Journal: Journal of Ovarian Research

Article Title: Notch2 improves granulosa cell functions in premature ovarian failure by activating the Wnt2/β-catenin pathway

doi: 10.1186/s13048-025-01745-9

Figure Lengend Snippet: Notch2 overexpression enhanced ovarian functions in premature ovarian failure (POF) mice by activating the Wnt/β-catenin pathway. ( A ) Quantitative real-time PCR (qPCR) for the validation of oe-Notch2 overexpression efficiency. ( B ) Representative images of ovarian size at the end of the study. ( C ) Quantification of ovarian weight at the end of the study. ( D ) Enzyme-linked immunosorbent assay (ELISA) for follicle-stimulating hormone (FSH), estradiol (E2) and anti-mullerian hormone (AMH) levels in serum. ( E ) qPCR for the expressions of steroidogenic enzyme-related genes (StARD1, Cyp11a1 and 3-β-HSD) in ovarian tissues. ( F ) Representative images of hematoxylin-eosin (HE) staining and quantification of primordial follicles, primary follicles, secondary follicles, antral follicles, atretic follicles and mature follicles for ovarian tissues. Magnification×200, scale bar = 100 μm; Magnification×40, scale bar = 500 μm. PF: primitive follicle; PrF: primary follicle; SF: secondary follicle; AnF: antral follicle; AF: atretic follicle; MF: mature follicle. △ P < 0.05 and △△ P < 0.01 vs. Normal; P < 0.05 and P < 0.01 vs. Control; # P < 0.05 and ## P < 0.01 vs. oe-NC; @ P < 0.05 and @@ P < 0.01 vs. oe-Notch2. Results were presented as mean ± standard deviation (SD)

Article Snippet: Notch2 , BIOSS , bs-21663R , 1:1000.

Techniques: Over Expression, Real-time Polymerase Chain Reaction, Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Staining, Control, Standard Deviation

Journal: Journal of Ovarian Research

Article Title: Notch2 improves granulosa cell functions in premature ovarian failure by activating the Wnt2/β-catenin pathway

doi: 10.1186/s13048-025-01745-9

Figure Lengend Snippet: Notch2 overexpression suppressed oxidative stress and ovarian cell apoptosis in POF mice by activating the Wnt/β-catenin pathway. ( A ) Levels of oxidative stress markers (malondialdehyde (MDA), glutathione (GSH) and superoxide dismutase (SOD)) in ovarian tissues. ( B ) Flow cytometry for cell apoptosis in ovarian tissues. ( C ) Western blot analysis was applied to measure the cleaved caspase-3/caspase-3 ratio in ovarian tissues. ( D ) qPCR for the expressions of cell cycle-related genes (CDK4, CCND2 and P21) and apoptosis-related genes (Bax and Bcl2) in ovarian tissues. P < 0.05 and P < 0.01 vs. Control; # P < 0.05 and ## P < 0.01 vs. oe-NC; @ P < 0.05 and @@ P < 0.01 vs. oe-Notch2. Results were presented as mean ± SD

Article Snippet: Notch2 , BIOSS , bs-21663R , 1:1000.

Techniques: Over Expression, Flow Cytometry, Western Blot, Control

Journal: Journal of Ovarian Research

Article Title: Notch2 improves granulosa cell functions in premature ovarian failure by activating the Wnt2/β-catenin pathway

doi: 10.1186/s13048-025-01745-9

Figure Lengend Snippet: Notch2 interacted with Wnt2 and β-catenin in POF mice. ( A ) qPCR was used to detect Wnt-2 and β-catenin mRNA expressions in the ovarian tissues. ( B ) Immunohistochemistry was applied to measure Notch2 protein expression in the ovarian tissues. Magnification×200, scale bar = 100 μm. ( C ) Western blot analysis was utilized to test Notch2 pathway-related protein expressions (Notch2, Jag1 and Hes2) and Wnt2/β-catenin pathway-related protein expressions (Wnt2, β-catenin, Axin2 and LEF1) in the ovaries. ( D ) Co-immunoprecipitation (Co-IP) was conducted to further verify the protein-protein interactions between Notch2 and Wnt2. P < 0.05 and P < 0.01 vs. Control; # P < 0.05 and ## P < 0.01 vs. oe-NC; @ P < 0.05 and @@ P < 0.01 vs. oe-Notch2. Results were presented as mean ± SD

Article Snippet: Notch2 , BIOSS , bs-21663R , 1:1000.

