Journal: Nature communications

Article Title: Presenilin 2 N141I mutation induces hyperactive immune response through the epigenetic repression of REV-ERBα.

doi: 10.1038/s41467-022-29653-2

Figure Lengend Snippet: Fig. 10 Chlorpromazine recovers REV-ERBα level by rectifying hypermethylation of the Nr1d1 promoter through the inhibition of PI3K/AKT1 pathway in Psen2 N141I KI/+ microglia. a Methylation-specific PCR analysis on Nr1d1 promoter of KI/+ microglia after CPZ (0.5 μM) treatment for 12 h. 5-Aza (10 μM) as a positive control was treated for 24 h. Genomic DNA extracted from microglia was conversed using bisulfite administration. The agarose gel image is representative of three independent experiments with similar results. b Comparison of relative mRNA expression levels of DNA (de)methylases between WT and KI/+ primary microglia (n = 3) (WT vs KI/+: #p = 0.0161, #p = 0.0447, and #p = 0.0459; unpaired t-test). c, d Relative Dnmt3a mRNA (c; n = 3) and protein levels (d; n = 3) after CPZ treatment for 12 h in WT and KI/+ microglia (untreated WT vs untreated KI/+: ##p = 0.0013 and ##p = 0.0083; untreated KI/+ vs CPZ-treated KI/+: †††p < 0.0001; one-way ANOVA). e Western blotting analyses of the phosphorylation levels of AKT1 at S473, MAPK1/3 at T202/Y204, and GSK3β at S9 in WT and KI/+ microglia. f CPZ inhibits PI3K/AKT pathway, but not MAPK1/3 or GSK3β. Cells were analyzed after CPZ (0.5 μM) treatment for 12 h. g LY (LY294002, 20 μM) inhibits PI3K/AKT pathway and downregulates DNMT3A leading to the restoration of REV-ERBα levels. The blots shown are representative of three experiments with similar results. h Relative Nr1d1 mRNA expression levels after LY treatment for 12 h in WT and KI/+ microglia (n = 4). mRNA levels were normalized to 18s rRNA (b, c, h). i Schematic diagram suggesting hyperactive immune responses caused by the Psen2 N141I mutation. The mutation activates PI3K/AKT pathway and upregulates DNMTs, which induces the epigenetic suppression of REV-ERBα and hyperactive immune response in innate immune cells. An antipsychotic, CPZ, rescues REV-ERBα expression by DNA demethylation through the inhibition of PI3K/AKT1 pathway, and prevents hyperimmunity and cognitive decline in Psen2N141I/+ mice. Data are mean ± SEM. †††p < 0.001 vs untreated KI/+ control. #p < 0.05, ##p < 0.01, and ###p < 0.001 for the indicated comparisons. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used: horseradish peroxidase–conjugated β-Actin (sc47778; 1:5000) from Santa Cruz Biotechnology; PSEN1 (5643; 1:1000), REV-ERBα (13418; 1:1000), DNMT3A (3598; 1:1000), AKT1 (2938; 1:2000), phosphorylated AKT1 (S473, 9271; 1:1000), MAPK1/3 (4969; 1:1000), phosphorylated MAPK1/3 (T203/Y205, 4370; 1:2000), GSK3β (9315; 1:1000) and phosphorylated GSK3β (9336; 1:1000) from Cell Signaling Technology; IBA-1 (019-19741; 1:250) from Wako Chemicals; N-cadherin (610920; 1:1000) from BD Bioscience; PSEN2 (ab51249; 1:2000) from Abcam; REV-ERBα (PA5-29865; 1:1000) from Thermo Fisher Scientific.

