mouse anti-h2av  (Active Motif)

Journal: BMB Reports

Article Title: Tau reduction impairs nephrocyte function in Drosophila

doi: 10.5483/BMBRep.2024-0047

Figure Lengend Snippet: Knockdown of dTau disrupts nuclear structure and forms senescence-associated heterochromatin foci (SAHF) in nephrocytes. (A) Nephrocytes were stained with DAPI (blue) to visualize DNA. SAHF, a marker of cellular senescence, was observed in the nephrocytes of dTau RNAi-expressing flies (arrowheads). (B) Quantification of DAPI foci-positive nephrocytes in adult flies. A significant increase in the number of DAPI foci-positive nephrocytes was observed in the dTau RNAi-expressing nephrocytes, compared to the control. Error bars represent the mean ± standard deviation (n = 4 adult flies for each genotype). Statistical significance was determined using Student’s t -test (***P < 0.001). (C) Pericardial nephrocytes (red) in the abdomen of mCD8−RFP-expressing flies under the control of snsGCN−Gal4. Nephrocytes dissected from (3 or 20)-d-old dTau RNAi-expressing flies were stained with anti-histone H2Av antibody (green) and DAPI (blue). dTau knockdown exhibits increased frequencies of γ–H2Av foci in the nephrocytes of 3-d-old flies (arrowheads). Dispersed γ–H2Av foci in 20-d-old dTau RNAi-expressing flies were detected in the cytoplasm of nephrocytes. (D, E) Quantification of nuclear and cytoplasmic γ–H2Av fluorescent signal in the nephrocytes. Error bars represent the mean ± standard deviation (n ≥ 8 nephrocytes for each genotype). Statistical significance was determined using Student’s t -test (**P < 0.01, ***P < 0.001).

Article Snippet: Subsequently, the samples were incubated with the primary antibodies: rabbit anti-DCP-1 antibody (1:100; Cell Signaling Technology, Cat#: 9578, Danvers, MA, USA), mouse anti-Histone H2Av antibody (1:100; DSHB, Cat#: UNC93-5.2.1, Iowa City, IA, USA), and mouse anti-Lamin B (Dm0) antibody (1:100; DSHB, Cat#: ADL67.10, Iowa City, IA, USA) for 12 h at 4°C.

Techniques: Knockdown, Staining, Marker, Expressing, Control, Standard Deviation

Journal: Cells

Article Title: Neuronal Progenitors Suffer Genotoxic Stress in the Drosophila Clock Mutant per 0

doi: 10.3390/cells13231944

Figure Lengend Snippet: DNA damage and chromosome aberrations in per 0 mutants. ( A ) Anti-γ-H2AV immune labelling (red) in CNS-squash preparations from third-instar male larvae. DNA is stained with DAPI (white). Size bar = 5 μm. ZT = 1. ( B ) Proportion of cells showing anti-γ-H2AV immune signal in CNS-squash preparations from CS and per 0 third-instar male larvae. Cells were considered ‘H2AV positive’ when the relative intensity of the anti-γ-H2AV immune signal in the nucleus [(signal-background)/background] was equal to or more than 1.5. The DAPI signal was used to identify nuclei. Fisher’s exact test, **** p < 0.0001. Total number of cells, n = 1323, 985, respectively. ZT = 1. ( C ) Mitotic metaphases in CNS-squash preparations from third-instar larvae. Left, CS , showing a normal metaphase. Right, chromosome aberrations in per 0 . The yellow arrow indicates a chromosome fragment (break). The white arrow points to a fusion. Numbers 2, 3, 4 identify the autosomes. X, Y label the sex chromosomes. Size bar = 5 μm. ZT = 2. ( D ) Frequency of chromosome aberrations [(abnormal metaphases/total metaphases) × 100] in CS and per 0 . Fisher’s exact test, **** p < 0.0001. ZT = 2. Total number of metaphases scored, N = 436, 527, respectively. ( E ) PER overexpression rescues chromosome aberrations in per 0 . The overexpression of PER using the pan-circadian per-GAL4 ( perG4 > PER, per 0 ) and tim-GAL4 ( timG4 > PER, per 0 ) drivers drastically reduced the frequency of aberrations in an otherwise per 0 background. Chi-square = 49.50, df = 3, **** p < 0.0001. Total number of metaphases scored (from left to right), n = 337, 463, 345, 1021. ZT = 2.

