Journal: Cell Proliferation

Article Title: Fam70A binds Wnt5a to regulate meiosis and quality of mouse oocytes

doi: 10.1111/cpr.12825

Figure Lengend Snippet: Fam70A interacts directly with oocyte‐predominant Wnt5a. A, SDS‐PAGE and silver staining of Fam70A antibody or control IgG IP complex showed many distinct bands; we cut and sent these distinct bands for IP‐MALDI to identify the potential interacting proteins of Fam70A (see also Figure ). B, Among all Wnt members, we found that Wnt5a (red dot‐line square) is the most abundant within mouse oocytes. C, Quantification of (B). D, Immunofluorescence showed that the Fam70A signal on the oocyte membrane highly overlapped with Wnt5a. Fam70A in green, Wnt5a in red, DNA in blue. E, Co‐IP in oocyte lysate showed that endogenous Fam70A and Wnt5a interacted with each other. F, Co‐IP in the lysate of NIH3T3 cells co‐transfected with Fam70A and Wnt5A plasmids showed that exogenous Fam70A and Wnt5a also interacted with each other. G, Yeast 2‐hybrid assays showed that Fam70A directly binds Wnt5a on both Trp/Leu‐deficient and Trp/Leu/His/Ade‐deficient Agar plate. H, Immuno‐EM showed that Fam70A localized close (<20 nm) to Wnt5a on oocyte membrane or microvillus. Fam70A and Wnt5a were labelled with 35 and 15 nm gold particles, respectively. Scale bar in E, 20 µm. Scale bar in F, 400 nm. GV, germinal vesicle; GVBD, germinal vesicle breakdown; MI, metaphase I; MII, metaphase II

Article Snippet: The following primary Abs were used: Rabbit anti‐Fam70a Ab (Cat#: bs‐11006R; Bioss); Rabbit anti‐Wnt5a Ab (Cat#: A12744; ABclonal); Rabbit anti‐adenomatous polyposis coli (APC) Ab (Cat#: AF0547; Affinity Biosciences); Rabbit anti‐Phospho Catenin Beta (Ser33/37/Thr41) Ab (Cat#: DF2989; Affinity Biosciences); Rabbit anti‐Phospho Akt (Thr308) Ab (Cat#: 13038; Cell Signaling Technology); Rabbit anti‐Phospho Akt (Ser473) Ab (Cat#: A5030; Bimake.Com); Mouse anti‐β‐actin Ab (Cat#: A5316‐100); Mouse anti‐β‐tubulin Ab (Cat#: sc‐5274); Mouse anti‐GAPDH Ab (Cat#: 30201ES60; YEASEN); Rabbit anti‐Beta‐Catenin Ab (Cat#: A5038; Bimake.Com); Mouse anti‐Strep II Tag Ab (Cat#: YFMA0054; YI FEI XUE BIOTECHNOLOGY); Mouse anti‐Flag Tag Ab (Cat#: YFMA0036; YI FEI XUE BIOTECHNOLOGY).

Techniques: SDS Page, Silver Staining, Immunofluorescence, Co-Immunoprecipitation Assay, Transfection

Journal: Cell Proliferation

Article Title: Fam70A binds Wnt5a to regulate meiosis and quality of mouse oocytes

doi: 10.1111/cpr.12825

Figure Lengend Snippet: Fam70A and Wnt5a regulate adenomatous polyposis coli (APC) level and MT stability. A, Western blot showed that APC protein remarkably increased after Fam70A depletion. B, Quantification of (A). C, Immunofluorescence showed that APC was enriched within spindles and remarkably increased after Fam70A depletion. APC in green, tubulin in red, DNA in blue. D, Western blot showed that Wnt5a was efficiently reduced by siRNA (Table ). E, Western blot showed that APC remarkably increased after Wnt5a knockdown. F, Quantification of (E). G, RT‐PCR showed that APC was efficiently reduced by siRNA (Table ). H, Fam70A depletion and Wnt5a knockdown both remarkably increased acetylated tubulin level within spindles, while APC knockdown remarkably reduced acetylated tubulin level. Acetylated tubulin in green, DNA in blue. I, Quantification of (H). Scale bar, 5 µm. * P < .05

