Journal: International journal of biological sciences

Article Title: SDF-1 in Mammary Fibroblasts of Bovine with Mastitis Induces EMT and Inflammatory Response of Epithelial Cells.

doi: 10.7150/ijbs.19591

Figure Lengend Snippet: Figure 3. INFs promote EMT in mammary epithelial cells. (A) Morphological changes of epithelial cells induced by NFs-condition medium (CM) and INFs-CM. The majority of epithelial cells cultured in INF-CM developed a spindle fibroblast-like morphology, while cells cultured in NF-CM or minimum serum medium (MSM, Control) maintained the typical epithelial cobblestone pattern. (B) Gene and (C) protein expression changes of α-SMA and cytokeratin in epithelial cells analyzed by RT-qPCR and western blot, respectively. GAPDH was used as internal controls. Expression of α-SMA at both mRNA and protein level were increased in epithelial cells cultured in INF-CM to a greater extent than those cultured in NF-CM or Control, while INF-CM downregulated cytokeratin expression in epithelial cells at both mRNA and protein level compared to NF-CM and Control. *P<0.01, **P<0.05 vs NF-CM and Control. The above results are given as mean ± SD, and the error bars represent the SD from three independent experiments

Article Snippet: After being blocked with 10% non-fat milk for 2 h, the membranes were probed with primary antibodies against MMP-1, collagen-1, vimentin (all 1:1000 diluted, Bioss, Beijing, China), Cytokeratin (1:1000 diluted, Sangon, Shanghai, China), p65 (1:500 diluted, Santa Cruz, Dallas, TX), p-p65 (1:500 diluted, Bioss), GAPDH (1:1000 diluted, TransGen) at 4 ̊C overnight and subsequently incubated with their corresponding secondary antibodies (1:2000 diluted, Beyotime) for 2 h at 37 ̊C.Unbound antibodies in each step were washed three times with TBST, per time ten minutes.

Techniques: Cell Culture, Control, Expressing, Quantitative RT-PCR, Western Blot

Journal: International journal of biological sciences

Article Title: SDF-1 in Mammary Fibroblasts of Bovine with Mastitis Induces EMT and Inflammatory Response of Epithelial Cells.

doi: 10.7150/ijbs.19591

Figure Lengend Snippet: Figure 6. SDF-1 is involved in INFs-induced EMT in epithelial cells through NF-κB pathway. (A) Morphological changes of epithelial cells stimulated by SDF-1 at the concentration of 0 (control), 20, 40, and 60 ng/ml. (B) Representative blots of α-SMA, vimentin and cytokeratin and (C) p-p65 and p65 in epithelial cells stimulated by SDF-1 analyzed by western blot. The quantitative analysis of each protein was relative to internal control. (D) The expression of p65 in cytoplasm and nucleus of epithelial with or without SDF-1 stimulation analyzed by western blot. GAPDH and Histone H3 served as internal controls for cytoplasm and nucleus, respectively. *P<0.01, **P<0.05 vs control.

Article Snippet: After being blocked with 10% non-fat milk for 2 h, the membranes were probed with primary antibodies against MMP-1, collagen-1, vimentin (all 1:1000 diluted, Bioss, Beijing, China), Cytokeratin (1:1000 diluted, Sangon, Shanghai, China), p65 (1:500 diluted, Santa Cruz, Dallas, TX), p-p65 (1:500 diluted, Bioss), GAPDH (1:1000 diluted, TransGen) at 4 ̊C overnight and subsequently incubated with their corresponding secondary antibodies (1:2000 diluted, Beyotime) for 2 h at 37 ̊C.Unbound antibodies in each step were washed three times with TBST, per time ten minutes.

