Journal: Aging Cell

Article Title: Age‐dependent integrity of the meiotic spindle assembly checkpoint in females requires Aurora kinase B

doi: 10.1111/acel.13489

Figure Lengend Snippet: SAC mediator and scaffolding proteins are reduced at kinetochores in Aurkb cKO oocytes from older females. (a, c, e, g, I, k) Representative confocal images of oocytes from wild‐type (WT) or conditional Aurkb knockouts (B cKO) from young and older females. Centromeres were detected by staining with antibodies against ACA (red) and chromosomes were detected with DAPI‐staining (blue). (a, c) Representative confocal images of Met I oocytes immunostained with antibody against MAD2 (gray) from young (a) or older females (c). (b) Quantification of MAD2 intensity at kinetochores in (a) ( p = 0.2711; number of oocytes examined, WT: 51, B cKO: 36; 4 mice/genotype). n.s.: not significant. (d) Quantification of MAD2 intensity at kinetochores in (c) (One‐way ANOVA, **** p < 0.0001; number of oocytes examined, WT: 52, B cKO: 47, C KO: 39; 3 mice/genotype). (e) Early pro‐metaphase I oocytes immunostained with antibodies against MAD2 (gray). (f) Quantification of MAD2 intensity at kinetochores in (e) (**** p < 0.0001; number of oocytes examined, WT: 40, B cKO: 35; 6 mice/genotype). (g) Late pro‐metaphase I oocytes immunostained with anti‐MPS1 (gray); (h) Quantification of MPS1 intensity at kinetochores of (g) ( p = 0.0514; number of oocytes examined, WT: 31, B cKO: 33; 3 mice/genotype). (i, k) Representative confocal images of metaphase I oocytes immunostained with (i) BUB1 (gray) or (k) ZW10 (gray). (j) Quantification of BUB1 intensity at kinetochores showed in (i) (**** p < 0.0001; number of oocytes examined, WT: 39, B cKO: 37; 3 mice/genotype). (l) Quantification of ZW10 intensity at kinetochores showed in (k) (* p <0.0121; number of oocytes examined, WT: 40, B cKO: 27; 3 mice/genotype). n.s.: not significant. Examples of individual bivalent (boxes) are magnified and shown in the zoom panels. Scale bars: 10 μm and 2 μm. Graph shows the mean value per oocyte of at least 30 kinetochores measured for each oocyte and includes the mean ±SEM from 3 experiments. Except for panels c‐d, Unpaired Students t‐Test, two‐tailed used

Article Snippet: Human MAD2 was amplified by PCR and cloned into the pIVT–Gfp vector (Addgene #16047).

Techniques: Scaffolding, Staining, Two Tailed Test

Journal: Aging Cell

Article Title: Age‐dependent integrity of the meiotic spindle assembly checkpoint in females requires Aurora kinase B

doi: 10.1111/acel.13489

Figure Lengend Snippet: Aurkb cKO oocytes from older females have reduced expression of MAD2, ZW10 and Securin. (a) Representative Western blot images of prophase I‐arrested oocytes from older wild‐type (WT) or conditional Aurkb knockout (B cKO) females detecting MAD2, ZW10 and Securin. (b) Quantification of MAD2 (** p < 0.0030), (c) Quantification of ZW10 (** p < 0.0016). (d) Quantification of Securin (* p < 0.0468). (e, g) Representative Western blots of prophase I‐arrested oocytes from young females detecting MAD2, Securin (d) and ZW10 (g). (f) Quantification of MAD2 ( p = 0.9774). Graph shows individual values plus the mean ±SEM from 3 independent experiments. (h) Quantification of ZW10 ( p = 0.1166). (i) Quantification of Securin (* p < 0.7505), MAD2, ZW10 and Securin signals were normalized to α‐Tubulin. Each lane contained 100 prophase I‐arrested oocytes. n.s.: not significant. Graph shows mean ±SEM from 2–3 independent experiments. (j) Representative confocal images of pro‐metaphase I oocytes expressing Gfp or MAD2‐gfp (green) DNA (blue). Scale bar: 10 μm. (k) Representative images of timing of PBE of Aurkb cKO oocytes from older females, matured in nocodazole expressing either Gfp or MAD2‐gfp. Red star indicates a PB. Scale bar: 20 μm. (l) Quantification of % of oocytes from (k) that undergo PBE in nocodazole. (* p = 0.0.0160; number of oocytes examined Gfp: 73, MAD2‐gfp: 59; 6 mice). Graph shows the mean ±SEM from 3 experiments. (m) Representative images of timing of PBE of Aurkb cKO oocytes from older females, matured in nocodazole expressing Securin‐gfp (green). Numbers of oocytes that expelled a PB relative to total number of oocytes analyzed is indicated to the right of the images. Unpaired Students t‐Test, two‐tailed was used

Article Snippet: Human MAD2 was amplified by PCR and cloned into the pIVT–Gfp vector (Addgene #16047).

