Journal: iScience

Article Title: JNK signaling coordinates epithelial cell turnover through exocytosis in Drosophila ribosomal protein mutants

doi: 10.1016/j.isci.2025.112587

Figure Lengend Snippet: Dying cells secrete Wg via exocytosis in the M/+ wing pouch (A and B’’) Wing discs of GFP-Wg/ + (A), or RpS3/+ , GFP-Wg/+ (B) flies, and dying cells in the wing pouch were visualized by anti-cleaved Dcp-1 staining (white). GFP-Wingless was visualized by anti-GFP staining (white). Arrowheads indicate massive cell death in the RpS3/+ wing pouch. Scale bar, 100 μm. (D–E”’) Immunofluorescent localization of the exosome marker CD63-mCherry, as visualized by anti-DsRed staining (magenta), and GFP-Wingless (white) in the wing pouch of GFP-Wg/+ (D), or RpS3/+ , GFP-Wg/+ (E) flies expressing CD63-mCherry. The nuclei were visualized by DAPI staining (blue). The images correspond to the area enclosed by rectangle 1 in the schematic diagram of the wing disc (C). Orange arrowheads indicate the colocalization of GFP-Wingless-positive puncta with CD63-mCherry-positive puncta. Scale bar, 20 μm. (F–J’) The membrane-tethered anti-GFP nanobody (Vhh4-CD8-HA) was expressed in the wing pouch of GFP-Wg/+ (F), RpS3/+ , GFP-Wg/+ (G), RpS3/+ , GFP-Wg/+ , nub-Gal4, UAS-Puc (H), RpS3/+ , GFP-Wg/+ , nub-Gal4 , UAS-unc-13-RNAi (I), or RpS3/+ , GFP-Wg/+ , nub-Gal4 , UAS-Dronc-RNAi (J) flies. GFP-Wingless was visualized by anti-GFP staining (white). Dying cells were visualized by anti-cleaved Dcp-1 staining in the wing discs (white). The images correspond to the area enclosed by the rectangle 2 in the schematic diagram of the wing disc (C). Immobilized GFP-Wg-positive puncta were frequently observed in the area of morphological dying cells in the RpS3/+ wing pouch (Indicated by orange arrowheads). Scale bar, 50 μm. (K) Boxplot with individual dots representing the number of GFP-wingless-positive puncta per rectangle-2 in the wing pouch (C). The respective genotypes are shown in (F) ( n = 9, number of wing pouches), (G) ( n = 14), (H) ( n = 11), (I) ( n = 11), and (J) ( n = 9). Thick line, median; ∗∗∗ p < 0.001, ∗∗ p < 0.01; Wilcoxon rank-sum test.

Article Snippet: Primary antibodies used are as follows; rat anti-GFP antibody (Nacalai Tesque, #04404-26, 1:1000) (Extracellular GFP, 1:300), rabbit anti-GFP antibody (Invitrogen, #A6455) (Extracellular GFP, 1:75), rabbit anti-cleaved PARP (Asp214) antibody (Cell Signaling Technology (CST), #9541, 1:200), rabbit anti-phospho-histone H3 (Ser10) antibody (CST, #9706, 1:100), mouse anti-β-galactosidase antibody (Sigma, #G8021, 1:500), rabbit anti-DsRed antibody (Takara Bio Clontech (CLN), #632496, 1:250) (Extracellular dsRed, 1:75), and rabbit anti-Cleaved Drosophila Dcp-1 (Asp216) antibody (CST, #9678, 1:100) for 3 days.

Techniques: Staining, Marker, Expressing, Membrane

Journal: iScience

Article Title: JNK signaling coordinates epithelial cell turnover through exocytosis in Drosophila ribosomal protein mutants