Techniques: Concentration Assay, Control, Western Blot, Expressing

Journal: Disease Models & Mechanisms

Article Title: Murine cell lines with defined mutations model different histological subtypes of epithelial ovarian cancer

doi: 10.1242/dmm.052177

Figure Lengend Snippet: Cell lines reproduce different ovarian cancer histotypes and have different chemokine secretion profiles. (A) Syngeneic mice were injected IP with the specified cell lines. Tumors obtained at necropsy were fixed in formalin and paraffin embedded. Four-micron sections were used for HE staining (top row), dual CD3 (brown)/CD20 (red) IHC (middle row) and chip cytometry with four fluorescent markers (WT1, red; HNF1β, blue; pan-cytokeratin, white; vimentin, green) (bottom row). Scale bars: 50 µm. (B) Heterogeneity of secreted cytokine and chemokines across 29 murine cell lines. Murine IP-10 (CXCL10) and eotaxin were measured in cell culture supernatants by Meso Scale Discovery. MCP-1 was measured by enzyme-linked immunosorbent assay. Values represent average concentration (in pg/ml) of two technical replicates. Key is shown on the right. Additional measurements are shown in .

Article Snippet: The following antibodies were used to stain the prepared tissue chips: anti-HNF1B polyclonal antibody (Proteintech, 12533-1-AP, 1:1500) with PE-donkey-anti-rabbit IgG (minimal x-reactivity; BioLegend, 406421, 1:300) as the secondary antibody, BUV395 mouse anti-human MUC1 (BD Biosciences, CD227, 1:300), Alexa Fluor ® 488 anti-vimentin (BioLegend, 677809, 1:600), Alexa Fluor ® 488 anti-WT1 (Abcam, ab202635, 1:200), PE-cytokeratin, pan antibody (Novus, C-11, 1:1500) and eFluorTM 570 anti-alpha-smooth muscle actin (eBioscience, 1A4, 1:600).

Techniques: Injection, Staining, Chip Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Scientific Reports

Article Title: Immunofluorescence Tomography: High-resolution 3-D reconstruction by serial-sectioning of methacrylate embedded tissues and alignment of 2-D immunofluorescence images

doi: 10.1038/s41598-018-38232-9

Figure Lengend Snippet: BMMA plastic sections can be immuno-labelled and imaged using multiple imaging modalities, such as non-linear optics. ( A ) Histological stains hematoxylin and eosin can be used to outline basic cell and tissue structures, such as muscle and hair follicles of the eyelid. ( B ) Immunofluorescence staining of proteins (keratin 1 – red) and second-harmonic imaging of collagen fibers (white) enables probing of protein expression and extra-cellular matrix structure, with cell nuclei distribution identified by DAPI staining (blue). ( C ) Endogenously expressed fluorescent proteins, such as the histone H2B-GFP fusion protein in the H2B-GFP/K5tTA mouse, were imaged for fluorescence intensity analysis of GFP and co-localization with antibody stained proteins, such as keratin 5 (red). ( D ) BMMA plastic sections cut at 100 nm were contrasted with heavy metal staining (uranyl acetate and phospho-tungstic acid) and imaged using a JOEL 1010 transmission electron microscope. Differentiated Meibomian gland cells undergoing lipogenesis and holocrine secretion are readily visible. ( E ) Darkfield micrograph of a BMMA section of calcified mouse femur. (Scale bars: A–C 100 µm; D 2 µm; E 100 µm).

Article Snippet: Primary antibodies were used at a 1/1000 concentration and included mouse anti-αSMA (Sigma Aldrich – a2547), monoclonal rabbit anti- keratin 5 (Abcam ab52635), keratin 6 (Abcam ab93279) and keratin 8 (Abcam ab53280), polyclonal rabbit anti- keratin 1 (Abcam ab185628), and Ki67 (Abcam ab15580).

Techniques: Imaging, Immunofluorescence, Staining, Expressing, Fluorescence, Transmission Assay, Microscopy

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Identification of Tengfu Jiangya Tablet Target Biomarkers with Quantitative Proteomic Technique

doi: 10.1155/2017/7594805

Figure Lengend Snippet: The molecular function, biological process, and expression levels of 20 candidate serum proteins quantified by iTRAQ technique.

Article Snippet: Human Retinol binding protein 4 (RBP4), Kininogen 1 (KNG1), Keratin 1 (KRT1) ELISA kit (Cusabio Biotech, Wuhan, Hubei, China), Human Serum amyloid protein A (SAA) ELISA kit (Abcam, Cambridge, MA, USA), and Human Myeloperoxidase (MPO) ELISA kit (Uscn Life Science, Wuhan, Hubei, China) were applied to determine the concentration of proteins in each serum sample in validation set (30 samples in EH group and 30 samples in TJT treated group).

