Journal: The Journal of Biological Chemistry

Article Title: Chemical Genetic Screen Reveals a Role for Desmosomal Adhesion in Mammary Branching Morphogenesis *

doi: 10.1074/jbc.M112.411033

Figure Lengend Snippet: Primary MECs branch in the presence of FGF2. A , aggregated primary MECs grown in Matrigel with 2.5 n m EGF underwent extensive reorganization to form a fluid-filled cyst between 72 and 96 h in culture. Continued growth was observed through 144 h. B , 2.5 n m FGF2 stimulated robust branching. Aggregates initially reorganized to form a cyst, which collapsed near 72 h in culture. Extensive branching and increased growth was observed through 144 h in culture. Representative time course DIC images are shown. C , dose-response analysis for EGF- and FGF2-induced branching over 144 h. D , quantification of percentage of branching in primary MECs grown for 144 h with 2.5 n m EGF or 2.5 n m FGF2. Statistical analysis was performed using Student's t test. Results are shown as mean ± S.D. ( error bars ); n = 3; ****, p < 0.0001. E , mammary ducts are composed of an epithelial bilayer, as shown by immunofluorescence staining of a cross-section of a duct from a virgin FVB/n mouse with a luminal epithelial marker, K8 ( green ), and a myoepithelial marker, K14 ( red ). F , immunofluorescence staining for K8 and K14 in our three-dimensional primary MEC culture model with 2.5 n m EGF. G , immunofluorescence staining for K8 and K14 in our three-dimensional primary MEC culture model with 2.5 n m FGF2. E–G , interactions between luminal epithelial and myoepithelial cells were similar to those observed in vivo . Nuclei were stained with DAPI (in blue ). The dashed white line defines the lumen. A , B , and E–G , scale bar , 40 μm.

Article Snippet: Aggregates were embedded as described above in 48-well tissue culture plates (BD Biosciences) with a 5-μl Matrigel base layer and 200 aggregates suspended in 10 μl of Matrigel on top.

Techniques: Immunofluorescence, Staining, Marker, In Vivo

Journal: Journal of Innate Immunity

Article Title: C3a and Its Receptor C3aR Are Detectable in Normal Human Epidermal Keratinocytes and Are Differentially Regulated via TLR3 and LL37

doi: 10.1159/000512547

Figure Lengend Snippet: TLR3 ligand Poly I:C up-regulates C3 mRNA and protein expression in NHEKs. Intracellular generation of the biologically active fragment of C3, the C3a neo-epitope, is enhanced in parallel via TLR3 in NHEKs. NHEKs were stimulated with Poly I:C (10 μg/mL) for 24 h (C3 mRNA and C3a generation) and for different time points as indicated (C3 protein) or left unstimulated. For a, d, and e, a selective and reversible inhibitor of human CTSL was used to prove if the enhanced generation of C3a neo-epitope is dependent on the presence of CTSL. (a) C3 mRNA expression was analysed by qPCR and tgt/ref ratio was calculated by the [delta] Ct method. Phosphoglycerate kinase 1 was used as reference gene. b Cell-free supernatants were collected after different time periods, and C3 concentrations were determined by ELISA. c–e C3a was stained intracellularly with a monoclonal mouse IgG1 antibody against a specific epitope of this cleavage product of C3 called neo-epitope. The expression of C3a was analysed by flow cytometry, and MFIs were calculated. f One representative histogram of intracellular C3a expression is shown. The dotted line represents the matched isotype control, the violet line represents the ns cells, and the green line represents the Poly I:C-stimulated cells. Significant differences, as determined by the Wilcoxon matched-pairs signed rank test (a, c, d) or by Friedman-Dunn's multiple comparison test selected pairs (b), are indicated as follows: +p < 0.05; ++p < 0.01; +++p < 0.001; medians are shown in graphs. n = 12 experiments from 6 independent donors (a); n = 7 experiments from 7 independent donors (b); n = 7 experiments from 4 independent donors (c); n = 8 experiments from 5 independent donors (d); n = 5 experiments from 4 independent donors (e). NHEK, normal human epidermal keratinocyte;ns, non-stimulated; MFI, mean fluorescence intensity; CTSL, cathepsin L; inh., inhibitor; tgt/ref, target/reference; C3a, active C3 fragment.

