experiments for all conditions in phosphatidylserine (PS) sensitivity, co-incidence detection, and anionic lipid selectivity assays; n = 12 independent experiments for WT, n = 3 independent experiments for K161A and R191A, and n = 6 independent experiments for K161A/R191A in the cholesterol sensitivity assay). DOPC, phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine); DOPS, phosphatidylserine (1,2-dioleoyl-sn-glycero-3-phospho-L-serine); Chol., cholesterol; PA, phosphatidic acid (1,2-dioleoyl-sn-glycero-3-phosphate); PI(4)P, phosphatidylinositol 4-phosphate; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PI, L-α-phosphatidylinositol.
Molecular basis of accessible plasma membrane cholesterol recognition by the GRAM domain of GRAMD1b (2021)
Figure 4

Figure 4. GRAMD1b carrying an intellectual disability mutation (R189W) is defective in membrane tethering and cholesterol transport in vitro

(A) Schematic of the dehydroergosterol (DHE) transfer assay in vitro. Plasma membrane-like liposomes (LPM) [10% phosphatidylserine (PS: DOPS), 30% cholesterol, 2.5% Dansyl-phosphatidylethanolamine (DNS-PE), 10% dehydroergosterol (DHE), 47.5% phosphatidylcholine (DOPC)] and endoplasmic reticulum-like liposomes (LER) [15% DGS-NTA (Ni), 20% phosphatidylethanolamine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/POPE), 65% DOPC] were incubated with either purified wild-type GRAMD1b proteins (WT) or purified mutant GRAMD1b proteins carrying GRAM domain mutations (1 mM total lipid, 0.2 μM final protein concentrations in 50 μl reaction volume unless otherwise noted). C-terminus of GRAMD1b, including the transmembrane domain, was replaced with a hexahistidine tag (His-tag) to attach purified GRAMD1b proteins to LER via His-tag DGS-NTA(Ni) interaction. A fluorometer was used to simultaneously monitor tethering of the liposomes in (B) and transfer of DHE from LPM to LER in (C).

(B) Liposome tethering by purified GRAMD1b proteins. Left: Representative time course of liposome tethering, as assessed by turbidity of mixtures containing both LPM and LER together with either wild-type GRAMD1b proteins (WT), mutant GRAMD1b R189W proteins (R189W), mutant GRAMD1b R189W/R191A proteins (RW/RA), or buffer alone as indicated. Turbidity, as assessed by measuring absorbance values at 405 nm, reflects clustering of liposomes due to tethering of LPM and LER. Right: Values of turbidity corresponding to the end of the experiment as indicated by the arrow (mean ± SEM, n = 6 independent experiments for WT, n = 3 independent experiments for buffer alone, R189W, and RW/RA; Dunnett's multiple comparisons test, **P < 0.0001)

(C) DHE transfer between liposomes by purified GRAMD1b proteins. Left: Representative time course of DHE transfer from LPM to LER mediated by either wild-type GRAMD1b proteins (WT), mutant GRAMD1b R189W proteins (R189W), mutant GRAMD1b R189W/R191A proteins (RW/RA), or buffer alone as indicated. Methyl-β-cyclodextrin (MCD) at 1 mM was used to determine DHE equilibration. Transfer of DHE from LPM to LER resulted in a decrease in fluorescence resonance energy transfer (FRET) between DHE and DNS-PE in LPM. A series of liposomes with different DHE mol% were prepared to plot a calibration curve to convert FRET signals to DHE molecules transferred (see Appendix Fig S2D and Materials and Methods). Right: Values of DHE transfer rate of GRAMD1b (WT), GRAMD1b (R189W), and GRAMD1b (RW/RA), as estimated by the FRET-based lipid transfer assay [mean ± SEM, n = 6 independent experiments for WT, n = 3 independent experiments for R189W and RW/RA; Dunnett's multiple comparisons test, **P = 0.0018 (WT vs. R189W), **P = 0.0002 (WT vs. RW/RA)].
Epigenetic gene-expression links heart failure to memory impairment (2020)
Figure 1

Fig. 1: Heart failure in CamkIIδc TG mice is linked to aberrant hippocampal gene-expression.

A. qPCR data comparing the expression of the CamkIIδc transgene in the brain and heart of 3 month old CamkIIδc TG mice; n=4/group; *P <0.05, Unpaired t-test, two-tailed. Note that the transgene is not detected in the brain.