Techniques: Immunohistochemistry, Expressing, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Protein-Protein interactions, Control

Journal: Journal of Ovarian Research

Article Title: Notch2 improves granulosa cell functions in premature ovarian failure by activating the Wnt2/β-catenin pathway

doi: 10.1186/s13048-025-01745-9

Figure Lengend Snippet: Notch2 knockdown worsened GC functions in POF models by suppressed Wnt/β-catenin pathway ( A ) qPCR revealed the knockdown efficiency of sh-Notch2 for KNG cells. ( B ) Cell counting kit-8 (CCK-8) assay revealed cell viability for KNG cells. ( C ) Flow cytometry displayed cell cycle distribution for KNG cells. ( D ) Flow cytometry displayed cell apoptosis for KNG cells. ( E ) Western blot analysis displayed the cleaved caspase-3/caspase-3 ratio for KNG cells. ( F ) qPCR for the expressions of CDK4, CCND2, P21, Bax and Bcl2 mRNAs for KNG cells. ( G ) ELISA exhibited FSH, E2 and AMH levels for KNG cells. ( H ) qPCR for the expressions of StARD1, Cyp11a1 and 3-β-HSD mRNAs for KNG cells. ( I ) MDA, GSH and SOD levels were detected for KNG cells. P < 0.05 and P < 0.01 vs. Normal; P < 0.05 and P < 0.01 vs. Control; * P < 0.05 and ** P < 0.01 vs. Model; # P < 0.05 and ## P < 0.01 vs. sh-NC; @ P < 0.05 and @@ P < 0.01 vs. sh-Notch2. Results were presented as mean ± SD

Article Snippet: Notch2 , BIOSS , bs-21663R , 1:1000.

Techniques: Knockdown, Cell Counting, CCK-8 Assay, Flow Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of Ovarian Research

Article Title: Notch2 improves granulosa cell functions in premature ovarian failure by activating the Wnt2/β-catenin pathway

doi: 10.1186/s13048-025-01745-9

Figure Lengend Snippet: Notch2 acted on the Wnt2/β-catenin pathway in POF cells. ( A ) qPCR analysis for the expressions Wnt2 and β-catenin mRNAs in KNG cells. ( B ) Western blot analysis for the expressions of Notch2, Jag1, Hes2 Wnt2, β-catenin, Axin2 and LEF1 proteins in KNG cells. P < 0.05 and P < 0.01 vs. Control; * P < 0.05 and ** P < 0.01 vs. Model; # P < 0.05 and ## P < 0.01 vs. sh-NC; @ P < 0.05 and @@ P < 0.01 vs. sh-Notch2. Results were presented as mean ± SD

Article Snippet: Notch2 , BIOSS , bs-21663R , 1:1000.

Techniques: Western Blot, Control

Journal: Journal of Ovarian Research

Article Title: Notch2 improves granulosa cell functions in premature ovarian failure by activating the Wnt2/β-catenin pathway

doi: 10.1186/s13048-025-01745-9

Figure Lengend Snippet: βcatenin knockdown worsened GC functions in POF cells treated with sh-Notch2 and SKL2001 ( A ) CCK-8 assay was used to measure cell viability for KNG cells among groups. ( B ) Flow cytometry was conducted to test cell cycle distribution for KNG cells among groups. ( C ) Flow cytometry was carried out to evaluate cell apoptosis for KNG cells among groups. ( D ) qPCR was performed to detect CDK4, CCND2, P21, Bax and Bcl2 mRNA expressions in KNG cells among groups. ( E ) ELISA was utilized to measure FSH, E2 and AMH levels for KNG cells among groups. ( F ) qPCR was performed to detect StARD1, Cyp11a1 and 3-β-HSD in KNG cells among groups. ( G ) MDA, GSH and SOD levels among groups were detected for KNG cells. P < 0.05 and P < 0.01 vs. Model; # P < 0.05 and ## P < 0.01 vs. sh-Notch2 + SKL2001 + sh-NC. Results were presented as mean ± SD

Article Snippet: Notch2 , BIOSS , bs-21663R , 1:1000.

Techniques: Knockdown, CCK-8 Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Journal of Ovarian Research

Article Title: Notch2 improves granulosa cell functions in premature ovarian failure by activating the Wnt2/β-catenin pathway

doi: 10.1186/s13048-025-01745-9

Figure Lengend Snippet: βcatenin knockdown inhibited Wnt2/β-catenin pathway in POF cells treated with sh-Notch2 and SKL2001 ( A ) qPCR analysis for the expressions Wnt2 and β-catenin mRNAs in KNG cells. ( B ) Western blot analysis for the expressions of Notch2, Jag1, Hes2 Wnt2, β-catenin, Axin2 and LEF1 proteins in KNG cells. # P < 0.05 and ## P < 0.01 vs. sh-Notch2 + SKL2001 + sh-NC. Results were presented as mean ± SD

Article Snippet: Notch2 , BIOSS , bs-21663R , 1:1000.