Article Snippet: To demonstrate the mode of action of the lysosomal protease CTSL, which plays a role in generation of C3a, NHEKs were treated with a selective CTSL inhibitor (40 ng/mL) (Enzo Life Science, Exeter, UK).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry, Fluorescence

Journal: Journal of Innate Immunity

Article Title: C3a and Its Receptor C3aR Are Detectable in Normal Human Epidermal Keratinocytes and Are Differentially Regulated via TLR3 and LL37

doi: 10.1159/000512547

Figure Lengend Snippet: TLR3 ligand Poly I:C up-regulates CTSL mRNA and protein expression in NHEKs. NHEKs were stimulated with Poly I:C (10 μg/mL) for 24 h (for mRNA expression) and 24 h or 48 h (protein production) or left unstimulated. a CTSL mRNA expression was analysed by qPCR and calculated by the [delta] [delta] Ct method normalized to the ns samples. Phosphoglycerate kinase 1 was used as reference gene. b Intracellular expression of CTSL was analysed by flow cytometry, and MFIs were calculated. c One representative histogram of intracellular CTSL expression is shown. The dotted line represents the matched isotype control, the violet line represents the ns cells, and the green line represents the Poly I:C-stimulated cells. Significant differences, as determined by the Wilcoxon matched-pairs signed rank test (a) or by Friedman-Dunn's multiple comparison test selected pairs (b), are indicated as follows: +p < 0.05; medians are shown in the graphs. n = 8 experiments from 8 independent donors (a); n = 7 experiments from 4 independent donors (b). NHEK, normal human epidermal keratinocyte; ns, non-stimulated; MFI, mean fluorescence intensity; CTSL, cathepsin L.

Article Snippet: To demonstrate the mode of action of the lysosomal protease CTSL, which plays a role in generation of C3a, NHEKs were treated with a selective CTSL inhibitor (40 ng/mL) (Enzo Life Science, Exeter, UK).

Techniques: Expressing, Flow Cytometry, Fluorescence

Journal: Journal of Innate Immunity

Article Title: C3a and Its Receptor C3aR Are Detectable in Normal Human Epidermal Keratinocytes and Are Differentially Regulated via TLR3 and LL37

doi: 10.1159/000512547

Figure Lengend Snippet: Cathelicidin antimicrobial peptide LL-37 potentiates Poly I:C-induced up-regulation of C3aR, C3, and CTSL mRNA expression in NHEKs. NHEKs were stimulated with (i) Poly I:C (10 μg/mL), (ii) LL-37 (1 μM), (iii) Poly I:C (10 μg/mL) in combination with LL-37 (1 μM) for 24 h or left unstimulated. C3aR mRNA expression (a), C3 mRNA expression (b), CTSL mRNA expression (c), C3a neo-epitope generation (d). mRNA expressions were analysed by qPCR and calculated by the [delta] Ct method. tgt/ref ratios are depicted. Phosphoglycerate kinase 1 was used as reference gene. The expression of C3a was analysed by flow cytometry, and MFIs were calculated. Significant differences, as determined by the Wilcoxon matched-pairs signed rank test are indicated as follows: +p < 0.05; medians are shown in the graphs. n = 5 experiments from 4 independent donors (a), n = 7 experiments from 6 independent donors (b), n = 7 experiments from 6 independent donors (c), n = 9 experiments from 3 independent donors (d). NHEK, normal human epidermal keratinocyte; tgt/ref, target/reference ratio; ns, non-stimulated; MFI, mean fluorescence intensity; CTSL, cathepsin L; C3aR, anaphylatoxin C3a receptor; C3a, active C3 fragment.