Article Snippet: Slides were transferred to 1xPBS for 5 min, permeabilized in 1% PBS-Tx for 10 min, and blocked in 1xPBS and 1% BSA for 30 min before incubation with mouse a-γ-H2AV antibodies (DSHB #UNC93-5.2.1) diluted 1:5 in 1xPBS and 1% BSA.

Techniques: Staining, Over Expression

Journal: Cells

Article Title: Neuronal Progenitors Suffer Genotoxic Stress in the Drosophila Clock Mutant per 0

doi: 10.3390/cells13231944

Figure Lengend Snippet: ROS buffering and reduction decrease DNA damage and chromosome aberrations in per 0 . Treatment with vitamin C (VitC, 40 mM in standard medium, from embryo) reduced anti-γ-H2AV immune labelling ( A – C ) and chromosome aberrations ( D ) in per 0 third-instar larva males. ( A ) Brain lobes (BLs) from third-instar per + and per 0 male larvae obtained by reciprocal crossing (♀ CS × ♂ per 0 and vice versa); anti-γ-H2AV on the whole mount. Confocal maximum intensity projections. Dashed lines outline the BLs. Size bar = 30 μm. ZT = 2. ( B ) Quantification of anti-γ-H2AV immune fluorescence intensity [relative signal intensity = (signal-background)/background] in whole-mount CNS from third-instar per + and per 0 male larvae as above. Normal distribution of data was confirmed with the Shapiro–Wilk test. Top, BL (one per individual). Two-way ANOVA, Genotype (F 1, 43 = 7.318, p = 0.0097), VitC treatment (F 1, 43 = 4.732, p = 0.0352), Genotype × VitC treatment (F 1, 43 = 1.617, p = 0.2104). Tukey’s multiple comparisons test, per + vs. per 0 , * p = 0.0282; per + VitC vs. per 0 , ** p = 0.0075; per 0 vs. per 0 VitC, * p = 0.0196. Bottom, VNC. Two-way ANOVA, Genotype (F 1, 33 = 23.75, p < 0.0001), VitC treatment (F 1, 33 = 5.241, p = 0.0286), Genotype × VitC treatment (F 1, 33 = 3.044, p = 0.0903). Tukey’s multiple comparisons test, per + vs. per 0 , *** p = 0.0003; per + VitC vs. per 0 , *** p = 0.0001; per 0 vs. per 0 VitC, ** p = 0.0066. ZT = 1. ( C ) Proportion of anti-γ-H2AV immune positive cells in CNS squash preparations from third-instar male larvae. Cells were considered ‘H2AV positive’ when the relative intensity of the anti-γ-H2AV immune signal in the nucleus [(signal-background)/background] was equal or more than 1.5. The DAPI signal was used to identify nuclei. Treatment with VitC did not affect CS (Fisher’s exact test, p = 0.9209) but lowered the proportion of anti-γ-H2AV immune labelled cells in per 0 (Fisher’s exact test, ** p = 0.0011). Total number of cells scored (from left to right), n = 912, 775, 295, 298. ZT = 2. ( D ) Proportion of chromosome aberrations [(abnormal metaphases/total metaphases) × 100] in CNS squash preparations from third-instar per + and per 0 male larvae obtained by reciprocal crossing (♀ CS × ♂ per 0 and vice versa). Treatment with VitC lowered the proportion of chromosome aberrations. The effect was small in per + (Fisher’s exact test, p = 0.1886) but highly significant in per 0 (Fisher’s exact test, *** p < 0.0005). Total number of metaphases scored (from left to right), n = 468, 440, 439, 633. ZT = 1. AOX overexpression rescues chromosome aberrations ( E , F ). ( E ) Cartoon showing the position of the Alternative Oxidase (AOX) in the electron transport chain. ( F ) The overexpression of AOX using the pan-circadian tim-GAL4 ( timG4 > AOX) driver reduced the frequency of aberrations. The effect was marginal in per + (Fisher’s exact test, p = 0.0663) but highly significant in per 0 (Fisher’s exact test, *** p < 0.0009). Total number of metaphases scored (from left to right), n = 397, 387, 456, 412. ZT = 1. Males were obtained by reciprocal crossing [♀ UAS-AOX ( per + ) × ♂ tim-GAL4 ( per 0 ) and vice versa].

