EMBO/SourceData huggingface dataset:
Figure 4B-C) Representative Ca2+ measurements in (B) wt vs BCL7A KO cortical neurons (CN, 10 days in vitro) and in (C) smNPC-derived parental and BCL7A KO neurons. Ca2+ changes were detected using Fluo-4 and upon treatment with (B) 50 μM glutamate (final concentration) and (C) 100 μM glutamate (final concentration). Upper right panels report Ca2+ peaks (F) relative to baseline (F0). In B, wt: n=18 cells; ko: n=23 cells (n=2 independent experiments). In C, P: n=27 cells; KO1: n=20 cells; KO2: n=19 cells (n=2 independent experiments).D) Immunoblot analysis of hippocampal tissues. Densitometries are shown on the right (ctr: n=4-5 mice; ko: n=3-4 mice).E) RT-PCR analysis of FOS expression upon glutamate (100 μM for 10 min) stimulation in smNPC-derived parental and BCL7A KO neurons (n=5-6 biological replicates).F) Immunofluorescence staining of cFos in the dentate gyrus of adult Bcl7afl/fl; Baf53b-Cretg/wt (ko) mice and control (ctr) littermates. Mice were either kept in standard housing conditions (upper images; ctr: n=3 mice; ko: n=3 mice) or exposed to an enriched environment (lower images; ctr: n=3 mice; ko: n=4 mice). Quantification of cFos+ cells are shown below.G-K) Behavioral characterization of Bcl7afl/fl; Nestin-Cretg/wt (ko) mice compared to control (ctr) littermates. Animals were tested for their locomotor activity and coordination in (H) the open field (ctr: n=13 mice; ko: n=11 mice) and (I) RotaRod test (ctr: n=12 mice; ko: n=7 mice). Cognitive function was assessed in (J) the Y-maze spontaneous alternation task (ctr: n=13 mice; ko: n=11 mice) and (K) fear conditioning paradigm (ctr: n=11 mice; ko: n=10 mice). The percentage of spontaneous alternations and freezing were used as measures for working and long-term memory function, respectively.L-Q) Behavioral characterization of adult Bcl7afl/fl; Baf53b-Cretg/wt (nKO) mice compared to control (nKO) littermates. Locomotor activity and coordination were assessed in (M) the open field (ctr: n=14 mice; nKO: n=12 mice) and (N) RotaRod paradigm (ctr: n=16 mice; nKO: n=12 mice). Cognitive function was tested in (O) the Y-maze (ctr: n=11 mice; nKO: n=11 mice) and (P-Q) Barnes maze test (ctr: n=11 mice; nKO: n=11 mice). The percentage of spontaneous alternations and freezing were used as measures for working and long-term memory function, respectively. Representative heatmaps of Barnes maze test are reported on the right. The time spent in target (T) and non-target quadrants during the probe trial are shown on the right.
Figure 5A) Heatmap of significantly dysregulated genes in BCL7A KO compared to wt eNPCs.E) Heatmap of significantly dysregulated genes encoding components of the Wnt and Notch signaling pathways.F) Immunoblots of spontaneously differentiated wt and BCL7A KO eNPCs in presence of 1 μM DAPT or 3 μM CHIR99021.G) Immunofluorescence staining of eNPCs undergoing spontaneous differentiation for 7 days. BCL7A KO eNPCs were incubated for 72 h (24 h in proliferation plus 48 h in differentiation medium) with DMSO, 1 μM DAPT, 3 μM CHIR99021 or a combination of 1 μM DAPT + 3 μM CHIR99021. Quantification of the β-III tubulin-positive or GFAP-positive cells is shown on the right (n=3-6 technical replicates from 3 independent experiments).H) Sholl analysis of β-III tubulin-positive immature neurons after 7 days of spontaneous differentiation (n=4 biological replicates with >30 cells per condition).I) mRNA expression levels of TUBB3 and GFAP at 15 and 25 days of spontaneously differentiating BCL7A KO compared parental smNPCs. BCL7A KO smNPCs were treated with DAPT, CHIR99021, DAPT + CHIR99021 or DMSO (as control= ctr) during the initial 7 days of spontaneous differentiation (n=3-7 biological replicates).J) Immunoblots for non-phospho (active) β-catenin and TCF4/TCF7L2 in proliferating and 48 h-differentiated wt and BCL7A KO eNPCs. Respective densitometries are shown on the right (n=5-8 biological replicates per condition).
