Journal: bioRxiv

Article Title: Age-dependent loss of cohesion protection in human oocytes

doi: 10.1101/2023.01.13.523952

Figure Lengend Snippet: (A) Sgo2 localization in GV, GVBD, metaphase I, telophase I, and metaphase II stage oocytes. Oocytes were immunostained with antibodies against Sgo2 (green), CenpC (inner kinetochores, magenta), a-tubulin (microtubules, orange) and counter stained with Hoechst (blue). White circles with dashed lines represent the oocyte circumference. White boxes with dashed lines represent chromosomes masses that have been further magnified below. (B) Representative line scans showing distinction between bridge and centromeric Sgo2 pools. A line was drawn as shown on individual sister chromatid pairs from metaphase II oocytes stained as in (A) and the fluorescence profile of Sgo2 is shown in the graph. (C) Comparison of Sgo2 localization at the pericentromeric bridge in metaphase II oocytes from women stratified by age (≤35 years or >35 years) undergoing ICSI or IVF treatment. Plots show median (dashed black line), 25th and 75th percentiles (dotted black lines). n.s. Not significant (Kruskal-Wallis test).

Article Snippet: Spreads were incubated in mouse monoclonal anti-PP2A Catalytic α antibody (1:50; BD Biosciences, 610556), rabbit polyclonal anti-SGOL2 antibody (1:200; Novus Biologicals, NBP1-83567), mouse polyclonal anti Rec8 antibody (1:50; Novus Biologicals, H00009985-B01P) human anti-CREST antibody/ antisera (1:50 Antibodies Incorporated, 15-235) and guinea pig polyclonal antibody anti-CenpC antibody (1:200; MBL, PB030) diluted in 3% BSA, 7% FBS in PBST.

Techniques: Staining, Fluorescence, Comparison

Journal: bioRxiv

Article Title: Age-dependent loss of cohesion protection in human oocytes

doi: 10.1101/2023.01.13.523952

Figure Lengend Snippet: (A-D) Human Sgo2 localizes to centromeric cups and an inter-sister pericentromere bridge in metaphase I oocytes and metaphase II oocytes of younger women, but is lost from the bridge with age. Representative images of human metaphase I (A and B) and metaphase II oocytes (C and D) from younger (age 30 and 29 years) and older women (age 40 and 39 years). Sgo2 (green), inner kinetochores (CenpC, magenta), microtubules (a-tubulin, orange) and chromosomes (Hoeschst, blue) are shown. White dashed line boxes indicate chromosomes further magnified in (B) metaphase I and (D) metaphase II. Centromeric localization of Sgo2 is bounded by dashed lines. Interpretations of localizations observed are indicated in the schematics below. (E and F) The percentage of chromatids per oocyte with Sgo2 localization at the pericentromeric bridge was scored relative to the woman’s age at MI (E) and MII (F). Data were fit to a Sigmodal, 4PL curve (metaphase I; R 2 = 0.84, metaphase II; R 2 = 0.67). Oocytes shown in (A-D) are labelled (green circles). (G) Centromeric Sgo2 signal is not significantly altered with age. The relative intensity of the centromeric pool of Sgo2 in metaphase II oocytes from younger (age ≤ 35 years) or older women (age >35 years) was measured in arbitrary units (A.U.) relative to the inner kinetochore (CENP-C) or centromere (CREST) markers. Plots show median (dashed black line), 25th and 75th percentiles (dotted black lines). P values were calculated using the Mann-Whitney test: CenpC ( P = 0.37) and CREST ( P = 0.15). n.s. = not significant. (H) Schematic summarises Sgo2 localization in younger women in metaphase I and II oocytes.

Article Snippet: Spreads were incubated in mouse monoclonal anti-PP2A Catalytic α antibody (1:50; BD Biosciences, 610556), rabbit polyclonal anti-SGOL2 antibody (1:200; Novus Biologicals, NBP1-83567), mouse polyclonal anti Rec8 antibody (1:50; Novus Biologicals, H00009985-B01P) human anti-CREST antibody/ antisera (1:50 Antibodies Incorporated, 15-235) and guinea pig polyclonal antibody anti-CenpC antibody (1:200; MBL, PB030) diluted in 3% BSA, 7% FBS in PBST.

Techniques: MANN-WHITNEY

Journal: bioRxiv

Article Title: Age-dependent loss of cohesion protection in human oocytes

doi: 10.1101/2023.01.13.523952

Figure Lengend Snippet: (A-F) Loss of Sgo2 at the pericentromeric bridge is associated with increased inter-sister kinetochore distance in metaphase I and metaphase II. (A) Inter-sister kinetochore distance (white dashed arrows) was determined on metaphase I chromosomes and related to the presence of the Sgo2 bridge. (B) Increase in inter-sister kinetochore distance at metaphase I with female age for chromosomes with a Sgo2 bridge (blue) or no bridge (green). Data was fit to a linear regression (R 2 = 0.81; P <0.0001). (C) Inter-sister kinetochore distance for sister chromatid pairs with a Sgo2 bridge or no bridge at metaphase I in younger (age ≤ 35 years) or older women (age >35 years). Plots show median (dashed black line), 25th and 75th percentiles (dotted black lines). Statistical analyses were performed using the Kruskal-Wallis test (**** P <0.0001). (D) Inter-sister kinetochore distance (white dashed arrows) was determined on metaphase II chromosomes and related to the presence of the Sgo2 bridge. (E) Increase in inter-sister kinetochore distance at metaphase II with female age for sister chromatid pairs with a Sgo2 bridge (blue) or no bridge (green). Data was fit to a linear regression (R 2 = 0.47; P <0.0001). (F) Inter-sister kinetochore distance where the Sgo2 bridge is present or absent at metaphase II in younger (age ≤ 35 years) or older women (age >35 years). Statistical analyses were performed using the Kruskal-Wallis test (**** P <0.0001, ** P = 0.0017). Plots show median (dashed black line), 25th and 75th percentiles (dotted black lines). (G) Centromeric Sgo2 does not correlate with inter-sister kinetochore distance. The relative intensity of the centromeric pool of Sgo2 metaphase II oocytes was measured in arbitrary units (A.U.) relative to the kinetochore markers CenpC ( P =0.23; R 2 = 0.0019) and CREST ( P = 0.052; R 2 = 0.012). Data was fit to a linear regression.

Article Snippet: Spreads were incubated in mouse monoclonal anti-PP2A Catalytic α antibody (1:50; BD Biosciences, 610556), rabbit polyclonal anti-SGOL2 antibody (1:200; Novus Biologicals, NBP1-83567), mouse polyclonal anti Rec8 antibody (1:50; Novus Biologicals, H00009985-B01P) human anti-CREST antibody/ antisera (1:50 Antibodies Incorporated, 15-235) and guinea pig polyclonal antibody anti-CenpC antibody (1:200; MBL, PB030) diluted in 3% BSA, 7% FBS in PBST.

Techniques:

Journal: bioRxiv

Article Title: Age-dependent loss of cohesion protection in human oocytes

doi: 10.1101/2023.01.13.523952

Figure Lengend Snippet: (A) Representative image showing presence of single chromatids (arrows) in human metaphase II oocytes from an older (age 40) women, compared to a younger (age 30) woman without single chromatids. White boxes with dashed lines indicate examples of paired and single chromosomes in oocyte 2. Sgo2 (green), inner kinetochores (CenpC; magenta), microtubules (a-tubulin; orange) and chromosomes (Hoechst; blue) are shown. (B) Increase of single chromatids in metaphase II oocytes with maternal age. The number of single chromatids was scored relative to woman’s age. Data were fit to a Sigmodal, 4PL curve (R 2 = 0.57). Oocytes used in representative images are labelled in the graphs. (C) Increased number of single chromatids in metaphase II oocytes with an increased fraction of sister chromatid pairs lacking a Sgo2 bridge. Data were fit to a Sigmodal, 4PL curve (R 2 = 0.60). (D) The relative intensity of the centromeric pool of Sgo2 between paired and single chromatids was measured only from oocytes that had single chromatids. Sgo2 intensity was measured in arbitrary units (A.U.) relative to the kinetochore markers CenpC ( P = 0.96, Mann-Whitney test) and CREST ( P = 0.24, Mann-Whitney test). Plots show median (dashed black line), 25th and 75th percentiles (dotted black lines). P values were calculated using the Mann-Whitney test. n.s., Not significant.

Article Snippet: Spreads were incubated in mouse monoclonal anti-PP2A Catalytic α antibody (1:50; BD Biosciences, 610556), rabbit polyclonal anti-SGOL2 antibody (1:200; Novus Biologicals, NBP1-83567), mouse polyclonal anti Rec8 antibody (1:50; Novus Biologicals, H00009985-B01P) human anti-CREST antibody/ antisera (1:50 Antibodies Incorporated, 15-235) and guinea pig polyclonal antibody anti-CenpC antibody (1:200; MBL, PB030) diluted in 3% BSA, 7% FBS in PBST.