Techniques: Inhibition, Methylation, Positive Control, Agarose Gel Electrophoresis, Comparison, Expressing, Western Blot, Phospho-proteomics, Mutagenesis, Control

Journal: Brain

Article Title: Efficacy of MEDI0618, a pH-dependent monoclonal antibody targeting PAR2, in preclinical models of migraine

doi: 10.1093/brain/awae344

Figure Lengend Snippet: Specificity and potency of anti-PAR2 monoclonal antibody MEDI0618 . ( A ) Live staining of PAR2-expressing (1321N1-hPAR2.cl8) or non-expressing (1321N1 parental) cell lines with MEDI0618 directly conjugated to Alexa Fluor 647. Scale bar = 20 µm. ( B ) Flow cytometry of hPAR2 overexpressing cells (1321N1-hPAR2.cl8) or non-expressing cells (1321N1 parental cell line) or A549 cells endogenously expressing hPAR2 live labelled with MEDI0618. ( C and D ) Calcium imaging from 1321N1-hPAR2 cells pretreated with MEDI0618 hIgG or an isotype control protein. ( C ) Exemplar raw calcium trace from a single well pretreated with MEDI0618 or isotype control antibody both at 1 nM, followed by PAR2 agonist stimulation with matriptase (10 nM). ( D ) Antibody titration of the anti-PAR2 antibodies MEDI0618 or PAR650097 or an isotype control antibody. Data show the matriptase (10 nM)-mediated calcium signal after preincubation with antibody and normalized to the matriptase response in the absence of antibody treatment. ( E ) Calcium imaging from A549 cells pretreated with MEDI0618, isotype control protein or PAR1 inhibitors (ATAP2 + WEDE15 mAbs) followed by 10 nM thrombin (PAR1 agonist) stimulation. Data are presented as mean ± standard error of the mean, n = 4. The concentration of the inhibitor is shown on the x -axis. Data are normalized to the peak thrombin calcium response in the absence of inhibitor pretreatment.

Article Snippet: Recombinant human matriptase (R&D Systems) was diluted in 50 mM Tris-HCl plus 10% glycerol at a concentration of 23 μM prior to final dilution in assay buffer.

Techniques: Staining, Expressing, Flow Cytometry, Imaging, Control, Titration, Concentration Assay

Journal: Brain

Article Title: Efficacy of MEDI0618, a pH-dependent monoclonal antibody targeting PAR2, in preclinical models of migraine

doi: 10.1093/brain/awae344

Figure Lengend Snippet: PAR2 functional expression in human and mouse cells relevant to migraine . Whole well calcium imaging from primary human dural fibroblasts (HDuF), human dural microvascular endothelial (HDuMEC) and mouse brain endothelial (bEnd.3) cells. ( A , D and G ) PAR2 agonists concentration response curve in ( A ) HDuF, ( D ) HDuMEC and ( G ) bEnd.3 cells. ( B , E and F ) Effect of MEDI0618 and isotype control protein (IgG) on inhibition of matriptase-induced calcium signalling at 30 nM in ( B ) HDuF, ( E ) HDuMEC and ( H ) bEnd.3 cells. ( C , F and I ) Representative calcium imaging traces of 30 nM matriptase-evoked activity following MEDI0618 or isotype control protein preincubation in ( C ) HDuF, ( F ) HDuMEC and ( I ) bEnd.3 cells. ( J and K ) Single-cell calcium imaging from mouse trigeminal neuron cultures. ( J ) Pseudocolour images of fura-2 ratio intensity show a subset of trigeminal neurons activated by treatment with 10 µM LIGRLO in comparison to 20 mM KCl treatment. Scale bar = 20 µm. ( K ) Representation of fura-2 traces recorded from two individual neurons during acute LIGRLO (10 µM) or KCl (20 mM) treatment.

Article Snippet: Recombinant human matriptase (R&D Systems) was diluted in 50 mM Tris-HCl plus 10% glycerol at a concentration of 23 μM prior to final dilution in assay buffer.

Techniques: Functional Assay, Expressing, Imaging, Concentration Assay, Control, Inhibition, Activity Assay, Comparison

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: ST14 expression in different datasets from patients with ovarian cancer. ( A ) Oncomine analysis of the mRNA expression levels of ST14 genes in different cancers. The differences in expression levels of genes between cancer and normal tissues are concluded. The thresholds are indicated in the colored cells. P < 0.05, fold-change > 2 and gene rank = 10% were considered statistically significant. Red cells represent overexpression of the target gene in tumor tissues compared to normal tissues, while blue cells indicate downregulation of the gene. Gene rank is depicted by the color depth in the cells. ( B ) UALCAN analysis of the mRNA expression levels of ST14 genes in different cancers. ( C ) GEPIA analysis of the mRNA expression levels of ST14 genes in different cancers. ( D ) ST14 DNA copy numbers based on chips for ovarian cancer research in TCGA Ovary. * P < 0.05. ( E ) Levels of ST14 mRNA in ovarian cancer based on research in the GEPIA websites (red for tumor, black for normal). The boxplot analysis showed the expression level by log2 (TPM + 1) on a log-scale. * P < 0.05. ( F ) Correlation between ST14 and TMEFF1 expression in ovarian cancer based on the GEPIA website. R = 0.32, *** P < 0.001. ( G ) The expression of ST14 and TMEFF1 in ovarian malignant tumor tissues and normal tissues detected by western blot. ( H ) Quantification of TMEFF1 normalized to GAPDH. Data are presented as the mean ± SEM ( n = 3 per group). * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: The working concentrations of ST14 and TMEFF1 primary antibodies were 1:300 (Proteintech, rabbit, catalog number 27176-1-AP) and 1:200 (Santa Cruz, mouse, catalog number 393,457), respectively.

Techniques: Expressing, Over Expression, Western Blot

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: Expression and co-localization of ST14 and TMEFF1 in different ovarian tissues. ( A ) Immunohistochemical staining of ovarian malignant tumors (i, v), borderline tumors (ii, vi), benign tumors (iii, vii), and normal ovarian tissues (vi, viii). ST14 (i-iv) and TMEFF1 (v-viii) staining is shown (original magnification, ×400). ( B ) Dual-labeled immunofluorescence technology was used to detect the co-localization of ST14 and TMEFF1 in different ovarian tissues. Blue represents the nucleus, red represents ST14, green represents TMEFF1, orange represents the co-localization of ST14 and TMEFF1 (original magnification, ×400). Pearson’s correlation coefficient (Rr) and Manders’ overlap coefficient (R) of the co-localization images: malignant tumors (Rr:0.64, R:0.87), borderline tumors (Rr:0.76,R:0.79), benign tumors (Rr:0.63,R:0.68), and normal ovarian tissues (Rr:0.76,R:0.92)

Article Snippet: The working concentrations of ST14 and TMEFF1 primary antibodies were 1:300 (Proteintech, rabbit, catalog number 27176-1-AP) and 1:200 (Santa Cruz, mouse, catalog number 393,457), respectively.

Techniques: Expressing, Immunohistochemical staining, Staining, Labeling, Immunofluorescence

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: Expression of TMEFF1 in different ovarian tissues

Article Snippet: The working concentrations of ST14 and TMEFF1 primary antibodies were 1:300 (Proteintech, rabbit, catalog number 27176-1-AP) and 1:200 (Santa Cruz, mouse, catalog number 393,457), respectively.

Techniques: Expressing

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: Association between TMEFF1 expression and pathological features in ovarian cancer

Article Snippet: The working concentrations of ST14 and TMEFF1 primary antibodies were 1:300 (Proteintech, rabbit, catalog number 27176-1-AP) and 1:200 (Santa Cruz, mouse, catalog number 393,457), respectively.

Techniques: Expressing

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: Univariate Kaplan-Meier prognostic analysis of ovarian cancer

Article Snippet: The working concentrations of ST14 and TMEFF1 primary antibodies were 1:300 (Proteintech, rabbit, catalog number 27176-1-AP) and 1:200 (Santa Cruz, mouse, catalog number 393,457), respectively.

Techniques: Biomarker Discovery

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: Univariate and multivariate Cox regression analysis of patients with ovarian cancer

Article Snippet: The working concentrations of ST14 and TMEFF1 primary antibodies were 1:300 (Proteintech, rabbit, catalog number 27176-1-AP) and 1:200 (Santa Cruz, mouse, catalog number 393,457), respectively.

Techniques: Biomarker Discovery

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: Relevance of ST14 and TMEFF1 expression in ovarian cancer

Article Snippet: The working concentrations of ST14 and TMEFF1 primary antibodies were 1:300 (Proteintech, rabbit, catalog number 27176-1-AP) and 1:200 (Santa Cruz, mouse, catalog number 393,457), respectively.

Techniques: Expressing

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: Co-localization and interaction of ST14 and TMEFF1 in ovarian cancer cells, and ST14 regulates the expression of TMEFF1. ( A ) Dual-labeled immunofluorescence technology was used to detect the co-localization of ST14 and TMEFF1 in ovarian cancer cell. Blue represents the nucleus, red represents ST14, green represents TMEFF1, and orange represents the co-localization of ST14 and TMEFF1 (original magnification, ×600). Pearson’s correlation coefficient (Rr) and Manders’ overlap coefficient (R) of the co-localization images are Rr:0.73, R:0.61. (B-C) The cell lysates of CAOV3, OVCAR3, and SKOV3 cells were immunoprecipitated with an anti-TMEFF1 antibody ( B ) and anti-ST14 antibody ( C ), and then, western blotting was performed with an anti-ST14 antibody and anti-TMEFF1 antibody. “Input” is the total cell lysate of CAOV3 cells. “IgG” is the negative control. ( D ) In the ovarian cancer cell lines CAOV3 and SKOV3, the expression of TMEFF1 decreased after knocking down the ST14 gene. ( E ) Quantification of ST14 and TMEFF1 normalized to GAPDH. Representative images and accompanying statistical plots are presented. Blank, blank control group, untreated original cells; siST14, ST14 gene knockdown group (through siRNA); NC, negative control group, negative gene (no sequence homology with ST14) knockdown group (through siRNA). Data are presented as the mean ± SEM ( n = 3 per group). * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: The working concentrations of ST14 and TMEFF1 primary antibodies were 1:300 (Proteintech, rabbit, catalog number 27176-1-AP) and 1:200 (Santa Cruz, mouse, catalog number 393,457), respectively.

Techniques: Expressing, Labeling, Immunofluorescence, Immunoprecipitation, Western Blot, Negative Control, Control, Knockdown, Sequencing

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: Interaction between ST14 and TMEFF1 promotes proliferation, invasion and migration of ovarian cancer. ( A , B , E , F ) The invasion capacities of ovarian cancer cells (CAOV3 and SKOV3) after downregulation of ST14 protein and addition of recombinant TMEFF1 active protein detected by Transwell assay. Number 1,2,3,4 respectively represents CAOV3-NC, CAOV3-siST14, CAOV3-NC + TMEFF1 and CAOV3-siST14 + TMEFF1; Number 5,6,7,8 respectively represents SKOV3-NC, SKOV3-siST14, SKOV3-NC + TMEFF1 and SKOV3-siST14 + TMEFF1. ( C , D , G , H ) The migration capacities of ovarian cancer cells (CAOV3 and SKOV3) after downregulation of ST14 protein and addition of recombinant TMEFF1 active protein detected by Wound healing assay. Number 1,2,3,4 respectively represents CAOV3-NC, CAOV3-siST14, CAOV3-NC + TMEFF1 and CAOV3-siST14 + TMEFF1; Number 5,6,7,8 respectively represents SKOV3-NC, SKOV3-siST14, SKOV3-NC + TMEFF1 and SKOV3-siST14 + TMEFF1. ( I - J ) The proliferation capacities of ovarian cancer cells (CAOV3 and SKOV3) after downregulation of ST14 protein and addition of recombinant TMEFF1 active protein detected by MTT assay. Data are presented as the mean ± SEM ( n = 3 per group). * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: The working concentrations of ST14 and TMEFF1 primary antibodies were 1:300 (Proteintech, rabbit, catalog number 27176-1-AP) and 1:200 (Santa Cruz, mouse, catalog number 393,457), respectively.

Techniques: Migration, Recombinant, Transwell Assay, Wound Healing Assay, MTT Assay