Figure 6G) Proximity ligation assay (PLA) for the OXPHOS subunits NDUFB8 and MTCO1 in adult hippocampal sections from Bcl7afl/fl; Baf53b-Cretg/wt (ko; n=4) and control littermates (ctr; n=4). Sections were co-stained with TOM20 and NeuN to label mitochondria and neurons, respectively. The total number of PLA dots was normalized to the mitochondrial area (panel on the lower right).
Figure 7A) OCR measurements in wt and BCL7A KO eNPCs treated with 3 μM of CHIR99021 or DMSO (as control, ctr) for 24 h. The average maximal respiration upon FCCP treatment is shown on the right (n= 5-7 biological replicates).B) OCR measurements in wt and BCL7A KO eNPCs following 48h of spontaneous differentiation. BCL7A KO eNPCs were treated for 72 h (24 h in proliferation plus 48 h in differentiation medium) with CHIR99021 or DMSO. The average maximal respiration upon FCCP treatment is shown on the right (n= 4 biological replicates).C) Immunoblot analysis of differentiated BCL7A KO eNPCs exposed to 3 μM CHIR99021, in presence or absence of 20 nM rotenone (rot). Antibodies against β-III tubulin and actin were used.D) Immunofluorescence staining of spontaneously differentiated eNPCs. BCL7A KO eNPCs were treated for 72 h (i.e., 24 h in proliferation plus 48 h in differentiation medium) with 3 μM CHIR99021, in presence or absence of 20 nM rotenone. Scale bar= 50 μm. Statistics are shown on the right (n=3-6 technical replicates from 3 independent experiments).E) Sholl analysis of β-III tubulin-positive immature neurons following 7 days of spontaneous differentiation. BCL7A KO eNPCs were exposed to 3 μM CHIR99021, in presence or absence of 20 nM rotenone (n=3 biological replicates).F) Representative OCR measurements in BCL7A KO eNPCs treated with 5 and 10 μM of pioglitazone or DMSO (as control, ctr) for 24 h. The average maximal respiration upon FCCP treatment is shown on the right (n= 3 biological replicates).G) Immunofluorescence staining of spontaneously differentiated eNPCs. BCL7A KO eNPCs were treated for 72 h (i.e., 24 h in proliferation plus 48 h in differentiation medium) with 5 μM pioglitazone. The percentage of β-III-tubulin-positive cells is shown on the right (n=3-8 technical replicates from 2 independent experiments).
Figure 1B. Representative immunoblot of transfected missense ROS1 TKD mutations in transfected HEK-293A lysates.C. Densitometry analysis of immunoblots (N = 3, biological replicates) as represented in (B). Fold-change in relative ROS1 phosphorylation is calculated by first calculating the ratio of phospho-ROS1 to total ROS1 protein for each variant and then normalizing to ROS1WT. One-way ANOVA and Sidak's test were used to control for multiple comparisons in C Error bars in figure represent mean ± SEM.D. Representative immunoblot of ROS1 D2113 position substitutions (E, G, Y, and Q as indicated) in HEK-293A cells. E. Densitometry of immunoblots (N = 3, biological replicates) as represented in (D). pROS1- phospho-ROS1, tROS1- total-ROS1. One-way ANOVA and Sidak's test were used to control for multiple comparisons in E. Error bars in figure represent mean ± SEM.
Figure 2A. Immunoblot analysis of phospho-ROS1 and total ROS1 protein expression of NIH-3T3 cells stably transduced with empty vector (EV) or indicated ROS1 TKD mutantsC. Soft-agar assay of NIH-3T3 ROS1D2113N compared with NIH-3T3 SLC-ROS1 fusion cells (positive control) or ROS1K1980E (kinase-dead, negative control). +/- lorlatinib treated samples were compared for significance using two-way RM ANOVA (matching across rows, N = 3, biological replicates) with Šídák's multiple comparisons test (P values are indicated in figure)D. Immunoblot analysis of phospho-ROS1 and total ROS1 protein expression in MCF10A cells stably transduced with indicated ROS1 variantsCell proliferation data showing effects of treatment with DMSO (Vehicle), crizotinib (250nM), lorlatinib (250 nM), and NVL-520 (250 nM) in MCF10A ROS1 wildtype (WT) (G), D2113N (H), D2113G (I) and SLC34A2-ROS1 (SLC-ROS1) (J) as indicated. Ordinary One-way ANOVA with Dunnett's multiple comparisons test was used to determine significant differences. N = 6 wells, biological replicates (H-I). Error bars in figure represent mean ± SEM.
Figure 4A-B. Volcano plots of statistical discoveries from IMAC-enriched (A) and phospho-tyrosine-enriched (B) phosphoproteomic spectral counts comparing ROS1D2113N data to ROS1WT. Select phosphosites linked to indicated cellular functions are annotated within the graph. Unpaired t tests of log2 transformed counts from ROS1WT and ROS1D2113N cells were conducted using Graphpad Prism with variance assumption of individual variance for each role (N=3, biological replicates). Multiple Comparison tests used False Discovery Rate (FDR) with two-stage step-up (Benjamini, Krieger, and Yekutieli) with desired FDR of 5%. X axis features log2-transformed ratio of normalized intensities of ROS1D2113N relative to ROS1WT while y axis features corresponding log10(q) values.
Figure 5A. Immunoblotting was performed on cell lysates generated via transient transfection of the following plasmids: pCX4 ROS1 wildtype (WT), ROS1D2113G (D2113G), ROS1D2113N (D2113N) and the ROS1 fusions, CD74-ROS1, EZR-ROS1 and SLC34A2-ROS1 (SLC-ROS1). The targets interrogated are indicated in the labels on the left side of the image panels and the catalog numbers as well as dilution for antibodies are reported in Methods. B. Immunoblot densitometry graphs to quantitatively compare activation of ROS1, SHP2, ERK1/2 and STAT3 (N = 2, biological replicates). Fusions are listed only by fusion partner: CD74-ROS1 (CD74), EZR-ROS1 (EZR), SLC34A2-ROS1 (SLC).C. Immunoblotting was performed on cell lysates generated from stable HEK293A ROS1D2113N, HEK293A CD74-ROS1 and NIH ROS1D2113N and NIH3T3 CD74-ROS1 after treatment with ROS1 TKI. HEK293A stable cell lines were treated with DMSO, 100 nM entrectinib and 100 nM lorlatinib, and NIH3T3 stable cell lines were treated with 25 nM and 100 nM lorlatinib. The targets interrogated are indicated in the labels on the left side of the image panels in HEK293 images and on the right side of image panels in NIH3T3 images. D. Immunoblot densitometry graphs to quantitatively compare extent of inhibition ROS1, SHP2, ERK1/2 and STAT3 and AKT1 phosphorylation from two independent replicate experiments. Pixel density of phosphorylated protein signal was divded by total protein signal, and this ratio was internally normalized to DMSO (Vehicle) treated cell ratio. Note that only the 100 nM lorlatinib condition that was common to the HEK293A and NIH3T3 experiments is depicted in graphs.
Figure 6A-C. Cell proliferation effects after treatment with SHP2 inhibitors (TNO155, RMC4550), MEK1/2 inhibitors (selumetinib, trametinib), and JNK1/2/3 inhibitor, SP600125 as indicated (N = 6, biological replicates).D-F. Cell proliferation effects after treatment with AKT1/2/3 inhibitor afuresertib, and MTOR1 inhibitor, everolimus, as indicated (N = 6, biological replicates).G-I. Cell proliferation effects after treatment with JAK1/2 inhibitor ruxolitinib, and TGFBR1 inhibitor galunisertib (N = 6, biological replicates). All inhibitors were used at 250 nM final concentration, with the exception of galunisertib, which was used at 500 nM final concentration. Note that the DMSO data are the same in for each cell line when compared to different class of inhibitors. Error bars in figure represent mean ± SEM.
Figure 7B. Kaplan-Meier survival curve of mice described in (A). Comparison of survival curves using Log-rank (Mantel-Cox) test was used to compare difference between EV (N = 5) and D2113N (N = 5), and EV and SLC34A2-ROS1 (N = 4); significance is indicated as P values within graph.F. Kaplan-Meier survival curve of mice described in (C).G. Immunoblot analysis of the phosphorylated (p) and total (t) proteins from NIH-3T3 ROS1D2113N P1 cell lysates prepared from cells treated with vehicle (DMSO) or 10, 100, or 500 nM of crizotinib, entrectinib, or lorlatinib for 4 hours.H-I. Immunoblot densitometry graphs to quantitatively compare extent of inhibition ROS1, SHP2 and ERK1/2 phosphorylation (panel H data) after treatment with indicated inhibitors from two independent biological replicate experiments.