Techniques: Knockdown, Western Blot

Journal: Aging Cell

Article Title: Deciphering the Contribution of Circular RNAs to Age‐Related Decline in Sertoli Cell Survivor

doi: 10.1111/acel.70023

Figure Lengend Snippet: Aging‐dependent effects on sperm circRNA content and SC survival. (A) A set of spermatic circRNAs tethering a group of miRNAs and mRNAs as targets, all involved in SC dynamics. Networks were built using Cytoscape. Hexagonal and rectangular symbols represent circRNAs and miRNAs, respectively. The arrow indicates the tethering activity of circRNAs toward miRNAs, while the dotted arrow indicates the pathways downstream of the miRNAs. (B–C) Expression analysis of (B) 6 circRNAs in Young and Aged cauda SPZ ( n = 6 in triplicate) and (C) circAbcb9 in testis, epididymis and cauda SPZ collected from Young and Aged mice ( n = 6 in triplicate). RT‐qPCR data were normalized using Cyclophilin , expressed as fold expression (nfe), and reported as mean value ± S.E.M; ** p < 0.01; * p < 0.05. (D) Senescence β‐gal immunofluorescence staining (green) of testicular tissues from Young and Aged mice ( n = 6 independent experiments); scale bar: 20 μm. Representative images of SC staining were shown on the right. Results were expressed as fold change (Fc) relative to the Young group and reported as mean ± SEM. ** p < 0.01. Nuclei were labeled with DAPI (blue). (E) Western blot analysis of SOX9, WT1, SOX8, and NOTCH2 in testis of Young and Aged mice ( n = 6 in triplicate). Signals were quantified by densitometry analysis and normalized against TUBULIN. Data were expressed in fold change (FC) OD values and reported as mean ± SEM; ** p < 0.01.

Article Snippet: The filters were blocked [5% nonfat milk, 0.25% Tween‐20 in Tris‐buffered saline (TBS, pH 7.6)] and incubated with different primary antibodies, all diluted 1:1000 [FUS antibody (PA5‐52610; Invitrogen, Milano, Italy), SOX9 (sc‐166505; Santa Cruz Biotechnology, Heidelberg, Germany), SOX8 (sc‐374445; Santa Cruz Biotechnology, Heidelberg, Germany), NOTCH2 (sc‐518049; Santa Cruz Biotechnology, Heidelberg, Germany), WT1 (sc‐7385; Santa Cruz Biotechnology, Heidelberg, Germany), DDX4 (MA5‐15565; Invitrogen, Milano, Italy), MAGE‐A4 (PA5‐72867; Invitrogen, Milano, Italy), SCP3 (PA1‐16766; Invitrogen, Milano, Italy), and TUBULIN (ab15246; Abcam, Cambridge, UK)].

Techniques: Activity Assay, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Labeling, Western Blot

Journal: Aging Cell

Article Title: Deciphering the Contribution of Circular RNAs to Age‐Related Decline in Sertoli Cell Survivor

doi: 10.1111/acel.70023

Figure Lengend Snippet: miR‐214‐3p modulates SC senescence. (A) A schematic representation of the in vitro experimental strategy. (B) Prediction of miR‐214‐3p binding sites on circAbcb9 sequence (miRNA seed sequence is indicated in red). (C) TM4 cells transfection with the luciferase‐based reporter constructs containing the wild‐type target sequence of miR‐214‐3p (WT—starting position number of the target pairing) or the control plasmids with inverted target sequence (I—starting position number of the target pairing) along with 50 nM of the miRNA mimic ( n = 6 independent experiments). The Renilla luciferase activity (Rl) was recorded and normalized to the firefly luciferase activity (Luc); values were reported as fold mean ± S.E.M. relative to RL/Luc recorded for transfection of controls (specific I construct + miRNA mimic) which were set to 1; ** p < 0.01. (D) Expression analysis of miR‐214‐3p in TM4 cells following transfection with an unrelated control (CTRL‐miR) or miR‐214‐3p mimic at 50 nM; ( n = 6 independent experiments); RT‐qPCR data were normalized using U6 snRNA , expressed as fold expression (nfe) and reported as mean value ± S.E.M; **** p < 0.0001. (E) Cell growth analysis in TM4 cells following miR‐214‐3p mimic transfection by using MTT assay ( n = 6 independent experiments). Data were normalized against to CTRL group, expressed as percentage of cell growth, and reported as mean ± SEM; ** p < 0.01; (F) Senescence β‐gal immunofluorescence staining (green) in TM4 cells following miR‐214‐3p mimic transfection ( n = 6 independent experiments). Data were expressed as fold change (Fc) relative to the CTRL group and reported as mean ± SEM. ** p < 0.01. Nuclei were labeled with DAPI (blue); scale bar: 50 μm. (G) Western blot analysis of SOX9, WT1, SOX8, and NOTCH2 proteins in TM4 cells following miR‐214‐3p mimic transfection ( n = 6 in triplicate). Signals were quantified by densitometry analysis and normalized against to TUBULIN. Data were expressed in fold change (FC) OD values and reported as mean ± SEM; ** p < 0.01.