Article Snippet: To demonstrate the mode of action of the lysosomal protease CTSL, which plays a role in generation of C3a, NHEKs were treated with a selective CTSL inhibitor (40 ng/mL) (Enzo Life Science, Exeter, UK).

Techniques: Expressing, Flow Cytometry, Fluorescence

Journal: Journal of Innate Immunity

Article Title: C3a and Its Receptor C3aR Are Detectable in Normal Human Epidermal Keratinocytes and Are Differentially Regulated via TLR3 and LL37

doi: 10.1159/000512547

Figure Lengend Snippet: C3aR, C3 the active fragment of C3, the anaphylatoxin C3a, and the protease CTSL are differentially expressed in normal human epidermis and in NHEKs cultured in low Ca2+ concentrations compared to NHEKs cultured in high Ca2+ concentrations. C3aR expression (rabbit polyclonal, Abcam, Cambridge, UK) (a), C3 expression (rabbit polyclonal, Abcam, Cambridge, UK) (b), C3a expression (monoclonal mouse IgG1, Hycult Biotech, Uden, The Netherlands) (c), and CTSL (monoclonal mouse IgG1, Abcam) (d) expression were detected in the epidermis in skin biopsies of normal human skin by immunohistochemistry. Staining with the corresponding isotypes showed no unspecific binding in the epidermis in skin biopsies of normal human skin by immunohistochemistry. m Mouse IgG1. n Rabbit polyclonal IgGs. One example for each target out of 4 different immunohistochemistry stainings of human skin is shown. NHEKs from different donors were cultured in chamber tek slides in medium containing 0.06 mM CaCL2 (low calcium conditions) and 1.36 mM CaCL2 (high calcium conditions) for 3 days and stained by immunohistochemistry. C3aR expression in high calcium conditions (e), C3aR expression in low calcium conditions (rabbit polyclonal, Abcam, Cambridge, UK) (i), C3 expression in high calcium conditions (f), C3 expression in low calcium conditions (rabbit polyclonal, Abcam, Cambridge, UK) (j), C3a expression in high calcium conditions (g), C3a expression in low calcium conditions (monoclonal mouse IgG1, Hycult Biotech, Uden, The Netherlands) (k), CTSL expression in high calcium conditions (h), CTSL expression in low calcium conditions (monoclonal mouse IgG1, Abcam) (l). Staining with the corresponding isotypes showed no unspecific binding by immunohistochemistry. o Mouse IgG1. p Rabbit polyclonal IgGs. One example for each target and condition out of 4 different immunohistochemistry stainings of NHEKs on chamber tek slides is shown. CTSL, cathepsin L; NHEK, normal human epidermal keratinocyte; C3aR, anaphylatoxin C3a receptor; C3a, active C3 fragment.

Article Snippet: To demonstrate the mode of action of the lysosomal protease CTSL, which plays a role in generation of C3a, NHEKs were treated with a selective CTSL inhibitor (40 ng/mL) (Enzo Life Science, Exeter, UK).

Techniques: Cell Culture, Expressing, Immunohistochemistry, Staining, Binding Assay

Journal: Journal of Innate Immunity

Article Title: C3a and Its Receptor C3aR Are Detectable in Normal Human Epidermal Keratinocytes and Are Differentially Regulated via TLR3 and LL37

