EMBO/sd-character-level-ner huggingface dataset:
SCC depend on USP28 expression for tumour induction and maintenance.Figure 5Schematic diagram of CRISPR/Cas9 mediated tumour modelling and targeting of p53∆; Lkb1∆; KRasG12D(KPL) or Usp28∆; p53∆; Lkb1∆:KRasG12D(KPLU) mouse lines in Rosa26Sor-CAGG-Cas9-IRES-GFP mice Representative haematoxylin and eosin (H&E) staining of tumour bearing animals 12 weeks post intratracheal infection. Boxes indicate highlighted tumour areas in (C) (a, b, a' and b'). Scale bar = 2000µm; nKPL= 6 and nKPLU = 5. H=heart. Representative IHC staining for ADC (TTF-1) and SCC (KRT5 and ∆Np63) marker expression as well as Usp28 abundance in KPL (n=6) and KPLU (n=5) lung tumours. Scale bar: 20µm. Quantification of % tumour area (normalised to total lung area) in KPL (n=6) and KPLU (n=5) animals. Quantification of lung tumour numbers in KPL (n=6) and KPLU (n=5) animals. Immunoblot of endogenous LKB1, USP28 and ∆Np63 in KPL and KPLU tumours. (VINCULIN as loading control). Western blot shown are representative of three independent experiments (n=3). Representative immunoblot of endogenous NOTCH1, c-MYC and c-JUN in KPL and KPLU (ACTIN as a control). n=3 Kaplan-Meier survival curves comparing KPL (n=6) and KPLU (n=5) animals upon AAV intratracheal infection. The p-value was calculated using a log-rank test. Schematic diagram of generating murine SCC tumour cells from the classic KP (lsl-KrasG12D: p53fl/fl) mouse model; Representative qPCR of SCC and ADC marker expression in two independent KP lung tumour clones, resulting in KPADC and KPSCC; (ACTIN served as loading control). Quantitative graph is represented as mean ± SD of three experiments (n=3). Immunoblot against endogenous USP28 in the murine KPSCC cell line upon targeting with two independent shRNA sequences. ACTIN served as loading control. Schematic diagram of orthotopic re-transplantation of cell lines into the lung of recipient wild type C57BL/6J mice. Representative haematoxylin and eosin (H&E) images of tumour bearing animals 8 weeks post intratracheal transplantation of 2 × 105 cells/animal. KPSCC sh-NTC (n=3); KPSCC sh-USP28 (n=3), Scale bar = 5000µm Quantification of % tumour area (top, normalised to total lung area) and lung tumour numbers (bottom) on KPSCC sh-NTC (n=6) and KPSCC sh-USP28 (n=6) animals. Representative IHC staining for ADC (TTF-1) and SCC (KRT5 and ∆Np63) marker expression as well as Usp28 and GFP abundance in KPSCC sh-NTC (n=3) and KPSCC sh-USP28 (n=3) lung tumours. Scale bar =50 µm
SH-BC-893 corrects metabolic dysfunction associated with HFD-induced obesity.Figure 7Representative H&E stained liver sections from the mice shown in Fig. 6F after 4 weeks of treatment with vehicle or 120 mg/kg SH-BC-893. Scale bar, 10 μm. Unbiased lipidomics performed on the livers of 4 randomly selected mice from Fig. 6F. Lipids that were significantly different in SD, HFD + vehicle, or HFD + 120 mg/kg SH-BC-893 shown. PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; Cer, ceramide; TAG, triacylglycerol; and LPC, lysophosphatidylcholine. Ceramide levels in livers of the same mice as in (B); n=4. Total triacylglycerol (TAG) levels in livers of the same mice as in (B); n=4. Fasting blood glucose from mice randomly selected from the cohorts in Fig. 6F after 25 d of treatment; n=4-6. Blood glucose levels (F) or area under the curve (AUC, (G)) during an oral glucose tolerance test performed in the same mice as in (E). Insulin levels in plasma collected 4 h after the indicated treatment with vehicle or SH-BC-893 from the mice in Fig. 5D,E or in 9 week old SD-fed mice treated with vehicle (same mice as in Fig. 5J,K, 0 weeks on HFD); n=8. Insulin levels in the plasma collected 4 h after treatment with vehicle or SH-BC-893 from the same mice as in Fig. 5A,B; n=8.