Figure 1B. Images of rosettes and seedlings (15 DAG) of Col-0, arf7/arf19, slr, and gLBD16-SRDX as well as the inducible lateral-root-less lines pGATA23::shy2-2-GR and pGATA23::slr1-GR grown on DMSO control medium or 30 µM Dexamethasone (DEX), Scale bar 1 cm. The rosettes were stained with a Lugol's Iodine solution for starch accumulation at the end of the dark period (representative images of n=3 biological replicates). C. Box plots of starch quantification in rosette tissues at 15 DAG (n=4 biological replicates). Comparison between samples was performed by one-way ANOVA and post-hoc Tukey HSD Test (α = 0.05); different letters indicate significant differences.
Figure 2E. Representative confocal sections of calcofluor counterstained (E, n=5 biological replicates) and differential interference contrast (DIC) images of GUS-stained root bends (F, n=16 biological replicates) in DR5:GUS seedlings treated as indicated 48h after gravistimulation. In E, orange arrows indicate the pericycle (marked in yellow). F. Fraction of root bends forming an LRP and showing DR5 GUS staining after treatment with either control or 2D containing agar blocks (n=16 biological replicates, ± SE).
Figure 3A. Representative western blot of root tissues of pUB10:S6K1-3xHA treated by the indicated combination of auxin (IAA, 10µM), sucrose (Suc, 110mM), 2-deoxy-d-glucose (2D, 20mM) and AZD8055 (AZD, 10µM) and probed with anti-S6K1/2, anti-HA or anti-S6K1-T449P. Blot is one of three biological replicates. B. Quantification of the relative S6K activation. Box plots show three biological replicates, and comparison between samples was performed by one-way ANOVA and post-hoc Tukey HSD Test (α = 0.05); different letters indicate significant differences.C. Representative DIC images showing RPT1B promoter expression at different stages of LR development in 10 DAG seedlings. Orange arrowheads indicate the accumulation of reporter signals in the dividing lateral root founder cells. Scale bars: 50 µm. D. Representative confocal section showing S6K1 expression in different stages of LR development in 10 DAG pS6K1:gS6K1-CFP seedlings. Orange arrowheads indicate the accumulation of reporter signals in the dividing lateral root founder cells. Scale bars: 50 µm.
Figure 4A-C. Box plots of emerged LR in TOR-oe (A), rpt1b (B), and lst8 (C) at 14 DAG. Comparison between samples was performed by one-way ANOVA and post-hoc Tukey HSD Test (α = 0.05); different letters indicate significant differences. The number of roots scored is indicated.H. Fraction of bends developing lateral root primordia and stained for GUS activity in primary root vasculature of UB10pro>>amiR-TOR/DR5:GUS, n=18 bends.
Figure 5E, F. Abundance of ARF19 (E) and LBD16 (F) transcripts in RNAseq samples. mRNA accumulation in response to auxin is comparable for both, whether TOR is knocked down. n=3 biological replicates.G, H. Relative expression levels (normalized to ACTIN) of ARF19 (G) and LBD16 (H) measured by RT-qPCR upon TOR activity inhibition with AZD8055. Comparison between samples was performed by one-way ANOVA. Different letters indicate significant differences based on a post-hoc Tukey HSD Test (n=5 biological replicates, α = 0.05).L, M. Total lysates prepared from lateral roots treated or not with IAA and AZD were fractionated through sucrose gradients, and the relative redistribution (percentage of total) of ACTIN, ARF7, ARF19, and LARP1 mRNAs in every 8 fractions were studied by RT-qPCR analysis. (L) Polysome profiles. 40S, small ribosomal subunit; 60S, large ribosomal subunit; 80S, mono-ribosome; polysomes, polyribosomes. AU is arbitrary units of RNA absorbance at A260 nanometers. (M) RT-qPCR analysis of mRNA redistribution through a sucrose gradient (8 fractions collected). Translation efficiency was computed as a percentage of mRNA in non-polysome fractions (40/60/80S; fractions 1-3) against both lights (fractions 4-5) and heavy polysomes (fractions 6-8). The plot is representative of three independently performed experiments with similar results.
Figure 6A. Abundance of WOX11 transcripts in RNAseq samples. mRNA accumulation in response to auxin is elevated when TOR is knocked down. n=3 biological replicates.B. Distribution of GUS-staining in UB10pro>>amiR-TOR/WOX11pro::GUS seedlings 6 days after transfer to 10µM Est. The proportion of root bends with the depicted phenotype is indicated.C. The arf7/arf19 mutant shows defective LR initiation. D. Formation of roots (arrows) close to the wound site of arf7/arf19 primary roots 6 days after excision (DAE). E. Blocking TOR-activity via exposure to 10µM AZD8055 (AZD) resulted in the loss of rooting capability on the wound site of arf7/arf19 primary roots at 6 DAE. SF, G. Close-ups indicate root formation location or absence of root formation on ctrl media and AZD-containing media, respectively, in arf7/arf19 primary roots at 6 DAE. The proportion of root wound sites with the depicted phenotype is indicated.
Figure 1(H Anti-FruM signals in 3-6h WTCS males are stronger than those in 4-6d WTCS males (H) n = 7 for each. Data information: Scale bars, 100 μm.(J and Anti-GFP signals are grossly similar in 3-6h and 4-6d UAS-myrGFP; fruGAL4 males (J), n = 6 for each. Data information: Scale bars, 100 μm.
Figure 2(A) Wing extension indices (zero) of isolated 3-6h juvenile w1118/Y; UAS-dTrpA1/+; fruGAL4/+, Met27/Y; UAS-dTrpA1/+; fruGAL4/+ and Gce2.5k/Y; UAS-dTrpA1/+; fruGAL4/+ males at 22 and 27℃. n = 20 for each. (B and C) Wing extension indices (B) and latencies (C) of isolated 4-6d adult w1118/Y; UAS-dTrpA1/+; fruGAL4/+, Met27/Y; UAS-dTrpA1/+; fruGAL4/+ and Gce2.5k/Y; UAS-dTrpA1/+; fruGAL4/+ males at 22 and 27℃. n = 20 for each. n.s., not significant, *p < 0.05, ***p < 0.001, Mann-Whitney U test for (B) and unpaired t test for (C). NA, not applicable for latency analysis. Data information: Error bars indicate SEM.(E and F) Tracking indices of 3-6h and 4-6d w1118/Y; UAS-dTrpA1/+; fruGAL4/+, Met27/Y; UAS-dTrpA1/+; fruGAL4/+ and Gce2.5k/Y; UAS-dTrpA1/+; fruGAL4/+ males toward moving dots at 22 and 27℃. n = 24 for each in (E). n = 22, 20, 24, 24, 24 and 24 from left to right in (F). n.s., not significant, ***p < 0.001, Mann-Whitney U test. Data information: Error bars indicate SEM.
Figure 3(A and B) qPCR experiments show that Met (A) and Gce (B) mRNA levels are significantly knocked down in actin-GAL4>UAS-Met::Gce-IR adult flies. n = 9 for each. ***p<0.001, unpaired t test. Data information: Error bars indicate SEM.Courtship behaviors of 3-6h juvenile and 4-6d adult males are not significantly affected by knocking down JH receptors Met and Gce (UAS-Met::Gce-IR) pan-neuronally or specifically in neurons driven by 57C10-GAL4 (C), fruGAL4 (D) N = 22 for each in (C), n = 24 for each in (D) n.s., not significant, Mann-Whitney U test. Data information: Error bars indicate SEM.Courtship behaviors of 3-6h juvenile and 4-6d adult males are not significantly affected by knocking down JH receptors Met and Gce (UAS-Met::Gce-IR) pan-neuronally or specifically in R41A01-GAL4 (E), NPF-GAL4 (F), TH-GAL4 (G) and R71G01-GAL4 (H) respectively. n = 24, 24, 21, and 22 from left to right in (E), n = 24, 24, 24, and 23 from left to right in (F), n = 24, 24, 20 and 24 from left to right in (G) and n = 24, 24, 23 and 24 from left to right in (H). n.s., not significant, Mann-Whitney U test. Data information: Error bars indicate SEM.
Figure 4(A-D) Expression pattern of R41A01-GAL4 (pCd, A), NPF-GAL4 (B), TH-GAL4 (C), and R71G01-GAL4 (P1, D) in adult male brains. Representative of three samples for each. Data information: Scale bars, 100 μm.(I-L) Courtship behaviors of 4-6d adult males toward virgin females are similarly intensive and not significantly influenced by activating pCd (I), NPF (J), TH (K) or P1 (L) neurons at 29℃. n = 24 for each in (I), n = 24, 24, 23, and 23 from left to right in (J), n = 24, 24, 24, and 23 from left to right in (K), and n = 24 for each in (L). n.s., not significant, **p < 0.01, ***p < 0.001, Mann-Whitney U test. Data information: Error bars indicate SEM.