Techniques: Expressing, Western Blot, Knock-Out, Two Tailed Test

Journal: Aging Cell

Article Title: Age‐dependent integrity of the meiotic spindle assembly checkpoint in females requires Aurora kinase B

doi: 10.1111/acel.13489

Figure Lengend Snippet: ROS levels are increased in Aurkb cKO oocytes from older females. (a) Representative images of CM‐H2DCFDA fluorescence in prophase I‐arrested oocytes from wild‐type (WT), conditional Aurkb knockouts (B cKO), or Aurkc knockout (C KO) females at different ages. (b) Quantification of CM‐H2DCFDA intensity showed in (a) **** p < 0.0001; number of oocytes examined: positive control: 29; young WT: 58, B cKO: 77, C KO: 92; older: WT: 60, B cKO: 36, C KO: 70; 3 mice/genotype). (c) Representative images of CM‐H2DCFDA fluorescence in prophase I‐arrested oocytes from wild‐type (WT), conditional Aurkb knockouts (B cKO), older females treated with and without NAC. Positive control: WT oocytes incubated with 200 μM H 2 O 2 . Scale bars: 20 μm. (d) Quantification of CM‐H2DCFDA intensity showed in (c) (** p = 0.0035; * p < 0.05; number of oocytes examined: WT: 20, B cKO: 25, B cKO +NAC:29; 3 mice/genotype). (e) Representative confocal images of WT and Aurkb cKO Met I oocytes from older females treated with or without NAC, immunostained with antibodies against ACA (red), MAD2 (gray) and DAPI (blue). (f) Quantification of MAD2 intensity at kinetochores in (e) (* p = 0.0464; **** p < 0.0001; n.s. not significant; number of oocytes examined: WT: 35, B cKO: 32, B cKO +NAC:28; 3 mice/genotype). Graph shows individual oocyte values plus the mean ±SEM from 3 experiments. One‐way ANOVA used

Article Snippet: Human MAD2 was amplified by PCR and cloned into the pIVT–Gfp vector (Addgene #16047).

Techniques: Fluorescence, Knock-Out, Positive Control, Incubation

Journal: Journal of Cardiology Cases

Article Title: Ostial left circumflex lesion with calcified nodule successfully treated with excimer laser coronary atherectomy and drug-coated balloon

doi: 10.1016/j.jccase.2020.04.004

Figure Lengend Snippet: Coronary angiography. (A) An angiogram at the baseline showed significant stenosis with a CN at the ostial LCX. (B) The systems are shown. Guide catheter: 6Fr VL3.5 guide catheter (Boston Scientific, Natick, MA, USA); LCX: a 0.014-in. guidewire (Sion Blue; Asahi Intec., Nagoya, Japan); LAD: a 0.014-in. guidewire (Runthrough NS; Terumo, Tokyo, Japan). (C) The balloon dilatation of a 1.75 mm × 15 mm PTCA catheter (canPass; Japan Lifeline Co., Tokyo, Japan). (D) A 0.9 mm concentric catheter of excimer laser coronary atherectomy. (E) A 1.4 mm concentric catheter of excimer laser coronary atherectomy. (F) The dilatation of a 2.5 mm × 13 mm scoring balloon catheter (Lacrosse NSE alpha; Goodman Co., Ltd., Nagoya, Japan). (G) The dilatation of 2.5 mm × 15 mm paclitaxel courted balloon catheter (Sequent Please; B Braun, Melsungen, Germany). (H) Final CAG at the PCI. (I) Follow-up CAG performed 3 months after the PCI. Restenosis at LCX was not identified without coronary flow limitation. (J) Follow-up CAG performed 3 years after the PCI. Restenosis at LCX was not identified without coronary flow limitation.

Article Snippet: The left coronary artery was engaged by a 6Fr VL3.5 guide catheter (Boston Scientific, Natick, MA, US) from a right radial artery approach ( B).

Techniques: