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sf9 cells (BPS Bioscience)

Name: sf9 cells
Brand: BPS Bioscience
Rating: 94 (3 reviews)

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BPS Bioscience sf9 cells
Sf9 Cells, supplied by BPS Bioscience, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/sf9 cells/product/BPS Bioscience
Average 94 stars, based on 3 article reviews

sf9 cells - by Bioz Stars, 2026-03

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(A) WB analysis of PRMT5 protein levels to reveal the knockout efficiency of Prmt5 f/f and Amhr2 cre Prmt5 f/f uteri. (B) IHC staining of PRMT5 to reveal the knockout specificity of Prmt5 f/f and Amhr2 cre Prmt5 f/f uteri. Scale bar, 100 μm. (C) qRT–PCR analysis of PRMT5 mRNA levels of primary EnSC of Prmt5 f/f and Amhr2 cre Prmt5 f/f uteri to reveal the knockout efficiency. (D) Average number of implantation sites in Prmt5 f/f and Amhr2 cre Prmt5 f/f mice on day 5 (D5) of pregnancy. Scale bar, 1 cm. (E) IHC staining of COX2 to show the embryo attachment reaction in implantation sites of Prmt5 f/f and Amhr2 cre Prmt5 f/f mice on D5 of pregnancy. COX2 positive cells are circled by a black dotted line. Scale bar, 100 μm. (F) Average number of implantation sites in Prmt5 f/f and Amhr2 cre Prmt5 f/f mice on D6 of pregnancy. Scale bar, 1 cm. (G) IHC staining of HAND2 to show the EnSC decidualization of Prmt5 f/f and Amhr2 cre Prmt5 f/f mice on D6 of pregnancy. The embryo is circled by a red dotted line. Scale bar, 100 μm. (H) Representative images of D8 uteri from Prmt5 f/f and Amhr2 cre Prmt5 f/f mice. Scale bar, 1 cm. (I) Gross morphology of unstimulated or oil-stimulated uterine side and the ratio of oil-stimulated to unstimulated uterine weight from Prmt5 f/f and Amhr2 cre Prmt5 f/f mice. Scale bar, 1 cm. (J) Alkaline phosphatase (ALP) activity staining of oil-stimulated uterine side from artificial decidualization model of Prmt5 f/f and Amhr2 cre Prmt5 f/f mice. Scale bar, 100 μm. (K) WB analysis of indicated protein levels to reveal impaired sDMA modification and decidualization regulators in oil-stimulated uterine side from artificial decidualization model of Amhr2 cre Prmt5 f/f mice. LE, luminal epithelium; GE, glandular epithelium; S, stroma; Em, embryo. Mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, Student’s t test.

Journal: bioRxiv

Article Title: PRMT5 deficiency disturbs Nur77 methylation to inhibit endometrial stromal cell differentiation in recurrent implantation failure

doi: 10.1101/2024.02.06.579055

Figure Lengend Snippet: (A) WB analysis of PRMT5 protein levels to reveal the knockout efficiency of Prmt5 f/f and Amhr2 cre Prmt5 f/f uteri. (B) IHC staining of PRMT5 to reveal the knockout specificity of Prmt5 f/f and Amhr2 cre Prmt5 f/f uteri. Scale bar, 100 μm. (C) qRT–PCR analysis of PRMT5 mRNA levels of primary EnSC of Prmt5 f/f and Amhr2 cre Prmt5 f/f uteri to reveal the knockout efficiency. (D) Average number of implantation sites in Prmt5 f/f and Amhr2 cre Prmt5 f/f mice on day 5 (D5) of pregnancy. Scale bar, 1 cm. (E) IHC staining of COX2 to show the embryo attachment reaction in implantation sites of Prmt5 f/f and Amhr2 cre Prmt5 f/f mice on D5 of pregnancy. COX2 positive cells are circled by a black dotted line. Scale bar, 100 μm. (F) Average number of implantation sites in Prmt5 f/f and Amhr2 cre Prmt5 f/f mice on D6 of pregnancy. Scale bar, 1 cm. (G) IHC staining of HAND2 to show the EnSC decidualization of Prmt5 f/f and Amhr2 cre Prmt5 f/f mice on D6 of pregnancy. The embryo is circled by a red dotted line. Scale bar, 100 μm. (H) Representative images of D8 uteri from Prmt5 f/f and Amhr2 cre Prmt5 f/f mice. Scale bar, 1 cm. (I) Gross morphology of unstimulated or oil-stimulated uterine side and the ratio of oil-stimulated to unstimulated uterine weight from Prmt5 f/f and Amhr2 cre Prmt5 f/f mice. Scale bar, 1 cm. (J) Alkaline phosphatase (ALP) activity staining of oil-stimulated uterine side from artificial decidualization model of Prmt5 f/f and Amhr2 cre Prmt5 f/f mice. Scale bar, 100 μm. (K) WB analysis of indicated protein levels to reveal impaired sDMA modification and decidualization regulators in oil-stimulated uterine side from artificial decidualization model of Amhr2 cre Prmt5 f/f mice. LE, luminal epithelium; GE, glandular epithelium; S, stroma; Em, embryo. Mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, Student’s t test.