Figure 8Impairment of mitochondrial function through mutation of nuo-6 or isp-1, which affect the mitochondrial electron transport chain, leads to the activation of the mitoUPR and nuclear localization of DAF-16. The mitoUPR transcription factor ATFS-1 can bind directly to innate immunity genes to increase their expression. Activation of ATFS-1 is also sufficient to decrease food consumption. As decreasing food consumption decreases activation of the p38-mediated innate immune signaling pathway, the ATFS-1-mediated decrease in food consumption should result in decreased signaling through the p38-mediated innate immune signaling pathway. This signaling pathway terminates with the ATF-7 transcription factor that can bind to the same genes as ATFS-1. ATFS-1 also acts to facilitate the nuclear localization of DAF-16, which modulates the expression of a separate set of genes that promotes survival of bacterial pathogens and longevity. DAF-16 acts to decrease food intake thereby decreasing signaling through the p38-mediated innate immune signaling pathway. DAF-16, ATFS-1, and all of the components of the p38-mediated innate immune signaling pathway (NSY-1, SEK-1, PMK-1 and ATF-7) are required for the increased resistance to bacterial pathogens and increased lifespan in nuo-6 and isp-1 mutants.
Figure 1(A) 786-0 or 769-P cells stably expressing shNT or shGDH1 were treated with or without aa starvation for the indicated times, and then cell viability was assessed. (B) 786-0 or 769-P cells stably expressing shNT or shGDH1 were reconstituted with or without various amounts of rGDH1 WT (3*WT indicates 3-fold expression WT) and then treated with aa starvation for the indicated time. Cell viability was subsequently assessed. ( Data information: The data are represented as means ± SEM of three independent experiments and all statistical analyses were conducted using unpaired t-test. (**p < 0.01; ***p < 0.001.) (C-E) RNA-seq analyses were performed in 786-0 cells stably expressing shNT or shGDH1 treated with or without aa starvation for 12 h. Gene ontology cellular components (C) and KEGG enrichment (D) analysis of differentially expressed genes was performed using shNT- and shGDH1-expressing 786-0 cells. The enrichment of differentially expressed ribosomal protein-coding genes was measured by GSEA (E). (F) Heatmap of all of the ribosomal protein-coding genes in shNT and shGDH1 cells. (G) Coomassie brilliant blue (CBB) staining and protein synthesis assays using the Cayman kit (601100) were performed in cells with or without GDH1. Western blotting (WB) was performed with the indicated antibodies. β-Actin was used as a loading control(left). The Click-iT HPG kit (C10428) is also used to detect newly synthesized proteins in cells with or without GDH1 (right). Data information: The data are represented as means ± SEM of three independent experiments and all statistical analyses were conducted using unpaired t-test. (**p < 0.01; ***p < 0.001.) (H) Boxplots of GDH1 expression between primary CRC tumors and adjacent normal tissues in the TCGA-KIRC cohort. In the boxplot, medians, 0.25/0.75 quantile, and min/max were represented by the central lines, the box limits, and the whiskers, respectively. (N: normal, n = 72; T: tumor, n = 534.) Unpaired t-test was used with ***p < 0.001. (I) IHC staining of GDH1 in primary KIRC tumors and adjacent normal tissues collected by us was performed (left). Quantitative analysis of the GDH1 protein levels was performed using unpaired t-test (right). ***p < 0.001. Scale bars: 50 μm. (J) Boxplots of GDH1 expression in four stages in the TCGA-KIRC cohort. In the boxplot, medians, 0.25/0.75 quantile, and min/max were represented by the central lines, the box limits, and the whiskers, respectively.(I: n = 267, II: n = 57, III: n = 123, IV: n = 84). One way ANOVA test was used with **p < 0.01. (K) Overall survival of patients in different tumor stages in the TCGA-KIRC cohort. The R package "survminer"-based maximally selected log-rank statistic was used to determine the best cutoff. The log-rank test was used to determine the p value.