Techniques: Expressing, Multiplex sample analysis, Binding Assay, Chemotaxis Assay, Activity Assay, Antioxidant Activity Assay, Protein Binding, Transduction, Activation Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Identification of Tengfu Jiangya Tablet Target Biomarkers with Quantitative Proteomic Technique

doi: 10.1155/2017/7594805

Figure Lengend Snippet: Validation of Kininogen-1, Keratin 1, Myeloperoxidase, Retinol binding protein 4, Serum amyloid A protein, and bradykinin in serum. (a) Levels of these candidate biomarkers and downstream substance were measured by ELISA in serum of EH patients ( n = 30) and TJT treated patients ( n = 30). p values were calculated with ANOVA test ( ∗ p = 0.000). (b) With Western Blot results, the expression of Kininogen 1 and Retinol binding protein 4 was significantly differentiated between two groups. Transferrin was applied as loading control.

Article Snippet: Human Retinol binding protein 4 (RBP4), Kininogen 1 (KNG1), Keratin 1 (KRT1) ELISA kit (Cusabio Biotech, Wuhan, Hubei, China), Human Serum amyloid protein A (SAA) ELISA kit (Abcam, Cambridge, MA, USA), and Human Myeloperoxidase (MPO) ELISA kit (Uscn Life Science, Wuhan, Hubei, China) were applied to determine the concentration of proteins in each serum sample in validation set (30 samples in EH group and 30 samples in TJT treated group).

Techniques: Biomarker Discovery, Binding Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Control

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Identification of Tengfu Jiangya Tablet Target Biomarkers with Quantitative Proteomic Technique

doi: 10.1155/2017/7594805

Figure Lengend Snippet: This map directly summarized the pathway of 5 target proteins (green ovals). The arrowheads represented variation trends of target protein after TJT treatment. KNG1, KRT1, and MPO were involved in bradykinin production; meanwhile MPO and RBP4 played vital role in vascular oxidative and inflammatory injury. SAA was an acute-phase marker for vascular injury.

Article Snippet: Human Retinol binding protein 4 (RBP4), Kininogen 1 (KNG1), Keratin 1 (KRT1) ELISA kit (Cusabio Biotech, Wuhan, Hubei, China), Human Serum amyloid protein A (SAA) ELISA kit (Abcam, Cambridge, MA, USA), and Human Myeloperoxidase (MPO) ELISA kit (Uscn Life Science, Wuhan, Hubei, China) were applied to determine the concentration of proteins in each serum sample in validation set (30 samples in EH group and 30 samples in TJT treated group).

Techniques: Marker

Journal: International Journal of Biological Sciences

Article Title: Stagewise keratinocyte differentiation from human embryonic stem cells by defined signal transduction modulators

doi: 10.7150/ijbs.44414

Figure Lengend Snippet: Early epidermal cell fate determination under the influence of multiple signaling pathways in hESCs. See also . A. Stagewise method for keratinocyte differentiation. B. The time course of epidermal differentiation under TGFβ inhibition. hESCs were treated with SB431542 (SB) in differentiation medium (See Materials and Methods), and the cells were harvested at specific time points for analysis of gene expression by RT-qPCR. The results were normalized to GAPDH. C. The emergence of TP63 under pulse treatment of TGFβ inhibitor. hESCs were treated with SB431542 (SB) for specified periods in differentiation medium followed by differentiation medium only until day 6. Cells were collected on day 6 for analysis of gene expression by RT-qPCR. The results were normalized to GAPDH. D. BMP4 promotes TP63 expression under TGF-β inhibition. hESCs were treated with SB431542 (SB) in differentiation medium, and BMP4 was added from day 1 till day 6 when cells were harvested for gene expression analysis by RT-qPCR. The results were normalized to hESC; Data are presented as the mean±SD of three independent experiments. *, p < 0.05. E. The impact of WNT pathway modulation on epidermal cell fate determination. hESCs were treated with SB431542 (D0-6) and BMP4 (D1-6) in differentiation medium, and CHIR99021(CHIR, 5 µM) or IWR-1 (1 μM) was added from day 1 to day 6. Cells were then harvested for gene expression analysis by RT-qPCR. The results were normalized to hESC; Data are presented as the mean±SD of three independent experiments. *, p < 0.05. ns, not significant.