Techniques: MANN-WHITNEY

Journal: bioRxiv

Article Title: Age-dependent loss of cohesion protection in human oocytes

doi: 10.1101/2023.01.13.523952

Figure Lengend Snippet: (A and B) Chromosome spreads of metaphase II-arrested human oocytes were stained with antibodies against Sgo2 (green), inner kinetochores (CenpC, magenta) and cohesin (Rec8, orange). Chromosomes were stained with Hoechst (blue). (A) Representative images with white dashed line boxes indicating representative chromosome figures shown at higher magnification on the right. (B) Localization of Rec8 in the pericentromere bridge was scored for sister chromatid pairs with and without a Sgo2 bridge. Schematic representations of the data are shown below. (C and D) Chromosome spreads of metaphase II-arrested human oocytes were stained with antibodies against Sgo2 (green), inner kinetochores (CenpC, magenta) and PP2A (orange). Chromosomes were stained with Hoechst (blue). (C) Representative images with white dashed line boxes indicating representative chromosome figures shown at higher magnification on the right. (D) Localization of PP2A in the pericentromere bridge was scored for sister chromatid pairs with and without a Sgo2 bridge. Schematic representations of the data are shown below.

Article Snippet: Spreads were incubated in mouse monoclonal anti-PP2A Catalytic α antibody (1:50; BD Biosciences, 610556), rabbit polyclonal anti-SGOL2 antibody (1:200; Novus Biologicals, NBP1-83567), mouse polyclonal anti Rec8 antibody (1:50; Novus Biologicals, H00009985-B01P) human anti-CREST antibody/ antisera (1:50 Antibodies Incorporated, 15-235) and guinea pig polyclonal antibody anti-CenpC antibody (1:200; MBL, PB030) diluted in 3% BSA, 7% FBS in PBST.

Techniques: Staining

Journal: bioRxiv

Article Title: Age-dependent loss of cohesion protection in human oocytes

doi: 10.1101/2023.01.13.523952

Figure Lengend Snippet: (A-D) Inhibition of Mps1 in metaphase II oocytes impairs Sgo2 localization. (A) Scheme of the experiment. Metaphase II eggs from women aged ≤ 36 years were treated with 500nM Reversine (to inhibit Mps1) or DMSO (control) overnight and fixed. (B) Representative images of control and reversine-treated metaphase II oocytes after immunostaining with antibodies against Sgo2 (green), CenpC (inner kinetochores, magenta) and a-tubulin (microtubules, orange). White boxes with dashed lines indicate chromosomes that have been further magnified below. (C) The percentage of chromatids per oocyte with Sgo2 localization at the pericentromeric bridge was scored in control and reversine-treated metaphase II oocytes (** P =0.0084; Welch’s t test). (D) The relative intensity of the centromeric pool of Sgo2 in metaphase II oocytes relative to CenpC in control and reversine-treated oocytes (**** P <0.0001; Mann-Whitney test). (E and F) Mps1 activity is required for Bub1 localization to kinetochores. Metaphase II oocytes treated with reversine as in (A) were immunostained with antibodies against Bub1 (green), CenpC (inner kinetochore; magenta), and counter stained with Hoechst (blue) to visualise chromosomes. (E) Representative images of Bub1 localisation in control and reversine-treated metaphase II oocytes. White boxes with dashed lines represent chromosomes that have been further magnified below. White dashed circles show examples of area selections for Bub1 intensity measurements. (F) The relative intensity of the centromeric Bub1 in metaphase II oocytes from control and reversine-treated oocytes relative to CenpC (**** P <0.0001; Mann-Whitney test). Plots show median (dashed black line), 25th and 75th percentiles (dotted black lines).

Article Snippet: Spreads were incubated in mouse monoclonal anti-PP2A Catalytic α antibody (1:50; BD Biosciences, 610556), rabbit polyclonal anti-SGOL2 antibody (1:200; Novus Biologicals, NBP1-83567), mouse polyclonal anti Rec8 antibody (1:50; Novus Biologicals, H00009985-B01P) human anti-CREST antibody/ antisera (1:50 Antibodies Incorporated, 15-235) and guinea pig polyclonal antibody anti-CenpC antibody (1:200; MBL, PB030) diluted in 3% BSA, 7% FBS in PBST.

Techniques: Inhibition, Control, Immunostaining, MANN-WHITNEY, Activity Assay, Staining

Journal: bioRxiv

Article Title: Age-dependent loss of cohesion protection in human oocytes

doi: 10.1101/2023.01.13.523952

Figure Lengend Snippet: Loss of Sgo2 localization upon inhibition of Bub1 in metaphase II oocytes. (A) Scheme of the experiment. Metaphase II oocytes from women aged ≤ 36 years were treated with 10μM Bay-320 (to inhibit Bub1 ) or DMSO (control) overnight and fixed. (B) Representative images of control and Bay-320-treated metaphase II oocytes after immunostaining with antibodies against Sgo2 (green), CenpC (inner kinetochores, magenta) and a-tubulin (microtubules, orange). White boxes with dashed lines indicate chromosomes that have been further magnified below. (C) The percentage of chromatids per oocyte with Sgo2 localization at the pericentromeric bridge was scored in control and Bay-320-treated metaphase II oocytes (**** P <0.0001, Mann-Whitney test). (D) The relative intensity of the centromeric pool of Sgo2 in metaphase II oocytes from control and Bay-320-treated oocytes were measured relative to CenpC. (****P <0.0001, Mann-Whitney test).Plots show median (dashed black line), 25th and 75th percentiles (dotted black lines).

Article Snippet: Spreads were incubated in mouse monoclonal anti-PP2A Catalytic α antibody (1:50; BD Biosciences, 610556), rabbit polyclonal anti-SGOL2 antibody (1:200; Novus Biologicals, NBP1-83567), mouse polyclonal anti Rec8 antibody (1:50; Novus Biologicals, H00009985-B01P) human anti-CREST antibody/ antisera (1:50 Antibodies Incorporated, 15-235) and guinea pig polyclonal antibody anti-CenpC antibody (1:200; MBL, PB030) diluted in 3% BSA, 7% FBS in PBST.

Techniques: Inhibition, Control, Immunostaining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Age-dependent loss of cohesion protection in human oocytes

doi: 10.1101/2023.01.13.523952

Figure Lengend Snippet: The impact of MPS inhibition on protection of sister chromatid cohesion. (A) Schematic of experiment. Oocytes from women undergoing ICSI aged ≤ 36 years provided at or prior to GVBD were treated with 500nM reversine or DMSO, allowed to mature to metaphase II for up 24 h and fixed. (B) Representative images of oocytes treated with reversine from GVBD stage alongside control oocytes after immunostaining with antibodies against Sgo2 (green), CenpC (inner kinetochore, magenta), a-tubulin (microtubules, orange) and counter staining with Hoechst (chromosomes, blue). White boxes with dashed lines represent chromosomes that have been further magnified below. (C) The percentage of chromatids per oocyte with Sgo2 localization at the pericentromeric bridge was scored (*** P = 0.0001; Mann-Whitney test). (D) The relative intensity of the centromeric pool of Sgo2 relative to CenpC. (**** P <0.0001; Mann-Whitney test). (E) The number of single chromatids observed in metaphase II oocytes after treatment with reversine from GBVD compared to controls (*** P <0.0003; Mann-Whitney test). (F) Centromeric Sgo2 was measured in metaphase II oocytes that had single chromatids after reversine treatment from GVBD stage onwards, and values for paired and single chromatids were compared. Sgo2 intensity was measured in arbitrary units relative to CenpC. Plots show median (dashed black line), 25th and 75th percentiles (dotted black lines) ( P =0.57; Mann-Whitney test). (G) Model for age-dependent loss of cohesin protection. In oocytes from younger women, cohesion is maintained between sister chromatids. Mps1 at the kinetochore recruits Bub1 which phosphorylates histone H2A Thr120 in the centromeric chromatin. This phosphorylation allows for the recruitment of Sgo2 at the centromere and the pericentromeric bridge coincident with cohesin. During anaphase I, Sgo2 protects cohesin within the pericentromeric bridge from separase activity to ensures that sister chromatids remain together at metaphase II and disjoin accurately only upon fertilisation-triggered anaphase II. In oocytes from older women, pericentromeric cohesin deteriorates. This both increases inter-sister kinetochore distances and results in loss of Sgo2 from the pericentromeric bridge. In the absence of Sgo2 at the bridge, residual pericentromeric cohesin is vulnerable to separase-dependent cleavage in anaphase I. Consequently, sister chromatids risk premature separation and such single chromatids at metaphase II which will disjoin randomly at fertilisation resulting in increased incidences of aneuploid conceptions in older women.

Article Snippet: Spreads were incubated in mouse monoclonal anti-PP2A Catalytic α antibody (1:50; BD Biosciences, 610556), rabbit polyclonal anti-SGOL2 antibody (1:200; Novus Biologicals, NBP1-83567), mouse polyclonal anti Rec8 antibody (1:50; Novus Biologicals, H00009985-B01P) human anti-CREST antibody/ antisera (1:50 Antibodies Incorporated, 15-235) and guinea pig polyclonal antibody anti-CenpC antibody (1:200; MBL, PB030) diluted in 3% BSA, 7% FBS in PBST.

Techniques: Inhibition, Control, Immunostaining, Staining, MANN-WHITNEY, Phospho-proteomics, Activity Assay

Journal: Biomarker Research

Article Title: SGOL2 is a novel prognostic marker and fosters disease progression via a MAD2-mediated pathway in hepatocellular carcinoma

doi: 10.1186/s40364-022-00422-z

Figure Lengend Snippet: Correlation between SGOL2 expression and clinicopathological characteristics in Cohort 1

Article Snippet: The following antibodies were used: SGOL2 antibody (Bethyl, A301–262A, for WB/Co-IP), MAD2 antibody (Bethyl, A300-301A, for WB/Co-IP), SGOL1 (Immunoway, YT4275), cyclin D1 antibody (CST, 55506), cyclin E1 antibody (CST, 20808), PCNA antibody (CST, 13110), normal rabbit IgG (CST, 2729), E-cadherin antibody (CST, 14472), N-cadherin antibody (CST, 13116), vimentin antibody (CST, 5741), MMP9 antibody (CST,13667), β-catenin antibody (CST, 8480), and fibronectin antibody (Abcam, ab268021), secondary antibody (CST,7074/7076), GAPDH antibody (CST, 5174).

Techniques: Expressing

Journal: Biomarker Research

Article Title: SGOL2 is a novel prognostic marker and fosters disease progression via a MAD2-mediated pathway in hepatocellular carcinoma

doi: 10.1186/s40364-022-00422-z

Figure Lengend Snippet: Differential expression of SGOL2 in liver cancer and adjacent tissues

Article Snippet: The following antibodies were used: SGOL2 antibody (Bethyl, A301–262A, for WB/Co-IP), MAD2 antibody (Bethyl, A300-301A, for WB/Co-IP), SGOL1 (Immunoway, YT4275), cyclin D1 antibody (CST, 55506), cyclin E1 antibody (CST, 20808), PCNA antibody (CST, 13110), normal rabbit IgG (CST, 2729), E-cadherin antibody (CST, 14472), N-cadherin antibody (CST, 13116), vimentin antibody (CST, 5741), MMP9 antibody (CST,13667), β-catenin antibody (CST, 8480), and fibronectin antibody (Abcam, ab268021), secondary antibody (CST,7074/7076), GAPDH antibody (CST, 5174).

Techniques: Quantitative Proteomics, Expressing

Journal: Biomarker Research

Article Title: SGOL2 is a novel prognostic marker and fosters disease progression via a MAD2-mediated pathway in hepatocellular carcinoma

doi: 10.1186/s40364-022-00422-z

Figure Lengend Snippet: Correlation between SGOL2 expression and clinicopathological characteristics in Cohort 1/2

Article Snippet: The following antibodies were used: SGOL2 antibody (Bethyl, A301–262A, for WB/Co-IP), MAD2 antibody (Bethyl, A300-301A, for WB/Co-IP), SGOL1 (Immunoway, YT4275), cyclin D1 antibody (CST, 55506), cyclin E1 antibody (CST, 20808), PCNA antibody (CST, 13110), normal rabbit IgG (CST, 2729), E-cadherin antibody (CST, 14472), N-cadherin antibody (CST, 13116), vimentin antibody (CST, 5741), MMP9 antibody (CST,13667), β-catenin antibody (CST, 8480), and fibronectin antibody (Abcam, ab268021), secondary antibody (CST,7074/7076), GAPDH antibody (CST, 5174).

Techniques: Expressing

Journal: Biomarker Research

Article Title: SGOL2 is a novel prognostic marker and fosters disease progression via a MAD2-mediated pathway in hepatocellular carcinoma

doi: 10.1186/s40364-022-00422-z

Figure Lengend Snippet: High expression of SGOL2 in HCC. A SGOL2 was overexpressed in HCC in Wurmbach liver database. B Protein expression of SGOL2 was elevated in HCC patients in HPA database. C Immunoblot analysis of SGOL2 in HCC samples and paracancerous tissues from patients in cohort 2, and GAPDH was used as a loading control. D - F SGOL2 staining of paired clinical specimens, and the statistic quantification results in cohorts 1 and 2( n = 202). Protein expression of SGOL2 in the poorly differentiated group was significantly higher than that in the well-differentiated group of cohort 2. G TCGA dataset analysis of the relationship between the SGOL2 expression levels and the prognosis of HCC patients ( n = 364). H Overall survival (OS) analysis of HCC patients with high SGOL2 expression or low SGOL2 expression in cohort 1 ( n = 100). I - J Identification of the optimal penalization coefficient lambda (λ) in the Lasso model in cohort 1. K The nomogram based on SGOL2 for predicting the prognosis of HCC patients in cohort 1. L ROC curve analysis was used to evaluate the performance of this nomogram for 3-year overall survival prediction in the training and validation groups. OS, overall survival; RFS, relapse-free survival; PFS, progression-free survival; DSS, disease-specific survival; HR, hazard ratio. The results are presented as the mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The following antibodies were used: SGOL2 antibody (Bethyl, A301–262A, for WB/Co-IP), MAD2 antibody (Bethyl, A300-301A, for WB/Co-IP), SGOL1 (Immunoway, YT4275), cyclin D1 antibody (CST, 55506), cyclin E1 antibody (CST, 20808), PCNA antibody (CST, 13110), normal rabbit IgG (CST, 2729), E-cadherin antibody (CST, 14472), N-cadherin antibody (CST, 13116), vimentin antibody (CST, 5741), MMP9 antibody (CST,13667), β-catenin antibody (CST, 8480), and fibronectin antibody (Abcam, ab268021), secondary antibody (CST,7074/7076), GAPDH antibody (CST, 5174).

Techniques: Expressing, Western Blot, Control, Staining, Biomarker Discovery

Journal: Biomarker Research

Article Title: SGOL2 is a novel prognostic marker and fosters disease progression via a MAD2-mediated pathway in hepatocellular carcinoma

doi: 10.1186/s40364-022-00422-z

Figure Lengend Snippet: Downregulation of SGOL2 expression inhibited the malignant behaviors of HCC cells in vitro. A The mRNA level of SGOL2 in liver and HCC cell lines. B - C , SK-HEP-1, and HEP3B cells were transfected with shNC or shSGOL2 lentivirus, and the knockdown of SGOL2 at the mRNA and protein levels was validated by RT–PCR and Western blots, respectively. GAPDH was used as a loading control. D - G Invasion, migration, sphere formation, and colony formation assays of the SGOL2-downregulated HCC cells were detected and analyzed. H - I Downregulation of SGOL2 expression induced cell cycle arrest in the G1/S phase and activated the apoptosis of HCC cells. J SK-HEP-1, and HEP3B cells were transfected with shNC or shSGOL2, and the proliferation of HCC cells was detected at Days 0, 1, 2, and 3 by CCK-8 assays. K Effect of SGOL2 on the EMT in HCC cell lines. SK-HEP-1 and HEP3B cells were transfected with shNC or shSGOL2, and Western blots were used to detect the levels of E-cadherin, N-cadherin, β-catenin, Vimentin, fibronectin, and MMP9. The results are presented as the mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The following antibodies were used: SGOL2 antibody (Bethyl, A301–262A, for WB/Co-IP), MAD2 antibody (Bethyl, A300-301A, for WB/Co-IP), SGOL1 (Immunoway, YT4275), cyclin D1 antibody (CST, 55506), cyclin E1 antibody (CST, 20808), PCNA antibody (CST, 13110), normal rabbit IgG (CST, 2729), E-cadherin antibody (CST, 14472), N-cadherin antibody (CST, 13116), vimentin antibody (CST, 5741), MMP9 antibody (CST,13667), β-catenin antibody (CST, 8480), and fibronectin antibody (Abcam, ab268021), secondary antibody (CST,7074/7076), GAPDH antibody (CST, 5174).

Techniques: Expressing, In Vitro, Transfection, Knockdown, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control, Migration, CCK-8 Assay

Journal: Biomarker Research

Article Title: SGOL2 is a novel prognostic marker and fosters disease progression via a MAD2-mediated pathway in hepatocellular carcinoma

doi: 10.1186/s40364-022-00422-z

Figure Lengend Snippet: SGOL2 dysregulated the cell cycle by regulating MAD2 in HCC cells. A SK-HEP-1 and HEP3B cells were transfected with shNC or shSGOL2, and the levels of PCNA, cyclin D1, cyclin E1, SGOL2, and MAD2 were detected by Western blots to study the effect of SGOL2 on cell cycle and MAD2. GAPDH was used as a loading control. B SK-HEP-1, and HEP3B cells were transfected with SGOL2 plasmid or vector control plasmid, and the levels of PCNA, cyclin D1, cyclin E1, SGOL2, and MAD2 were detected by Western blots. GAPDH was used as a loading control. C - D , Invasion and sphere formation of SGOL2-upregulated HCC cells with or without M2I-1 treatment were detected and analyzed. The results are presented as the mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The following antibodies were used: SGOL2 antibody (Bethyl, A301–262A, for WB/Co-IP), MAD2 antibody (Bethyl, A300-301A, for WB/Co-IP), SGOL1 (Immunoway, YT4275), cyclin D1 antibody (CST, 55506), cyclin E1 antibody (CST, 20808), PCNA antibody (CST, 13110), normal rabbit IgG (CST, 2729), E-cadherin antibody (CST, 14472), N-cadherin antibody (CST, 13116), vimentin antibody (CST, 5741), MMP9 antibody (CST,13667), β-catenin antibody (CST, 8480), and fibronectin antibody (Abcam, ab268021), secondary antibody (CST,7074/7076), GAPDH antibody (CST, 5174).

Techniques: Transfection, Western Blot, Control, Plasmid Preparation

Journal: Biomarker Research

Article Title: SGOL2 is a novel prognostic marker and fosters disease progression via a MAD2-mediated pathway in hepatocellular carcinoma

doi: 10.1186/s40364-022-00422-z

Figure Lengend Snippet: Overexpression of MAD2 reversed the knockdown effects of SGOL2-shRNA in HCC. A - D , F Invasion, migration, sphere formation, and colony formation assays of SGOL2 knockdown HCC cells with or without MAD2 overexpression were performed. E SK-HEP-1, and HEP3B cells were transfected with shSGOL2 or MAD2 plasmid, and the proliferation of HCC cells was detected at Days 3 by CCK-8 assays. The results are presented as the mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The following antibodies were used: SGOL2 antibody (Bethyl, A301–262A, for WB/Co-IP), MAD2 antibody (Bethyl, A300-301A, for WB/Co-IP), SGOL1 (Immunoway, YT4275), cyclin D1 antibody (CST, 55506), cyclin E1 antibody (CST, 20808), PCNA antibody (CST, 13110), normal rabbit IgG (CST, 2729), E-cadherin antibody (CST, 14472), N-cadherin antibody (CST, 13116), vimentin antibody (CST, 5741), MMP9 antibody (CST,13667), β-catenin antibody (CST, 8480), and fibronectin antibody (Abcam, ab268021), secondary antibody (CST,7074/7076), GAPDH antibody (CST, 5174).

Techniques: Over Expression, Knockdown, shRNA, Migration, Transfection, Plasmid Preparation, CCK-8 Assay

Journal: Biomarker Research

Article Title: SGOL2 is a novel prognostic marker and fosters disease progression via a MAD2-mediated pathway in hepatocellular carcinoma

doi: 10.1186/s40364-022-00422-z

Figure Lengend Snippet: SGOL2 exerted its effect by forming a SGOL2-MAD2 complex. A SK-HEP-1 and HEP3B cells were transfected with shSGOL2 lentivirus or SGOL2 plasmid. The colocalization between SGOL2 (Red) and MAD2 (Green) was visualized as yellow fluorescence in the merged panel by Confocal microscopy. B The endogenous interaction between SGOL2 and MAD2 was detected by IP assays in HCC cells

Article Snippet: The following antibodies were used: SGOL2 antibody (Bethyl, A301–262A, for WB/Co-IP), MAD2 antibody (Bethyl, A300-301A, for WB/Co-IP), SGOL1 (Immunoway, YT4275), cyclin D1 antibody (CST, 55506), cyclin E1 antibody (CST, 20808), PCNA antibody (CST, 13110), normal rabbit IgG (CST, 2729), E-cadherin antibody (CST, 14472), N-cadherin antibody (CST, 13116), vimentin antibody (CST, 5741), MMP9 antibody (CST,13667), β-catenin antibody (CST, 8480), and fibronectin antibody (Abcam, ab268021), secondary antibody (CST,7074/7076), GAPDH antibody (CST, 5174).

Techniques: Transfection, Plasmid Preparation, Fluorescence, Confocal Microscopy

Journal: Biomarker Research

Article Title: SGOL2 is a novel prognostic marker and fosters disease progression via a MAD2-mediated pathway in hepatocellular carcinoma

doi: 10.1186/s40364-022-00422-z

Figure Lengend Snippet: Upregulated MAD2 expression predicted poor prognosis in HCC and hub gene analysis positively related to both SGOL2 and MAD2. A MAD2 mRNA level is higher in HCC tissues than that in normal liver tissues (UALCAN). B MAD2 has a positive relation with SGOL2 in HCC (GEPIA). C Highly expressed MAD2 predicted poor prognosis in HCC. D Forty-seven genes positively correlated with both SGOL2 and MAD2, as shown by Venn diagram analysis. E The interaction network of the 47 genes. F KEGG enrichment of the 47 genes. G The graph shows the interaction network of the top 15 hub genes. H - I GO analysis and KEGG enrichment of the top 15 hub genes. HR: hazard ratio. J The role and mechanism of SGOL2 in HCC cells. SGOL2 forms a SGOL2-MAD2 complex and further regulates MAD2, resulting in dysregulation of the cell cycle and finally enhancing HCC malignant behaviors

Article Snippet: The following antibodies were used: SGOL2 antibody (Bethyl, A301–262A, for WB/Co-IP), MAD2 antibody (Bethyl, A300-301A, for WB/Co-IP), SGOL1 (Immunoway, YT4275), cyclin D1 antibody (CST, 55506), cyclin E1 antibody (CST, 20808), PCNA antibody (CST, 13110), normal rabbit IgG (CST, 2729), E-cadherin antibody (CST, 14472), N-cadherin antibody (CST, 13116), vimentin antibody (CST, 5741), MMP9 antibody (CST,13667), β-catenin antibody (CST, 8480), and fibronectin antibody (Abcam, ab268021), secondary antibody (CST,7074/7076), GAPDH antibody (CST, 5174).

Techniques: Expressing

Journal: Biomarker Research

Article Title: SGOL2 is a novel prognostic marker and fosters disease progression via a MAD2-mediated pathway in hepatocellular carcinoma

doi: 10.1186/s40364-022-00422-z

Figure Lengend Snippet: SGOL2 knockdown inhibited HCC growth and metastasis in vivo. A - B Xenograft model was set up to study the effects of SGOL2 on HCC tumor growth in vivo. Mice were divided into two groups and inoculated with SK-HEP-1 shNC or SK-HEP-1 shSGOL2 cells (s.c. n = 3, i.v. n = 7). Images of the isolated livers and tumors from sacrificed mice are presented, and the hepatic replacement area (HRA%) and the tumor volumes and tumor weights of the indicated groups were analyzed and compared. Loss of SGOL2 in SK-HEP-1 cells contributed to the reduction in tumorigenesis. C - D , HE staining of metastatic tumors in liver and lung tissues. Representative images and quantitative analysis results are shown

Article Snippet: The following antibodies were used: SGOL2 antibody (Bethyl, A301–262A, for WB/Co-IP), MAD2 antibody (Bethyl, A300-301A, for WB/Co-IP), SGOL1 (Immunoway, YT4275), cyclin D1 antibody (CST, 55506), cyclin E1 antibody (CST, 20808), PCNA antibody (CST, 13110), normal rabbit IgG (CST, 2729), E-cadherin antibody (CST, 14472), N-cadherin antibody (CST, 13116), vimentin antibody (CST, 5741), MMP9 antibody (CST,13667), β-catenin antibody (CST, 8480), and fibronectin antibody (Abcam, ab268021), secondary antibody (CST,7074/7076), GAPDH antibody (CST, 5174).

