iv jw55 tocris bioscience pre clinical cas 664993 53 7  (Tocris)

Journal: Journal of Advanced Research

Article Title: Tankyrase activity is essential for asymmetric division and chromosome segregation in oocyte meiosis

doi: 10.1016/j.jare.2025.07.008

Figure Lengend Snippet: TNKS affects oocyte meiotic progression with asymmetric division and chromosome segregation defects. (A) Representative images of (large) PB1 extrusion after 12 h of culture in the control and treatments oocytes with JW55 at concentrations of 50 μM, 100 μM, and 200 μM. Bar = 80 μm. A polar body with a diameter exceeding one-third of the oocyte is considered a large PB1. (B) The percentage of PB1 or large PB1 extrusion in the control (n = 136) and treatment groups with JW55 at concentrations of 50 μM (n = 148), 100 μM (n = 159), and 200 μM (n = 154). A concentration of 200 μM JW55 was selected for subsequent experiments. ns, P > 0.05. *, P < 0.05. **, P < 0.01. (C). Representative images of oocytes meiotic progression from 7 h to 12 h during in vitro culture in the control and JW55 treatments groups. Bar = 80 μm. (D) The percentage of PB1 extrusion from 7 h to 12 h during in vitro culture in the control (n = 167) and JW55 treatment (n = 182) mouse oocytes. The PB1 extrusion includes both normal small PB1 and large PB1. The rates of normal and large PB1 were further calculated based on the all the oocytes in each group. The pink significance marker indicated the difference in large polar body extrusion in the JW55-treated groups at different culture times. ns, P > 0.05. *, P < 0.05. **, P < 0.01. (E) Representative images and percentage of asymmetric cortical granule (CG) distribution in MI stage oocytes from the control (n = 48) and JW55 treatment (n = 44) groups. The arrows highlight the CG-free domain (CGFD) overlaid chromosomes. Green, CG. Blue, DNA. Bar = 20 μm. **, P < 0.01. (F) Quantitative analysis of chromosome position relative to the cortex in the control (n = 48) and JW55 treatment (n = 51) oocytes. We defined the distance from the side of chromosome closest to the cortex as L and the dimeter of oocytes as D. The ratio of L/D was significantly increased in JW55-treated oocytes compared to control groups. Green, CG. Black, DNA. Bar = 20 μm. ***, P < 0.001. (G) Representative images of pS19-Myosin II; in MI or TI stage oocytes from the control and JW55 treatment groups. Chromosome positions were categorized into center, center-one quadrant, and one quadrant of the oocyte. The arrows highlight the accumulation of pS19-Myosin II; in the cortex overlying the chromosomes. Green, pS19-Myosin II;. Black, DNA. Bar = 20 μm. (H) The percentage of asymmetric cortical p-Myosin II; distribution in the control (n = 52) and JW55 treatment (n = 56) oocytes. Accumulation of pS19-Myosin II; in the cortex overlying the chromosome was defined as asymmetric. ***, P < 0.001. (I) The percentage of chromosome position pattern in the control (n = 52) and JW55 treatment (n = 56) oocytes. **, P < 0.01. ***, P < 0.001. (J) Representative images and percentage of aneuploidy in control (n = 58) and JW55 treatment (n = 61) oocytes at the MII; stage. Red, ACA. Blue, DNA. Bar = 5 μm. *, P < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: JW55 (HY-13968), XAV939 (HY-15147), and IWR-1 (HY-12238) was from MedChemExpress.

Techniques: Control, Concentration Assay, In Vitro, Marker

Journal: Journal of Advanced Research

Article Title: Tankyrase activity is essential for asymmetric division and chromosome segregation in oocyte meiosis

