Journal: The Journal of Experimental Medicine

Article Title: The Cxxc1 subunit of the Trithorax complex directs epigenetic licensing of CD4 + T cell differentiation

doi: 10.1084/jem.20201690

Figure Lengend Snippet: Loss of Cxxc1 results in the enhanced production of Th1 cytokines via dysregulation of Trib3 expression in CD4 + T cells. (A) The pathways that were changed in Cxxc1-deficient Th1 cells compared with WT controls are shown. Dashed ellipses (added manually) indicate groups of similar GO terms. The node color indicates the gene set score. (B) The pathways changed in both Cxxc1-deficient Th1 cells and Th1 cells that received prolonged TCR/coreceptor stimulation are shown. (C) Representative staining profiles of phosphorylated (p)-AKT (T308), p-AKT (S473), p-p38, p-S6, and p-STAT4 in WT (with or without prolonged TCR/coreceptor stimulation) and Cxxc1-deficient Th1 cells are shown. The bar graphs show mean fluorescence intensity (MFI) with SDs from three independent experiments (**, P < 0.01). (D) Naive CD4 + T cells from the indicated mice were cultured under Th1 cell–inducing conditions with the indicated concentrations of IL-12. The cultured cells were restimulated with PMA plus ionomycin for 4 h. The intracellular staining profiles of IFNγ and T-bet were analyzed by flow cytometry. The bar graph shows the T-bet MFI with SDs from three independent experiments (**, P < 0.01). (E) The protein expression of IFNγ from WT Th1 cells, Cxxc1-deficient Th1 cells, or Th1 cells that received prolonged TCR/coreceptor stimulation. IFNγ secreted by Th1 cells stimulated with anti-TCRβ for 24 h was measured by ELISA. Data from three independent experiments are shown as the mean values and SDs (**, P < 0.01). (F) The MA plot depicts 10,298 genes in WT versus Cxxc1-deficient Th1 cells based on RNA-seq. The blue and red points indicate the Cxxc1-dependent group 4 genes that were down-regulated or up-regulated, respectively (greater than twofold) in the absence of Cxxc1. (G) A genome browser view of ChIP-seq signals in Th1 cells for 3xFlag-Cxxc1 and H3K4Me3 is shown for the Trib3 gene. Retroviral transduction of 3xFlag-Cxxc1 into Th1 cells was performed before the ChIP-seq analysis. ATAC-seq signals of naive (0 h) and differentiation process (48 h and 120 h) of WT or Cxxc1-deficient cells are also shown. (H) Flow cytometry of Cxxc1-deficient Th1 cells, which were transduced with retroviral vector encoding Trib3 . Representative profiles of p-AKT (T308), p-AKT (S473), p-p38, p-STAT4, and T-bet expression are shown. Bar graphs show data from three independent experiments with mean values and SDs (**, P < 0.01). (I) Intracellular staining profiles of IFNγ and IL-4 in the indicated cells restimulated with PMA plus ionomycin for 4 h are shown. The bar graph shows the percentage of IFNγ-positive cells from three independent experiments with mean values and SDs (**, P < 0.01). n.s., not significant.

Article Snippet: The antibodies used for cytoplasmic and cell surface staining were as follows: BV421-conjugated anti-IFNγ mAb (XMG1.2; BioLegend), allophycocyanin-conjugated anti–IL-4 mAb (11B11; BioLegend), PE-conjugated anti–IL-5 mAb (TRFK5; BioLegend), Alexa Fluor 488–conjugated anti–IL-13 mAb (eBio13A; eBioscience), allophycocyanin-conjugated anti–IL-17A mAb (TC11-18H10.1; BioLegend), Alexa Fluor 647–conjugated anti-Foxp3 mAb (MF23; BioLegend), Alexa Fluor 647–conjugated anti-GATA3 (560068; BD PharMingen), PE-conjugated anti-GATA3 (4B10; BD PharMingen), BV421-conjugated anti-CD4 mAb (GK1.5; BioLegend), FITC-conjugated anti-CD90.1 mAb (HIS51; BD PharMingen), PE-conjugated anti-CD212 mAb (114; BD PharMingen), PE-conjugated anti-CD124 mAb (mIL4R-M1; BD PharMingen), allophycocyanin-conjugated anti-CD25 mAb (3C7; BioLegend), FITC-conjugated anti-CD44 mAb (IM7; BD PharMingen), PE-conjugated anti-Akt (pT308) mAb (J1-223.371; BD PharMingen), PE-conjugated anti-Akt (pS473; M89-61; BD PharMingen), Alexa Fluor 647–conjugated anti-p38 (pT180/Y182) mAb (36/p38 [pT180/Y182]; BD PharMingen), Alexa Fluor 647–conjugated anti-Stat4 (pY693) mAb (38/p-Stat4; BD PharMingen), Alexa Fluor 647–conjugated anti-S6 (pS235/pS236) mAb (N7-548; BD PharMingen), and Alexa Fluor 647–conjugated anti-Irf4 mAb (IRF4.3E4; BioLegend).

