Journal: Molecular Biology of the Cell

Article Title: APC FZR1 prevents nondisjunction in mouse oocytes by controlling meiotic spindle assembly timing

doi: 10.1091/mbc.E12-05-0352

Figure Lengend Snippet: Premature spindle elongation is independent of CDK1 activity or TPX2, HURP, and Eg5 protein level in Fzr1 −/− oocytes. (A) CDK1 activity measured using an enzyme-linked immunosorbent assay in Fzr1 −/− and control fl/fl oocytes at 2.5 h after GVB. Activity levels were significantly higher in Fzr1 −/− oocytes (* p = 0.037, one-way ANOVA with Tukey's test). Treatment with 2.5 μM flavopiridol reduced CDK1 activity to levels that were not significantly different from control oocytes. (B) Spindle length for control and Fzr1 −/− oocytes, with and without flavopiridol treatment, measured 2.5 h after GVB. Flavopiridol treatment of Fzr1 −/− oocytes did not significantly alter spindle length. (C) Representative immunoblots for TPX2, HURP, and Eg5 on protein from GV-arrested oocytes collected from Fzr1 −/− mice or controls, with n = 50–100 oocytes per lane and at least two separate experiments performed. (D) Densitometric analysis of immunoblots in C. Error bars show SD.

Article Snippet: Immunoblotting was performed using antibodies for FZR1 (ab3242; Abcam, Cambridge, United Kingdom), gylceraldehyde-3-phosphate dehydrogenase (G9545; Sigma-Aldrich), Aurora A kinase (ab13824; Abcam), cyclin B1 (ab72; Abcam), securin (ab3305; Abcam), CDC20 (sc8358; Santa Cruz Biotechnology, Santa Cruz, CA), TPX2 (ab32795; Abcam), Hurp (sc98809; Santa Cruz Biotechnology), Eg5 (NB100-8467; Novus Biologicals, Littleton, CO), γ-tubulin (T6557; Sigma-Aldrich), and secondary immunoglobulin horseradish peroxidase (Dako, Glostrup, Denmark).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Control, Western Blot

Journal: Biomedicines

Article Title: Unmasking Protein Phosphatase 2A Regulatory Subunit B as a Crucial Factor in the Progression of Dilated Cardiomyopathy

doi: 10.3390/biomedicines12081887

Figure Lengend Snippet: Characterization of the ISO-induced DCM mouse model. ( A ) Representative images of DCM mode echocardiography in each group. ISO (Low): 30 mg/kg/day; ISO (high): 80 mg/kg/day. ( B ) Quantitative analysis of the echo parameters of the left ventricle of each group. LVEF: left ventricular ejection fraction, LVFS: left ventricular fraction shortening, LVEDV: LV end-diastolic volume, LVESV: LV end-systolic volume, LEVDD: left ventricular end-diastolic dimension, LEVSD: left ventricular end-systolic dimension ( n = 3). ( C ) Masson stain of the short-axis section of hearts. Scale bar: 1 mm. ( D ) The expression of Ppp2r5d mRNA in the cardiac ventricles of DCM mice and MI mice was compared with that of sham mice using RT-qPCR ( n = 4). ( E ) GO enrichment analysis of DEGs in Ppp2r5d knockdown with MCMs vs. control MCMs. ( F ) KEGG pathways of DEGs in Ppp2r5d knockdown with MCMs vs. control MCMs. Data are presented as the mean ± SD with p values indicated. * p < 0.05.

Article Snippet: The following primary antibodies were used: anti-Phospho-STAT3 (Y705) (9145, CST, New York, NY, USA); anti-Phospho-STAT3 (S727) (9136, CST, New York, NY, USA); anti-STAT3 (4904; CST, New York, NY, USA); anti- Ppp2r5d (B56δ) (12068-1-AP, Proteintech, Wuhan, China); anti-GADPH-HRP (Pab001H, ESscience, China); anti-VDAC1/2 (10866-1-AP, Proteintech, Wuhan, China); and anti-Total OXPHOS Rodent WB antibody Cocktail (ab110413, Abcam, Cambridge, UK).