Article Snippet: To perform the experiment, 0.5µg of GST-Nur77-NT/DBD/LBD, either wild or mutant type, and recombinant PRMT5/MEP50 (Sigma-Aldrich, #SRP0146) was mixed with 0.5 µg in a buffer solution.

Techniques: Knock-Out, Immunohistochemistry, Quantitative RT-PCR, Activity Assay, Staining, Modification

(A) IHC staining of PRMT5 protein expression in proliferative endometrium (n = 24) and mid-secretory endometrium (n = 24) from normal fertile women. Scale bars, 100 μm. The integrated optical density (IOD) of each area from endometrium is analyzed using Image-Pro Plus 6.0. LE, luminal epithelium; GE, glandular epithelium; S, stroma. (B) WB analysis of PRMT5 and sDMA modified protein levels in proliferative endometrium (n = 6) and mid-secretory endometrium (n = 6) from normal fertile women. (C) WB analysis of PRMT5 protein level in human endometrial stromal cells (EnSC) treated with medroxyprogesterone acetate (MPA) and 8-bromo-cyclic adenosine monophosphate (8Br-cAMP; cAMP) for 0, 12, 24, 48 and 72 hours, respectively. (D) WB analysis of PRMT5 protein level in human EnSC infected with transfected with indicated multiplicity of infection (moi) of adenoviruses harboring shPRMT5 (Ad-shPRMT5). (E, F) qRT–PCR analysis of IGFBP1 and PRL mRNA levels of human EnSC transfected with Ad-shPRMT5 upon MPA and cAMP treatment for 3 days. (G) ELISA analysis of secreted PRL concentration in supernatant of human EnSC transfected with Ad-shPRMT5 upon MPA and cAMP treatment for 3 days. (h) IF staining of F-actin with phalloidin (green) and PRMT5 (red) in human EnSC transfected with Ad-shPRMT5 upon MPA and cAMP treatment for 3 days. Ad-Ctrl was used as the control virus. Scale bar, 50 μm. Mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. Student’s t test in (A) and (B), ANOVA with Tukey’s multiple comparisons test in (C), (E) and (F).

Journal: bioRxiv

Article Title: PRMT5 deficiency disturbs Nur77 methylation to inhibit endometrial stromal cell differentiation in recurrent implantation failure

doi: 10.1101/2024.02.06.579055

Figure Lengend Snippet: (A) IHC staining of PRMT5 protein expression in proliferative endometrium (n = 24) and mid-secretory endometrium (n = 24) from normal fertile women. Scale bars, 100 μm. The integrated optical density (IOD) of each area from endometrium is analyzed using Image-Pro Plus 6.0. LE, luminal epithelium; GE, glandular epithelium; S, stroma. (B) WB analysis of PRMT5 and sDMA modified protein levels in proliferative endometrium (n = 6) and mid-secretory endometrium (n = 6) from normal fertile women. (C) WB analysis of PRMT5 protein level in human endometrial stromal cells (EnSC) treated with medroxyprogesterone acetate (MPA) and 8-bromo-cyclic adenosine monophosphate (8Br-cAMP; cAMP) for 0, 12, 24, 48 and 72 hours, respectively. (D) WB analysis of PRMT5 protein level in human EnSC infected with transfected with indicated multiplicity of infection (moi) of adenoviruses harboring shPRMT5 (Ad-shPRMT5). (E, F) qRT–PCR analysis of IGFBP1 and PRL mRNA levels of human EnSC transfected with Ad-shPRMT5 upon MPA and cAMP treatment for 3 days. (G) ELISA analysis of secreted PRL concentration in supernatant of human EnSC transfected with Ad-shPRMT5 upon MPA and cAMP treatment for 3 days. (h) IF staining of F-actin with phalloidin (green) and PRMT5 (red) in human EnSC transfected with Ad-shPRMT5 upon MPA and cAMP treatment for 3 days. Ad-Ctrl was used as the control virus. Scale bar, 50 μm. Mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. Student’s t test in (A) and (B), ANOVA with Tukey’s multiple comparisons test in (C), (E) and (F).

Techniques: Immunohistochemistry, Expressing, Modification, Infection, Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Concentration Assay, Staining, Virus

(A) WB analysis of sDMA, aDMA and MMA modified protein levels and (B) qRT-PCR analysis of PRMT5 and PRMT9 mRNA levels in mid-secretory endometrium from infertile women with RIF (n = 6) and normal controls (n = 6). (C) qRT-PCR analysis of PRMT5 mRNA levels in mid-secretory endometrium from infertile women with RIF (n = 22) and normal controls (n = 22). (D) WB analysis and (E) immunohistochemistry staining of PRMT5 protein expression in mid-secretory endometrium from infertile women with RIF (n = 24) and normal controls (n = 24). (F) WB analysis of PRMT5 protein level in human EnSC infected with transfected with indicated moi of adenoviruses harboring PRMT5 (Ad-PRMT5). qRT–PCR analysis of (G) PRL and (H) IGFBP1 mRNA of human EnSC from infertile women with RIF and normal controls transfected with Ad-shPRMT5 upon MPA and cAMP treatment for 3 days. (I) ELISA analysis of secreted PRL concentration in supernatant of human EnSC from infertile women with RIF and normal controls transfected with Ad-shPRMT5 upon MPA and cAMP treatment for 3 days and 6 days. Ad-LacZ was used as the control virus. Scale bar, 100 μm. The integrated optical density (IOD) of each area from endometrium is analyzed using Image-Pro Plus 6.0. LE, luminal epithelium; GE, glandular epithelium; S, stroma. Mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, Student’s t test. ANOVA with Tukey’s multiple comparisons test in (G) and (H). Two-way ANOVA with the Bonferroni multiple comparisons test in (J).

Journal: bioRxiv

Article Title: PRMT5 deficiency disturbs Nur77 methylation to inhibit endometrial stromal cell differentiation in recurrent implantation failure

doi: 10.1101/2024.02.06.579055

Figure Lengend Snippet: (A) WB analysis of sDMA, aDMA and MMA modified protein levels and (B) qRT-PCR analysis of PRMT5 and PRMT9 mRNA levels in mid-secretory endometrium from infertile women with RIF (n = 6) and normal controls (n = 6). (C) qRT-PCR analysis of PRMT5 mRNA levels in mid-secretory endometrium from infertile women with RIF (n = 22) and normal controls (n = 22). (D) WB analysis and (E) immunohistochemistry staining of PRMT5 protein expression in mid-secretory endometrium from infertile women with RIF (n = 24) and normal controls (n = 24). (F) WB analysis of PRMT5 protein level in human EnSC infected with transfected with indicated moi of adenoviruses harboring PRMT5 (Ad-PRMT5). qRT–PCR analysis of (G) PRL and (H) IGFBP1 mRNA of human EnSC from infertile women with RIF and normal controls transfected with Ad-shPRMT5 upon MPA and cAMP treatment for 3 days. (I) ELISA analysis of secreted PRL concentration in supernatant of human EnSC from infertile women with RIF and normal controls transfected with Ad-shPRMT5 upon MPA and cAMP treatment for 3 days and 6 days. Ad-LacZ was used as the control virus. Scale bar, 100 μm. The integrated optical density (IOD) of each area from endometrium is analyzed using Image-Pro Plus 6.0. LE, luminal epithelium; GE, glandular epithelium; S, stroma. Mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, Student’s t test. ANOVA with Tukey’s multiple comparisons test in (G) and (H). Two-way ANOVA with the Bonferroni multiple comparisons test in (J).

Techniques: Modification, Quantitative RT-PCR, Immunohistochemistry, Staining, Expressing, Infection, Transfection, Enzyme-linked Immunosorbent Assay, Concentration Assay, Virus

(A) Silver staining of proteins immunoprecipitated by IgG antibody and PRMT5 antibody using lysates of human EnSC treated with MPA and cAMP for 3 days. (B) Venn plot shows 104 proteins identified with confidence in the PRMT5 antibody group. (B) Co-IP and WB analysis of the interaction of exogenous OFP-PRMT5 with Flag-NR4A2 or Flag-NR4A3 in HEK293T cells. (D) Co-IP and WB analysis of the interaction of exogenous OFP-PRMT5 with Myc-Nur77 in HEK293T cells. (E) IF staining array for the localization of endogenous Nur77 (green) and PRMT5 (red) in human EnSC treated with MAP and cAMP. (F) Co-IP and WB analysis of the interaction of endogenous Nur77 and PRMT5 human EnSC treated with MAP and cAMP. (G) WB analysis of the expression level of Nur77 and PRMT5 in human EnSC transfected with Ad-siNur77 and Ad-PRMT5. (H) IF staining of F-actin with phalloidin (green) and PRMT5 (red) in human EnSC transfected with Ad-siNur77 and Ad-PRMT5 with MPA and cAMP treatment. (I, J) qRT–PCR analysis of PRL and IGFBP1 mRNA and (K) ELISA analysis of secreted PRL concentration of supernatant in human EnSC transfected with Ad-shPRMT5 and Ad-Flag-Nur77 with MPA and cAMP treatment. Ad-Ctrl and Ad-LacZ were used as the control virus. Scale bars, 50 μm. Mean ± SEM. * P < 0.05, ** P < 0.01. ANOVA with Tukey’s multiple comparisons test.

Journal: bioRxiv

Article Title: PRMT5 deficiency disturbs Nur77 methylation to inhibit endometrial stromal cell differentiation in recurrent implantation failure

doi: 10.1101/2024.02.06.579055

Figure Lengend Snippet: (A) Silver staining of proteins immunoprecipitated by IgG antibody and PRMT5 antibody using lysates of human EnSC treated with MPA and cAMP for 3 days. (B) Venn plot shows 104 proteins identified with confidence in the PRMT5 antibody group. (B) Co-IP and WB analysis of the interaction of exogenous OFP-PRMT5 with Flag-NR4A2 or Flag-NR4A3 in HEK293T cells. (D) Co-IP and WB analysis of the interaction of exogenous OFP-PRMT5 with Myc-Nur77 in HEK293T cells. (E) IF staining array for the localization of endogenous Nur77 (green) and PRMT5 (red) in human EnSC treated with MAP and cAMP. (F) Co-IP and WB analysis of the interaction of endogenous Nur77 and PRMT5 human EnSC treated with MAP and cAMP. (G) WB analysis of the expression level of Nur77 and PRMT5 in human EnSC transfected with Ad-siNur77 and Ad-PRMT5. (H) IF staining of F-actin with phalloidin (green) and PRMT5 (red) in human EnSC transfected with Ad-siNur77 and Ad-PRMT5 with MPA and cAMP treatment. (I, J) qRT–PCR analysis of PRL and IGFBP1 mRNA and (K) ELISA analysis of secreted PRL concentration of supernatant in human EnSC transfected with Ad-shPRMT5 and Ad-Flag-Nur77 with MPA and cAMP treatment. Ad-Ctrl and Ad-LacZ were used as the control virus. Scale bars, 50 μm. Mean ± SEM. * P < 0.05, ** P < 0.01. ANOVA with Tukey’s multiple comparisons test.

Techniques: Silver Staining, Immunoprecipitation, Co-Immunoprecipitation Assay, Staining, Expressing, Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Concentration Assay, Virus

(A) Schematic representation of Flag-tagged human Nur77 truncation derivatives, including NT, DBD and LBD. Co-IP and WB analysis of the interaction between full-length PRMT5 and Nur77 fragments in HEK293T cell. Three domains of Nur77 interact with PRMT5. (B) Exogenous Myc-Nur77 was immunoprecipitated from HEK293T cell transfected OFP-PRMT5 plasmid by Myc antibody, and the arginine methylation status of Nur77 was examined with a sDMA antibody. (C) Electrophoretic Mobility Shift Assay of binding capacity of extracts from human EnSC transfected with Ad-shPRMT5 to cy5 labeled NBRE DNA probe, unlabeled NBRE DNA probe was used as competitor. (D) Human EnSCs transfected with Ad-Flag-Nur77 and Ad-shPRMT5 and (E) EnSCs from Prmt5 f/f and Amhr2 cre Prmt5 f/f mice were subjected to the detection of NBRE-luciferase activity. (F) Human EnSCs transfected with Ad-shPRMT5 were treated with 50 μg/mL cycloheximide (CHX) for 2, 4, and 8 hours. The cell extracts were subjected to Western blotting. The level of the remaining Nur77 was normalized to that of GAPDH and plotted relative to the level at the 0-hour time point. (G) IF staining of Nur77 and PRMT5 shows the intracellular localization of Nur77 in human EnSCs transfected with Ad-shPRMT5. (H) WB analysis of immunoprecipitated proteins, obtained from 293T cells transiently transfected with plasmids expressing Myc tagged Nur77, Nur77 mutants including R266K, R345K, R346K, and R348K. (I) Purified GST-Nur77 truncation derivatives, including NT, DBD, LBD and DBD R346K mutant, Flag-PRMT5 and Flag-MEP50 fusion protein were subjected to in vitro methylation analysis with a sDMA antibody to detect Nur77 methylation. Histone4 protein was used as the positive control. (J) Alignment of the consensus Nur77 amino acid sequences around the arginine 346 residue highlighted in red among various species. (K) NBRE-luciferase activity analysis of HEK293T exogenously expressing Myc-Nur77 and Myc-Nur77-R346K. (L) HEK293T exogenously expressing Myc-Nur77 and Myc-Nur77-R346K were treated with CHX for 2, 4, and 8 hours, then subjected to Western blotting. The level of the remaining Myc tagged protein was normalized to that of GAPDH and plotted relative to the level at the 0-hour time point. Mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. Student’s t test in E. ANOVA with Tukey’s multiple comparisons test in (D) and (K). Two-way ANOVA with the Bonferroni multiple comparisons test in (F) and (L).

Journal: bioRxiv

Article Title: PRMT5 deficiency disturbs Nur77 methylation to inhibit endometrial stromal cell differentiation in recurrent implantation failure