Article Snippet: The filters were blocked [5% nonfat milk, 0.25% Tween‐20 in Tris‐buffered saline (TBS, pH 7.6)] and incubated with different primary antibodies, all diluted 1:1000 [FUS antibody (PA5‐52610; Invitrogen, Milano, Italy), SOX9 (sc‐166505; Santa Cruz Biotechnology, Heidelberg, Germany), SOX8 (sc‐374445; Santa Cruz Biotechnology, Heidelberg, Germany), NOTCH2 (sc‐518049; Santa Cruz Biotechnology, Heidelberg, Germany), WT1 (sc‐7385; Santa Cruz Biotechnology, Heidelberg, Germany), DDX4 (MA5‐15565; Invitrogen, Milano, Italy), MAGE‐A4 (PA5‐72867; Invitrogen, Milano, Italy), SCP3 (PA1‐16766; Invitrogen, Milano, Italy), and TUBULIN (ab15246; Abcam, Cambridge, UK)].

Techniques: In Vitro, Binding Assay, Sequencing, Transfection, Luciferase, Construct, Control, Activity Assay, Expressing, Quantitative RT-PCR, MTT Assay, Immunofluorescence, Staining, Labeling, Western Blot

Journal: Aging Cell

Article Title: Deciphering the Contribution of Circular RNAs to Age‐Related Decline in Sertoli Cell Survivor

doi: 10.1111/acel.70023

Figure Lengend Snippet: FUS protein regulates sertolian circAbcb9 biogenesis and in turn cellular senescence. (A) A schematic representation of the in vitro experimental strategy. (B and F). CircAbcb9 enrichment levels in the products of RIP assay (FUS‐IP vs IgG‐IP) in (B) TM4 cell line ( n = 6 independent experiments) and (F) TM4 cells treated with siCTRL or siFUS (50 nM) ( n = 6 in triplicate) by RT‐qPCR. Data were expressed in fold change (FC) and reported as mean ± SEM; ** p < 0.01. (C‐D) Expression analysis of (C) Fus transcript and (D) FUS protein in TM4 cells treated with siCTRL or siFUS (25 and 50 nM) ( n = 6 in triplicate); RT‐qPCR data were normalized using Cyclophilin , expressed as fold expression (nfe), Western blot signals were quantified by densitometry analysis, normalized against to TUBULIN, expressed in fold change (FC) OD values and reported as mean ± SEM; ** p < 0.01. (E) CircAbcb9 levels in TM4 cells treated with siCTRL or siFUS (50 nM) ( n = 6 in triplicate). Data were normalized using Cyclophilin , expressed as nfe, and reported as mean ± S.E.M; ** p < 0.01. (G) Cell growth analysis in TM4 cells using MTT assay following transfection with siCTRL or siFUS (50 nM) ( n = 6 independent experiments). Data were normalized against to siCTRL group, expressed as percentage of cell growth, and reported as mean ± SEM; ** p < 0.01; (H) Senescence β‐gal immunofluorescence staining (green) in TM4 cells treated with siCTRL or siFUS (50 nM) ( n = 6 independent experiments). Data were expressed as fold change (Fc) relative to the siCTRL group, and reported as mean ± SEM. ** p < 0.01; scale bar: 50 μm. (I) Western blot analysis of SOX9, WT1, SOX8, and NOTCH2 in TM4 cells treated with siCTRL or siFUS (50 nM) ( n = 6 in triplicate). Signals were quantified by densitometry analysis and normalized against TUBULIN. Data were expressed in fold change (FC) OD values and reported as mean ± SEM; ** p < 0.01.

Article Snippet: The filters were blocked [5% nonfat milk, 0.25% Tween‐20 in Tris‐buffered saline (TBS, pH 7.6)] and incubated with different primary antibodies, all diluted 1:1000 [FUS antibody (PA5‐52610; Invitrogen, Milano, Italy), SOX9 (sc‐166505; Santa Cruz Biotechnology, Heidelberg, Germany), SOX8 (sc‐374445; Santa Cruz Biotechnology, Heidelberg, Germany), NOTCH2 (sc‐518049; Santa Cruz Biotechnology, Heidelberg, Germany), WT1 (sc‐7385; Santa Cruz Biotechnology, Heidelberg, Germany), DDX4 (MA5‐15565; Invitrogen, Milano, Italy), MAGE‐A4 (PA5‐72867; Invitrogen, Milano, Italy), SCP3 (PA1‐16766; Invitrogen, Milano, Italy), and TUBULIN (ab15246; Abcam, Cambridge, UK)].

Techniques: In Vitro, Quantitative RT-PCR, Expressing, Western Blot, MTT Assay, Transfection, Immunofluorescence, Staining