Journal: Cell Death & Disease

Article Title: Ablation of beta subunit of protein kinase CK2 in mouse oocytes causes follicle atresia and premature ovarian failure

doi: 10.1038/s41419-018-0505-1

Figure Lengend Snippet: a Immunoblotting analysis of DNA damage response signaling in ovaries of Ck2β fl/fl and Ck2β fl/fl ;GCre + mice at 2 weeks after birth. The ovary lysates were collected at least from three mice of each genotype and immunoblotted for γH2AX, H2AX, p-CHK2 (T68), CHK2, p63, p-p53 (S15), p53 and β-actin. Level of β-actin was detected as internal control. Each experiment was repeated at least 2–3 times. Molecular mass is given in kilo Daltons. b Immunoblotting analysis of the expression of p53 and p63 in ovaries of Ck2β fl/fl and Ck2β fl/fl ;GCre + at 4 weeks after birth. The ovary lysates were obtained from at least three mice of each genotype and immunoblotted for p63, p-p53 (S15), p53 and β-actin. Level of β-actin was used as internal control. Each experiment was repeated at least three times. Molecular mass is given in kilo Daltons. c MVH and γH2AX immunofluorescent staining of 2-week-old ovarian sections from Ck2β fl/fl and Ck2β fl/fl ;GCre + mice. Green: γH2AX; Red: MVH; Blue: DAPI. At least three mice of each genotype were used in this assay. Scale bar: 50 μm

Article Snippet: Rabbit monoclonal anti-CK2β antibody (AJ1128b; ABGENT); mouse monoclonal anti-β-actin antibody (sc-47778; Santa Cruz); mouse monoclonal anti-MVH antibody (ab27591; abcam); rabbit monoclonal anti-p-Akt (S473) antibody (4046; Cell Signaling Technology, Inc.); rabbit monoclonal anti-p-Akt (T308) (13038; Cell Signaling Technology, Inc.); rabbit monoclonal anti-p-Akt (S129) (ab133458; abcam); rabbit monoclonal anti-AKT1/2/3 antibody (ab32505; abcam); rabbit monoclonal anti-PTEN antibody (9559; Cell Signaling Technology, Inc.); rabbit polyclonal anti-p-TSC2 (S1387) antibody (5584; Cell Signaling Technology, Inc.); rabbit monoclonal anti-TSC2 antibody (4308; Cell Signaling Technology, Inc.); rabbit monoclonal anti-p-mTOR (S2448) antibody (5536; Cell Signaling Technology, Inc.); rabbit monoclonal anti-p-S6K (T389) antibody (9234; Cell Signaling Technology, Inc.); rabbit monoclonal anti-p-rpS6 (S240/244) antibody (5364; Cell Signaling Technology, Inc.); rabbit polyclonal anti-GSK3α/β (S21/9) antibody (9331; Cell Signaling Technology, Inc.); rabbit monoclonal anti-GSK3α/β antibody (5676; Cell Signaling Technology, Inc.); rabbit polyclonal anti-p-FOXO1 (T24)/FOXO3a (T32) antibody (9464; Cell Signaling Technology, Inc.); rabbit monoclonal anti-FOXO1 antibody (2880; Cell Signaling Technology, Inc.); rabbit monoclonal anti-γH2AX antibody (9718; Cell Signaling Technology, Inc.); rabbit monoclonal anti-H2AX antibody (7631; Cell Signaling Technology, Inc.); rabbit polyclonal anti-p-CHK2 (T68) antibody (BS4043; Bioworld Technology, Inc.); rabbit polyclonal anti-CHK2 antibody (BS6791; Bioworld Technology, Inc.); rabbit monoclonal anti-p-p53 (S15) (12571; Cell Signaling Technology, Inc.); rabbit polyclonal anti-p53 antibody (BS3156; Bioworld Technology, Inc.); rabbit monoclonal anti-p63 (ab124762; abcam); mouse monoclonal anti-CK2α antibody (ab70774; abcam); rabbit polyclonal anti-CK2α’ antibody (BS6571; Bioworld Technology, Inc.); rabbit monoclonal anti-phospho-CK2 substrate [(pS/pT)DXE] (8738; Cell Signaling Technology, Inc.); secondary antibodies were purchased from Zhongshan Golden Bridge Biotechnology Co, Ltd (Beijing).

Techniques: Western Blot, Expressing, Staining

Journal: Cells

Article Title: Protective Effect of Follicle-Stimulating Hormone on DNA Damage of Chicken Follicular Granulosa Cells by Inhibiting CHK2/p53

doi: 10.3390/cells11081291

Figure Lengend Snippet: Antisense primers for siRNA.

Article Snippet: The primary antibodies used for the immunofluorescence were as follows: rabbit anti-FSHR (1:200, GB11275-1, Servicebio, Wuhan, China), rabbit anti-γH2AX (1:200, A11463, Abclonal, Wuhan, China), rabbit anti-CDKN1A (1:200, ER1906-07) and rabbit anti-CHK2 (1:200, ET1610-52, HUABIO, Hangzhou, China).

Techniques:

Journal: Cells

Article Title: Protective Effect of Follicle-Stimulating Hormone on DNA Damage of Chicken Follicular Granulosa Cells by Inhibiting CHK2/p53

doi: 10.3390/cells11081291

Figure Lengend Snippet: Primers for PCR analysis.

Article Snippet: The primary antibodies used for the immunofluorescence were as follows: rabbit anti-FSHR (1:200, GB11275-1, Servicebio, Wuhan, China), rabbit anti-γH2AX (1:200, A11463, Abclonal, Wuhan, China), rabbit anti-CDKN1A (1:200, ER1906-07) and rabbit anti-CHK2 (1:200, ET1610-52, HUABIO, Hangzhou, China).

Techniques: Sequencing

Journal: Cells

Article Title: Protective Effect of Follicle-Stimulating Hormone on DNA Damage of Chicken Follicular Granulosa Cells by Inhibiting CHK2/p53

doi: 10.3390/cells11081291

Figure Lengend Snippet: Morphological changes between SWFs and ASWFs. ( A ) The expression of FSHR in SWFs or ASWFs. Scale bar: 20 µm. GL: Granulosa layer; TL: Theca layer. ( B ) β-galactosidase staining of SWFs or ASWFs. Scale bar: 50 µm. ( C ) The expression of γH2AX in SWFs or ASWFs. Scale bar: 20 µm. ( D ) The expression of CDKN1A in SWFs or ASWFs. Scale bar: 20 µm. ( E ) The expression of CHK2 in SWFs or ASWFs. Scale bar: 20 µm. ( F ) The expression of p-CHK2 in SWFs or ASWFs. Scale bar: 20 µm. ( G ) The ultrastructure of SWFs or ASWFs in hens aged 280 days. Scale bar: 1 µm (left) or 500 nm (right). ( H ) Relative expression of proteins related to DNA damage in the follicles. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibodies used for the immunofluorescence were as follows: rabbit anti-FSHR (1:200, GB11275-1, Servicebio, Wuhan, China), rabbit anti-γH2AX (1:200, A11463, Abclonal, Wuhan, China), rabbit anti-CDKN1A (1:200, ER1906-07) and rabbit anti-CHK2 (1:200, ET1610-52, HUABIO, Hangzhou, China).

Techniques: Expressing, Staining

Journal: Cells

Article Title: Protective Effect of Follicle-Stimulating Hormone on DNA Damage of Chicken Follicular Granulosa Cells by Inhibiting CHK2/p53

doi: 10.3390/cells11081291

Figure Lengend Snippet: Effect of FSH on DNA damage of cultured GCs via CHK2 activation. GCs were treated with 0.01 IU/mL FSH and/or 50 μM ACNU for 24 h. ( A ) Changes in cell viability using the CCK-8 assay. Values are the means ± SEM ( n = 3). Bars with different superscripts are statistically different ( p < 0.05). ( B ) Relative expression of proteins and mRNA related to DNA damage in the follicles. Bars with different superscripts are statistically different ( p < 0.05). ( C ) The expression of CHK2 in GCs after treatment of ACNU and/or FSH. Scale bar: 20 µm.

Article Snippet: The primary antibodies used for the immunofluorescence were as follows: rabbit anti-FSHR (1:200, GB11275-1, Servicebio, Wuhan, China), rabbit anti-γH2AX (1:200, A11463, Abclonal, Wuhan, China), rabbit anti-CDKN1A (1:200, ER1906-07) and rabbit anti-CHK2 (1:200, ET1610-52, HUABIO, Hangzhou, China).

Techniques: Cell Culture, Activation Assay, CCK-8 Assay, Expressing

Journal: Cells

Article Title: Protective Effect of Follicle-Stimulating Hormone on DNA Damage of Chicken Follicular Granulosa Cells by Inhibiting CHK2/p53

doi: 10.3390/cells11081291

Figure Lengend Snippet: The interactions of CHK2 with FoxK or FoxO3a. ( A ) The cell lysates were processed for coimmunoprecipitation with anti-CHK2, followed by probing with anti-FoxO3a, FoxK and FoxO4. IP, immunoprecipitation. ( B ) GCs transfected with CHK2 siRNA or scrambled control siRNA for 24 h/48 h were cultured for 24 h and then the cells were rinsed with PBS. The expression of CHK2 was determined by qPCR and the expression of CHK2 protein was determined by Western blot. Bars with different superscripts are statistically different ( p < 0.05). ( C ) GCs transfected with CHK2 siRNA for 24 h were cultured in media containing ACNU. 24 h later, the cells were rinsed with PBS and the expressions of p53, p-p53, Rad51, CDKN1A, CCND1, LC3B and Atg7 were determined by Western blot. Bars with different superscripts are statistically different ( p < 0.05).

Article Snippet: The primary antibodies used for the immunofluorescence were as follows: rabbit anti-FSHR (1:200, GB11275-1, Servicebio, Wuhan, China), rabbit anti-γH2AX (1:200, A11463, Abclonal, Wuhan, China), rabbit anti-CDKN1A (1:200, ER1906-07) and rabbit anti-CHK2 (1:200, ET1610-52, HUABIO, Hangzhou, China).

Techniques: Immunoprecipitation, Transfection, Cell Culture, Expressing, Western Blot

Journal: Cells

Article Title: Protective Effect of Follicle-Stimulating Hormone on DNA Damage of Chicken Follicular Granulosa Cells by Inhibiting CHK2/p53

doi: 10.3390/cells11081291

Figure Lengend Snippet: Reduced DNA damage after FSH treatment in ASWFs. ( A ) GCs from ASWFs were treated with 0.01 IU/mL FSH in culture. Cell viability using the CCK-8 assay in GCs from ASWFs treated with FSH in culture. Values are the means ± SEM ( n = 3). Bars with different superscripts are statistically different ( p < 0.05). ( B ) Relative expression of proteins related to DNA damage in GCs from ASWFs treated with FSH. Bars with different superscripts are statistically different ( p < 0.05). ( C ) ASWFs were treated with 0.1 IU/mL FSH for 72 h. Representative morphology and the expression of FSHR of ASWFs treated with FSH in culture. Scale bar in H&E staining: 50 µm; Scale bar in IF staining: 20 µm. GL: Granulosa layer; TL: Theca layer. ( D ) Relative expression of γH2AX, FSHR, caspase3, PCNA, Rad51 and CHK2 proteins in cultured ASWFs treated with FSH. Bars with different superscripts are statistically different ( p < 0.05).

Article Snippet: The primary antibodies used for the immunofluorescence were as follows: rabbit anti-FSHR (1:200, GB11275-1, Servicebio, Wuhan, China), rabbit anti-γH2AX (1:200, A11463, Abclonal, Wuhan, China), rabbit anti-CDKN1A (1:200, ER1906-07) and rabbit anti-CHK2 (1:200, ET1610-52, HUABIO, Hangzhou, China).

Techniques: CCK-8 Assay, Expressing, Staining, Cell Culture