B. Representative M-mode images from left ventricle from CamkIIδc TG and control mice. ESD: Left ventricle end systolic diameter, EDD: Left ventricle diastolic diameter.

C. Ejection fraction, cardiac output and index are significantly decreased in CamkIIδc TG mice (n =8) when compared to control mice (n=5; *P <0.05, , **P<0.01, ***P<0.001, Unpaired t-test, two-tailed). Heart to body ratio, left ventricle to body weight ratio, and lung to body weight ratio are increased in CamkIIδc TG (n=8) compared to control (n=5; *P <0.05, **P<0.01, Unpaired t-test; two-tailed).

D. Experimental scheme for RNA-seq analysis that was performed from hippocampal CA1 region of CamkIIδc TG mice (n=6) and control mice (n=5) at 3 months of age. Right panel shows principal component analysis (PCA) of the gene-expression data. The first principal component (PC1) can explain 42% of the variation between two groups.

E. Volcano plot showing differentially expressed genes (FDR<0.05). Red color indicates up-regulation while green represents down-regulation of transcripts.

F. Hypergeometric overlap analysis comparing genes deregulated in CamkIIδc TG mice to genes uniquely expressed in neurons, astrocytes or microglia. Numbers represent the P value after multiple adjustments with Benjamini-Hochberg (BH) method. Fisher's hypergeometric test; color represents fold enrichment.

G. Dot plot showing Top GO biological processes after removing redundant GO terms using Rivago.

H. qPCR quantification of selected genes reflecting ER-stress or protein methylation-related processes in CamkIIδc TG mice (n=4) and control mice (n=5). *p<0.05, ns = non-significant, Unpaired t-test; two-tailed. Data is normalized to Hprt1 expression.

I-K. Hypergeometric overlap analysis comparing genes deregulated in CamkIIδc TG mice to genes deregulated under (I) hypoxia conditions and (J) in response to tunicamycin-induced ER-stress and (K) in hippocampal tissue from animal models of memory impairment and neurodegeneration. Benjamini-Hochberg (BH) adjusted P values after are denoted as numbers and fold enrichment is represented as color heatmap. Fisher's hypergeometric test.

Data information: Bars and error bars indicate average ± standard error mean.
Epigenetic gene-expression links heart failure to memory impairment (2020)
Figure 2

Fig. 2: CamkIIδc TG mice display impaired hippocampus-dependent memory function.

A. The distance traveled (left panel), the speed (middle panel) and the time spent in the center region (right panel) during a 5 min open field test was similar amongst 3 months old CamkIIδc TG (n = 16) and control mice (n = 13). Unpaired t-test, two-tailed.

B. The time to enter the escape hole during training sessions of the Barnes maze test is impaired in old CamkIIδc TG (n = 16) and control mice (n = 13; two-way ANOVA, *p<0.05).

C. Plots showing the different search strategies of CamkIIδc TG (n = 16) and control mice (n = 13) across training trials. Each strategy is labeled with a unique color.

D. The cumulative score of hippocampus-dependent search strategies during Barnes maze training is impaired in CamkIIδc TG (n = 16) when compared to control mice (n = 13; two-tailed, unpaired t-test, *p<0.05).

E. Number of visits to escape hole during probe test to assay memory retrieval was impaired in CamkIIδc TG (n = 16) when compared to control mice (n = 13; two-tailed, unpaired t-test, *p<0.05).

Data information: Bars and error bar indicates mean ± SEM.
Epigenetic gene-expression links heart failure to memory impairment (2020)
Figure 3

Fig. 3: Neuronal H3K4m3 is impaired in the hippocampus of CamkIIδc TG mice

A. Hypergeometric overlap analysis comparing genes deregulated in CamkIIδc TG mice to genes differentially expressed in the hippocampal CA1 region of Kat2A, Kmt2A and Kmt2b knock out mice. Fisher's hypergeometric test, Benjamini-Hochberg (BH) correction.

B. Experimental scheme for ChIP-seq analysis. NeuN+ neuronal nuclei were FACS sorted and used to perform chromatin immunoprecipitation (ChIP) for H3K4me3

C. Pie chart showing the distribution of H3K4me3 peaks in the neurons of the hippocampal CA1 region from CamkIIδc TG mice.