Techniques: Knockdown, In Vivo, Isolation, Staining

Journal: Biomarker Research

Article Title: SGOL2 is a novel prognostic marker and fosters disease progression via a MAD2-mediated pathway in hepatocellular carcinoma

doi: 10.1186/s40364-022-00422-z

Figure Lengend Snippet: Downregulation of SGOL2 expression promoted apoptosis in vivo. A - B IHC staining of CD34, Ki-67, PCNA, and EMT-related markers. Representative images and quantitative analysis results are shown. C The apoptotic area was dramatically increased in the shSGOL2 group compared with the shNC group. The results are presented as the mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The following antibodies were used: SGOL2 antibody (Bethyl, A301–262A, for WB/Co-IP), MAD2 antibody (Bethyl, A300-301A, for WB/Co-IP), SGOL1 (Immunoway, YT4275), cyclin D1 antibody (CST, 55506), cyclin E1 antibody (CST, 20808), PCNA antibody (CST, 13110), normal rabbit IgG (CST, 2729), E-cadherin antibody (CST, 14472), N-cadherin antibody (CST, 13116), vimentin antibody (CST, 5741), MMP9 antibody (CST,13667), β-catenin antibody (CST, 8480), and fibronectin antibody (Abcam, ab268021), secondary antibody (CST,7074/7076), GAPDH antibody (CST, 5174).

Techniques: Expressing, In Vivo, Immunohistochemistry

Journal: Journal of Cancer

Article Title: High SGO2 predicted poor prognosis and high therapeutic value of lung adenocarcinoma and promoted cell proliferation, migration, invasion, and epithelial-to-mesenchymal transformation

doi: 10.7150/jca.86285

Figure Lengend Snippet: SGO2 increases the risk of poor prognosis for LUAD. (A, E, and H) Based on the TCGA database, SGO2 expression, ROC curve, and survival analysis in LUAD. (B, F, and I) Based on the GEO database, the validation dataset GSE30219 was used to verify the SGO2 expression, ROC curve, and survival analysis in LUAD. (C) The expression of SGO2 in LUAD and normal tissue. (D) The protein expression of SGO2 in LUAD and normal tissue. (G and J) Immunohistochemical staining of SGO2 in LUAD and normal tissue.

Article Snippet: The sections were incubated with a 1:1000 rabbit anti-human SGO2 antibody (Cat. # NBP1-83567; Novus, USA) overnight at a 4 °C in wet box.

Techniques: Expressing, Biomarker Discovery, Immunohistochemical staining, Staining

Journal: Journal of Cancer

Article Title: High SGO2 predicted poor prognosis and high therapeutic value of lung adenocarcinoma and promoted cell proliferation, migration, invasion, and epithelial-to-mesenchymal transformation

doi: 10.7150/jca.86285

Figure Lengend Snippet: High SGO2 is associated with a higher TNM stage of LUAD. (A) Based on the TCGA and GSE30219, the expression of SGO2 in T1 vs T2. (B) Based on the TCGA and GSE30219, the expression of SGO2 in N0 vs N1. (C) Based on the TCGA and GSE31210, the expression of SGO2 in stage I vs stage II + III. (D and G) Based on IHC, the protein expression and staining of SGO2 in T1 vs T2. (E and H) Based on IHC, the protein expression and staining of SGO2 in N0 vs N1. (F and I) Based on IHC, the protein expression and staining of SGO2 in stage I vs stage II + III.

Article Snippet: The sections were incubated with a 1:1000 rabbit anti-human SGO2 antibody (Cat. # NBP1-83567; Novus, USA) overnight at a 4 °C in wet box.

Techniques: Expressing, Staining

Journal: Journal of Cancer

Article Title: High SGO2 predicted poor prognosis and high therapeutic value of lung adenocarcinoma and promoted cell proliferation, migration, invasion, and epithelial-to-mesenchymal transformation

doi: 10.7150/jca.86285

Figure Lengend Snippet: SGO2 silencing inhibits the proliferation of lung cancer cells. (A)SGO2 expression after knockdown in A549 and H1299. (B)CCK8 assays were performed to detect A549 and H1299 proliferation. (C)Edu assays were performed to detect A549 and H1299 proliferation.

Article Snippet: The sections were incubated with a 1:1000 rabbit anti-human SGO2 antibody (Cat. # NBP1-83567; Novus, USA) overnight at a 4 °C in wet box.

Techniques: Expressing, Knockdown

Journal: Journal of Cancer

Article Title: High SGO2 predicted poor prognosis and high therapeutic value of lung adenocarcinoma and promoted cell proliferation, migration, invasion, and epithelial-to-mesenchymal transformation

doi: 10.7150/jca.86285

Figure Lengend Snippet: The downregulation of SGO2 affects migration, invasion, and EMT. (A) A Transwell assay was performed to examine the effect of SGO2 knockdown on A549 and H1299 cells. (C) A wound healing assay was performed to examine the effect of SGO2 knockdown on A549 and H1299 cells. (B and D) WB assay the expression levels of E-cadherin, N-cadherin, Vimentin, and Cytokeratin in A549 and H1299 to evaluate the effect on EMT after SGO2 knockdown.

Article Snippet: The sections were incubated with a 1:1000 rabbit anti-human SGO2 antibody (Cat. # NBP1-83567; Novus, USA) overnight at a 4 °C in wet box.

Techniques: Migration, Transwell Assay, Knockdown, Wound Healing Assay, Expressing

Journal: Journal of Cancer

Article Title: High SGO2 predicted poor prognosis and high therapeutic value of lung adenocarcinoma and promoted cell proliferation, migration, invasion, and epithelial-to-mesenchymal transformation

doi: 10.7150/jca.86285

Figure Lengend Snippet: SGO2 expression and tumor immune infiltration. (A) the expression of SGO2 was positively correlated with the infiltration of Memory B cells, Activated CD4+ memory T cells, and CD8+ T cells, while the infiltration of Memory B cells and Tregs decreased with increasing SGO2 expression. (B) Correlation of SGO2 expression with 28 distinct types of tumor-infiltrating immune cells based on ssGSEA.

Article Snippet: The sections were incubated with a 1:1000 rabbit anti-human SGO2 antibody (Cat. # NBP1-83567; Novus, USA) overnight at a 4 °C in wet box.

Techniques: Expressing

Journal: Journal of Cancer

Article Title: High SGO2 predicted poor prognosis and high therapeutic value of lung adenocarcinoma and promoted cell proliferation, migration, invasion, and epithelial-to-mesenchymal transformation

doi: 10.7150/jca.86285

Figure Lengend Snippet: High SGO2 has multiple therapeutic benefits. (A) Comparison of mutational landscapes of SGO between high cluster and low cluster, and Comparison of tumor mutation burden (TMB) between two clusters. (B) Comparison of first-line chemotherapy and targeted therapy drug targets of high and low SGO2 clusters. (C) Comparison of immunomodulatory drug targets of high and low SGO2 clusters.

Article Snippet: The sections were incubated with a 1:1000 rabbit anti-human SGO2 antibody (Cat. # NBP1-83567; Novus, USA) overnight at a 4 °C in wet box.

Techniques: Comparison, Mutagenesis

Journal: Reproductive Medicine and Biology

Article Title: Effects of post‐ovulatory aging on centromeric cohesin protection in murine MII oocytes

doi: 10.1002/rmb2.12433

Figure Lengend Snippet: Comparison of SGO2 expression in mouse oocytes. A, Localization of SGO2 in mouse oocytes. Immunofluorescent staining of SGO2 (green) in the 12‐h aged and fresh oocytes. DNA (blue) was stained with Hoechst 33258. D‐PBS was used as the negative control instead of the primary antibody. Scale bar = 10 μm. B, Comparison of brightness values of SGO2 signal in the 12‐h aged and fresh oocytes. C, Relative expression of SGO2 to α‐tubulin. Bars with different superscripts indicate significant differences ( p < 0.05). D, Western blotting for SGO2 protein in MII oocyte. α‐tubulin was used as a loading control

Article Snippet: The oocyte lysates were diluted with an equal volume of 2× Laemmli sample buffer (Bio‐Rad) containing 5% 2‐mercaptoethanol (Wako) and heated to 100°C for 5 min. Proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) with a stacking gel containing 4% acrylamide (Wako) and a separating gel containing 7.5–10% acrylamide run for 50 min at 200 V. Proteins were then electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes (GE Healthcare) for 30 min at 15 V. Each membrane was blocked with PVDF blocking reagent (TOYOBO Co., Ltd.) for 1 h at room temperature, followed by incubation overnight at 4 °C with rabbit anti‐p‐REC8 antibody (1:1,000 dilution, LS‐C47114; LifeSpan Biosciences, Inc.), mouse anti‐PP2A‐Aα/β antibody (1:1,000 dilution; Santa Cruz), rabbit anti‐SGO2 antibody (1:1,000 dilution; My BioSource), mouse anti‐BUB1 antibody (1:500 dilution; Santa Cruz), mouse anti‐ARK‐2 antibody (1:500 dilution; Santa Cruz), or goat anti‐MAD2 antibody (1:500 dilution; Santa Cruz) as the primary antibody.