Figure 5(A-F) Munc18-1 protein levels were measured in glia free cultures of munc18-1null mutant neurons expressing M18WT, M18S241A or M18S241D.(A) Typical examples showing Munc18-1 protein localization and intensity in neurites of M18WT, M18S241A or M18S241Dneurons. MAP2 in blue, VAMP2 in red and Munc18-1 in green. Scale bar represents 2 m.(B) Mean somatic Munc18-1 intensity (M18WT: 1682 ± 206 a.u., n = 46; M18S241A: 1719 ± 238 a.u., n = 36; M18S241D: 582 ± 98 a.u., n = 33; *** p < 0.001).(C) Mean synapticMunc18-1 intensity (M18WT: 1281 ± 161 a.u.; M18S241A: 1390 ± 180 a.u.; M18S241D: 210 ± 30 a.u.; *** p < 0.001).(D) Mean synapticMunc18-1 intensity as a function of radial distance from the soma. (A-F) Munc18-1 protein levels were measured in glia free cultures of munc18-1 null mutant neurons expressing M18WT, M18S241A or M18S241D.(E) Mean synaptic Munc18-1 intensity in cultures with 3 times the standard number of neurons to increase network activity (M18WT: 1072 ± 123 a.u., n = 36; M18S241A: 1480 ± 170 a.u., n = 32; M18S241D: 251 ± 70 a.u., n = 34; * p < 0.05, *** p < 0.001). (A-F) Munc18-1 protein levels were measured in glia free cultures of munc18-1 null mutant neurons expressing M18WT, M18S241A or M18S241D.(F) Mean synaptic Munc18-1 intensity in cultures treated with GABAA receptor antagonist Bicuculline (40 μM, 48 h). (M18WT: 761 ± 121 a.u., n = 26; M18S241A: 1482 ± 150 a.u., n = 31, ** p < 0.01). (G) Mean synaptic syntaxin intensity in autaptic cultures (M18WT: 875 ± 35, n = 51; M18S241A: 1058 ± 42, n = 46; M18S241D: 458 ± 19, n=40, ** p < 0.01, ***p<0.001). (H) Western Blot of lysates from high-density neuronal cultures from munc18-1 null mutant mice rescued with M18WT, M18S241A or M18S241D. Prior to lysis, cells were treated for 6 h with MG132 (10 M) or DMSO as control. Western Blots were stained for Munc18-1 and tubulin as loading control.(I) Quantification of (H). Munc18-1 levels were normalized to tubulin levels. Effect of proteasome inhibition was quantified as ratio between MG132 and DMSO treatment of each mutant (M18WT: 1.62 ± 0.41; M18S241A: 1.26 ± 0.16; M18S241D: 7.53 ± 1.17, n = 3; **p<0.01). (J) Ubiquitination of different Munc18-1 mutants in HEK293T cells. Western Blot of denatured Munc18-1 immuno-precipitations from cells expressing Munc18-1 mutants and HA-tagged ubiquitin. Prior to lysis, cells were treated for 8 h with MG132 (10 M). Ubiquitin covalently attached to Munc18-1 can be observed as a smear above the characteristic 68 kD Munc18-1 band on Western blots stained for HA-tag.(K) Quantification of immuno-precipitation in (J). Level of Munc18-1 ubiquitination was quantified as ratio of immunoprecipitated ubiquitin and Munc18-1 levels. M18WT was set to 1 (M18WT: 1 ± 0.06; M18S241A: 0.84 ± 0.57; M18S241D: 2.70 ± 0.07, n = 3; **p<0.01). All data are expressed as mean ± SEM.