doi: 10.1016/j.isci.2025.112587

Figure Lengend Snippet: JNK signaling universally triggers exocytosis-mediated Wg secretion (A–D) Eye disc bearing eyFLP-induced MARCM clones of UAS-yellow-RNAi (A), UAS-unc-13-RNAi (B), UAS-Eiger W + UAS-yellow-RNAi (C), or UAS-Eiger W +UAS-unc-13-RNAi (D) cells. Scale bar, 100 μm. (E) Boxplot with individual dots representing the clone size (% of total clone area per eye disc area) in genotypes shown in (A) ( n = 23, number of eye discs), (B) ( n = 26), (C) ( n = 32), and (D) ( n = 17). Thick line, median; ∗∗∗ p < 0.001; n.s., not significant; Wilcoxon rank-sum test. (F–H) Eye disc bearing eyFLP-induced MARCM clones of UAS-CD63-mCherry (F), UAS-Eiger W + UAS-CD63-mCherry (G), or UAS-Eiger W +UAS-unc-13-RNAi + UAS-CD63-mCherry (H) cells, stained with anti-DsRed antibody (magenta) to detect extracellular CD63-mCherry. Scale bar, 10 μm. (I) Boxplot with individual dots representing the relative extracellular CD63-mCherry area in the eye disc for genotypes shown in (F) ( n = 17, number of clones), (G) ( n = 21), and (H) ( n = 17). Thick line, median; ∗∗∗ p < 0.001; Wilcoxon rank-sum test. (J–K’’’) Eye disc bearing eyFLP-induced MARCM clones of GFP-Wg/ + (J), or UAS-Eiger KB , GFP-Wg/ + (K) cells stained with anti-GFP antibody (white). Scale bar, 20 μm. (L) Boxplot with individual dots representing the relative GFP-Wg area per clone area of the eye disc for genotypes shown in (J) ( n = 11, number of clones), and (K) ( n = 9). Thick line, median; ∗∗∗, p < 0.001; Wilcoxon rank-sum test. (M–O) Eye disc bearing eyFLP-induced MARCM clones of wild-type (M), UAS-Eiger KB (N), or UAS-Eiger KB , wg/+ (O) cells. Scale bar, 100 μm. (P) Boxplot with individual dots representing the clone size (% of total clone area per eye disc area) in genotypes shown in (M) ( n = 12, number of eye discs), (N) ( n = 11), and (O) ( n = 9). Thick line, median; ∗∗∗ p < 0.001, ∗∗ p < 0.01; Wilcoxon rank-sum test.

Article Snippet: Primary antibodies used are as follows; rat anti-GFP antibody (Nacalai Tesque, #04404-26, 1:1000) (Extracellular GFP, 1:300), rabbit anti-GFP antibody (Invitrogen, #A6455) (Extracellular GFP, 1:75), rabbit anti-cleaved PARP (Asp214) antibody (Cell Signaling Technology (CST), #9541, 1:200), rabbit anti-phospho-histone H3 (Ser10) antibody (CST, #9706, 1:100), mouse anti-β-galactosidase antibody (Sigma, #G8021, 1:500), rabbit anti-DsRed antibody (Takara Bio Clontech (CLN), #632496, 1:250) (Extracellular dsRed, 1:75), and rabbit anti-Cleaved Drosophila Dcp-1 (Asp216) antibody (CST, #9678, 1:100) for 3 days.

Techniques: Clone Assay, Staining

Journal: eLife

Article Title: Executioner caspase is proximal to Fasciclin 3 which facilitates non-lethal activation in Drosophila olfactory receptor neurons

doi: 10.7554/eLife.99650

Figure Lengend Snippet: ( A ) Representative expression patterns of C-terminally V5::TurboID knocked-in tagged Drice (streptavidin [SA]; magenta) and Fasciclin 3 (Fas3, anti-Fasciclin 3 antibody staining; green) in the adult male brains. Arrows indicate the antennal lobe (AL), optic lobe (OL), and subesophageal ganglion (SOG), respectively. Scale bar: 50 µm. ( B ) Representative expression patterns of C-terminally V5::TurboID knocked-in tagged Drice (SA; magenta) and Fas3 (anti-GFP antibody staining; green) in the ALs of the adult male brains. Scale bar: 50 µm. ( C ) Schematic protein structures of Fas3 isoforms. Intracellular regions differ from each other. A peptide region used to raise anti-Fas3G antibody is shown. Signal peptides (SPs), immunoglobulin-like (Ig-like) domains, and transmembrane (TM) domains are shown in boxes. ( D ) IDR predictions of intracellular regions of each protein isoform. Regions wherein the score is more than 0.5 are predicted to be disordered. ( E ) Western blot of proteins extracted from adult heads overexpressing each 3xFLAG-tagged Fas3 isoform . Anti-Fas3G antibody specifically detects Fas3 isoform G. ( F ) Western blot of endogenous Fas3 expressed in adult male heads. Drice-proximal proteins biotinylated by C-terminally knocked-in tagged V5::TurboID are purified using NeutrAvidin. ( G ) Western blot of 3xFLAG-tagged Fas3 isoforms overexpressed in adult head. Drice-proximal proteins biotinylated by C-terminally knocked-in tagged V5::TurboID are purified using NeutrAvidin. Figure 2—source data 1. Uncropped raw blot images of . Figure 2—source data 2. Uncropped annotated blot images of .

Article Snippet: The primary antibodies used were mouse anti-Fasciclin 3 (7G10) antibody (1:20, #AB_528238, DSHB), rat anti-GFP (GF090R) monoclonal antibody (1:100, #04404-26, nacalai Tesque), mouse anti-FLAG M2 monoclonal antibody (1:500, F1804, Sigma), and rat anti-mCD8 monoclonal antibody (1:50, #MCD0800, Invitrogen).

Techniques: Expressing, Staining, Western Blot, Purification