Article Snippet: The following primary antibodies were used: anti-TP63 antibody (BA1887, Boster); Cytokeratin 14 (sc-58724, Santa Cruz); Cytokeratin 1 (sc-65999, Santa Cruz); Cytokeratin 10 (sc-23877, Santa Cruz); Involucrin (sc-21748, Santa Cruz); Filaggrin (sc-30229, Santa Cruz); KRT18 (sc-6259, Santa Cruz).

Techniques: Protein-Protein interactions, Inhibition, Gene Expression, Quantitative RT-PCR, Expressing

Journal: International Journal of Biological Sciences

Article Title: Stagewise keratinocyte differentiation from human embryonic stem cells by defined signal transduction modulators

doi: 10.7150/ijbs.44414

Figure Lengend Snippet: Optimization of epidermal cell fate determination at early stage. See also . A. The time course of primitive ectoderm induction. Cells were treated with SB431542 single treatment for 0, 1, 2 or 3 days in differentiation medium, followed by combination treatment with SB431542, BMP4 and CHIR99021 until day 6, and collected on day 6 for TP63 gene expression analysis by RT-qPCR. The results were normalized to hESC; Data are presented as the mean±SD of three independent experiments. *, p < 0.05. B. The impact of prolonged exposure to TGFβ inhibitor. H1 cells were treated with TGFβ inhibitor SB431542 (SB) for 2, 4 or 7 days in the presence of BMP4 (Day 1 to 7) and CHIR99021 (CHIR, Day 1 to 5) in differentiation medium, and gene expression was analyzed on day 7 by RT-qPCR. The results were normalized to control (no SB4315432 treatment); Data are presented as the mean±SD of three independent experiments. *, p < 0.05. C. The effect of BMP4 timing on TP63 induction. In the presence of SB431542 (SB, Day 0-6) and CHIR99021 (CHIR, Day 1-5), BMP4 was added for specific time periods, and gene expression was analyzed on day 7 by RT-qPCR. The results were normalized to control (no BMP4 treatment); Data are presented as the mean±SD of three independent experiments. ns, not significant (one-way ANOVA with post hoc multiple comparisons). D. The effect of WNT activation timing on epidermal differentiation. WNT activator CHIR99021 (CHIR) was added in the presence of SB431542 (SB, Day 0-6) and BMP4 (Day 1-7) for specified periods of time, and gene expression was analyzed by RT-qPCR on day 7. The results were normalized to control (no CHIR treatment); Data are presented as the mean±SD of three independent experiments. *, p < 0.05. E. Impact of NOTCH inhibition during epidermal differentiation. Epidermal differentiation was carried out with SB431542 (SB, Day 0-6), CHIR99021 (CHIR, Day 1-6) and BMP4 (Day 1-6). NOTCH inhibitor DAPT was added between Day 4-6, and the gene expression was analyzed by qPCR on Day 6. The results were normalized to hESC; Data are presented as the mean±SD of three independent experiments. *, p < 0.05. F. The impact of WNT activation along with NOTCH inhibition on epidermal cell fate determination. Cells in epidermal differentiation were treated with DAPT with or without CHIR99021 (CHIR) between day 4 to 6, and the samples were collected on Day 6 for RT-qPCR. The results were normalized to hESC; Data are presented as the mean±SD of three independent experiments. *, p < 0.05.

Article Snippet: The following primary antibodies were used: anti-TP63 antibody (BA1887, Boster); Cytokeratin 14 (sc-58724, Santa Cruz); Cytokeratin 1 (sc-65999, Santa Cruz); Cytokeratin 10 (sc-23877, Santa Cruz); Involucrin (sc-21748, Santa Cruz); Filaggrin (sc-30229, Santa Cruz); KRT18 (sc-6259, Santa Cruz).