doi: 10.1016/j.jare.2025.07.008

Figure Lengend Snippet: TNKS regulates ARP2/Rab11a/Fascin for chromosome migration in mouse oocytes. (A) Representative images and relative intensity of cytoplasmic actin filaments in MI stage oocytes from the control (n = 39) and JW55 treatment (n = 38) groups. Green, α-Tubulin. Red, F-Actin. Blue, DNA. Bar = 10 μm. ***, P < 0.001. (B) Representative images and relative intensity of cortical actin filaments in MI stage oocytes from the control (n = 44) and JW55 treatment (n = 40) groups. Black, F-Actin. Blue, DNA. Bar = 20 μm. ***, P < 0.001. (C) GO enrichment analysis of TNKS-associated proteins based on mass spectrometry data. (D) Protein-protein interaction network analysis of actin-related proteins identified by mass spectrometry upon STRING database. Different node color indicated core and non-core nodes, while edge thickness edge represented interaction strength. (E) Band intensity analysis of N-WASP, Fascin, ARP2, and Ran in the MI stage oocytes from control and JW55 treatment groups. *, P < 0.05. (F) Co-IP analysis with an anti-TNKS antibody. The immunoblots were probed with antibodies against Fascin, Ran, and pS19-Myosin II;. (G) Representative images and relative intensity of Ran in MI stage oocytes from the control (n = 50) and JW55 treatment (n = 47) groups. Red, Ran. Blue, DNA. Bar = 10 μm. **, P < 0.01. (H) Representative images of ARP2 in MI or TI stage oocyte from the control and JW55 treatment groups. Chromosome positions were categorized into center, center-one quadrant, and one quadrant of the oocyte. The arrow highlighted the accumulation of ARP2 in the cortex overlying the chromosomes. Red, ARP2. Black, DNA. Bar = 20 μm. (I) The percentage of asymmetric cortical ARP2 distribution in the control (n = 42) and JW55 treatment (n = 43) oocytes. Accumulation of ARP2 in the cortex overlying the chromosome was defined as asymmetric. **, P < 0.01. (J) The percentage of chromosome position patterns in the control (n = 42) and JW55 treatment (n = 43) oocytes. *, P < 0.05. **, P < 0.01. ***, P < 0.001. (K) Representative images and relative intensity of Fascin in MI stage oocytes from the control (n = 61) and JW55 treatment (n = 51) groups. Green, Fascin. Blue, DNA. Bar = 20 μm. ***, P < 0.001. (L) Representative images and relative intensity of Rab11a in MI stage oocytes from the control (n = 54) and JW55 (n = 49) treatment groups. Red, Ran. Blue, DNA. Bar = 10 μm. *, P < 0.05. (M) Co-IP analysis with an anti-TNKS antibody. The immunoblots were probed with an anti-Rab11a antibody. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: JW55 (HY-13968), XAV939 (HY-15147), and IWR-1 (HY-12238) was from MedChemExpress.

Techniques: Migration, Control, Mass Spectrometry, Co-Immunoprecipitation Assay, Western Blot

Journal: Journal of Advanced Research

Article Title: Tankyrase activity is essential for asymmetric division and chromosome segregation in oocyte meiosis

doi: 10.1016/j.jare.2025.07.008

Figure Lengend Snippet: TNKS affects distributions of Formin2 and ER for chromosome migration in mouse oocytes. (A) Representative images of Formin2 in MI stage oocyte from the control and JW55 treatment groups. Green, Formin2. Cyan, DNA. Bar = 20 μm. (B) Relative intensity of Formin2 at spindle poles in oocytes from the control (n = 47) and JW55 treatment (n = 51) groups. ***, P < 0.001. (C) Band intensity analysis of Formin2 expression in the MI stage oocytes from control and JW55 treatment groups. *, P < 0.05. (D) Representative images of ER-tracker in MI stage oocyte from the control and JW55 treatment groups. Black, ER-tracker. Bar = 20 μm. (E) The percentage of abnormal ER distribution in oocytes from the control (n = 50) and JW55-treated (n = 56) groups. *, P < 0.05. (F) Relative intensity of ER-tracker in oocytes from the control (n = 50) and JW55-treated (n = 56) groups. ***, P < 0.001. (G) Western blot of GRP78 in MI stage oocytes from control and JW55 treatment groups. (H) Band intensity analysis of GRP78 in the control and JW55 treatment groups. *, P < 0.05. (I) Representative images of a relative position change between chromosomes and Formin2 in ATI stage oocytes from the control and JW55 treatment groups. Green, Formin2. Cyan, DNA. Bar = 20 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: JW55 (HY-13968), XAV939 (HY-15147), and IWR-1 (HY-12238) was from MedChemExpress.

Techniques: Migration, Control, Expressing, Western Blot

Journal: Journal of Advanced Research

Article Title: Tankyrase activity is essential for asymmetric division and chromosome segregation in oocyte meiosis