doi: 10.1101/2024.02.06.579055

Figure Lengend Snippet: (A) Schematic representation of Flag-tagged human Nur77 truncation derivatives, including NT, DBD and LBD. Co-IP and WB analysis of the interaction between full-length PRMT5 and Nur77 fragments in HEK293T cell. Three domains of Nur77 interact with PRMT5. (B) Exogenous Myc-Nur77 was immunoprecipitated from HEK293T cell transfected OFP-PRMT5 plasmid by Myc antibody, and the arginine methylation status of Nur77 was examined with a sDMA antibody. (C) Electrophoretic Mobility Shift Assay of binding capacity of extracts from human EnSC transfected with Ad-shPRMT5 to cy5 labeled NBRE DNA probe, unlabeled NBRE DNA probe was used as competitor. (D) Human EnSCs transfected with Ad-Flag-Nur77 and Ad-shPRMT5 and (E) EnSCs from Prmt5 f/f and Amhr2 cre Prmt5 f/f mice were subjected to the detection of NBRE-luciferase activity. (F) Human EnSCs transfected with Ad-shPRMT5 were treated with 50 μg/mL cycloheximide (CHX) for 2, 4, and 8 hours. The cell extracts were subjected to Western blotting. The level of the remaining Nur77 was normalized to that of GAPDH and plotted relative to the level at the 0-hour time point. (G) IF staining of Nur77 and PRMT5 shows the intracellular localization of Nur77 in human EnSCs transfected with Ad-shPRMT5. (H) WB analysis of immunoprecipitated proteins, obtained from 293T cells transiently transfected with plasmids expressing Myc tagged Nur77, Nur77 mutants including R266K, R345K, R346K, and R348K. (I) Purified GST-Nur77 truncation derivatives, including NT, DBD, LBD and DBD R346K mutant, Flag-PRMT5 and Flag-MEP50 fusion protein were subjected to in vitro methylation analysis with a sDMA antibody to detect Nur77 methylation. Histone4 protein was used as the positive control. (J) Alignment of the consensus Nur77 amino acid sequences around the arginine 346 residue highlighted in red among various species. (K) NBRE-luciferase activity analysis of HEK293T exogenously expressing Myc-Nur77 and Myc-Nur77-R346K. (L) HEK293T exogenously expressing Myc-Nur77 and Myc-Nur77-R346K were treated with CHX for 2, 4, and 8 hours, then subjected to Western blotting. The level of the remaining Myc tagged protein was normalized to that of GAPDH and plotted relative to the level at the 0-hour time point. Mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. Student’s t test in E. ANOVA with Tukey’s multiple comparisons test in (D) and (K). Two-way ANOVA with the Bonferroni multiple comparisons test in (F) and (L).

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Transfection, Plasmid Preparation, Methylation, Electrophoretic Mobility Shift Assay, Binding Assay, Labeling, Luciferase, Activity Assay, Western Blot, Staining, Expressing, Purification, Mutagenesis, In Vitro, Positive Control, Residue

(A) KEGG enrichment pathway analysis of differential expression genes between control and PRMT5 knockdown human EnSC treated with MPA and cAMP. (B) The “PI3K-AKT signaling” gene set was enriched in PRMT5 knockdown decidualized EnSC group according to GSEA. WB analysis of phosphorylated AKT Ser473 (pAKT S473), pNur77 S341 and pNur77 S351 in (C) Ad-shPRMT5 transfected human EnSC with treatment of MPA and 8Br-cAMP and (D) Amhr2 cre Prmt5 f/f mice uteri. (E) IHC analysis of Ki67 positive EnSC in Prmt5 f/f and Amhr2 cre Prmt5 f/f mice uteri on day 6 of pregnancy. Ki67 positive cells are circled by a black dotted line, and the embryo is circled by a red dotted line. Scale bar, 100 μm. (F) IP and WB assay for Amhr2 cre Prmt5 f/f mice uteri to reveal the relationship of Nur77-sDMA and pNur77 S351. (G) Co-IP and WB assay for HEK293T transfected with AKT1-HA and Myc-Nur77 or Myc-Nur77-346K to reveal the role of R346 mutant on the interaction between Nur77 and AKT or pAKT(S473). (H) Co-IP and WB assay for HEK293T transfected with OFP-PRMT5 and Myc-Nur77 or Myc-Nur77-346K to reveal the role of R346 mutant on the interaction between Nur77 and PRMT5. (I) Co-IP and WB assay for human EnSC transfected with Ad-Flag-Nur77 and Ad-shPRMT5 to reveal the role of PRMT5 knockdown on the interaction between Nur77 and AKT or pAKT(S473).