Article Snippet: Slides were transferred to 1xPBS for 5 min, permeabilized in 1% PBS-Tx for 10 min, and blocked in 1xPBS and 1% BSA for 30 min before incubation with mouse a-γ-H2AV antibodies (DSHB #UNC93-5.2.1) diluted 1:5 in 1xPBS and 1% BSA.

Techniques: Fluorescence, Over Expression

Journal: bioRxiv

Article Title: Neuronal progenitors suffer genotoxic stress in the Drosophila clock mutant per 0

doi: 10.1101/2024.08.13.607601

Figure Lengend Snippet: a) Anti-γ-H2AV immune labelling (red) in CNS-squash preparations from 3 rd instar male larvae. DNA is stained with DAPI (white). Size bar = 5 μm. ZT=1. b) Proportion of cells showing anti-γ-H2AV immune signal in CNS-squash preparations from CS and per 0 3 rd instar male larvae. Fisher’s exact test, ****P<0.0001. Total number of cells, N = 1323, 985, respectively. ZT=1. c) Mitotic metaphases in CNS-squash preparations from 3 rd instar larvae. Left, CS , showing a normal metaphase. Right, chromosome aberration in per 0 . The yellow arrow indicates a chromosome fragment (break). The white arrow points to a fusion. 2, 3, 4 identify the autosomes. X, Y label the sex chromosomes. Size bar = 5 μm. ZT=2. d) Frequency of chromosome aberrations in CS and per 0 . Fisher’s exact test, ****P<0.0001. ZT=2. Total number of metaphases scored, N = 436, 527, respectively. e) PER overexpression rescues chromosome aberrations in per 0 . The overexpression of PER ( UASper16 ) using the pan-circadian per- and tim-GAL4 drivers drastically reduced the frequency of aberrations in an otherwise per 0 background. Chi-square= 49.50, df=3, ****P<0.0001. Total number of metaphases scored (from left to right), N = 337, 463, 345, 1021. ZT=2.

Article Snippet: Slides were transferred to 1XPBS for 5 min, permeabilized in 1% PBS-Tx for 10 min, and blocked in 1X PBS, 1% BSA, for 30 min before incubation with mouse α-γ-H2AV antibodies (DSHB #UNC93-5.2.1) diluted 1:5 in 1X PBS, 1% BSA.

Techniques: Staining, Over Expression

Journal: bioRxiv

Article Title: Neuronal progenitors suffer genotoxic stress in the Drosophila clock mutant per 0