D. MA plot showing the number of significantly altered neuronal H3K4me3 peaks when comparing CamkIIδc TG and control mice.

E. NGS plot showing H3K4me3 peaks at the TSS of genes down-regulated in the hippocampus of CamkIIδc TG mice. Inset shows statistical analysis, (unpaired t-test, two-tailed, ***P < 0.001). Bars and error bars indicate mean ± SEM. H3K4me3 ChIP analysis was performed on 4 replicates/group.
Epigenetic gene-expression links heart failure to memory impairment (2020)
Figure 5

Fig. 5: Vorinostat ameliorates pathological hippocampal gene-expression in CamkIIδc TG mice.

A. Schematic outline of the experimental design.

B. Scheme for WGCNA analysis.

C. Upper panel: Expression of RNA module 1 among the three experimental groups. Kruskal-Wallis test. Lower panel: Gene ontology analysis of genes that are part of RNA module 1.

D. Network representing top 30 hub genes of the gene network based on RNA module 1.

E. H3K4me3 at TSS region of genes from RNA module1. Inset shows statistical comparisons among groups (One-way ANOVA, ***P<0.001, ****P<0.0001). Bars and error bars indicate mean ± SEM. Number of replicates analyzed for H3K4me3 profile at TSS (Wildtype vehicle: 3; CamkIIδc TG-vehicle n = 4; CamkIIδc TG-Vorinostat n = 4).

F. Upper panel: RNA module 2 and its expression among the three experimental groups. Kruskal-Wallis test. Lower panel: Functional annotations of the genes that are part of RNA module 2. Wildtype vehicle: n = 10; CamkIIδc TG-vehicle n = 9; CamkIIδc TG-Vorinostat n = 9.

G. Gene correlation network of the top hub genes (n =30) of RNA module 2.

H. Left panel: Schematic outline of the analysis of microRNA-sequencing data. Right panel: Expression of microRNA module 2 amongst experimental groups. Kruskal-Wallis test. Wildtype vehicle: n = 9; CamkIIδc TG-vehicle n = 7; CamkIIδc TG-Vorinostat n = 10;

I. Heatmap showing significant negative correlation (FDR < 0.05) between microRNA members of microRNA module 2 and hub genes from RNA module 2 (see panel G).

In the boxplots in C, F, and H, the center line indicates the median, while the upper and lower lines represent the 75th and 25th percentiles, respectively. The whiskers represent the smallest and largest values in the 1.5× interquartile range.
A multifaceted small RNA modulates gene expression upon glucose limitation in Staphylococcus aureus (2019)
Figure 1

Figure 1: RsaI responds to glucose through the transcriptional factor CcpA.

(A) Northern blot experiments show the expression of RsaI during growth phase in the HG001 strain in MHB medium with or without the addition of 1,5 g/L of D-glucose. Glucose was added either at the beginning of growth (+ glucose, left panel) or after 3h of growth (right panel).

(B) Northern blot experiment shows the expression of RsaI during growth phase in the HG001, ∆ccpA mutant strain, and ∆codY mutant strain, in MHB medium with (+) or without (-) the addition of 1,5 g/L of D-glucose.

(C) Northern blot experiment shows the expression of RsaI in the HG001 and ∆ccpA mutant strains. Total RNA was prepared after 2, 3, 4, 5 and 6h of culture in BHI medium at 37°C.

(D) Northern blot analysis of RsaI in the HG001 strain grown in MHB medium with or without the addition of 1 g/L of glucose, fructose or xylose. For all the experiments, loading controls were done using the expression of 5S rRNA (5S) as revealed after hybridization of the membranes with a specific probe. However for these controls, we used aliquots of the same RNA preparations but the migration of the samples was performed in parallel to the experiments on a separate agarose gel because RsaI and 5S rRNA have very similar sizes.
A multifaceted small RNA modulates gene expression upon glucose limitation in Staphylococcus aureus (2019)
Figure 2

Figure 2: RsaI binds to icaR, glcU_2, fn3K mRNA and the sRNA RsaG.

(A) Secondary structure model of RsaI. In red, are the nucleotides deleted in the RsaI mutants (mut1 to mut4), and the nucleotides, which were substituted (mut5). The potential base-pairings between RsaI and RsaG are shown. Squared nucleotides are conserved sequences in RsaI.