Journal: bioRxiv

Article Title: PRMT5 deficiency disturbs Nur77 methylation to inhibit endometrial stromal cell differentiation in recurrent implantation failure

doi: 10.1101/2024.02.06.579055

Figure Lengend Snippet: (A) KEGG enrichment pathway analysis of differential expression genes between control and PRMT5 knockdown human EnSC treated with MPA and cAMP. (B) The “PI3K-AKT signaling” gene set was enriched in PRMT5 knockdown decidualized EnSC group according to GSEA. WB analysis of phosphorylated AKT Ser473 (pAKT S473), pNur77 S341 and pNur77 S351 in (C) Ad-shPRMT5 transfected human EnSC with treatment of MPA and 8Br-cAMP and (D) Amhr2 cre Prmt5 f/f mice uteri. (E) IHC analysis of Ki67 positive EnSC in Prmt5 f/f and Amhr2 cre Prmt5 f/f mice uteri on day 6 of pregnancy. Ki67 positive cells are circled by a black dotted line, and the embryo is circled by a red dotted line. Scale bar, 100 μm. (F) IP and WB assay for Amhr2 cre Prmt5 f/f mice uteri to reveal the relationship of Nur77-sDMA and pNur77 S351. (G) Co-IP and WB assay for HEK293T transfected with AKT1-HA and Myc-Nur77 or Myc-Nur77-346K to reveal the role of R346 mutant on the interaction between Nur77 and AKT or pAKT(S473). (H) Co-IP and WB assay for HEK293T transfected with OFP-PRMT5 and Myc-Nur77 or Myc-Nur77-346K to reveal the role of R346 mutant on the interaction between Nur77 and PRMT5. (I) Co-IP and WB assay for human EnSC transfected with Ad-Flag-Nur77 and Ad-shPRMT5 to reveal the role of PRMT5 knockdown on the interaction between Nur77 and AKT or pAKT(S473).

Techniques: Expressing, Transfection, Co-Immunoprecipitation Assay, Mutagenesis

(A) WB analysis of PRMT5, pNur77(S351), pAKT(S473) and sDMA modified Nur77 and (B) IF staining of Ki67 and PRMT5 in the human EnSC cultured in 10% FBS, 2.5% FBS and 2.5% FBS with MAP+8Br-cAMP. (C) IHC staining of Ki67 and PRMT5 in human endometrium from fertile women sampled in LH+3, LH+5 and LH+9. (D) IF staining of Ki67 and PRMT5 in the human endometrium from fertile women. (E) WB analysis of PRMT5, pNur77(S351), pAKT(S473) and sDMA modified Nur77 in human endometrium from fertile women sampled in LH+3, LH+5 and LH+9. (F) IHC analysis of mid-secretory endometrial PRMT5 and pNur77(S351) protein expression in women with RIF versus normal controls. (G) WB analysis of PRMT5, pNur77(S351), pAKT(S473) and Nur77-sDMA protein levels in mid-secretory endometrium from infertile women with RIF and normal controls. Scale bars, 100 μm.

Journal: bioRxiv

Article Title: PRMT5 deficiency disturbs Nur77 methylation to inhibit endometrial stromal cell differentiation in recurrent implantation failure

doi: 10.1101/2024.02.06.579055

Figure Lengend Snippet: (A) WB analysis of PRMT5, pNur77(S351), pAKT(S473) and sDMA modified Nur77 and (B) IF staining of Ki67 and PRMT5 in the human EnSC cultured in 10% FBS, 2.5% FBS and 2.5% FBS with MAP+8Br-cAMP. (C) IHC staining of Ki67 and PRMT5 in human endometrium from fertile women sampled in LH+3, LH+5 and LH+9. (D) IF staining of Ki67 and PRMT5 in the human endometrium from fertile women. (E) WB analysis of PRMT5, pNur77(S351), pAKT(S473) and sDMA modified Nur77 in human endometrium from fertile women sampled in LH+3, LH+5 and LH+9. (F) IHC analysis of mid-secretory endometrial PRMT5 and pNur77(S351) protein expression in women with RIF versus normal controls. (G) WB analysis of PRMT5, pNur77(S351), pAKT(S473) and Nur77-sDMA protein levels in mid-secretory endometrium from infertile women with RIF and normal controls. Scale bars, 100 μm.

