Journal: bioRxiv

Article Title: Hormonal control of postmitotic neuronal identity

doi: 10.64898/2026.01.28.702037

Figure Lengend Snippet: (A-E) Intersectional genetic labeling strategy to assess neurotransmitter and neuropeptide expression in 84C10 dFB neurons. Flies carrying UAS-FLP; 84C10-LexA; LexAop-FRT-STOP-FRT-GFP were crossed to neurotransmitter- or neuropeptide-specific GAL4 driver lines. GAL4-driven FLP excision permanently activates GFP expression in 84C10 neurons that express the corresponding marker at any point during development or adulthood. (A) vGLUT-GAL4 labels the majority of 84C10 dFB neurons, indicating a predominantly glutamatergic identity. (B-D) Smaller subsets of 84C10 dFB neurons are labeled by Dh44-GAL4 (B), AstC-GAL4 (C), and Lkr-GAL4 (D), revealing neuropeptidergic heterogeneity within the population. (E) Trh-GAL4 labels a substantial subset of 84C10 dFB neurons, indicating the capacity for serotonergic biosynthesis. (F) Quantification summarizing the number of 84C10 dFB neurons labeled by each neurotransmitter or neuropeptide driver. The majority of neurons are vGLUT-positive, with smaller subsets expressing Dh44, AstC, Lkr, or Trh. Drivers for Dh31, MIP, NPF, sNPF, and ChAT did not label 84C10 neurons. (G-G”) Fluorescent in situ hybridization (FISH) for vGLUT mRNA in adult brains reveals robust postmitotic expression of vGLUT in 84C10 dFB neurons, confirming maintenance of glutamatergic identity in adulthood. Error bars represent mean ± SD. Scale bars, 20μm. n=10 adult hemibrains per genotype. White arrows indicate 84C10 dFB neuron cell bodies.

Article Snippet: Fluorescent in situ hybridization probes (Molecular Instruments) were used to detect vGlut , DH44 , and AstC transcripts.

Techniques: Labeling, Expressing, Marker, In Situ Hybridization

Journal: bioRxiv

Article Title: Hormonal control of postmitotic neuronal identity

doi: 10.64898/2026.01.28.702037

Figure Lengend Snippet: (A-A″) Representative confocal images showing control adult brains in which 84C10 dFB neurons are labeled with myrGFP (green) and analyzed by fluorescent in situ hybridization (FISH) for vGLUT mRNA. Robust vGLUT expression is detected in the cell bodies of 84C10 dFB neurons. (B-B″) Representative confocal images of adult brains following postmitotic E93 knockdown in 84C10 dFB neurons (UAS-myrGFP; GAL80ts; 84C10-GAL4 > E93 RNAi). vGLUT mRNA signal is markedly reduced in surviving 84C10 neurons compared with controls. (C) Quantification of vGLUT fluorescence intensity in individual 84C10 dFB neuron cell bodies. Postmitotic E93 knockdown significantly reduces vGLUT expression relative to controls, indicating loss of glutamatergic identity in surviving neurons. (D) Quantification of total 84C10 dFB neuron number per adult hemibrain in the same animals analyzed in (A-C). Postmitotic E93 knockdown results in a significant reduction in neuron number, consistent with earlier analyses. For neuronal analyses (C, D), n = 14 adult hemibrains per genotype. Error bars represent mean ± SD. Statistical significance was assessed using unpaired two-tailed Student’s t-tests with Welch’s correction. Asterisks denote significance levels: p < 0.05; p < 0.01; p < 0.001; p < 0.0001; NS, not significant. Scale bars, 20 μm. White arrows indicate neuronal cell bodies. (E) Triglyceride (TAG) levels normalized to total protein content in age-matched adult male flies subjected to dietary manipulation. Flies of the following genotypes were analyzed: w¹¹¹⁸ Canton-S (control), 84C10-GAL4 > w¹¹¹⁸CS, 84C10-GAL4 > UAS-Dicer; E93 RNAi, 84C10-GAL4 > UAS-E93, Pointed-GAL4 > w¹¹¹⁸CS, and Pointed-GAL4 > UAS-Dicer; E93 RNAi. Flies were placed on a control or 30% sugar diet for 7 days. Data are shown as mean ± SD. n = 16-24 flies per condition. Statistical significance was assessed using two-way ANOVA followed by Tukey’s multiple comparisons test, with comparisons made relative to the control-diet condition. Asterisks denote significance levels as indicated.

Article Snippet: Fluorescent in situ hybridization probes (Molecular Instruments) were used to detect vGlut , DH44 , and AstC transcripts.

Techniques: Control, Labeling, In Situ Hybridization, Expressing, Knockdown, Fluorescence, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: ecDNA replication is disorganized and vulnerable to replication stress

doi: 10.1093/nar/gkaf711

Figure Lengend Snippet: Characterization of ecDNA in COLO 320DM and its isolation by FACS. ( A ) Representative DNA FISH image of c- MYC localization in RPE-1, COLO 320HSR, and COLO 320DM. ecDNA or the HSR were labelled by FISH with a c- MYC probe. Slides were stained with DAPI, 5-fluorescein (centromeric region of chromosome 8) and 5-TAMRA ( c-MYC ). Although a previous study had reported that the FISH for c- MYC is found only on ecDNAs in COLO 320DM , we observed some c-MYC signal within chromosomes at a frequency of < ∼0.04 (Fig. , right panel, white arrows) which likely represent ecDNA reintegration events. Scale bar, 10 μm. The cartoons provide a graphic representation of the MYC amplification in COLO 320HSR and DM. ( B ) Composite graph depicting the ecDNA structure in COLO 320DM generated using short reads and optical genome mapping with Amplicon Reconstructor. G4 calls from G4Hunter above threshold 1.5 (purple dots); grey dotted lines are shown at G4Hunter scores 2.0 and 3.0); GC content (blue line); grey dotted line shown at 50%. ( C ) Experimental workflow of the FACS-based protocol for ecDNA isolation involving cell preparation, DNA release and FACS sorting. ( D ) Flow cytometry plots of chromosomes (top) and the region containing ecDNA and debris (bottom) from RPE-1 (left), COLO 320HSR (centre), and COLO 320DM (right) cell lines. The scales represent linear mean fluorescence intensity but do not reflect detector voltage gains. Chromosome detection was performed using a detector gain of 622 V (DAPI) and 651 V (Chromomycin A3) for COLO 320DM; 622 V (DAPI) and 604V (Chromomycin A3) for RPE-1; and 668V (DAPI) and 713 V (Chromomycin A3) for COLO 320HSR. ecDNA detection was set using a gain of 750 V (DAPI) and 750V (Chromomycin A3) for COLO 320DM; 750 V (DAPI) and 750 V (Chromomycin A3) for RPE-1; and 801 V (DAPI) and 875 V (Chromomycin A3) for COLO 320HSR. ( E ) Ratio of ONT long read counts of sequencing reads aligned to the ecDNA region. Left: Whole genome results excluding adaptive sampling sequencing, right: following FACS-based ecDNA purification. n indicates number of biological replicates. Error bars represent standard deviation.

Article Snippet: Fluorescence in situ hybridization (FISH) probes (Empire Genomics; green CHR08-10-GR for chromosome 8 and orange MYC-20-OR for c-Myc) were applied to the slides, which were then sealed with rubber cement.

Techniques: Isolation, Staining, Amplification, Generated, Flow Cytometry, Fluorescence, Sequencing, Sampling, Purification, Standard Deviation