Techniques: Expressing, Staining, Fluorescence, Cell Culture, Flow Cytometry, Enzyme-linked Immunosorbent Assay, RNA Sequencing Assay, ChIP-sequencing, Transduction, Plasmid Preparation

Journal: Virchows Archiv

Article Title: Epithelial-mesenchymal transition in undifferentiated carcinoma of the pancreas with and without osteoclast-like giant cells

doi: 10.1007/s00428-020-02889-3

Figure Lengend Snippet: A representative case of undifferentiated carcinoma of the pancreas with osteoclast-like giant cells, with an associated ductal adenocarcinoma, is shown. a Hematoxylin-eosin staining reveals an undifferentiated carcinoma with atypical cells and the presence of multinucleated osteoclast-like giant cells; neoplastic glands of the associated ductal adenocarcinoma are also evident (original magnification: × 20). b Snai2 is expressed by undifferentiated neoplastic cells, while neoplastic glands are totally negative (original magnification: × 20). c Twist1 is expressed by undifferentiated neoplastic cells, while neoplastic glands are totally negative (original magnification: × 20). d E-cadherin expression is lost by undifferentiated neoplastic cells, while it is retained by neoplastic glands (original magnification: × 20)

Article Snippet: Four micrometer (μm) formalin-fixed paraffin-embedded sections were immunostained with antibodies for E-cadherin (clone: NCH-38, 1:20 dilution, Dako/USA), Twist1 (Twist2C1a, 1:80, Santacruz/USA), and Snai2 (rabbit, 1:350, Xeptagen/Italy), as previously described [ , ].

Techniques: Staining, Expressing

Journal: Virchows Archiv

Article Title: Epithelial-mesenchymal transition in undifferentiated carcinoma of the pancreas with and without osteoclast-like giant cells

doi: 10.1007/s00428-020-02889-3

Figure Lengend Snippet: A representative case of undifferentiated carcinoma of the pancreas with osteoclast-like giant cells (without an associated ductal adenocarcinoma) is shown. a Hematoxylin-eosin staining reveals an undifferentiated carcinoma with atypical cells and the presence of multinucleated osteoclast-like giant cells (original magnification: × 20). b Snai2 is expressed by undifferentiated neoplastic cells, while multinucleated osteoclast-like giant cells are totally negative (original magnification: × 20). c Twist1 is expressed by undifferentiated neoplastic cells, multinucleated osteoclast-like giant cells are totally negative (original magnification: × 20). d E-cadherin expression is lost by undifferentiated neoplastic cells (original magnification: × 20)

Article Snippet: Four micrometer (μm) formalin-fixed paraffin-embedded sections were immunostained with antibodies for E-cadherin (clone: NCH-38, 1:20 dilution, Dako/USA), Twist1 (Twist2C1a, 1:80, Santacruz/USA), and Snai2 (rabbit, 1:350, Xeptagen/Italy), as previously described [ , ].

Techniques: Staining, Expressing

Journal: Virchows Archiv

Article Title: Epithelial-mesenchymal transition in undifferentiated carcinoma of the pancreas with and without osteoclast-like giant cells

doi: 10.1007/s00428-020-02889-3

Figure Lengend Snippet: A representative case of undifferentiated carcinoma of the pancreas is shown. a Hematoxylin-eosin staining reveals an undifferentiated carcinoma with atypical cells lacking any glandular organization. On the left, there is a thin band of normal pancreatic parenchyma (original magnification: × 20). b Snai2 is expressed by most of neoplastic cells and not expressed by normal pancreatic parenchyma (original magnification: × 20). c Twist1 is totally negative in neoplastic and normal tissues (original magnification: × 20); d E-cadherin expression is lost by neoplastic cells, with the normal pancreatic parenchyma as an internal positive control (original magnification: × 20)

Article Snippet: Four micrometer (μm) formalin-fixed paraffin-embedded sections were immunostained with antibodies for E-cadherin (clone: NCH-38, 1:20 dilution, Dako/USA), Twist1 (Twist2C1a, 1:80, Santacruz/USA), and Snai2 (rabbit, 1:350, Xeptagen/Italy), as previously described [ , ].