Techniques: Expressing, Staining, Negative Control, Western Blot

Journal: Scientific reports

Article Title: Shugosin 2 is a biomarker for pathological grading and survival prediction in patients with gliomas.

doi: 10.1038/s41598-021-97119-4

Figure Lengend Snippet: Figure 2. The expression level of SGO2 is related to the survival of patients with high-grade gliomas. The Kaplan–Meier survival curve analyzed from GEO profile (GDS1816/230165_at/SGO2) (a), TCGA (b), and CGGA (c). Data showed that patients with high expression of SGO2 had unfavorable survival outcome. (GDS1816/230165_at/SGO2, n = 77, p = 0.0011 by log-rank test, 95% CI 1.000–1.00, hazard ratio 1.001; TCGA, n = 343, p < 1 × 10–15 by log-rank test, 95% CI 0.87–5.30, hazard ratio 3.90; CGGA, n = 209, p = 1.23 × 10–11 by log- rank test, 95% CI 2.13–4.25, hazard ratio 3.01).

Article Snippet: The tissue microarrays were incubated with a polyclonal rabbit antihuman SGO2 antibody (HPA035163, Atlas Antibodies, Stockholm.

Techniques: Expressing

Journal: Scientific reports

Article Title: Shugosin 2 is a biomarker for pathological grading and survival prediction in patients with gliomas.

doi: 10.1038/s41598-021-97119-4

Figure Lengend Snippet: Figure 1. The expression level of SGO2 is related to pathological grading of gliomas. The SGO2 mRNA level in different grade of gliomas and non-tumor brain tissue from GEO profile (GDS1962/230165_at/SGO2) (a), TCGA (b), and CCGA (c). SGO2 expression was significantly higher in high-grade gliomas (Grade III and IV) than in low-grade gliomas (Grade II) and non-tumor control. The Y-axis indicates the SGO2 mRNA expression. The p value was adjusted by Bonferroni method in R software (version 3.0.1) between each group.

Article Snippet: The tissue microarrays were incubated with a polyclonal rabbit antihuman SGO2 antibody (HPA035163, Atlas Antibodies, Stockholm.

Techniques: Expressing, Control, Software

Journal: Scientific reports

Article Title: Shugosin 2 is a biomarker for pathological grading and survival prediction in patients with gliomas.

doi: 10.1038/s41598-021-97119-4

Figure Lengend Snippet: Figure 4. Validation of SGO2 protein expression in human gliomas and non-tumor brain tissue. Hematoxylin and eosin staining of non-tumor brain tissue (a), low grade (b) and high grade gliomas (c). The immunohistochemical staining of SGO2 on non-tumor brain tissue (d), low grade (e), and high grade gliomas (f) (scale bar: 50 μm). (g–i) The SGO2 immunostaining scores in normal brain tissue, low-grade glioma and high-grade glioma were statistically analyzed. The adjusted p value was calibrated between each group.

Article Snippet: The tissue microarrays were incubated with a polyclonal rabbit antihuman SGO2 antibody (HPA035163, Atlas Antibodies, Stockholm.

Techniques: Biomarker Discovery, Expressing, Staining, Immunohistochemical staining, Immunostaining

Journal: Scientific reports

Article Title: Shugosin 2 is a biomarker for pathological grading and survival prediction in patients with gliomas.

doi: 10.1038/s41598-021-97119-4

Figure Lengend Snippet: Figure 3. Validation of SGO2 mRNA and protein levels in glioma cell lines and normal brain tissue (a) qRT- PCR was performed to examine SGO2 mRNA expression and the quantitative results are shown in glioma cell lines. The relative expressions were normalized with normal brain. Bars mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.005 showed significant differences. Data are representative of three independent experiments. (b) Protein lysates of glioma cell lines, including U87MG, LN229, GBM8401, and U118MG were applied to SDS-PAGE and Western blot analysis to quantitate SGO2 protein expression (full length blot is presented in Supplementary Fig. 1). GAPDH served as a loading control.

Article Snippet: The tissue microarrays were incubated with a polyclonal rabbit antihuman SGO2 antibody (HPA035163, Atlas Antibodies, Stockholm.

Techniques: Biomarker Discovery, Quantitative RT-PCR, Expressing, SDS Page, Western Blot, Control

Journal: Scientific reports

Article Title: Shugosin 2 is a biomarker for pathological grading and survival prediction in patients with gliomas.

doi: 10.1038/s41598-021-97119-4

Figure Lengend Snippet: Figure 5. The effect of SGO2 on cell proliferation and apoptosis (a) The SGO2 knockdown model constructed by siRNA 25 nM transfection into LN229 and GBM8401 cell lines. The knockdown efficiency of SGO2 siRNA or control siRNA in infected LN229 and GBM8401 cells measured by RT-qPCR. Bars, mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.005 showed significant differences. Data are representative of three independent experiments. (b) LN229 and GBM8401 cells were transfected with 25 nM siRNA or siControl. Cell count was determined at the indicated time points. The data are expressed as the mean ± s.d.; n = 3; **p < 0.01, and ***p < 0.001. (c) LN229 and GBM8401 cell with siSGO2 or siControl transfection were labeled with BrdU then proceeded analysis by flow cytometry (**p < 0.01, ***p < 0.005). (d) Cell cycle analysis of LN229 and GBM8401 siSGO2 cells was determined by propidium iodide (PI) stain and flow cytometry. The data are expressed as the mean ± s.d.; n = 3; *p < 0.05, **p < 0.01. (e) Cell apoptosis analysis of LN 229 and GBM8401 siSGO2 cells were determined by tetraethylbenzimidazolylcarbocyanine iodide (JC-1) dye and flow cytometry.

Article Snippet: The tissue microarrays were incubated with a polyclonal rabbit antihuman SGO2 antibody (HPA035163, Atlas Antibodies, Stockholm.

Techniques: Knockdown, Construct, Transfection, Control, Infection, Quantitative RT-PCR, Cell Counting, Labeling, Flow Cytometry, Cell Cycle Assay, Staining

Journal: Scientific reports

Article Title: Shugosin 2 is a biomarker for pathological grading and survival prediction in patients with gliomas.

doi: 10.1038/s41598-021-97119-4

Figure Lengend Snippet: Figure 6. The effect of SGO2 knockdown on cell migration detected by wound-healing assays. Images and Quantitative analysis of LN229 (a) and GBM8401 (b) cells in the wound-healing assay. Data are presented as the mean ± SD (n = 3). *p < 0.05, **p < 0.01.

Article Snippet: The tissue microarrays were incubated with a polyclonal rabbit antihuman SGO2 antibody (HPA035163, Atlas Antibodies, Stockholm.

Techniques: Knockdown, Migration, Wound Healing Assay

Journal: Scientific reports

Article Title: Shugosin 2 is a biomarker for pathological grading and survival prediction in patients with gliomas.

doi: 10.1038/s41598-021-97119-4

Figure Lengend Snippet: Figure 7. The SGO2 protein–protein interaction (PPI) network. (a) In the PPI network established by STRING dataset, SGO2 is a hub protein. (b) The STRING dataset also predicted the association between SGO2, ARUKB, and FOXM1. (c) Protein lysates of LN229 and GBM8401 were applied to SDS-PAGE and Western blot to investigate the protein expression of AURKB and FOXM1(full length blot is presented in Supplementary Fig. 2). α-actinin served as a loading control.

Article Snippet: The tissue microarrays were incubated with a polyclonal rabbit antihuman SGO2 antibody (HPA035163, Atlas Antibodies, Stockholm.