Techniques: Staining, Expressing, Quantitative RT-PCR, Knockdown, Control

Journal: Biomedicines

Article Title: Unmasking Protein Phosphatase 2A Regulatory Subunit B as a Crucial Factor in the Progression of Dilated Cardiomyopathy

doi: 10.3390/biomedicines12081887

Figure Lengend Snippet: The effects of Ppp2r5d -knockdown MCMs on mitochondrial dysfunctions. ( A ) Representative histogram plots show the ROS levels in MCMs after treatment. ( B ) Quantification of DCFH-DA staining (Mean ± SD, n = 5). ( C ) Representative histogram plots show the mitochondria levels in MCMs after treatment. ( D ) Quantification of MitoTracker green staining (Mean ± SD, n = 3). ( E ) ATP production in MCMs was measured with an ATP kit (Mean ± SD, n = 6). ( F ) Mitochondrial respiration in MCMs was measured with the Seahorse assay. MCMs were sequentially incubated with oligomycin (1.5 μM), FCCP (1 μM), and antimycin (10 μM). ( G ) The mitochondrial functional parameters from F (basal respiration, maximal respiration, ATP production, spare respiratory capacity, non-mitochondrial respiration, and proton leak). n = 7. Data are presented as the mean ± SD with p values indicated.

Article Snippet: The following primary antibodies were used: anti-Phospho-STAT3 (Y705) (9145, CST, New York, NY, USA); anti-Phospho-STAT3 (S727) (9136, CST, New York, NY, USA); anti-STAT3 (4904; CST, New York, NY, USA); anti- Ppp2r5d (B56δ) (12068-1-AP, Proteintech, Wuhan, China); anti-GADPH-HRP (Pab001H, ESscience, China); anti-VDAC1/2 (10866-1-AP, Proteintech, Wuhan, China); and anti-Total OXPHOS Rodent WB antibody Cocktail (ab110413, Abcam, Cambridge, UK).

Techniques: Knockdown, Staining, Incubation, Functional Assay

Journal: Biomedicines

Article Title: Unmasking Protein Phosphatase 2A Regulatory Subunit B as a Crucial Factor in the Progression of Dilated Cardiomyopathy

doi: 10.3390/biomedicines12081887

Figure Lengend Snippet: The effects of cardiomyocyte-specific Ppp2r5d knockdown on the pathogenesis of DCM and heart failure (HF) in vivo. ( A ) Workflow of the animal experiments. ( B ) Representative echocardiographic images at 8 weeks after removal of the osmotic mini-pump are shown. ( C ) Quantitation of the left ventricles’ parameters in each group. LVEF: left ventricular ejection fraction; LVFS: left ventricular fraction shortening; LVEDV: LV end-diastolic volume; LVESV: LV end-systolic volume; LEVDD: left ventricular end-diastolic dimension; LEVSD: left ventricular end-systolic dimension. n = 6. ( D ) Representative anatomical images of the hearts. Scale bar: 1 mm. ( E ) Heart weight/body weight ratio and heart weight/tibia length ratio in each group of mice. n = 6. ( F ) Relative mRNA expression of Nppa , Nppb , and Myh7 in the ventricles of sham mice, AAV-cTnT-shRNA-NC & ISO mice, and AAV-cTnT-shRNA- Ppp2r5d & ISO mice. n = 6. ( G ) Masson stain of the collagen deposition of the lateral cross-section of hearts (Mag 1.25× and 20×). ( H ) Quantification of fibrosis area percentage. ( I ) WGA stain of the lateral cross-section of hearts (Mag 63×). ( J ) Quantification of cell area (μm 2 ). ( K ) Representative transmission electron microscopy (TEM) images of the left ventricular (LV) and right ventricular (RV) section ultrastructure from sham mice (left), AAV-cTnT-shRNA-NC & ISO mice (middle), and AAV-cTnT-shRNA- Ppp2r5d & ISO mice (right). Magnification: 5000×; scale bar: 1 μm. Red arrows: vacuolated mitochondria; blue arrows: broken myocardial muscle fibers. ( L ) Quantification of mitochondria-related parameters from the panel: the number of mitochondria per field, the mean area of the mitochondria in each image, and the percentage of the abnormal mitochondria per field, respectively. Data are presented as the mean ± SD with p values indicated.

Article Snippet: The following primary antibodies were used: anti-Phospho-STAT3 (Y705) (9145, CST, New York, NY, USA); anti-Phospho-STAT3 (S727) (9136, CST, New York, NY, USA); anti-STAT3 (4904; CST, New York, NY, USA); anti- Ppp2r5d (B56δ) (12068-1-AP, Proteintech, Wuhan, China); anti-GADPH-HRP (Pab001H, ESscience, China); anti-VDAC1/2 (10866-1-AP, Proteintech, Wuhan, China); and anti-Total OXPHOS Rodent WB antibody Cocktail (ab110413, Abcam, Cambridge, UK).

Techniques: Knockdown, In Vivo, Quantitation Assay, Expressing, shRNA, Staining, Transmission Assay, Electron Microscopy

Journal: Biomedicines

Article Title: Unmasking Protein Phosphatase 2A Regulatory Subunit B as a Crucial Factor in the Progression of Dilated Cardiomyopathy

doi: 10.3390/biomedicines12081887

Figure Lengend Snippet: Knockdown of Ppp2r5d impaired the activity of the mitochondrial respiratory chain complex in MCMs. ( A ) Relative mRNA expression of the mitochondrial genes in each group. ( B ) Protein expression of mitochondrial complexes assessed by Western blot in each group. Experiments were repeated three independent times. ( C ) The relative band intensity of NDUFB8, SDHB, UQCRC2, MTCO1, ATP5a, and B56δ were analyzed by Image J software (version 1.4.3.67) and normalized to total VDAC1. ( D ) Mitochondria were isolated from MCMs, and complex I activity was measured. The enzymatic activity is presented as milliunits (nmol/min) per milligram (mg) of mitochondrial protein. Data are presented as the mean ± SD with p values indicated.

Article Snippet: The following primary antibodies were used: anti-Phospho-STAT3 (Y705) (9145, CST, New York, NY, USA); anti-Phospho-STAT3 (S727) (9136, CST, New York, NY, USA); anti-STAT3 (4904; CST, New York, NY, USA); anti- Ppp2r5d (B56δ) (12068-1-AP, Proteintech, Wuhan, China); anti-GADPH-HRP (Pab001H, ESscience, China); anti-VDAC1/2 (10866-1-AP, Proteintech, Wuhan, China); and anti-Total OXPHOS Rodent WB antibody Cocktail (ab110413, Abcam, Cambridge, UK).

Techniques: Knockdown, Activity Assay, Expressing, Western Blot, Software, Isolation

Journal: Biomedicines

Article Title: Unmasking Protein Phosphatase 2A Regulatory Subunit B as a Crucial Factor in the Progression of Dilated Cardiomyopathy

doi: 10.3390/biomedicines12081887

Figure Lengend Snippet: Knockdown Ppp2r5d in MCMs affected the status of STAT3 phosphorylation in the DCM model. ( A ) The expressions of the STAT3-related proteins were determined by Western blot, and representative images are shown. Total STAT3 and β-Actin were used as internal controls. The fold changes in protein expression level were normalized to the internal control and determined by densitometric analysis. Experiments were repeated three independent times. ( B ) The relative band intensity of pSTAT3 (Y705) and pSTAT3 (S727) was analyzed by ImageJ software (version 1.4.3.67) and normalized to Total STAT3. ( C ) Knockdown of Ppp2r5d resulted in upregulation of IL6 expression in mouse cardiomyocytes. Relative mRNA level of IL6 and Ppp2r5d in MCMs transfected with siRNA-NC or siRNA- Ppp2r5d after ISO treatment for 18 h, normalized to β-Actin expression ( n = 6). ( D ) The protein level of IL6 in culture supernatants of MCMs among individual groups ( n = 3). ( E ) Relative mRNA levels of IL6 and Ppp2r5d in NMCMs transfected with siRNA-NC or siRNA- Ppp2r5d after ISO treatment, normalized to β-Actin expression ( n = 6). ( F ) The IL6 protein levels in culture supernatants of NMCMs were determined by an ELISA kit in each group ( n = 3). ( G ) Relative IL6 mRNA level in MCMs transfected with siRNA-NC or siRNA- Ppp2r5d after ISO or S3I-201 treatment for 18 h. The values were normalized to β-Actin mRNA levels ( n = 6). ( H ) The IL6 protein levels in culture supernatants of NMCMs were determined with an ELISA kit in each group ( n = 3). Data are presented as the mean ± SD with p values indicated.

Article Snippet: The following primary antibodies were used: anti-Phospho-STAT3 (Y705) (9145, CST, New York, NY, USA); anti-Phospho-STAT3 (S727) (9136, CST, New York, NY, USA); anti-STAT3 (4904; CST, New York, NY, USA); anti- Ppp2r5d (B56δ) (12068-1-AP, Proteintech, Wuhan, China); anti-GADPH-HRP (Pab001H, ESscience, China); anti-VDAC1/2 (10866-1-AP, Proteintech, Wuhan, China); and anti-Total OXPHOS Rodent WB antibody Cocktail (ab110413, Abcam, Cambridge, UK).

Techniques: Knockdown, Phospho-proteomics, Western Blot, Expressing, Control, Software, Transfection, Enzyme-linked Immunosorbent Assay