Techniques: Staining, Expressing, Positive Control

Journal:

Article Title: ? 3 -adrenoceptor deficiency blocks nitric oxide-dependent inhibition of myocardial contractility

doi:

Figure Lengend Snippet: Abundance of NOS3 protein and β3–/– mRNA in myocardium. (a) Western blot of NOS3 from mouse heart tissue. Equal amounts of protein extracts were resolved on agarose gels, transferred to nitrocellulose, and exposed to anti-NOS3 Ab or anti–p-38 Ab. The FVB and β3–/– mice had similar NOS3 abundance relative to p-38 MAP kinase, and NOS3 was absent in the NOS3–/– mice. (b) Representative ethidium-stained agarose gel demonstrating expression of β3-adrenoceptor mRNA in different mice strains: mRNA is expressed in both heart (H) and epididymal fat (F) of NOS3–/– and FVB control mice, but not β3–/– mice. Little or no PCR product is amplified in reactions lacking reverse transcriptase (H-), confirming minimal genomic contamination of mRNA. (c) Autoradiograph of RNase protection assay confirming the expression of β3-adrenoceptor in the myocardium of FVB and NOS3–/– mouse hearts. β3AR P, β3-adrenoceptor probe; β3AR PF, β3-adrenoceptor–protected fragment.

Article Snippet: The membranes were then incubated with either monoclonal anti-NOS3 (1:1000 dilution; Transduction Laboratories, Lexington, Kentucky, USA), monoclonal anti-actin (1:1000 dilution; Sigma-Aldrich, St. Louis, Missouri, USA), or polyclonal anti–p-38 (1:3000 dilution; Santa Cruz Biotechnology Inc., Santa Cruz, California, USA) Ab in 5% nonfat dry milk for 1 hour.

Techniques: Western Blot, Staining, Agarose Gel Electrophoresis, Expressing, Amplification, Autoradiography, Rnase Protection Assay

Journal: Journal of Pharmaceutical Analysis

Article Title: Epimedin B exhibits pigmentation by increasing tyrosinase family proteins expression, activity, and stability

doi: 10.1016/j.jpha.2023.09.006

Figure Lengend Snippet: Epimedin B (EB) increases tyrosinase family of proteins (TYRs) expression through microphthalmia-associated transcription factor (MITF)-mediated p-AKT (referred to as protein kinase B (PKB))/glycogen synthase kinase 3β (GSK3β)/β-catenin, p-p70 S6 kinase cascades, and protein 38 (p38)/mitogen-activated protein (MAP) kinase (MAPK) and extracellular regulated protein kinases (ERK)/MAPK pathways. (A) Representative images of immunofluorescence staining on primary human melanocytes (green: tyrosinase (TYR), blue: 4′,6-diamidino-2-phenylindole (DAPI); EB: 100 μM treated for 72 h), and fluorescence intensity statistics for TYR. (B) The protein expression levels of p-MITF, MITF, TYR, TYR-related protein 1 (TYRP1), dopachrome tautomerase (DCT), and melanoma antigen (Melan-A) (72 h on B16F10 cells). (C) Relative protein levels for p-MITF, MITF, TYR, TYRP1, DCT, and Melan-A expression. (D) The messenger RNA (mRNA) expression levels of TYR , TYRP1 , DCT , MITF , premelanosome protein ( PMEL ), and Melan-A ( MLANA ) on B16F10 cells. (E) The mRNA expression levels of OCA2 , SLC45A2 , and SLC24A5 on B16F10 cells. (F) The mRNA expression levels of Ap1g1 , Ap2m1 , and Ap3m1 on B16F10 cells. (G) The mRNA expression levels of Blocls1 and Blocls2 on B16F10 cells. For graphical representation, data are presented as mean ± standard error of the mean ( n ≥ 3 in each group). P < 0.05, P < 0.01, and P < 0.001 vs. control (CTRL) are considered to be significant. Representative image from 3 independent experiments is shown.

Article Snippet: The membrane was incubated with anti-p-MITF (ImmunoWay Biotechnology Company, Plano, TX, USA), anti-MITF (Abcam Trading Co., Ltd.), anti-TYR (Abcam Trading Co., Ltd.), anti-TYRP1 (Abcam Trading Co., Ltd.), anti-DCT (Abcam Trading Co., Ltd.), anti-melanoma antigen (Melan-A) (Abcam Trading Co., Ltd.), anti-Actin (Sigma-Aldrich), anti-protein kinase A (PKA) (Cell Signaling Technology, Inc., Boston, MA, USA), anti-phosphoinositide 3-kinase (PI3K) (Cell Signaling Technology, Inc.), anti-p-AKT (referred to as protein kinase B (PKB)) (Cell Signaling Technology, Inc.), anti-AKT (Cell Signaling Technology, Inc.), anti-glycogen synthase kinase 3β (GSK3β; Cell Signaling Technology, Inc.), anti-β-catenin (ImmunoWay Biotechnology Company), anti-p-p70 S6 (Cell Signaling Technology, Inc.), anti-p70 S6 (Cell Signaling Technology, Inc.), anti-p-c-Jun N-terminal kinase (JNK)/JNK (Cell Signaling Technology, Inc.), anti-p-protein 38 (p38)/p38 (Cell Signaling Technology, Inc.), anti-p-extracellular regulated protein kinases (ERK)/ERK (Cell Signaling Technology, Inc.), anti-Ras-related protein Rab-27A (Rab27a) (Cell Signaling Technology, Inc.), anti-Ras homolog family member A (RhoA) (Abcam Trading Co., Ltd.), and anti-cell division cycle 42 (CDC42) (Abcam Trading Co., Ltd.) at 4 °C overnight, and then, the membrane was probed with the corresponding secondary antibody at room temperature for 1 h. The signals were visualized using Tanon 4600SF (Tanon Technology Co., Ltd., Shanghai, China) and determined by quantitative analysis of digital images of gels using ImageJ (version 1.8.0).