Figure 5(A and B) Anti-GFP signals are grossly similar in 3-6h and 4-6d UAS-myrGFP/+; dsxGAL4/+ males (A), which are quantified in (B). n = 6 and 8, respectively. n.s., not significant, Mann-Whitney U test. Data information: Scale bars, 50 μm. Error bars indicate SEM.(C and D) Anti-DsxM signals in 3-6h WTCS males are stronger than those in 4-6d WTCS males (C), which are quantified in (D). n = 6 for each. *p < 0.05, Mann-Whitney U test. Data information: Scale bars, 50 μm. Error bars indicate SEM.(E-L) Synaptic transmission levels are visualized by syb-GRASP signals (GFP). (E and F) syb-GRASP signals from NPF neurons to dsx neurons are only observed in 4-6d adult males but not in 3-6h or 1d males (E), which are quantified in (F). The genotype is LexAop-nSyb-spGFP1-10, UAS-CD4-spGFP11/+; NPF-lexA/dsxGal4. n = 12, 7 and 5 from left to right. (G and H) syb-GRASP signals from dsx neurons to NPF neurons in 4-6d adult males are much stronger than those in 3-6h and 1d males. syb-GRASP signals in (G) are quantified in (H). The genotype is UAS-nSyb-spGFP1-10, LexAop-CD4-spGFP11/+; NPF-lexA/dsxGal4. n = 13, 7 and 6 from left to right. (I and J) syb-GRASP signals from TH neurons to dsx neurons increase with age from 3-6h juvenile males to 4-6d adult males (I), which are quantified in (J). The genotype is LexAop-nSyb-spGFP1-10, UAS-CD4-spGFP11/TH-LexA; dsxGal4/+. n = 12, 5 and 6, respectively. (K and L) syb-GRASP signals from dsx neurons to TH neurons gradually increase from juvenile males to adult males. syb-GRASP signals in (K) are quantified in (L). The genotype is UAS-nSyb-spGFP1-10, LexAop-CD4-spGFP11/TH-LexA; dsxGal4/+. n = 14, 6 and 6, respectively. White arrowheads indicate syb-GRASP signals in the same position in brains of juvenile males and adult males. n.s., not significant, **p < 0.01, ***p < 0.001, Mann-Whitney U test. Data information: Scale bars, 50 μm. Error bars indicate SEM.
Figure 6(A-D) Activation of NPF neurons with dTrpA1 induced Ca2+ rise in pC1 dsx neurons in 4-6d males but not in 3-6h males. (A and B) Ca2+ imaging of pC1 neurons from 22˚C to 27˚C and 30˚C in 3-6h (A) and 4-6d (B) males. (C) Time-course of fluorescent changes in pC1 neurons upon activation of NPF neurons in 3-6h and 4-6d males (UAS-GCaMP6m/LexAop-dTrpA1; NPF-lexA/dsxGAL4), and in 4-6d control males (UAS-GCaMP6m/+; dsxGAL4/+) without NPF neuronal activation, which are quantified in (D). n = 8 for 4-6d control males, 15 for 3-6h and 8 for 4-6d males with dTrpA1. n.s., not significant, *p < 0.05, **p < 0.01, ***p<0.001, unpaired t test. Data information: Scale bars, 10 μm. Error bars indicate SEM.(E-H) Activation of TH neurons with dTrpA1 triggered Ca2+ increase in pC1 dsx neurons in both 3-6h and 4-6d males. (E and F) Ca2+ imaging of pC1 neurons from 22˚C to 27˚C and 30˚C in 3-6h (E) and 4-6d (F) males. (G) Time-course of fluorescent changes in pC1 neurons upon activation of TH neurons in 3-6h and 4-6d males (UAS-dTrpA1/LexAop-GCaMP6s; dsxLexA/TH-GAL4), and in 4-6d control males (LexAop-GCaMP6s/+; dsxLexA/+) without activating TH neurons, which are quantified in (H). n = 8 for each. n.s., not significant, *p < 0.05, Mann-Whitney U test. Data information: Scale bars, 10 μm. Error bars indicate SEM.