Article Snippet: The following primary Abs were used: Rabbit anti‐Fam70a Ab (Cat#: bs‐11006R; Bioss); Rabbit anti‐Wnt5a Ab (Cat#: A12744; ABclonal); Rabbit anti‐adenomatous polyposis coli (APC) Ab (Cat#: AF0547; Affinity Biosciences); Rabbit anti‐Phospho Catenin Beta (Ser33/37/Thr41) Ab (Cat#: DF2989; Affinity Biosciences); Rabbit anti‐Phospho Akt (Thr308) Ab (Cat#: 13038; Cell Signaling Technology); Rabbit anti‐Phospho Akt (Ser473) Ab (Cat#: A5030; Bimake.Com); Mouse anti‐β‐actin Ab (Cat#: A5316‐100); Mouse anti‐β‐tubulin Ab (Cat#: sc‐5274); Mouse anti‐GAPDH Ab (Cat#: 30201ES60; YEASEN); Rabbit anti‐Beta‐Catenin Ab (Cat#: A5038; Bimake.Com); Mouse anti‐Strep II Tag Ab (Cat#: YFMA0054; YI FEI XUE BIOTECHNOLOGY); Mouse anti‐Flag Tag Ab (Cat#: YFMA0036; YI FEI XUE BIOTECHNOLOGY).

Techniques: Western Blot, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction

Journal: Cell Proliferation

Article Title: Fam70A binds Wnt5a to regulate meiosis and quality of mouse oocytes

doi: 10.1111/cpr.12825

Figure Lengend Snippet: Fam70A and Wnt5a regulate β‐catenin activity. A, Co‐IP showed that Fam70A interacted with β‐catenin within oocytes. B, Co‐IP showed that Wnt5a interacted with β‐catenin within oocytes. C, Western blot showed that Fam70A depletion remarkably increased p‐β‐catenin level. D, Quantification of (C). E, Immunofluorescence showed that Fam70A depletion remarkably increased p‐β‐catenin at spindle poles. F, Western blot showed that Wnt5a knockdown remarkably increased p‐β‐catenin level. G, Quantification of (F). H, Western blot showed that APC knockdown remarkably decreased p‐β‐catenin level. I, Quantification of (H). Scale bar, 5 µm. * P < .05

Article Snippet: The following primary Abs were used: Rabbit anti‐Fam70a Ab (Cat#: bs‐11006R; Bioss); Rabbit anti‐Wnt5a Ab (Cat#: A12744; ABclonal); Rabbit anti‐adenomatous polyposis coli (APC) Ab (Cat#: AF0547; Affinity Biosciences); Rabbit anti‐Phospho Catenin Beta (Ser33/37/Thr41) Ab (Cat#: DF2989; Affinity Biosciences); Rabbit anti‐Phospho Akt (Thr308) Ab (Cat#: 13038; Cell Signaling Technology); Rabbit anti‐Phospho Akt (Ser473) Ab (Cat#: A5030; Bimake.Com); Mouse anti‐β‐actin Ab (Cat#: A5316‐100); Mouse anti‐β‐tubulin Ab (Cat#: sc‐5274); Mouse anti‐GAPDH Ab (Cat#: 30201ES60; YEASEN); Rabbit anti‐Beta‐Catenin Ab (Cat#: A5038; Bimake.Com); Mouse anti‐Strep II Tag Ab (Cat#: YFMA0054; YI FEI XUE BIOTECHNOLOGY); Mouse anti‐Flag Tag Ab (Cat#: YFMA0036; YI FEI XUE BIOTECHNOLOGY).

Techniques: Activity Assay, Co-Immunoprecipitation Assay, Western Blot, Immunofluorescence

Journal: Cell Proliferation

Article Title: Fam70A binds Wnt5a to regulate meiosis and quality of mouse oocytes

doi: 10.1111/cpr.12825

Figure Lengend Snippet: Fam70A working model. At cell membrane, Fam70A directly binds Wnt5a, which results in the inhibition of APC and activation of Akt (p‐Akt). APC promotes the phosphorylation (inactive form) of β‐catenin and stabilizes microtubules as well. β‐Catenin (active form) can get into the nucleus to promote gene transcription (but not in fully grown oocytes). Proper microtubule stability and p‐Akt together promote meiosis and quality of mouse oocytes