doi: 10.1101/2024.08.13.607601

Figure Lengend Snippet: Treatment with vitamin C (VitC, 40 mM in standard medium, from embryo) reduced anti-γ-H2AV immune labelling (a-d) and chromosome aberrations (e) in per 0 3 rd instar larva males (a-e). a) Brain lobes (BLs) from 3 rd instar per + and per 0 male larvae obtained by reciprocal crossing (♀ CS x ♂ per 0 and vice versa ); anti-γ-H2AV on whole mount. Confocal maximum intensity projections. Dashed lines outline BLs. Size bar = 30 μm. ZT=2. b, c) Quantification of anti-γ-H2AV immune fluorescence intensity [relative signal intensity = (signal-background)/background] in whole mount CNS from 3 rd instar per + and per 0 male larvae as above. b & c show two independent experiments (imaged on different microscopes). Normal distribution of data was confirmed with the Shapiro-Wilk test. b) Top, BL (one per individual). Two-way ANOVA, Genotype (F 1, 43 =7.318, **P=0.0097), VitC treatment (F 1, 43 =4.732, *P=0.0352), Genotype x VitC treatment (F 1, 43 =1.617, P=0.2104). Tukey’s multiple comparisons test, per + vs . per 0 , *P=0.0282; per + VitC vs . per 0 , **P=0.0075; per 0 vs . per 0 VitC, *P=0.0196. Bottom, VNC. Two-way ANOVA, Genotype (F 1, 33 =23.75, ****P<0.0001), VitC treatment (F 1, 33 =5.241, *P=0.0286), Genotype x VitC treatment (F 1, 33 =3.044, P=0.0903). Tukey’s multiple comparisons test, per + vs . per 0 , ***P=0.0003; per + VitC vs . per 0 , ***P=0.0001; per 0 vs . per 0 VitC, **P=0.0066. ZT=1. c) Top, BL (one per individual). Two-way ANOVA, Genotype (F 1, 14 =21.03, ***P=0.0004), VitC treatment (F 1, 14 =40.05, ****P<0.0001), Genotype x VitC treatment (F 1, 14 =39.53, ****P<0.0001). Tukey’s multiple comparisons test, per + vs . per 0 , ****P<0.0001; per + VitC vs . per 0 , ****P<0.0001; per 0 vs . per 0 VitC, ****P<0.0001. Bottom, VNC. Two-way ANOVA, Genotype (F 1, 14 =46.94, ****P<0.0001), VitC treatment (F 1, 14 =12.97, **P=0.0029), Genotype x VitC treatment (F 1, 14 =7.040, *P=0.0189). Tukey’s multiple comparisons test, per + vs . per 0 , ***P=0.0001; per + VitC vs . per 0 , ****P<0.0001; per + VitC vs . per 0 , *P=0.0236; per 0 vs . per 0 VitC, **P=0.0011. Points show individual samples. Error bars = SD. ZT=1. d) Proportion of γ-H2AV immune positive cells in CNS squash preparations from 3 rd instar male larvae. Treatment with VitC did not affect CS (Fisher’s exact test, P=0.9209) but lowered the proportion of γ-H2AV immune labelled cells in per 0 (Fisher’s exact test, **P=0.0011). Total number of cells scored (from left to right), N = 912, 775, 295, 298. ZT=2. e) Proportion of chromosome aberrations in CNS squash preparations from 3 rd instar per + and per 0 male larvae obtained by reciprocal crossing (♀ CS x ♂ per 0 and vice versa ). Treatment with VitC lowered the proportion of chromosome aberrations. The effect was small in per + (Fisher’s exact test, P=0.1886) but highly significant in per 0 (Fisher’s exact test, ***P<0.0005). Total number of metaphases scored (from left to right), N = 468, 440, 439, 633. ZT=1. AOX overexpression rescues chromosome aberrations (f, g). f) Cartoon showing the position of the Alternative Oxidase (AOX) in the electron transport chain. g) The overexpression of AOX using the pan-circadian tim-GAL4 driver reduced the frequency of aberrations. The effect was marginal in per + (Fisher’s exact test, P=0.0663) but highly significant in per 0 (Fisher’s exact test, ***P<0.0009). Total number of metaphases scored (from left to right), N = 397, 387, 456, 412. ZT=1. Males obtained by reciprocal crossing [♀ UAS-AOX (per + ) x ♂ tim-GAL4 ( per 0 ) and vice versa ].

Article Snippet: Slides were transferred to 1XPBS for 5 min, permeabilized in 1% PBS-Tx for 10 min, and blocked in 1X PBS, 1% BSA, for 30 min before incubation with mouse α-γ-H2AV antibodies (DSHB #UNC93-5.2.1) diluted 1:5 in 1X PBS, 1% BSA.

Techniques: Fluorescence, Over Expression