Journal: Nature Communications

Article Title: DNase II mediates a parthanatos-like developmental cell death pathway in Drosophila primordial germ cells

doi: 10.1038/s41467-021-22622-1

Figure Lengend Snippet: a DNA of dying PGCs becomes highly fragmented. A WT embryo at ES 13 stained to visualize the PGCs (Vasa; red) and the DNA (Hoechst; blue). The area of the midline PGCs is magnified in the right panels. Shown, are two dying midline PGCs during early (white circle) and late (yellow circle) cell death stages. Hoechst is readily detected during the early PGC death stage, and its signal fades away at advanced PGC death stages, typical of extensive DNA fragmentation. The asterisk indicates gonadal PGCs. Scale bars, 10 μm. b - d Dying PGCs exhibit DNase II-dependent DNA breaks. Images of the midline areas of WT ( b ) and dnaseII mutant ( c ) embryos, at ES 11, stained to visualize phosphorylation of H2Av (γH2Av, green; an early event following DSBs), PGCs (Vasa; red), and DNA (Hoechst; blue). Circled are three dying midline PGCs at different cell death stages displaying varying levels of γH2Av ( b ). In contrast, ectopically surviving midline PGCs in dnaseII mutants displayed no γH2Av signal (circled in c ). The asterisks indicate gonadal PGCs. Scale bars, 20 μm. d Corresponding quantifications of the percentage of embryos displaying γH2Av staining. The percent value is indicated above each column. n , number of examined embryos. e – g Developmentally dying PGCs, but not ectopically surviving dnaseII mutant PGCs or dying apoptotic PGCs, exhibit DNase II-type DNA breaks. Representative images of WT ( e ) and dnaseII mutant ( f ) embryos, at ES 13, as well as an ES 10 embryo with PGC-specific OE of hid ( g ), all labeled by a Top I-mediated ligation assay to visualize DNase II-type DNA cuts (green). PGCs (Vasa; red). The outlined areas (yellow squares) are magnified in the right panels. Circled, is a dying PGC positively labeled for DNase II-type DNA cuts. An arrowhead is pointing at unlabeled ectopically surviving PGCs. Asterisks indicate gonadal PGCs. Note that Hid-induced apoptotic PGCs readily stain for TUNEL and cleaved caspase (Supplementary Fig. ). Scale bars, 50 μm.

Article Snippet: The primary antibodies used in this study were polyclonal rabbit anti-Vasa (1:250, sc-30210; Santa Cruz), mouse anti-PAR (1:20, ab14459; Abcam), rabbit anti-cleaved Dcp-1 (1:100, 9578 S; Cell Signaling), rabbit anti-Hb9 (1:100; from J. Skeath, Haverford College, PA), and the following antibodies from the Hybridoma Bank (DSHB): polyclonal rabbit anti-Arl8 (1:20) and monoclonal mouse anti-Vasa (direct, 46F11) and mouse anti-γH2Av (direct, UNC93-5.2.1).

Techniques: Staining, Mutagenesis, Labeling, Ligation, TUNEL Assay

Journal: Nature

Article Title: Senescent glia link mitochondrial dysfunction and lipid accumulation

doi: 10.1038/s41586-024-07516-8

Figure Lengend Snippet: a , Lifespan of genomic AP1 reporter line ( TRE -dsRed; n = 200 flies). b , Representative images of fly brains showing age-onset AP1 activity (dsRed; top) is mostly in glial cells (repo; bottom). c , Quantification of dsRed intensity shows that AP1 activity is higher the central brain (left) versus optic lobes (right) ( TRE -dsRed; n = 93 brains). See Extended Data Fig. for high-magnification images and quantification of dsRed colocalization with glial versus neuronal markers. a.u., arbitrary units. d , e , Representative images showing SA-β-Gal activity increases with age ( d ) with quantification ( e ) ( TRE -dsRed; n = 119 brains). f , Brain γH2Av levels increase with age ( TRE -dsRed; n = 8 brains per replicate). For gel source data, see Supplementary Fig. . g , Bulk RNA-seq of brains shows that senescence-associated genes increase with age ( w 1118 ; n = 20 brains per replicate). h , Cells were FACS-isolated from 40-day-old brains for bulk RNA-seq ( repo -GAL4 > TRE -dsRed; UAS -GFP; n = 500 cells per replicate). i , j , Expression of AP1 subunits ( dFos , dJun ), AP1-target genes ( i ) and senescence-associated genes ( j ) is highest in AP1 + glia. See Supplementary Data for differential expression genes. k , Most AP1 + glia are non-dividing by EdU labelling at 40 days ( TRE -dsRed; n = 39 brains). l , Analysis of live FACS-isolated cells from 40-day-old brains shows that AP1 + glia are larger (left) with normal DNA content (right) ( n = 4,748 neurons, n = 14,326 AP1 neg glia, n = 490 AP1 + glia). m , Distribution of γH2Av staining in fixed FACS-isolated cells from 40-day-old brains ( n = 8,609 neurons, n = 845 AP1 neg glia, n = 302 AP1 + glia). For all bar graphs, data shown are means. Each point in a microscopy experiment represents one brain; in immunoblot or bulk RNA-seq experiments it represents one biological replicate. All data were collected from two or three independent experiments. Pearson’s correlation ( c , e , f ). Precise n and P values are in the . * P -adjusted < 0.05 for sequencing data; *** P < 0.001; ** P < 0.01, * P < 0.05 for all other data. All scale bars, 100 μm.