Techniques: SDS Page, Western Blot, Expressing, Control

Journal: Molecular cell

Article Title: Phospho-H1 decorates the inter-chromatid axis and is evicted along with Shugoshin by SET during mitosis

doi: 10.1016/j.molcel.2017.07.008

Figure Lengend Snippet: (A) Silver stain of Tandem Affinity Purification (TAP) of SET-complexes from iMEFs. CYTO: cytosol, SNE: soluble nuclear extract, CHR: chromatin pellet, NFH-SET: N-terminal FLAG-HA-SET, ENDO-SET: endogenous SET. (B) Venn diagram depicting top proteins identified from TAP-MS analysis of cytosolic and nuclear extracts prepared from mouse (iMEFs) and human (hTERT-RPE1) cells. Raw peptide count numbers are presented in Table S1. Only proteins with a cumulative peptide count of 10 or higher were included in the Venn diagram. (C) Volcano plot of quantitative-MS analysis of SET-associated proteins from mitotic extracts. SGOL1 and SGOL2 are highlighted in ellipses. Note that the plot represents proteins enriched in cytosolic and nuclear fractions combined. Histones enriched in the nuclear extracts are depicted separately in Figure 1H. (D) IF analysis on human chromosomes from WT RPE1 cells depicting co-localization of SET and SGOL2 at centromeres. Chromosomal arm staining is also observed for SET. Also see Figure S1E for another zoomed in chromosome. Scale bar =5 µm. (E) Glycerol gradient analysis of SET-complexes indicating co-elution of SET and SGOL2 at fraction 7, independent of PP2A (fraction 3). Traces of peak normalized intensities for each fraction is shown below. Also see Figure S1E for the same analysis with an alternate PP2A-B subunit. (F) (Top) Domain organization and constructs of SGOL2 and SET used in the pull-down assay. D-box, KEN-box: putative APC/C recognition domains, SGO: SGO domain. The asterisks in SET DIM represent point mutations that impaired dimerization. (Bottom) In vitro pull-down assay between GST-tagged N-terminal (1–650 a.a.) or C-terminal (651–1265 a.a.) regions of SGOL2 with SET, either WT or mutant in its dimerization domain (DIM) or in its acidic tail (ACID), indicating direct association of SET with the N-terminal region of SGOL2. Dimerization, but not the acidic tail, is required for SET binding. REP1 and REP2: experimental replicates. (G) Glycerol gradient separation of H1.2 complexes indicating co-elution with SET at fractions 7–9. Traces of peak normalized intensities for each fraction is shown below. Fraction 23 was omitted for clarity. (See also Figure S1I). (H) Quantitative MS data for histones co-purifying with SET in mitotic extracts. Linker histones are the most abundant histones associated with SET in the nuclear extracts. (See also Figure S1).

Article Snippet: METHOD DETAILS Antibodies Antibodies for western blots were used at the following dilutions: rabbit anti-SET and goat anti-SET (Santa Cruz, 1:1000), rabbit anti-SGOL2 (Bethyl, 1:1000), mouse anti-SGOL1 (Abcam, 1:1000), rabbit anti-PP2A-B (Cell Signaling, 1:1000), rabbit anti-PP2A-B’ (Bethyl, 1:1000), mouse anti-H1 (Santa Cruz, 1:2500), rabbit anti-H3 and anti-H4 (Abcam, 1:5000), mouse anti-H3S10ph (Millipore, 1:1000), mouse anti-GAPDH (Genetex, 1:5000), rabbit anti-REPIN1 (Sigma, 1:1000), mouse anti-MPM2 (Millipore, 1:1000), rabbit anti-H1S/T18ph (Abcam, 1: 1000), rabbit anti-PAF1 and anti-LEO1 (Reinberg lab, 1:1000), Rabbit anti-H1S27ph (Sigma 1:1000), anti-H1S35ph (genetex, 1:1000).

Techniques: Silver Staining, Affinity Purification, Staining, Co-Elution Assay, Construct, Pull Down Assay, In Vitro, Mutagenesis, Binding Assay

Journal: Molecular cell

Article Title: Phospho-H1 decorates the inter-chromatid axis and is evicted along with Shugoshin by SET during mitosis

doi: 10.1016/j.molcel.2017.07.008

Figure Lengend Snippet: (A) IF analysis on WT RPE1 cells highlighting SET localization during the cell-cycle. Nuclear SET increases dramatically during G2-prophase, followed by its cytosolic presence at metaphase. Centrosomal/spindle pole localization is observed at all stages as depicted by co-staining with the centrosomal marker, γ-tubulin. Scale bar = 5 µm. (B) (Top) Schematic to study SET-SGOL2 complexes in FLAG-HA-SET expressing RPE1 cells, at different mitotic stages: Nocodazole (prometaphase arrest), BI2536 (PLK1 inhibitor; prophase and prometaphase arrest). (Bottom) FLAG-SET IP from cells treated with nocodazole (NOCO) and BI2536 indicating complex formation with SGOL2 in the cytosol (NOCO and BI2536) and nuclear fractions (BI2536-nucleus/chromosome). Quantification of the same is shown on the right. (C) (Left) Schematic to synchronize cells at prophase by double-thymidine block and release. (Right) FLAG-SET IP from prophase synchronized cells indicating SGOL2 complex formation predominantly in the nucleus (D) (Left) Schematic to synchronize cells at prometaphase by double-thymidine block and release into nocodazole. Cells were harvested by mitotic shake-off (Right) FLAG-SET IP showing that the SET-SGOL2 complex is largely cytosolic at this stage. (E) (Left) Schematic to study SET-H1 complexes using Barasertib and BI2536 (AURKB inhibitor; prophase and prometaphase arrest). (Right) FLAG-SET IP showing the enrichment of H1 complexes in the nucleus. (F) (Left) Schematic to study cell-cycle dependency of SET-H1 interaction using Roscovitine (CDK inhibitor; G1/S and G2/M arrest). (Right) FLAG H1.2 IP from RPE1 cells treated with Roscovitine show ablation of SET association with H1, but not of PAF1/LEO1, highlighting that progression through the cell-cycle is necessary for SET-H1 interaction. All complexes in (B), (C), (D), (E) and (F) were natively eluted using FLAG peptide. (See also Figure S2).

Article Snippet: METHOD DETAILS Antibodies Antibodies for western blots were used at the following dilutions: rabbit anti-SET and goat anti-SET (Santa Cruz, 1:1000), rabbit anti-SGOL2 (Bethyl, 1:1000), mouse anti-SGOL1 (Abcam, 1:1000), rabbit anti-PP2A-B (Cell Signaling, 1:1000), rabbit anti-PP2A-B’ (Bethyl, 1:1000), mouse anti-H1 (Santa Cruz, 1:2500), rabbit anti-H3 and anti-H4 (Abcam, 1:5000), mouse anti-H3S10ph (Millipore, 1:1000), mouse anti-GAPDH (Genetex, 1:5000), rabbit anti-REPIN1 (Sigma, 1:1000), mouse anti-MPM2 (Millipore, 1:1000), rabbit anti-H1S/T18ph (Abcam, 1: 1000), rabbit anti-PAF1 and anti-LEO1 (Reinberg lab, 1:1000), Rabbit anti-H1S27ph (Sigma 1:1000), anti-H1S35ph (genetex, 1:1000).

Techniques: Staining, Marker, Expressing, Blocking Assay

Journal: Molecular cell

Article Title: Phospho-H1 decorates the inter-chromatid axis and is evicted along with Shugoshin by SET during mitosis

doi: 10.1016/j.molcel.2017.07.008

Figure Lengend Snippet: (A) Schematic of CHREA assay to study mitotic protein eviction from chromosomal clusters. (B) (Top) Western blot of evicted proteins reveals that addition of SET, but not BSA, or NAP1 (Figure S3), results in a dose-dependent eviction of SGOL1 and SGOL2 from chromosomes. Additional controls are shown in Figure S3A. Histones H3 and H4 are not evicted. Immunoblot for H3 from the ensuing pellets of the reaction serves as loading control to gauge chromosomal input across samples. (Bottom) H3-pellet and peak normalized intensities from left. (C) (Top) Chaperone assay with SET mutants indicating that both dimerization and acidic tail domains are required for chaperone activity. (Bottom) H3-pellet and peak normalized intensities from left. (D) (Top) NFH-SET RPE1 cells treated with increasing concentrations of BI2536 (20, 100 and 500 nM) display a dose-dependent retention of SET-SGOL2, but not SET-PP2A complexes in the nucleus. (Bottom) Quantification of SET-normalized intensities. Two lines reflect replicates of the experiment. See also Figure S3D for total levels of SGOL2 and SET under these conditions. (E) Schematic of the two-step CHREA in the presence of PLK1. (F) Results of a kinase assay [step 1 of (E)] visualized by autoradiography along with the corresponding Coomassie Blue staining. PLK1 phosphorylates chromosomal clusters used in the chaperone assay. (G) (Left) CHREA performed as in step 2 of (E) indicating that SET evicts SGOL1 and SGOL2 from PLK1 pre-phosphorylated chromosomal clusters, with higher efficiency than those phosphorylated with PLK1+BI2536. (Right) H3-pellet and peak normalized intensities from top. (H) PLK1 kinase assay with purified N-terminal and C-terminal domains of SGOL2. The C-terminal region of SGOL2 is preferentially phosphorylated by PLK1. (See also Figure S3).

Article Snippet: METHOD DETAILS Antibodies Antibodies for western blots were used at the following dilutions: rabbit anti-SET and goat anti-SET (Santa Cruz, 1:1000), rabbit anti-SGOL2 (Bethyl, 1:1000), mouse anti-SGOL1 (Abcam, 1:1000), rabbit anti-PP2A-B (Cell Signaling, 1:1000), rabbit anti-PP2A-B’ (Bethyl, 1:1000), mouse anti-H1 (Santa Cruz, 1:2500), rabbit anti-H3 and anti-H4 (Abcam, 1:5000), mouse anti-H3S10ph (Millipore, 1:1000), mouse anti-GAPDH (Genetex, 1:5000), rabbit anti-REPIN1 (Sigma, 1:1000), mouse anti-MPM2 (Millipore, 1:1000), rabbit anti-H1S/T18ph (Abcam, 1: 1000), rabbit anti-PAF1 and anti-LEO1 (Reinberg lab, 1:1000), Rabbit anti-H1S27ph (Sigma 1:1000), anti-H1S35ph (genetex, 1:1000).

