Journal: Journal of Traditional Chinese Medicine

Article Title: Tongqiao Yizhi granule (通窍益智颗粒) repress the nuclear factor kappa-b/nucleotide oligomerization domain-like receptors 3/caspase-1 pyroptosis pathway in the hippocampus to counter vascular dementia in rats

doi: 10.19852/j.cnki.jtcm.20240515.005

Figure Lengend Snippet: A: expression of caspase-1 protein in rat hippocampus (× 200); A1: Sham; A2: Model; A3: NMDP; A4: TQYZKL of 6.2 g·kg-1·d-1; A5: TQYZKL of 12.4 g·kg-1·d-1; A6: TQYZKL of 24.8 g·kg-1·d-1; B: expression of GSDMD protein in rat hippocampus (× 200), B1: Sham; B2: Model; B3: NMDP; B4: TQYZKL of 6.2 g·kg-1·d-1; B5: TQYZKL of 12.4 g·kg-1·d-1; B6: TQYZKL of 24.8 g·kg-1·d-1; Dyeing method of all pictures are the immunohistochemical method; C: expression of IL-18 and IL-1β in serum of rats. Sham and Model group: treated with physiological saline; NMDP group: treated with nimodipine (6.25 mg·kg-1·d-1); 6.2, 12.4, 24.8 group: treated with TQYZKL (6.2 g·kg-1·d-1), TQYZKL (12.4 g·kg-1·d-1), TQYZKL (24.8 g·kg-1·d-1) respectively. TQYZKL: Tongqiao Yizhi granule; NF-κB: nuclear factor kappa-B; NLRP3: nucleotide oligomerization domain-like receptors 3; IHC: immunohistochemistry; ELISA: enzyme-linked immunosorbent assay; IL-18: interleukin-18; IL-1β: interleukin-1β; NMDP: nimodipine. Data are presented as mean ± standard error of the mean (n = 4). Significant differences compared with Sham group were designated as aP < 0.05, with Model group as bP < 0.05 and with NMDP group with cP < 0.05.

Article Snippet: Anti-Golgi peripheral membrane protein p65 (P65) antibody (8242, CST, Boston, USA); anti-NLRP3 antibody (AF2155, Beyotime, Shanghai, China); anti-caspase-1 antibody (22915-1-AP, Proteintech, Chicago, IL, USA); Anti-Gasdermin D (GSDMD) antibody (AF4012, Affinity Biosciences, Cincinnati, OH, USA); anti-β-actin antibody (4967S, CST, Boston, MA, USA); horseradish peroxidase-conjugated anti-rabbit secondary antibody (7074S, CST, Boston, MA, USA); reverse transcription kit (R2028, UE, Beijing, China); real-time fluorescence quantitative polymerase chain reaction (PCR) kit (S2024, UE, Beijing, China); Tunel kit (C1090, Beyotime, Shanghai, China); Hematoxylin-eosin (HE) staining kit (C0105S, Beyotime, Shanghai, China); immunohistochemical staining kit (PV-9001, Zhongshan Golden Bridge, Beijing, China); enzyme-linked immunosorbent assay (ELISA) rat Interleukin-18 (IL-18) ELISA kit (ERC010.96, eBioscience, San Diego, CA, USA); ELISA rat Interleukin-1β (IL-1β) ELISA kit (ERC007.96, eBioscience, San Diego, CA, USA).

Techniques: Expressing, Immunohistochemical staining, Saline, Immunohistochemistry, Enzyme-linked Immunosorbent Assay

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: This was followed by the immunohistochemical staining kits provided by Zhongshan Golden Bridge.

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

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).

Article Snippet: This was followed by the immunohistochemical staining kits provided by Zhongshan Golden Bridge.

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

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).

Article Snippet: This was followed by the immunohistochemical staining kits provided by Zhongshan Golden Bridge.

Techniques: Quantitative Proteomics, Control, Knockdown, Transfection, Co-Immunoprecipitation Assay, Mutagenesis

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.

Article Snippet: This was followed by the immunohistochemical staining kits provided by Zhongshan Golden Bridge.

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