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence, Control

Journal: Journal of Pharmaceutical Analysis

Article Title: Epimedin B exhibits pigmentation by increasing tyrosinase family proteins expression, activity, and stability

doi: 10.1016/j.jpha.2023.09.006

Figure Lengend Snippet: Epimedin B (EB) increases tyrosinase family of proteins (TYRs) expression through microphthalmia-associated transcription factor (MITF)-mediated p-AKT (referred to as protein kinase B (PKB))/glycogen synthase kinase 3β (GSK3β)/β-catenin, p-p70 S6 kinase cascades, and protein 38 (p38)/mitogen-activated protein (MAP) kinase (MAPK) and extracellular regulated protein kinases (ERK)/MAPK pathways. (A) Diagram of signal pathways in melanogenesis: adenylyl cyclase (AC), rat sarcoma (RAS), Raf protein kinase (Raf), and MAP kinase (MEK). (B) The protein expression levels of protein kinase A (PKA), phosphoinositide 3-kinase (PI3K), p-AKT, AKT, GSK-3β, β-catenin, p-p70 S6, p70 S6, p-c-Jun N-terminal kinase (JNK), JNK, p-p38, p38, p-ERK, and ERK (24 h on B16F10 cells). (C) Statistics for cellular cyclic AMP (cAMP) contents for 0, 0.5, 1, 3, and 6 h; PKA, PI3K, p-AKT, AKT, GSK-3β, β-catenin, p-p70 S6, p70 S6, p-JNK, JNK, p-p38, p38, p-ERK, and ERK expression. For graphical representation, data are presented as mean ± standard error of the mean ( n ≥ 3 in each group). P < 0.05, P < 0.01, and P < 0.001 vs. control (CTRL) are considered to be significant. TYRP1: TYR-related protein 1; DCT: dopachrome tautomerase.

Article Snippet: The membrane was incubated with anti-p-MITF (ImmunoWay Biotechnology Company, Plano, TX, USA), anti-MITF (Abcam Trading Co., Ltd.), anti-TYR (Abcam Trading Co., Ltd.), anti-TYRP1 (Abcam Trading Co., Ltd.), anti-DCT (Abcam Trading Co., Ltd.), anti-melanoma antigen (Melan-A) (Abcam Trading Co., Ltd.), anti-Actin (Sigma-Aldrich), anti-protein kinase A (PKA) (Cell Signaling Technology, Inc., Boston, MA, USA), anti-phosphoinositide 3-kinase (PI3K) (Cell Signaling Technology, Inc.), anti-p-AKT (referred to as protein kinase B (PKB)) (Cell Signaling Technology, Inc.), anti-AKT (Cell Signaling Technology, Inc.), anti-glycogen synthase kinase 3β (GSK3β; Cell Signaling Technology, Inc.), anti-β-catenin (ImmunoWay Biotechnology Company), anti-p-p70 S6 (Cell Signaling Technology, Inc.), anti-p70 S6 (Cell Signaling Technology, Inc.), anti-p-c-Jun N-terminal kinase (JNK)/JNK (Cell Signaling Technology, Inc.), anti-p-protein 38 (p38)/p38 (Cell Signaling Technology, Inc.), anti-p-extracellular regulated protein kinases (ERK)/ERK (Cell Signaling Technology, Inc.), anti-Ras-related protein Rab-27A (Rab27a) (Cell Signaling Technology, Inc.), anti-Ras homolog family member A (RhoA) (Abcam Trading Co., Ltd.), and anti-cell division cycle 42 (CDC42) (Abcam Trading Co., Ltd.) at 4 °C overnight, and then, the membrane was probed with the corresponding secondary antibody at room temperature for 1 h. The signals were visualized using Tanon 4600SF (Tanon Technology Co., Ltd., Shanghai, China) and determined by quantitative analysis of digital images of gels using ImageJ (version 1.8.0).

Techniques: Expressing, Control