sEcGFP as a pH sensor.Figure 5A) Fluorescence emission spectrum of mt-sEcGFP in HeLa cells in situ at different pH values (λex.=405 nm, λem.=415-705 nm; λ-emission scan of transfected cells with 10 nm steps using a cLSM microscope). Before and after the isosbestic point (indicated), the pH dependence of the emission is reversed, allowing for a readout of pH-dependent fluorescence emission in the two indicated channels and ratiometric imaging (λ511/λ464). The λ range for the first channel is larger than the λ range for the second channel, allowing a better match of the signal intensities of the two different channels. B) Scheme showing the positions of the pH sensor fused to subunit of different OXPHOS complexes and MPP in the matrix. CIV: Cox8a (CoxVIIIa)-sEcGFP; CV SU e-sEcGFP, CV SU γ-sEcGFP. Matrix: MPP-sEcGFP. CI: complex I, CII: complex II, CIII: complex III, CIV: complex VI, CV2: dimeric complex V. ICS: intracristal space, CM: crista membrane. C) Images showing matrix pH equilibration with extracellular pH. Shown are ratiometric fluorescence images of MPP-sEcGFP before and after addition of the uncouplers and inhibitors FCCP, nigericin and oligomycin (plus 0.002 % w/v Triton X-100 where indicated). False color images (λ511/λ464) indicate ratio values. D) Ratiometric imaging of mitochondrial pH after equilibration with extracellular pH values pH=8, pH=7.4 and pH=6.5. Scale bars = 10 µm. E E) Calibration curves for the different sensor-fusion proteins. Ratiometric response of sEcGFP to the local pH. Extracellular buffers with different pH values used: MES (2-(N-morpholino) ethanesulfonic acid, 20 mM, pH 5.6-6.1), BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 20 mM, pH 6.1-7.6) or HEPPSO (4-(2-hydroxyethyl)-piperazine-1-(2-hydroxy)-propanesulfonic acid, 20 mM, pH 7.1-8.0) in 125 mM KCl, 20 mM NaCl, 0.5 mM CaCl2, 0.5 mM MgSO4 plus FCCP (10 µM), nigericin (1 µM) and oligomycin (5 µg/mL) Each data point represents n=2 independent experiments and mean values ± s.d. of at least 15 cells. NFO: nigericin + FCCP + oligomycin; NFOD: nigericin + FCCP + oligomycin + digitonin; NFOT: nigericin + FCCP + oligomycin + Triton X-100. F) One-way ANOVA comparison of pH values at SU γ of CV at the matrix site after treating cells with the cocktail (FCCP, nigericin and oligomycin) for pH equilibration. Box (75%) and whisker plots, median (horizontal line in box), mean (box in box). ***, p<0.001; **, p<0.01; *, p<0.5, n.s.: non-significant (N=3 technical replicates, ≥10 cells analyzed). NFO: nigericin + FCCP + oligomycin; NFOD: nigericin + FCCP + oligomycin + digitonin (0.006 % w/v); NFOT: nigericin + FCCP + oligomycin + Triton X-100 (0.002 % w/v). G) Local pH values at CoxVIIIa, SU e, SU γ and in the matrix bulk in cells supplied with high glucose (High Glc, 25 mM) before and after the addition of FCCP, nigericin and oligomycin, demonstrating successful pH equilibration with extracellular pH 7.3. Three technical replicates from same clones. Bars = mean, error bars ± SD. Statistics: one-way ANOVA with post hoc Scheffé test.
Figure 1(A) Quantitative analysis of Rnf114 mRNA by realtime-PCR in various mouse tissues and organs shows that Rnf114 was predominately expressed in oocytes. (B) Immunohistochemistry analysis of RNF114 in mouse ovary shows that RNF114 was mainly expressed in the oocytes (Scale bar = 100um). (C) Knockdown efficiency of Rnf114 in the 2-cell stage embryos derived from Rnf114 siRNA #1 or siRNA #2 injected zygotes was verified by realtime-PCR and western blotting. NC indicates negative control injected with non-silencing siRNA. Three independent experiment replicates were performed for realtime-PCR, error bars represent s.e.m.; **:P<0.01, ***:P<0.01 in unpaired two-tailed t-test. (D) RNF114 depletion impaired the early embryonic development. Rnf114 siRNA #1 or siRNA #2 injected zygotes were cultured and quantified (siRNA1#, n = 120; siRNA2#, n = 128; three independent experiments); NC indicates negative control (n = 193; six independent experiments). The error bars represent s.d., *:P<0.05, **:P<0.01, ***:P<0.001 in unpaired two-tailed t-test. (E) Representative images to show early embryonic development defects. The embryos arrested at two-cell stage are indicated by arrows (Scale bar = 100um).