Techniques: Gene Expression, Quantitative RT-PCR, Control, Activation Assay, Inhibition

Journal: International Journal of Biological Sciences

Article Title: Stagewise keratinocyte differentiation from human embryonic stem cells by defined signal transduction modulators

doi: 10.7150/ijbs.44414

Figure Lengend Snippet: Optimization of keratinocyte maturation conditions. See also . A. BMP4 and NOTCH inhibitor (DAPT) treatments are sufficient to support keratinocyte maturation from day 6 to day 8. Beyond Day 6 of differentiation, cells were treated with or without SB431542 (SB) or CHIR99021 (CHIR) under BMP4 and NOTCH inhibitor treatment for two extra days, and the gene expression was examined by RT-qPCR. The results were normalized to hESC; Data are presented as the mean±SD of three independent experiments. *, p < 0.05. B. Immunostaining of TP63 expression on day 0 and day 8. Scale bar, 50 µm. C. The impact of calcium concentration on the expression of keratinocyte markers. After eight days of differentiation, cells were maintained in different calcium concentrations (1 mM versus 0.06 mM) between day 9 and day 11, and analyzed for gene expression. The results were normalized to hESC; Data are presented as the mean±SD of three independent experiments. *, p < 0.05. D. Immunostaining of TP63 (green) and KRT14 (red) expression on D8 and D11. Scale bar, 50 µm. White arrow, KRT14 expression. E. Flow cytometry analysis of TP63 on Day 8 and Day 11 of differentiation. Blue peak represents undifferentiated hESCs; pink peak represents keratinocyte. F. Quantification of TP63-positive cells on Day 0, Day 8 and Day 11 by flow cytometric analysis. Data shown are mean±SD of three independent experiments. *, p < 0.05.

Article Snippet: The following primary antibodies were used: anti-TP63 antibody (BA1887, Boster); Cytokeratin 14 (sc-58724, Santa Cruz); Cytokeratin 1 (sc-65999, Santa Cruz); Cytokeratin 10 (sc-23877, Santa Cruz); Involucrin (sc-21748, Santa Cruz); Filaggrin (sc-30229, Santa Cruz); KRT18 (sc-6259, Santa Cruz).

Techniques: Gene Expression, Quantitative RT-PCR, Immunostaining, Expressing, Concentration Assay, Flow Cytometry

Journal: International Journal of Biological Sciences

Article Title: Stagewise keratinocyte differentiation from human embryonic stem cells by defined signal transduction modulators

doi: 10.7150/ijbs.44414

Figure Lengend Snippet: Keratinocyte derivation procedure in defined conditions. See also . A. Schematic drawing of the optimized keratinocyte differentiation protocol. B. Cell morphology changes in the differentiation process. Scale bar,100µm. C. Stagewise keratinocyte gene expression by RT-qPCR. The results were normalized to hESC (D0); Data are representative of 3 independent experiments. D. Immunostaining of TP63 (red), KRT14 (green), KRT1 (green) and KRT10 (green) on day 24 and day 32 of differentiation. Scale bar, 50µm. E. Flow cytometry analysis of KRT14 (left panel) and KRT1 (right panel) on Day 28 of differentiation. Blue peak represents undifferentiated hESCs; pink peak represents keratinocyte. F. Keratinocyte differentiation from multiple hESC (H1, H9) and hiPSC (ND1-4, NL-1, NL-4) lines. Gene expression was analyzed on day 20 of differentiation and compared with HaCaT keratinocyte cell line, primary human foreskin keratinocytes (HFK-1, HFK-2) and undifferentiated hESCs (H1). The results were normalized to hESC (H1); Data are representative of 3 independent experiments.

Article Snippet: The following primary antibodies were used: anti-TP63 antibody (BA1887, Boster); Cytokeratin 14 (sc-58724, Santa Cruz); Cytokeratin 1 (sc-65999, Santa Cruz); Cytokeratin 10 (sc-23877, Santa Cruz); Involucrin (sc-21748, Santa Cruz); Filaggrin (sc-30229, Santa Cruz); KRT18 (sc-6259, Santa Cruz).

Techniques: Gene Expression, Quantitative RT-PCR, Immunostaining, Flow Cytometry