doi: 10.1159/000512547

Figure Lengend Snippet: mRNA and protein expression of C3aR, C3/C3a neo-epitope, and protease CTSL are differentially regulated in NHEKs cultured in low Ca2+ concentrations compared to NHEKs cultured in high Ca2+ concentrations. NHEKs from different donors were cultured in medium containing 0.06 mM CaCL2 (low calcium conditions) and 1.36 mM CaCL2 (high calcium conditions) (Ca2+) for 3 days. a C3aR mRNA tgt/ref ratio was analysed. b C3 mRNA tgt/ref ratio was analysed. c CTSL mRNA tgt/ref ratio was analysed. C3aR expression (d), C3a neoepitope expression (e), and CTSL expression were measured intracellularly by flow cytometry (f). g Keratin 14 mRNA and filaggrin mRNA tgt/ref ratios were analysed. Keratin 14 is highly expressed in cells cultured in low calcium conditions when compared to filaggrin. h Filaggrin mRNA tgt/ref ratio was analysed. Filaggrin mRNA is moderately expressed in undifferentiated cells cultured in low Ca2+ concentrations and up-regulated in cells differentiated in the presence of high Ca2+ concentrations. Phosphoglycerate kinase 1 was used as reference gene. Significant differences, as determined by the Wilcoxon matched-pairs signed rank test, are indicated as follows: +p < 0.05; g medians are shown in the graph. a–c, g, hn = 6 experiments from 5 independent donors); (d–f, n = 9 experiments from 9 independent donors). NHEK, normal human epidermal keratinocyte; ns, non-stimulated; MFI, mean fluorescence intensity; CTSL, cathepsin L; C3aR, anaphylatoxin C3a receptor; tgt/ref, target/reference; C3a, active C3 fragment.

Article Snippet: To demonstrate the mode of action of the lysosomal protease CTSL, which plays a role in generation of C3a, NHEKs were treated with a selective CTSL inhibitor (40 ng/mL) (Enzo Life Science, Exeter, UK).

Techniques: Expressing, Cell Culture, Flow Cytometry, Fluorescence

Journal: Journal of Cellular and Molecular Medicine

Article Title: CCL18 promotes the metastasis of squamous cell carcinoma of the head and neck through MTDH‐NF‐κB signalling pathway

doi: 10.1111/jcmm.14168

Figure Lengend Snippet: CCL18 promotes migration and invasion of SCCHN cells in vitro. Relative expression level of PITPNM3 protein in (A) five different SCCHN cell lines and (B) an immortalized non‐malignant cell line derived from oral mucosa (DOK). C, Expression of PITPNM3 protein in SCCHN cell lines. D‐G, Effect of rhCCL18 on migration (D and F) and invasion (E and G) of SCCHN cells. (* P < 0.05, ** P < 0.01, *** P < 0.001 vs vector). CCL18, chemokine (C‐C motif) ligand 18; DOK, dysplastic oral keratinocyte; rhCCL18, recombinant human CCL18; SCCHN, squamous cell carcinoma of the head and neck

Article Snippet: EMT was induced in Tu686 and 6‐10B cells by incubating them with 20 ng/mL recombinant human CCL18 (rhCCL18) protein (Abnova, Taibei, Taiwan), while FaDu cells were incubated with 40 ng/mL of rhCCL18 for 48 hours.

Techniques: Migration, In Vitro, Expressing, Derivative Assay, Plasmid Preparation, Recombinant

Journal: Journal of Cellular and Molecular Medicine

Article Title: CCL18 promotes the metastasis of squamous cell carcinoma of the head and neck through MTDH‐NF‐κB signalling pathway

doi: 10.1111/jcmm.14168

Figure Lengend Snippet: rhCCL18 promotes EMT in SCCHN cells. A, Effect of rhCCL18 on EMT markers at mRNA and (B and C) protein level (** P < 0.01, *** P < 0.001 vs vector). EMT, epithelial‐mesenchymal transition; rhCCL18, recombinant human CCL18; SCCHN, squamous cell carcinoma of the head and neck

Article Snippet: EMT was induced in Tu686 and 6‐10B cells by incubating them with 20 ng/mL recombinant human CCL18 (rhCCL18) protein (Abnova, Taibei, Taiwan), while FaDu cells were incubated with 40 ng/mL of rhCCL18 for 48 hours.