(A) His pull-down assay was performed with His-EV- or His-fused CARS protein and HA-tagged AMPK subunit proteins overexpressed in 293T cells.Figure 3(A) His pull-down assay was performed with His-EV- or His-fused CARS protein and HA-tagged AMPK subunit proteins overexpressed in 293T cells. GST pull-down assay was performed with GST-EV, GST-AMPKγ1, GST-AMPKγ2 (B) or GST-EV, GST-AMPKγ2, GST-AMPKγ2-N, GST-AMPKγ2-C (C) proteins and His-tagged CARS protein purified from E. coli. The segments of AMPKγ2 are shown in the bar graph above (C). The red arrows indicate the target bands. (D, E) WB analysis of p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor, AMPKγ2 protein expression in 293T cells transfected with shRNAs targeting AMPKγ2 that were further treated with cystine-deficient medium for 8 h (D) or transfected with Flag-CARS for 48 h (E). Actin served as the loading control. (F) 293T cells were transfected with HA-AMPKγ2 alone or with Flag-CARS for 48 h and then cultured with cystine-deficient medium or complete medium for 8 h. Cell lysates were immunoprecipitated with anti-Flag, and a WB analysis was performed. (G) 293T cells were transfected with HA-AMPKγ2 for 48 h and then cultured with cystine-deficient medium or complete medium for 8 h. Cell lysates were immunoprecipitated with anti-CARS, followed by WB analysis with anti-HA and anti-CARS. (H) GST pull-down of His-CARS by GST-AMPKγ2 using protein purified from E. coli, followed by WB analysis. Cysteine (0 μM, 2 μM, 10 μM, 50 μM and 250 μM) was added to the experimental buffer. (I) The schematic diagram shows that cystine deficiency-induced AMPK activation is mediated by CaMKK2, which is recruited by CARS and the AMPKγ2 complex.
Fig.Figure 9(A) Influence diagram of the network controlling the phosphorylation of mitotic substrates. The auto-activation of CycB:Cdk1 and PP2A:B55 is controlled by inhibitory phosphorylation and by the Greatwall-ENSA pathway, respectively. CycA:Cdk1 helps CycB:Cdk1 auto-activation and inhibits PP2:B55 activity through Greatwall phosphorylation. Three different classes of mitotic substrates (early, intermediate and late) are distinguished based on their sensitivity to CycA:Cdk1, CycB:Cdk1 and PP2A:B55. (B) Final level of mitotic regulators and Cdk1-substrates in response to cyclin synthesis. The heat map is based on model simulations starting with initial conditions without cyclins (Interphase, top row) until high and constant cyclin levels caused by APC/C inhibition. (C) Numerical simulation of mitotic substrate phosphorylation network in response to rising cyclin levels. The model simulates mitotic entry experiments where cyclin levels become stabilized by APC/C inhibition. Each row corresponds to one of the experimental conditions used in this work (control, CycA depletion etc.). Left columns: cyclin levels and phosphorylated mitotic substrates (early, intermediate and late). Right columns: temporal changes of some mitotic regulators. Changes of Cdc25-P and Gwl-P (not shown) are similar to phosphorylated ENSA (pENSA).
Figure 7A Activity-dependent polyspiking of population spikes (PS) during 1 Hz stimulation is reduced by VPS34IN1 and facilitated by Roscovitine compared to DMSO-treated control slices. Top, representative traces of the 1st and 30th stimulus responses with polyspikes indicated by arrows. N = 12 slices per condition from 12 mice. B Quantification of polyspiking amplitudes in response to the 30th stimulus in the presence of DMSO, VPS34IN1, or Roscovitine. Polyspiking amplitudes are increased by Roscovitine and reduced by VPS34IN1. N = 12 slices per condition from 12 mice; One-way ANOVA. Data information: Data presented as mean ± SEM ; *p < 0.05; ***p < 0.001. C The relative changes in the PS and polyspike areas in the presence of picrotoxin (50 µM) and a subsequent treatment with DMSO or VPS34IN1 (10 µM). VPS34IN1 significantly reduced the PS and polyspike area compared to DMSO. N = 6 slices per condition from 6 mice. D Mean area of PS and polyspikes in picrotoxin-treated slices 40 min post-application of DMSO or VPS34IN1. N ≥ 6 slices per condition from ≥ 6 mice; Student's t-test. Data information: Data presented as mean ± SEM ; *p < 0.05; ***p < 0.001. E A working-model for the feedback regulation of synaptic activity. Prolonged neuronal excitation downregulates VSP34-mediated synthesis of endosomal PI(3) P. PI(3) P depletion causes the activation of Calpain 2 and Cdk5, resulting in repression of SV recycling, neurotransmission and kinetic impairment of SV endocytosis to limit SV re-supply and downregulate neuronal network activity.