Article Snippet: The following primary Abs were used: Rabbit anti‐Fam70a Ab (Cat#: bs‐11006R; Bioss); Rabbit anti‐Wnt5a Ab (Cat#: A12744; ABclonal); Rabbit anti‐adenomatous polyposis coli (APC) Ab (Cat#: AF0547; Affinity Biosciences); Rabbit anti‐Phospho Catenin Beta (Ser33/37/Thr41) Ab (Cat#: DF2989; Affinity Biosciences); Rabbit anti‐Phospho Akt (Thr308) Ab (Cat#: 13038; Cell Signaling Technology); Rabbit anti‐Phospho Akt (Ser473) Ab (Cat#: A5030; Bimake.Com); Mouse anti‐β‐actin Ab (Cat#: A5316‐100); Mouse anti‐β‐tubulin Ab (Cat#: sc‐5274); Mouse anti‐GAPDH Ab (Cat#: 30201ES60; YEASEN); Rabbit anti‐Beta‐Catenin Ab (Cat#: A5038; Bimake.Com); Mouse anti‐Strep II Tag Ab (Cat#: YFMA0054; YI FEI XUE BIOTECHNOLOGY); Mouse anti‐Flag Tag Ab (Cat#: YFMA0036; YI FEI XUE BIOTECHNOLOGY).

Techniques: Inhibition, Activation Assay

Journal: Oncotarget

Article Title: Increased high mobility group A 2 expression promotes transition of cervical intraepithelial neoplasm into cervical cancer

doi: 10.18632/oncotarget.24080

Figure Lengend Snippet: DIPS-PCR results

Article Snippet: The following primary antibodies used: rabbit anti-GAPDH (1:2000, Antgene, Wuhan, China), rabbit anti- HMGA2 (1:200, A12743, ABclonal), rabbit anti-Bcl-2 (1:1000, 12789-1-AP, Proteintech), and rabbit anti-Caspase 3 (1:1000, A11953, ABclonal).

Techniques:

Journal: Oncotarget

Article Title: Increased high mobility group A 2 expression promotes transition of cervical intraepithelial neoplasm into cervical cancer

doi: 10.18632/oncotarget.24080

Figure Lengend Snippet: Patient data

Article Snippet: The following primary antibodies used: rabbit anti-GAPDH (1:2000, Antgene, Wuhan, China), rabbit anti- HMGA2 (1:200, A12743, ABclonal), rabbit anti-Bcl-2 (1:1000, 12789-1-AP, Proteintech), and rabbit anti-Caspase 3 (1:1000, A11953, ABclonal).

Techniques: Expressing

Journal: Oncotarget

Article Title: Increased high mobility group A 2 expression promotes transition of cervical intraepithelial neoplasm into cervical cancer

doi: 10.18632/oncotarget.24080

Figure Lengend Snippet: Representative HMGA2 staining in normal cervix ( A ), CIN ( B ) and cervical cancer samples ( C ) (×200). Scale bars, 20 μm. ( D ) HPV signals (red) in cervical lesions (×1000). ( E ) and ( F ) Variation in HMGA2 signals (green) in different cervical lesions (×1000). Scale bars, 10 μm.

Article Snippet: The following primary antibodies used: rabbit anti-GAPDH (1:2000, Antgene, Wuhan, China), rabbit anti- HMGA2 (1:200, A12743, ABclonal), rabbit anti-Bcl-2 (1:1000, 12789-1-AP, Proteintech), and rabbit anti-Caspase 3 (1:1000, A11953, ABclonal).

Techniques: Staining

Journal: Oncotarget

Article Title: Increased high mobility group A 2 expression promotes transition of cervical intraepithelial neoplasm into cervical cancer

doi: 10.18632/oncotarget.24080

Figure Lengend Snippet: Statistical analysis of HPV copy number ( A ), HMGA2 copy number ( B ) and HMGA2 protein expression ( C ) in normal cervical biopsies, CIN and cervical cancer samples. HPV copy number ( D ), HMGA2 copy number ( E ) and HMGA2 protein expression ( F ) in cervical lesions of different stages. The original data are shown in .

Article Snippet: The following primary antibodies used: rabbit anti-GAPDH (1:2000, Antgene, Wuhan, China), rabbit anti- HMGA2 (1:200, A12743, ABclonal), rabbit anti-Bcl-2 (1:1000, 12789-1-AP, Proteintech), and rabbit anti-Caspase 3 (1:1000, A11953, ABclonal).