(B) Gel retardation assays show the formation of the complex between RsaI and icaR, glcU_2 and fn3K mRNAs. The 5' end-labeled wild-type RsaI (RsaI), RsaI mutant 3 (RsaI mut3, deletion of the two G-track motifs), and RsaI mutant 4 (RsaI mut4, deletion of the C/U rich unpaired interhelical region) were incubated with increasing concentrations of mRNAs: lane 1, 0 nM; lane 2, 25 nM; lane 3, 50 nM; lane 4, 100 nM and lane 5, 200 nM. Below the gels, the predicted interactions between RsaI and its targets are shown. Translation start codons are in green and SD is for Shine Dalgarno sequence. Graphs represented the % of complex formed between either RsaI or its two mutant forms (RsaI mut3 and RsaI mut4) and the target mRNA (icaR, glcU_2, fn3K) as the function of mRNA concentrations.

(C) Gel retardation assays show the formation of the complex between RsaI and RsaG. The 5' end-labeled wild-type RsaI (RsaI), RsaI mutant 2 (RsaI mut2), RsaI mutant 4 (RsaI mut4), and RsaI mutant 1 (RsaI mut1) were incubated with increasing concentrations of RsaG given in nM on the top of the autoradiographies.
A multifaceted small RNA modulates gene expression upon glucose limitation in Staphylococcus aureus (2019)
Figure 4

Figure 4: Interaction of RsaI to icaR mRNA induces biofilm production

(A) Secondary structure model of icaR mRNA. The nucleotides in red are involved in the long-range interaction between the 3' and 5'-UTRs or with RsaI. SD and anti-SD are respectively for the Shine and Dalgarno sequence and for the sequence complementary to the SD sequence.

(B) Gel retardation assays show the formation of the complex between RsaI and icaR full length, icaR SUBST, icaR-5'UTR, and icaR-3'UTR. The 5' end-labeled of RsaI was incubated with increasing concentrations of the various mRNAs. UTR is for untranslated region, SUBST stands for the substitution of UCCCCUG sequence by AGGGGAC. This mutation, which is located in the 3'UTR of icaR, significantly destabilizes the long-range interaction, and enhances icaR translation. Lane C represents binding between radiolabelled RsaI and full-length icaR mRNA (50 nM). Due to the rather short migration of the gel, the full-length mRNA is still observed in the pocket.

(C) In vivo effect of RsaI on PIA-PNAG synthesis in the S. aureus wild-type (WT) 132 strain, the ∆rsaI mutant, and the strain carrying a deletion of icaR 3'UTR (∆3'UTR). This last mutant strain has been transformed with the pES plasmids expressing rsaI or rsaI mut5. The PIA-PNAG exopolysaccharide biosynthesis was quantified using dot-blot assays. Serial dilutions (1/5) of the samples were spotted onto nitrocellulose membranes and PIA-PNAG production was detected with specific anti-PIA-PNAG antibodies. RsaI was detected in the same samples by Northern blot using a probe directed against RsaI. Ethidium bromide staining of rRNA was used as loading controls of the same gel.
Epigenetic regulator Stuxnet modulates octopamine effect on sleep through a Stuxnet-Polycomb-OctB2R cascade (2020)
Figure 1

Figure 1. Stuxnet (stx) is involved in sleep regulation in adult Drosophila.