Article Snippet: Membranes were blocked in 3% bovine serum albumin in 1× Tris-buffered saline, 0.1% Tween 20 detergent, incubated in primary antibody overnight at 4 °C (1:200 mouse anti-γH2Av, DSHB UNC93-5.2.1; 1:2,000 mouse anti-tubulin, DSHB AA4.3).

Techniques: Activity Assay, RNA Sequencing Assay, Isolation, Expressing, Staining, Microscopy, Western Blot, Sequencing

Journal: Nature

Article Title: Senescent glia link mitochondrial dysfunction and lipid accumulation

doi: 10.1038/s41586-024-07516-8

Figure Lengend Snippet: a , Bulk RNA-sequencing of brains shows that neuronal loss of ND42 increases AP1 subunits and AP1-target genes ( n = 20 brains per replicate). Supplementary Data for DE genes. b , ND42 and ND30 levels are selectively reduced with neuronal UAS -RNAi. c , Neuronal loss of ND42 increases γH2Av protein levels at 10 d age ( n = 8 brains per replicate). d , Real-time qPCR of 10 d brains shows that neuronal knockdown of mitochondrial genes increases Irbp while reducing Vglut and Dop1R ( n = 20 brains per replicate). e , Feeding TRE -dsRed flies the anti-oxidant drug AD4 reduces brain AP1 activity by dsRed at 20 d with representative images in ( f ). FACS-isolated and bulk RNA-sequenced 10 ddsRed+ and dsRed neg cells from two AP1-activating inner complex RNAi lines showing ( g ) expression of neuronal and glial marker genes, ( h ) AP1 subunits and AP1 target genes and ( j ) RNAi-targeted genes ( n = 500 cells per replicate). See Supplementary Data for DE genes. For all bar graphs, data are mean. Each point in a microscopy experiment represents one brain, and in western immunoblot, real-time qPCR and bulk RNA-sequencing experiments it one biological replicate. All data were collected from two or three independent experiments. Precise n and P values are provided in the . * P -adjusted<0.05 for sequencing data; *** P < 0.001; ** P < 0.01, * P < 0.05 for all other data.

Article Snippet: Membranes were blocked in 3% bovine serum albumin in 1× Tris-buffered saline, 0.1% Tween 20 detergent, incubated in primary antibody overnight at 4 °C (1:200 mouse anti-γH2Av, DSHB UNC93-5.2.1; 1:2,000 mouse anti-tubulin, DSHB AA4.3).

Techniques: RNA Sequencing Assay, Activity Assay, Isolation, Expressing, Marker, Microscopy, Western Blot, Sequencing

Journal: Nature

Article Title: Senescent glia link mitochondrial dysfunction and lipid accumulation