Techniques: Expressing

Journal: Oncotarget

Article Title: Increased high mobility group A 2 expression promotes transition of cervical intraepithelial neoplasm into cervical cancer

doi: 10.18632/oncotarget.24080

Figure Lengend Snippet: ( A – C ) Fusion signals for HPV and HMGA2 (×1000). (A) HPV signal (red) in sample T-025; (B) HMGA2 signal (green) in sample T-025; (C) Fusion image of (A) and (B). Arrows shows merged signals (yellow). ( D – F ) Samples without HPV integration showed no fusion signals (×1000). (D) HPV signal (red) in sample T-011; (E) HMGA2 signal (green) in sample T-011; (F) Fusion image of (D) and (E). Scale bars, 10 μm.

Article Snippet: The following primary antibodies used: rabbit anti-GAPDH (1:2000, Antgene, Wuhan, China), rabbit anti- HMGA2 (1:200, A12743, ABclonal), rabbit anti-Bcl-2 (1:1000, 12789-1-AP, Proteintech), and rabbit anti-Caspase 3 (1:1000, A11953, ABclonal).

Techniques:

Journal: Oncotarget

Article Title: Increased high mobility group A 2 expression promotes transition of cervical intraepithelial neoplasm into cervical cancer

doi: 10.18632/oncotarget.24080

Figure Lengend Snippet: ( A ) ROC curve for HPV copy number. The AUC is 0.848 with a 95% CI of 0.772–0.923. ( B ) ROC curve for HMGA2 protein expression. The AUC is 0.910 with a 95% CI of 0.844–0.976.

Article Snippet: The following primary antibodies used: rabbit anti-GAPDH (1:2000, Antgene, Wuhan, China), rabbit anti- HMGA2 (1:200, A12743, ABclonal), rabbit anti-Bcl-2 (1:1000, 12789-1-AP, Proteintech), and rabbit anti-Caspase 3 (1:1000, A11953, ABclonal).

Techniques: Expressing

Journal: Oncotarget

Article Title: Increased high mobility group A 2 expression promotes transition of cervical intraepithelial neoplasm into cervical cancer

doi: 10.18632/oncotarget.24080

Figure Lengend Snippet: Western blot analysis of HMGA2, Bcl-2 , and Caspase 3 protein expression in S12 ( A ), SiHa ( B ), and CaSki ( C ) cells and control cells after treatment with HMGA2 overexpression plasmid or siRNAs. Real-time PCR assay of HMGA2, Bcl-2 , and Caspase 3 in expression in S12 ( D ), SiHa ( E ), and CaSki ( F ) cells and control cells after treatment with HMGA2 overexpression plasmid or siRNAs. The data represent the mean ± SD ( n = 3). * P < 0.01.

Article Snippet: The following primary antibodies used: rabbit anti-GAPDH (1:2000, Antgene, Wuhan, China), rabbit anti- HMGA2 (1:200, A12743, ABclonal), rabbit anti-Bcl-2 (1:1000, 12789-1-AP, Proteintech), and rabbit anti-Caspase 3 (1:1000, A11953, ABclonal).

Techniques: Western Blot, Expressing, Control, Over Expression, Plasmid Preparation, Real-time Polymerase Chain Reaction

Journal: Oncotarget

Article Title: Increased high mobility group A 2 expression promotes transition of cervical intraepithelial neoplasm into cervical cancer

doi: 10.18632/oncotarget.24080

Figure Lengend Snippet: ( A ) Representative cell apoptosis images of S12, SiHa, and CaSki cells 48 hours after transfection with HMGA2 overexpression plasmid or siRNAs were obtained using flow-cytometry. ( B ) Representative cell clone formation images of S12, SiHa, and CaSki cells 2 weeks after transfection with HMGA2 overexpression plasmid or siRNAs.

Article Snippet: The following primary antibodies used: rabbit anti-GAPDH (1:2000, Antgene, Wuhan, China), rabbit anti- HMGA2 (1:200, A12743, ABclonal), rabbit anti-Bcl-2 (1:1000, 12789-1-AP, Proteintech), and rabbit anti-Caspase 3 (1:1000, A11953, ABclonal).

Techniques: Transfection, Over Expression, Plasmid Preparation, Flow Cytometry