Techniques: Western Blot, Activity Assay, Kinase Assay, Autoradiography, Staining, Purification

Journal: Molecular cell

Article Title: Phospho-H1 decorates the inter-chromatid axis and is evicted along with Shugoshin by SET during mitosis

doi: 10.1016/j.molcel.2017.07.008

Figure Lengend Snippet: (A) (Top) Giemsa staining of metaphase chromosomes showing unresolved sister-chromatids upon SET KO. Scale bar = 10 µm. (Bottom) Inter-chromatid distances are significantly reduced and percentage of metaphases with resolution defects are markedly increased. (B) FLAG-SET IP from BI2536-treated cells showing specific association with H1S/T18ph but not with other mitotic marks, H1S27ph or H1S25ph. Also see Figure S6C. CYTO: Cytosol; NUC: Nuclear Extract. (C) (Left) Giemsa staining of metaphase chromosomes isolated from cells overexpressing H1E_WT or H1E_T18A showing defective chromatid resolution in the latter case. Quantification is shown on right. (D) (Left) Dynamics of H1S/T18ph through mitosis was monitored by IF analysis. Images were captured on the same frame for comparing the different stages. Scale bar = 20 µm in the low magnification image and 10 µm on the zoomed panels. (Right) Quantification of H1S/T18ph expression by measurement of corrected total cell fluorescence. (E) Schematic to study H1 eviction from chromosomes upon SET KO. (F) Western blot analysis of chromosomes from control and SET KO cells depicting a dramatic increase in chromosomal H1S/T18ph upon SET KO. A modest increase in SGOL2 was also observed. Levels of other chromosomal proteins are largely unaltered. (G) IF analysis on metaphase chromosomes showing that H1S/T18ph is not evicted from the inter-chromatid axis upon SET KO. ACA: Anti centromere antigen. Scale bar = 10 µm. Quantification is shown on the right. The staining intensity with H1S/T18ph antibody was weak and the green channel was enhanced equally for all control and SET KO images. (H) Chromosome IF analysis of SET (Left) and H1S/T18ph (Right) in RPE1 WT cells depicting strong inter-chromatid axial staining. In addition, SET also localizes to centromeres. Scale bar = 10 µm. For quantification panels in 5A, 5C, 5D and 5G: N numbers are indicated on the y-axis. *** = p<0.0001, Fisher’s exact test. (See also Figures S5 and S6).

Article Snippet: METHOD DETAILS Antibodies Antibodies for western blots were used at the following dilutions: rabbit anti-SET and goat anti-SET (Santa Cruz, 1:1000), rabbit anti-SGOL2 (Bethyl, 1:1000), mouse anti-SGOL1 (Abcam, 1:1000), rabbit anti-PP2A-B (Cell Signaling, 1:1000), rabbit anti-PP2A-B’ (Bethyl, 1:1000), mouse anti-H1 (Santa Cruz, 1:2500), rabbit anti-H3 and anti-H4 (Abcam, 1:5000), mouse anti-H3S10ph (Millipore, 1:1000), mouse anti-GAPDH (Genetex, 1:5000), rabbit anti-REPIN1 (Sigma, 1:1000), mouse anti-MPM2 (Millipore, 1:1000), rabbit anti-H1S/T18ph (Abcam, 1: 1000), rabbit anti-PAF1 and anti-LEO1 (Reinberg lab, 1:1000), Rabbit anti-H1S27ph (Sigma 1:1000), anti-H1S35ph (genetex, 1:1000).

Techniques: Staining, Isolation, Expressing, Fluorescence, Western Blot

Journal: Molecular cell

Article Title: Phospho-H1 decorates the inter-chromatid axis and is evicted along with Shugoshin by SET during mitosis

doi: 10.1016/j.molcel.2017.07.008

Figure Lengend Snippet: (A) Schematic of CHREA to study H1S/T18ph eviction. (B) (Top) Western blot analysis of evicted proteins showing eviction of H1S/T18ph (as well as SGOL2) upon rescue with WT, but not ACID or DIM SET. REP1 and REP2 are two replicates of the experiment. (Bottom) Quantification of western blot on top. (C) Schematic of CHREA for analyzing effect of phosphorylation on H1 eviction by treatment with λ phosphatase (PPASE) or λ phosphatase with phosphatase inhibitors (PPASE+ Inhibitors). (D) (Right) Western blot analysis of evicted proteins showing a significant reduction in H1 eviction upon phosphatase treatment. Note that recognition by the H1S/T18ph mark is lost upon phosphatase treatment. (Left) Quantification of evicted proteins. (E) Model depicting the function of SET as a mitotic chaperone. See discussion for description.

Article Snippet: METHOD DETAILS Antibodies Antibodies for western blots were used at the following dilutions: rabbit anti-SET and goat anti-SET (Santa Cruz, 1:1000), rabbit anti-SGOL2 (Bethyl, 1:1000), mouse anti-SGOL1 (Abcam, 1:1000), rabbit anti-PP2A-B (Cell Signaling, 1:1000), rabbit anti-PP2A-B’ (Bethyl, 1:1000), mouse anti-H1 (Santa Cruz, 1:2500), rabbit anti-H3 and anti-H4 (Abcam, 1:5000), mouse anti-H3S10ph (Millipore, 1:1000), mouse anti-GAPDH (Genetex, 1:5000), rabbit anti-REPIN1 (Sigma, 1:1000), mouse anti-MPM2 (Millipore, 1:1000), rabbit anti-H1S/T18ph (Abcam, 1: 1000), rabbit anti-PAF1 and anti-LEO1 (Reinberg lab, 1:1000), Rabbit anti-H1S27ph (Sigma 1:1000), anti-H1S35ph (genetex, 1:1000).

Techniques: Western Blot

Journal: Molecular cell

Article Title: Phospho-H1 decorates the inter-chromatid axis and is evicted along with Shugoshin by SET during mitosis

doi: 10.1016/j.molcel.2017.07.008

Figure Lengend Snippet: (A) (Left) Schematic of CRISPR-Cas9 mediated SET knockout (SET KO) in RPE1 cells. (Right) Western blot of whole-cell lysate confirming the knockout. (B) Growth curve of control and SET KO cells indicating severe growth-impairment upon SET KO. Number of replicates = 3 for each time point. *** = p <0.0001, two-way ANOVA test. (C) (Left) Panels from live-cell imaging of control and SET KO cells expressing H2B-RFP. Mitoses marked with asterisks are zoomed on the insets. Scale bar = 10 µm. SET KO cells display a significant delay in prophase-metaphase transition and increased frequencies of abnormal mitoses (quantification shown on right), including the presence of lagging chromosomes (arrowheads on SET KO panel- 80 min) and occurrence of micronuclei (arrows on SET KO panels −100 min and 110 min). (D) IF analysis at various mitotic stages showing that SET KO results in SGOL2 and SGOL1 (Figure S4C), persistence beyond anaphase into telophase and interphase. Scale bar = 5 µm. Quantification is shown on the right. For all quantification panels in (C) and (D): ***=p<0.0001, Fisher’s exact test, N numbers are indicated on the y-axis. (See also Figure S4, movies S1 and S2).

Article Snippet: METHOD DETAILS Antibodies Antibodies for western blots were used at the following dilutions: rabbit anti-SET and goat anti-SET (Santa Cruz, 1:1000), rabbit anti-SGOL2 (Bethyl, 1:1000), mouse anti-SGOL1 (Abcam, 1:1000), rabbit anti-PP2A-B (Cell Signaling, 1:1000), rabbit anti-PP2A-B’ (Bethyl, 1:1000), mouse anti-H1 (Santa Cruz, 1:2500), rabbit anti-H3 and anti-H4 (Abcam, 1:5000), mouse anti-H3S10ph (Millipore, 1:1000), mouse anti-GAPDH (Genetex, 1:5000), rabbit anti-REPIN1 (Sigma, 1:1000), mouse anti-MPM2 (Millipore, 1:1000), rabbit anti-H1S/T18ph (Abcam, 1: 1000), rabbit anti-PAF1 and anti-LEO1 (Reinberg lab, 1:1000), Rabbit anti-H1S27ph (Sigma 1:1000), anti-H1S35ph (genetex, 1:1000).

Techniques: CRISPR, Knock-Out, Western Blot, Live Cell Imaging, Expressing