(A-E) Statistical analysis of stx mutant stx34 sleep profile compared with that of the Canton-S control. (A) 24-hour-sleep curve (y-axis was average sleep time in every 30 minutes), n=16. (B) total sleep (From left to right mean±SEM: 275.8±17.08, n=47; 358.6±23.47, n=39; 517.0±16.49, n=47; 519.7±15.89, n=39). (C) sleep bout duration (From left to right mean±SEM: 13.04±1.287, n=47; 18.01±2.212, n=39; 66.70±9.569, n=47; 59.41±6.909, n=39). (D) number of sleep bouts (From left to righ mean±SEM: 23.61.5±0.882, n=47; 25.95±1.353, n=39; 12.64±0.713, n=47; 12.44±0.888, n=39). (E) sleep latency (From left to right mean±SEM: 49.00±2.041, N=4; 33.09±5.100, N=4). (F-J) Statistical analysis of stx mutant stxd77 sleep profile compared with control w1118. (F) 24-hour-sleep curve, n=16. (G) total sleep (From left to right mean±SEM: 359.0±12.91, n=64; 444.5±10.98, n=74; 478.9±10.76, n=64; 458.1±13.98, n=74). (H) sleep bout duration (From left to right mean±SEM: 21.01±1.312, n=64; 37.63±2.988, n=74; 58.81±5.202, n=64; 61.0±6.555, n=74), (I) number of sleep bouts (From left to right mean±SEM: 20.23±0.627, n=64; 16.38±0.693, n=74; 11.66±0.605, n=64; 11.93±0.556, n=74). (J) sleep latency (From left to right mean±SEM: 52.20±5.496, N=7; 22.76±2.621, N=9). (K-N) Sleep deprivation test of stx mutants. (K-L) 12-hour mechanical night sleep deprivation was performed in stx34 and Canton-S control. (For K, from left to right mean±SEM: 34.53±9.85, n=53; 334.2±14.34, n=53; 378.8±14.20, n=53; 108.8±20.73, n=47; 361.1±16.71, n=47; 450.8±14.84, n=47; for L, from left to right mean±SEM: 45.60±13.43, n=53; 104.9±17.70, n=47). (M-N) 12-hour mechanical night sleep deprivation was performed in stxd77 and w1118 control. Sleep amount calculation of deprived night sleep (Deprivation), the day sleep amount of previous day (Baseline) and the day sleep amount after the deprivation (Recovery) (For M, from left to right mean±SEM: 35.06±10.76, n=50; 370.8±16.07, n=50; 434.5±17.42, n=50; 59.36±11.59, n=53; 420.3±15.60, n=53; 523.9±15.12, n=53; For N, from left to right mean±SEM: 61.86±13.35, n=50; 104.0±11.86, n=53) (K, M). Increased sleep amount in the Recovery sleep comparing to the Baseline (L, N), which was calculated by the subtraction of the day sleep amount after the deprivation (Recovery) by the day sleep amount of previous day (Baseline).

Data information: Bar graphs are presented as mean ± SEM. (B-D, G-I, K, M) Statistical differences were measured using non-parametric test with two-tailed Mann-Whitney test, n.s. indicates no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001. (E, J, L, N) Statistical differences were measured using unpaired Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001. n indicates the number of tested flies, N indicates the number of biological repetitions. Horizontal white and black boxes along the x axis indicate light and dark periods under LD, respectively. All the p-values are listed in supplementary table (Table EV3).
Epigenetic regulator Stuxnet modulates octopamine effect on sleep through a Stuxnet-Polycomb-OctB2R cascade (2020)
Figure 2

Figure 2. Characterization of stx-expressing neurons.

(A-D') Colocalization of Stx represented by antibody staining with EB1-Gal4. Scale bar: 10um. (E-H') Colocalization of stx antibody staining with stx-Gal4 expression pattern. (I) stx-Gal4 expression pattern shown by crossing with UAS-mCD8GFP. (J-O) Dendrite (RFP) and axon (GFP) pattern of stx expressing neurons. The dendrite is highlighted by Denmark (RFP), and the axons are highlighted by syt-eGFP. (P-Q) Rescue experiment of stx mutant. The stx mutant phenotype can be rescued by stx over expression driven by stx-Gal4 (in female flies, P) or EB1-Gal4 (Q). For P, from left to right females mean±SEM: 226.0±11.76, n=49; 336.6±14.10, n=86; 382.7±16.43, n=50; 357.8±16.55, n=72; 296.2±16.04, n=68; 484.9±12.97, n=49; 507.6±13.83, n=86; 539.5±12.99, n=50. 577.0±11.34, n=72; 520.7±14.96, n=68; For Q, from left to right mean±SEM; 398.3±9.535, n=63; 378.4±23.78, n=32; 324.4±12.33, n=45; 495.8±16.43, n=28; 478.5±14.65, n=53; 328.7±16.99, n=34; 540.7±7.690, n=63; 489.9±21.39, n=32; 490.9±13.65, n=45; 494.7±11.99, n=28; 537.8±12.07, n=53; 450.3±15.47, n=34. (R) Colocalization of stx-Gal4 with EB1-Gal4. Scale bar showed was 10um.

Data information: Bar graphs are presented as mean ± SEM. (P, Q) 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. n indicates the number of tested flies. Horizontal white and black boxes along the x axis indicate light and dark periods under LD, respectively.