Spheroplasts were generated from cells expressing either HA-Apg12 or HA-Apg5.figf6Spheroplasts were generated from cells expressing either HA-Apg12 or HA-Apg5. Their lysates were mixed and layered on top of a 10-step (18-54 % w/w) sucrose gradient, and centrifuged at 174,000g for 2.5 h (ref. 29). Fifteen fractions were collected and the positions of free Apg5, free Apg12 and Apg5/Apg12 conjugate were examined by western blotting. The peak fractions of alcohol dehydrogenase (ADH)(cytosol), ALP (vacuole), Kex2 (Golgi) and Sec12 (endoplasmic reticulum) are indicated by arrows.
Figure 5(A) Principal component analysis (PCA) plot of the first two components of the analyzed samples showing separation between the three treatments: far-red illuminated wild-type, far-red illuminated pXccBphP (XccBphP overexpressing) and dark wild-type Xcc treatments. (B) Venn diagram showing overlap of differentially expressed (DE) genes between treatments (P-values < 0.05), bubbles are drawn to scale. A total of 1451 DE genes was found in comparisons between all treatments and relative percentages to this number are indicated. (C) Gene Ontology (GO) enrichment analysis. GO enrichment was evaluated at three different levels: Biological Processes (BP), Molecular Function (MF) and Cellular Component (CC). Relevant categories showing enrichment of DE genes are depicted. Bubble size correlates with enrichment factor values, grey bubbles represent P-values > 0.05.
Figure 1A. Three possible mechanisms explaining toxicity after over-expression of conventional shRNAs (see text for details). Orange, shRNA sense strand; blue, shRNA antisense strand. B. Improved shRNA expression strategies to circumvent the mechanisms in panel A. C. Four principal vector designs compared in this study. Symbols on the left indicate the AAV vector backbone; numbers denote serotype origin of the ITRs (inverted terminal repeats, serving as AAV DNA packaging signals; asterisk indicates a deletion in the AAV4 ITR creating the self-complementary [sc]AAV genotype). Six thymines (6xT) serve as termination signal for H1/U6 promoters; bGH, bovine growth hormone polyadenylation signal; TTR, transthyretin promoter.
Regulation of ft/Hpo levels is functionally relevant to neurodegeneration by Atrophins.Figure 3(A) qPCR analysis of the fold changes±s.d. of ft transcription. Downregulation starts at 2 days and progresses at 14 days. (B) qPCR analysis of the enrichment±s.d. of different regions of the ft regulatory elements in ChIP for Atro from BG3neuronal cell extracts. Enrichment is calculated as the percentage of DNAimmunoprecipitated from extracts of cells in which Atro expression has been induced, with respect to the amount immunoprecipitated from uninduced cell extracts according to the (Atro-No Ab)induced/(Atro−No Ab)uninduced formula. The DNA region from IV chromosome has been previously shown not to be immunoprecipitated in ChIP for Atro ( Haecker et al, 2007) and has been used as a negative control. (C) Histograms showing the number of ommatidia with a full complement (7) of PR in flies expressing Atro75QN with the Rhodopsin1 driver in either a control (w1118) or different mutant backgrounds and aged at 29°C for 28 days. Heterozygosis for two independent ft alleles and a sav allele significantly enhances the loss of PR. Mild overexpression of Wts via a GMR-wts transgene, which does not display any strong phenotype per se, significantly suppresses the loss of PR. No interaction was detected in this assay with wtsx1 and ykiB5 alleles in heterozygosity. N=430-963 from at least four eyes. (D) Tangential eye sections and histograms showing the degeneration of GMR>Atro75QN, Ubi-Gal80ts flies in combination with different UAS transgenes and aged 14 days as in Figure 1A. UAS-EGFP is used as a negative control. Either Ft or Wts overexpression strongly suppresses loss of PR caused by Atro75QN. Lower panels are provided as examples of quantification. A more modest but still significant rescue is observed by overexpressing a Yki RNAi contruct or Hpo; however, in this case it is to be considered that the overexpression of Hpoper se brings about the loss of at least one PR in ∼5% of ommatidia (data not shown). No effect is detected with overexpression of Sav. Two‐tailed t‐test: *P0.05; **P0.01. The wtsEPG4808 is a previously uncharacterised EP element insertion at the 5′ of the wts gene. Rare homozygous escapers display larger eyes, whereas in combination with GMR-GMR this line gives rise to smaller rough eyes, and, finally, GMR analysis of GMR>wtsEPG4808 indicates a 10‐fold increase in the head content of wts mRNA (data not shown).