doi: 10.1016/j.jare.2025.07.008

Figure Lengend Snippet: TNKS regulates p-PLK1 and microtubule stability for spindle assembly in mouse oocyte. (A) Representative images and percentage of abnormal spindle in MI stage oocyte from the control (n = 47) and JW55 treatment (n = 42) groups. Green, α-Tubulin. Blue, DNA. Bar = 20 μm. *, P < 0.05. (B) Representative images and percentage of abnormal spindle in MII; stage oocyte from the control (n = 57) and JW55 treatment (n = 45) groups. Green, α-Tubulin. Blue, DNA. Bar = 20 μm. **, P < 0.01. (C) Quantitative analysis of chromosome alignment in MI stage oocyte from the control (n = 50) and JW55 treatment (n = 34) groups. We defined the length on both sides of chromosomes as L and the diameter of oocytes as D. The ratio of L/D was markedly increased in the JW55-treated oocytes compared to the control groups. White, DNA. Bar = 20 μm. *, P < 0.05. (D) Representative images and relative intensity of H3S10ph in MI stage oocyte from the control (n = 56) and JW55 treatment (n = 58) groups. Red, H3S10ph. Blue, DNA. Bar = 5 μm. *, P < 0.05. (E) Representative images of γ-Tubulin distribution in MI or MII; stage oocytes from the control and JW55 treatment groups. Magenta, γ-Tubulin. Green, α-Tubulin. Blue, DNA. Bar = 20 μm. (F) The percentage of abnormal γ-Tubulin distribution in MI/MII; stage oocytes from the control (n = 36/58) and JW55 treatment (n = 43/42) groups. *, P < 0.05. **, P < 0.01. (G) Band intensity analysis of p-PLK1 in MI stage oocytes from the control and JW55 treatment groups. *, P < 0.05. (H) Protein-protein interaction network analysis of microtubule-related proteins identified by mass spectrometry of TNKS and PLK1 upon STRING database. Different node color indicated core and non-core nodes, while edge thickness edge represented interaction strength. (I) Co-IP analysis with an anti-TNKS antibody. The immunoblots were probed with an anti‐p-PLK1 antibody. (J) Western blot results of Ac-Tubulin expression in MI stage oocytes from the control and JW55 treatment groups. (K) Relative intensity of Ac-Tubulin in the MI stage oocytes from the control and JW55 treatment groups. *, P < 0.05. (L) Representative images and relative intensity of Ac-Tubulin in MI stage oocytes from the control (n = 50) and JW55 treatment (n = 47) groups. Red, Ac-Tubulin. Blue, DNA. Bar = 10 μm. ***, P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: JW55 (HY-13968), XAV939 (HY-15147), and IWR-1 (HY-12238) was from MedChemExpress.

Techniques: Control, Mass Spectrometry, Co-Immunoprecipitation Assay, Western Blot, Expressing

Journal: Journal of Advanced Research

Article Title: Tankyrase activity is essential for asymmetric division and chromosome segregation in oocyte meiosis

doi: 10.1016/j.jare.2025.07.008

Figure Lengend Snippet: TNKS regulates BubR1/MPF activity, and Securin accumulation in mouse oocyte. (A) Representative images of BubR1 signals at the kinetochore in oocytes from the control and JW55 treatment groups. Green, BubR1. Red, ACA. Blue, DNA. Bar = 5 μm. (B) The percentage of positive BubR1 signals at the kinetochore in oocytes from the control (n = 53) and JW55 (n = 50) treatment groups. ***, P < 0.001. (C) Representative images of oocytes PB1 extrusion after 12 h culture in the Control, control + Milri, control + 10 μM ETOP, JW55 + 10 μM ETOP and JW55 groups. GV oocytes were exposed to etoposide (ETOP) at concentrations of 1 μM and 10 μM for 30 min in the presence 4 μM milrinone (Milri) to prevent GVBD during this treatment. Bar = 80 μm. (D) The percentage of PB1 or large PB1 extrusion in the control (n = 94), control + Milri (n = 89), control + 1 μM ETOP (n = 98), control + 10 μM ETOP (n = 97), JW55 + 1 μ M ETOP (n = 128), JW55 + 10 μM ETOP (n = 120), and JW55 treatment (n = 114) oocytes after ETOP pretreatment. ns, P > 0.05. **, P < 0.01. ***, P < 0.001. (E) Representative images of γ-H2AX in MI stage oocyte from the control + 10 μM ETOP and JW55 + 10 μM ETOP oocytes. Red, γ-H2AX. Blue, DNA. Bar = 5 μm. (F) Relative intensity of γ-H2AX in MI stage oocyte from the control + 10 μM ETOP (n = 47) and JW55 + 10 μM ETOP (n = 39) oocytes. ***, P < 0.001. (G) Western blot of Cyclin B1 and Securin in oocytes from the control and JW55 treatment groups after 6 h of culture. (H) Western blot of pT161-CDK1 and CDC20 in oocytes from the control and JW55 treatment groups after 6 h of culture, and band intensity analysis of their protein levels in oocytes from the control and JW55 treatment groups. *, P < 0.05. **, P < 0.01. (I) Band intensity analysis of pT161-CDK1, pY15-CDK1, and Securin in oocytes from the control and JW55 treatment groups after 3 h of culture. ns, P > 0.05. *, P < 0.05. (J) Band intensity analysis of CDC25C, Cyclin B1, and pT14-CDK1 in oocytes from the control and JW55 treatment groups after 3 h of culture. *, P < 0.05. **, P < 0.01. (K) Protein-protein interaction network analysis of cell cycle-related proteins identified by mass spectrometry and MPF/Securin/CDC20/CDC25C upon STRING database. Different node color indicated core and non-core nodes, while edge thickness edge represented interaction strength. (L) Co-IP analysis with an anti-TNKS antibody. The immunoblot was probed with anti-CDC20 and anti-CDC25C antibodies. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: JW55 (HY-13968), XAV939 (HY-15147), and IWR-1 (HY-12238) was from MedChemExpress.

Techniques: Activity Assay, Control, Western Blot, Mass Spectrometry, Co-Immunoprecipitation Assay