Techniques: Plasmid Preparation, Recombinant

Journal: Journal of Cellular and Molecular Medicine

Article Title: CCL18 promotes the metastasis of squamous cell carcinoma of the head and neck through MTDH‐NF‐κB signalling pathway

doi: 10.1111/jcmm.14168

Figure Lengend Snippet: rhCCL18 promotes the expression of MTDH. A, Effect of CCL18 on the expression of MTDH at mRNA and (B and C) protein level (*** P < 0.001 vs vector). CCL18, chemokine (C‐C motif) ligand 18; MTDH, metadherin; rhCCL18, recombinant human CCL18

Article Snippet: EMT was induced in Tu686 and 6‐10B cells by incubating them with 20 ng/mL recombinant human CCL18 (rhCCL18) protein (Abnova, Taibei, Taiwan), while FaDu cells were incubated with 40 ng/mL of rhCCL18 for 48 hours.

Techniques: Expressing, Plasmid Preparation, Recombinant

Journal: Journal of Cellular and Molecular Medicine

Article Title: CCL18 promotes the metastasis of squamous cell carcinoma of the head and neck through MTDH‐NF‐κB signalling pathway

doi: 10.1111/jcmm.14168

Figure Lengend Snippet: rhCCL18 promotes migration and invasion of SCCHN cells in an MTDH‐dependent manner. A and C, Migratory changes were examined and quantified by wound healing assay. B and D, Invasive ability was assayed and quantified by Transwell invasion assay. (** P < 0.01, *** P < 0.001) MTDH, metadherin; rhCCL18, recombinant human CCL18; SCCHN, squamous cell carcinoma of the head and neck

Article Snippet: EMT was induced in Tu686 and 6‐10B cells by incubating them with 20 ng/mL recombinant human CCL18 (rhCCL18) protein (Abnova, Taibei, Taiwan), while FaDu cells were incubated with 40 ng/mL of rhCCL18 for 48 hours.

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

Journal: Journal of Cellular and Molecular Medicine

Article Title: CCL18 promotes the metastasis of squamous cell carcinoma of the head and neck through MTDH‐NF‐κB signalling pathway

doi: 10.1111/jcmm.14168

Figure Lengend Snippet: Knockdown of MTDH impaired the rhCCL18‐induced EMT in SCCHN cells. A, mRNA levels and (B) protein levels. (** P < 0.01) EMT, epithelial‐mesenchymal transition; MTDH, metadherin; rhCCL18, recombinant human CCL18; SCCHN, squamous cell carcinoma of the head and neck

Article Snippet: EMT was induced in Tu686 and 6‐10B cells by incubating them with 20 ng/mL recombinant human CCL18 (rhCCL18) protein (Abnova, Taibei, Taiwan), while FaDu cells were incubated with 40 ng/mL of rhCCL18 for 48 hours.

Techniques: Knockdown, Recombinant

Journal: Journal of Cellular and Molecular Medicine

Article Title: CCL18 promotes the metastasis of squamous cell carcinoma of the head and neck through MTDH‐NF‐κB signalling pathway

doi: 10.1111/jcmm.14168

Figure Lengend Snippet: NF‐κB activation is essential for CCL18/MTDH‐mediated cell migration, invasion and EMT in SCCHN cells. A, Representative immunofluorescence images show that CCL18 induces nuclear translocation of p65. B, Expression of NF‐κB‐associated protein subsequent to rhCCL18 stimulation. C and D, rhCCL18 enhanced the EMT property featured by E‐cadherin and Vimentin expression (** P < 0.01). This effect of CCL18 is reduced by repressing NF‐κB activation in Tu686 cells. (C) mRNA levels and (D) protein levels. CCL18, chemokine (C‐C motif) ligand 18; EMT, epithelial‐mesenchymal transition; MTDH, metadherin; rhCCL18, recombinant human CCL18; SCCHN, squamous cell carcinoma of the head and neck

Article Snippet: EMT was induced in Tu686 and 6‐10B cells by incubating them with 20 ng/mL recombinant human CCL18 (rhCCL18) protein (Abnova, Taibei, Taiwan), while FaDu cells were incubated with 40 ng/mL of rhCCL18 for 48 hours.

Techniques: Activation Assay, Migration, Immunofluorescence, Translocation Assay, Expressing, Recombinant