Techniques: Modification, Staining, Cell Culture, Immunohistochemistry, Expressing

qRT-PCR analysis of (A) IGFBP1 and (B) PRL mRNA level to reveal the role of exogenous overexpressing Nur77 on the differentiation capacity of human EnSC from RIF patients. (C) NBRE-luciferase activity analysis of human EnSC upon Cytosporone B (CsnB) treatment. qRT-PCR analysis of (D) IGFBP1 and (E) PRL mRNA level to reveal the role of CsnB on the differentiation capacity of human EnSC from RIF patients. (F) WB assay for pAKT(S374) and pNur77 (S351) in human EnSC transfected with Ad-shPRMT5 upon MK2206 treatment. (G) NBRE-luciferase activity analysis of human EnSC upon MK2206 treatment. (H) Co-IP and WB for human EnSC overexpressing Flag-Nur77 with MK2206 treatment to reveal the role of MK2206 on the interaction between Nur77 and PRMT5, and regulation of Nur77-sDMA and pNur77 S351. qRT-PCR analysis of (I) IGFBP1 and (J) PRL mRNA level to reveal the role of MK2206 or CsnB on the differentiation capacity of human EnSC with PRMT5 knockdown. (K) Schematic representation of Nur77-derived peptides including Pep1, Pep2 and Pep2-mut. (L) NBRE-luciferase activity analysis of human EnSC upon Nur77-derived peptides treatment. RT-PCR analysis of (M) IGFBP1 and (N) PRL mRNA level to reveal the role of Nur77-derived peptides on the differentiation capacity of human EnSC with MPA and 8Br-cAMP treatment. (O) WB analysis of PRMT5, pNur77(S351), pAKT(S473) and Nur77-sDMA protein levels in human EnSC treated with Nur77-derived peptide Pep2-mut. Mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. Student’s t test in C and G. ANOVA with Tukey’s multiple comparisons test in (A, B, D, E, I, J, L, M, and N).

Journal: bioRxiv

Article Title: PRMT5 deficiency disturbs Nur77 methylation to inhibit endometrial stromal cell differentiation in recurrent implantation failure

doi: 10.1101/2024.02.06.579055

Figure Lengend Snippet: qRT-PCR analysis of (A) IGFBP1 and (B) PRL mRNA level to reveal the role of exogenous overexpressing Nur77 on the differentiation capacity of human EnSC from RIF patients. (C) NBRE-luciferase activity analysis of human EnSC upon Cytosporone B (CsnB) treatment. qRT-PCR analysis of (D) IGFBP1 and (E) PRL mRNA level to reveal the role of CsnB on the differentiation capacity of human EnSC from RIF patients. (F) WB assay for pAKT(S374) and pNur77 (S351) in human EnSC transfected with Ad-shPRMT5 upon MK2206 treatment. (G) NBRE-luciferase activity analysis of human EnSC upon MK2206 treatment. (H) Co-IP and WB for human EnSC overexpressing Flag-Nur77 with MK2206 treatment to reveal the role of MK2206 on the interaction between Nur77 and PRMT5, and regulation of Nur77-sDMA and pNur77 S351. qRT-PCR analysis of (I) IGFBP1 and (J) PRL mRNA level to reveal the role of MK2206 or CsnB on the differentiation capacity of human EnSC with PRMT5 knockdown. (K) Schematic representation of Nur77-derived peptides including Pep1, Pep2 and Pep2-mut. (L) NBRE-luciferase activity analysis of human EnSC upon Nur77-derived peptides treatment. RT-PCR analysis of (M) IGFBP1 and (N) PRL mRNA level to reveal the role of Nur77-derived peptides on the differentiation capacity of human EnSC with MPA and 8Br-cAMP treatment. (O) WB analysis of PRMT5, pNur77(S351), pAKT(S473) and Nur77-sDMA protein levels in human EnSC treated with Nur77-derived peptide Pep2-mut. Mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. Student’s t test in C and G. ANOVA with Tukey’s multiple comparisons test in (A, B, D, E, I, J, L, M, and N).

Techniques: Quantitative RT-PCR, Luciferase, Activity Assay, Transfection, Co-Immunoprecipitation Assay, Derivative Assay, Reverse Transcription Polymerase Chain Reaction

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