doi: 10.1038/s41586-024-07516-8

Figure Lengend Snippet: a , Reactome pathway enrichment shows neuronal mitochondrial function decreases with age. See Supplementary Data and for differential expression genes and gene ontology (GO) terms. b , Quantification of dsRed intensity shows knockdown of inner complex genes in neurons increases (red) or decreases AP1 activity (black) relative to control (white) ( TRE -dsRed; elav -GS> UAS- RNAi as indicated on the x axis; n = 11–19 brains per genotype). c , d , Neuronal knockdown of inner complex genes elicits AP1 + glia ( c ) similar to natural ageing ( d ). See Extended Data Fig. for quantification and high-magnification images. e , PCA of bulk RNA-sequenced brains at 10 days of age ( n = 20 brains per replicate). f – i , Neuronal loss of ND42 reduces neuron-specific processes ( f ) and genes ( g ) with increased DNA damage pathways ( h ) and genes ( i ). See Supplementary Data and for enriched terms and differential expression genes ( TRE -dsRed; elav -GS> UAS -ND42-RNAi versus > UAS -mCherry-RNAi). j , Representative immunoblot showing neuronal ND42 knockdown increases brain γH2Av levels, legend as in e ; quantification in Extended Data Fig. . For gel source data, see Supplementary Fig. . k , Real-time qPCR of 10-day-old brains shows neuronal loss of ND75 and NP15.6 (two AP1-activating RNAi lines, b ) increases Irbp while reducing Vglut and Dop1R ( TRE -dsRed; elav -GS> UAS- RNAi as indicated on the x axis; n = 20 brains per replicate). l , FACS-isolated and bulk RNA-sequenced 10-day-old RNAi-induced dsRed + cells express senescence-associated genes ( n = 500 cells per replicate). See Supplementary Data for differential expression genes. m , PCA shows 10-day-old RNAi-induced dsRed + cells cluster with naturally occurring AP1 + glia. For bar graphs ( g , i , l ), data shown are means. Each point in a microscopy experiment represents one brain, and in immunoblot, real-time PCR and bulk RNA-seq experiments it represents one biological replicate. All data were collected from two or three independent experiments. A two sample t -test was used ( b , k ). Precise n and P values are in the . * P -adjusted <0.05 for sequencing data; *** P < 0.001; ** P < 0.01, * P < 0.05 for all other data. All scale bars, 100 μm.

Article Snippet: Membranes were blocked in 3% bovine serum albumin in 1× Tris-buffered saline, 0.1% Tween 20 detergent, incubated in primary antibody overnight at 4 °C (1:200 mouse anti-γH2Av, DSHB UNC93-5.2.1; 1:2,000 mouse anti-tubulin, DSHB AA4.3).

Techniques: Expressing, Activity Assay, Western Blot, Isolation, Microscopy, Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Sequencing

Journal: Journal of Molecular Cell Biology

Article Title: Impaired dNKAP function drives genome instability and tumorigenic growth in Drosophila epithelia

doi: 10.1093/jmcb/mjad078

Figure Lengend Snippet: dNKAP localizes to the nucleus and functions to maintain genome stability. ( A – B’ ) Nuclear localization of dNKAP in mitotic wing disc ( A and A’ ) and polyploid salivary gland ( B and B’ ) cells. Scale bar, 50 μm. ( C – D’ ) Accumulation of R-loops in dNKAP -deficient wing disc cells. Scale bar, 20 μm. ( E ) Quantification of S9.6 signals from C and D . n = 10. ( F – G’ ) Increased γH2Av signal in dNKAP -deficient wing disc cells. Scale bar, 50 μm. ( H ) Quantification of γH2Av signals from F and G . n = 10. ( I – J’ ) Increased γH2Av signal in dNKAP -deficient salivary gland cells. Scale bar, 50 μm. ( K ) Quantification of γH2Av signals from I and J . n = 9. ( L – M’ ) Increased Mre11 protein level in dNKAP -deficient wing disc cells. Scale bar, 50 μm. ( N ) Quantification of Mre11 signals from L and M . n = 6. Note that three different regions with the same size were calculated per disc. ( O – R’ ) Chromosome abnormalities in dNKAP -deficient wing disc cells. Note the chromosome bridge and micronucleus phenotype in dNKAP -deficient cells. Scale bar, 150 μm. GFP was used to mark the knockdown domain. Panels C – D’ and I – J’ show single confocal sections of third-instar wing imaginal discs with posterior side to the right and dorsal side up. Panels F and G show the maximum projections across image slices. **** P < 0.0001.

Article Snippet: The primary antibodies used were chicken anti-GFP (1:2000; Abcam, ab13970), mouse anti-β-galactosidase (1:2000; Abcam), mouse anti-armadillo (1:100; DSHB, N2 7A1), mouse anti-MMP1 (1:50; DSHB, 3A6B4), rat anti-E-cadherin (1:100; DSHB, DCAD2-S), mouse anti-NICD (1:100; DSHB, C17.9C6), rabbit anti-cleaved caspase 3 (1:100; Cell Signaling Technology), rabbit anti-aPKC (1:1000, Santa Cruz, sc216), mouse anti-Dlg (1:100, DSHB, 4F3), mouse S9.6 (1:1000, Kerafast, ENH001), mouse anti-γH2Av (1:500; DSHB, Unc93), rabbit anti-γH2Av (1:1000; Rockland, 600-401-914), rabbit anti-Mre11 (1:1000, a gift from Xiaolin Bi), and guinea pig anti-Nbs (1:200, a gift from Yikang Rong).

Techniques: