Journal: Respiratory Research

Article Title: Fibroblast-epithelial cell interactions drive epithelial-mesenchymal transition differently in cells from normal and COPD patients

doi: 10.1186/s12931-015-0232-4

Figure Lengend Snippet: Primers for quantitative real-time RT-PCR analyses of gene transcript expression

Article Snippet: Paraffin-embedded lung sections were stained with specific antibodies for E-cadherin (a mouse anti-human E-cadherin antibody, 1:300 dilution, ab1416, Abcam, Cambridge, MA) or snail1 (1:250 dilution, a rabbit anti-Snail + Slug, ab85936, Abcam, Cambridge, MA) or N-cadherin (a rabbit polyclonal anti-human N-cadherin antibody, 1:300 dilution, ab18203, Abcam, Cambridge, MA).

Techniques: Quantitative RT-PCR

Journal: Respiratory Research

Article Title: Fibroblast-epithelial cell interactions drive epithelial-mesenchymal transition differently in cells from normal and COPD patients

doi: 10.1186/s12931-015-0232-4

Figure Lengend Snippet: Basal mRNA expression of mesenchymal markers was increased in DHBE cells. Basal mRNA expression of EMT-related markers in non-COPD (NHBE, open bar, n = 3) and COPD (DHBE, closed bar, n = 3) human bronchial epithelial cells were analyzed by real-time RT-PCR. Results were normalized to NHBE and expressed as mean (SE) of three independent experiments. Data were analysed by Student t test, * p < 0.05, compared with NHBE. ( a ) E-cadherin ( b ) N-cadherin ( c ) vimentin ( d ) Ratio of E-/N-cadherin. ns: not significant

Article Snippet: Paraffin-embedded lung sections were stained with specific antibodies for E-cadherin (a mouse anti-human E-cadherin antibody, 1:300 dilution, ab1416, Abcam, Cambridge, MA) or snail1 (1:250 dilution, a rabbit anti-Snail + Slug, ab85936, Abcam, Cambridge, MA) or N-cadherin (a rabbit polyclonal anti-human N-cadherin antibody, 1:300 dilution, ab18203, Abcam, Cambridge, MA).

Techniques: Expressing, Quantitative RT-PCR

Journal: Respiratory Research

Article Title: Fibroblast-epithelial cell interactions drive epithelial-mesenchymal transition differently in cells from normal and COPD patients

doi: 10.1186/s12931-015-0232-4

Figure Lengend Snippet: Snail1 and E-cadherin protein expression in non-COPD and COPD lungs. Immunohistochemical localization of EMT markers in the airway epithelium of non-COPD ( a , c , e ) and COPD ( b , d , f ) lungs ( n = 3). Immunostaining for E-cadherin ( a , b ) and Snail1 transcription factor ( c , d ) and N-cadherin ( e , f ). The dark brown color is indicative of strong expression (denoted by red arrows) of E-cadherin in non-COPD ( a ), Snail1 in COPD ( d ) and N-cadherin in COPD ( f ) lung. Negative control was stained with isotype-matched control antibody (data not shown). Non-COPD subjects are non smokers. aw: airway lumen

Article Snippet: Paraffin-embedded lung sections were stained with specific antibodies for E-cadherin (a mouse anti-human E-cadherin antibody, 1:300 dilution, ab1416, Abcam, Cambridge, MA) or snail1 (1:250 dilution, a rabbit anti-Snail + Slug, ab85936, Abcam, Cambridge, MA) or N-cadherin (a rabbit polyclonal anti-human N-cadherin antibody, 1:300 dilution, ab18203, Abcam, Cambridge, MA).

Techniques: Expressing, Immunohistochemical staining, Immunostaining, Negative Control, Staining

Journal: Respiratory Research

Article Title: Fibroblast-epithelial cell interactions drive epithelial-mesenchymal transition differently in cells from normal and COPD patients

doi: 10.1186/s12931-015-0232-4

Figure Lengend Snippet: mRNA expression of EMT markers in HBE cells after 3-day treatment with CM from HLF. HBE cells from non-COPD (N) or COPD patients (D) were stimulated with 100 % CM (black bars), or without CM (white bars) collected from human lung fibroblasts (HLF) derived from non-smokers (N) or COPD subjects (D) for 3 days and total RNA was extracted. The mRNA expression of EMT markers were determined by real-time RT-PCR and normalized to GAPDH mRNA expression in the same origin of HBE cells. ( a ) E-cadherin, ( b ) N-cadherin, ( c ) vimentin, and ( d ) the ratio of E-/N-cadherin were calculated. Data were analysed and expressed as mean (SE) of from six to nine independent experiments by one-way ANOVA with Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, compared between the indicated groups

Article Snippet: Paraffin-embedded lung sections were stained with specific antibodies for E-cadherin (a mouse anti-human E-cadherin antibody, 1:300 dilution, ab1416, Abcam, Cambridge, MA) or snail1 (1:250 dilution, a rabbit anti-Snail + Slug, ab85936, Abcam, Cambridge, MA) or N-cadherin (a rabbit polyclonal anti-human N-cadherin antibody, 1:300 dilution, ab18203, Abcam, Cambridge, MA).

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR

Journal: Respiratory Research

Article Title: Fibroblast-epithelial cell interactions drive epithelial-mesenchymal transition differently in cells from normal and COPD patients

doi: 10.1186/s12931-015-0232-4

Figure Lengend Snippet: Effects of CM from HLF on protein expression of EMT markers in HBE cells. HBE cells from normal subjects (N) or COPD patients (D) were lysed after stimulation with or without 100 % CM collected from human lung fibroblasts of N or D for 5 days. Total protein was prepared analysed by western blotting, using 40 μg of protein per lane. The membranes were probed with anti-E-cadherin, anti-N-cadherin, anti-vimentin, or anti-GAPDH antibodies. The intensities of the bands for target protein were normalized to the corresponding GAPDH bands for the each treated cells. ( a ) E-cadherin, ( b ) N-cadherin, ( c ) vimentin, and ( d ) the ratio of E-/N-cadherin were calculated. Data were analysed and expressed as mean (SE) of from seven to ten independent experiments by one-way ANOVA with Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, compared between the indicated groups

Article Snippet: Paraffin-embedded lung sections were stained with specific antibodies for E-cadherin (a mouse anti-human E-cadherin antibody, 1:300 dilution, ab1416, Abcam, Cambridge, MA) or snail1 (1:250 dilution, a rabbit anti-Snail + Slug, ab85936, Abcam, Cambridge, MA) or N-cadherin (a rabbit polyclonal anti-human N-cadherin antibody, 1:300 dilution, ab18203, Abcam, Cambridge, MA).

Techniques: Expressing, Western Blot

Journal: iScience

Article Title: Benzyl isothiocyanate suppresses development of thyroid carcinoma by regulating both autophagy and apoptosis pathway

doi: 10.1016/j.isci.2024.110796

Figure Lengend Snippet: Effect of BITC on EMT markers in an orthotopic model of ATC The immunohistochemical assay revealed a significant increase of N-cadherin expression in the ATC group (B) compared to Sham mice (A); this expression was reduced following treatment with BITC at doses of 10 mg/kg (D) and 30 mg/kg (E) compared to ATC group (B). BITC at the dose of 5 mg/kg did not show significant effect (C, see score panel F). At the same time the staining revelated a significant reduction of E-cadherin expression in the ATC group (H), compared to Sham mice (G); 10 mg/kg (J) and 30 mg/kg (K) BITC treatments caused an important increase of E-cadherin expression compared to ATC group (H). BITC at the dose of 5 mg/kg did not show significant effect (I, see score panel L). The data are representative of at least three independent experiments. The sections were observed and photographed at 20× magnification. Values are means ± SEM. One-way ANOVA test followed by Bonferroni post hoc test for multiple comparisons. ∗∗∗ p < 0.001 vs. Sham; # p < 0.05 vs. ATC; ## p < 0.01 vs. ATC; ### p < 0.001 vs. ATC.

Article Snippet: Immunohistochemical localization for E-Cadherin (1:100; sc-8426 Santa Cruz Biotechnology, Dallas, TX), N-Cadherin (1:100; sc-8424 Santa Cruz Biotechnology, Dallas, TX), and S100 antibodies (1:100; sc-393919 Santa Cruz Biotechnology, Dallas, TX), was made as previously described.

Techniques: Immunohistochemical staining, Expressing, Staining

Journal: iScience

Article Title: Benzyl isothiocyanate suppresses development of thyroid carcinoma by regulating both autophagy and apoptosis pathway

doi: 10.1016/j.isci.2024.110796

Figure Lengend Snippet: Effect of BITC on S100 expression in an orthotopic model of ATC (A, B, D, and E) The immunohistochemical assay revealed a significant increase of S100 expression in the ATC group (B) compared to Sham mice (A); this expression was reduced following treatment with BITC at doses of 5 mg/kg (C),10 mg/kg (D) and 30 mg/kg (E) compared to ATC group (B) (see score F). The data are representative of at least three independent experiments. The sections were observed and photographed at 20× magnification. Values are means ± SEM. One-way ANOVA test followed by Bonferroni post hoc test for multiple comparisons. ∗∗∗ p < 0.001 vs. Sham; # p < 0.05 vs. ATC.

Article Snippet: Immunohistochemical localization for E-Cadherin (1:100; sc-8426 Santa Cruz Biotechnology, Dallas, TX), N-Cadherin (1:100; sc-8424 Santa Cruz Biotechnology, Dallas, TX), and S100 antibodies (1:100; sc-393919 Santa Cruz Biotechnology, Dallas, TX), was made as previously described.

Techniques: Expressing, Immunohistochemical staining

Journal: Cancer Science

Article Title: Overexpression of Tetraspanin31 contributes to malignant potential and poor outcomes in gastric cancer

doi: 10.1111/cas.15342

Figure Lengend Snippet: Overexpression of Tetraspanin31 (TSPAN31) in gastric cancer. (A) Expression of TSPAN31 in seven gastric cancer (GC) cell lines compared with the fibroblast cell strain WI‐38. The level of TSPAN31 mRNA was determined by quantitative RT‐PCR in a panel of GC cell lines. Results shown are means ± SD (bars). Black bars represent cell lines, in which upregulation of TSPAN31 mRNA expression was observed and compared with that in WI‐38. The status of a TP53 mutation was the reported status of a TP53 mutation in the database ( http://p53.free.fr/index.html ). M, mutant TP53 ; W, WT TP 53. Note that among TP53 ‐mutated GC cell lines, KATO‐III and HGC27 cells had a p53 gene deletion and a frameshift mutation, respectively. (B) Specific immunostaining of TSPAN31 in a representative primary tumor sample. Based on this result, the intensity scores for TSPAN31 staining were determined as follows: 0, negative; 1, weak; 2, moderate; 3, strong. Kaplan–Meier plots depending on the intensity (C) and proportion (D) scores of specific immunostainings of TSPAN31. The log–rank test was used for statistical analysis; p < 0.05 was considered to be statistically significant. (E) For the scoring of TSPAN31 expression, the intensity and percentage of total cell population (proportion score; 0 < 10%, 10% ≤ 1 < 33%, 33% ≤ 2 < 66%, 66% ≤ 3 ≤ 100%) that expressed TSPAN31 were evaluated for each case. Expression of TSPAN31 was graded as high expression (intensity plus proportion scores ≥4 for tumor cells showing immunopositivity) or low expression (intensity plus proportion scores ≤3 for tumor cells showing immunopositivity). (F) Cancer‐specific survival rates of patients with GC (as determined by Kaplan–Meier plots), depending on TSPAN31 expression

Article Snippet: The abundance of mRNA was measured by quantitative PCR using a StepOnePlus PCR System (Applied Biosystems), and cycle threshold (Ct) values were calculated with StepOne Software version 2.0 (Applied Biosystems) using TaqMan Gene Expression Assays (Hs00919595_g1 for TSPAN31 , Hs00170423_m1 for E ‐ cadherin , Hs00958111_m1 for Vimentin , Hs00960489_m1 for ABCC2 ; Applied Biosystems) according to the manufacturer’s instructions.

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Mutagenesis, Immunostaining, Staining

Journal: Cancer Science

Article Title: Overexpression of Tetraspanin31 contributes to malignant potential and poor outcomes in gastric cancer

doi: 10.1111/cas.15342

Figure Lengend Snippet: Associations between clinicopathologic features and expression of Tetraspanin31 (TSPAN31) in gastric cancer patients

Article Snippet: The abundance of mRNA was measured by quantitative PCR using a StepOnePlus PCR System (Applied Biosystems), and cycle threshold (Ct) values were calculated with StepOne Software version 2.0 (Applied Biosystems) using TaqMan Gene Expression Assays (Hs00919595_g1 for TSPAN31 , Hs00170423_m1 for E ‐ cadherin , Hs00958111_m1 for Vimentin , Hs00960489_m1 for ABCC2 ; Applied Biosystems) according to the manufacturer’s instructions.

Techniques: Expressing

Journal: Cancer Science

Article Title: Overexpression of Tetraspanin31 contributes to malignant potential and poor outcomes in gastric cancer

doi: 10.1111/cas.15342

Figure Lengend Snippet: Multivariate analysis using Cox proportional hazards model for cancer‐specific survival among gastric cancer patients who received gastrectomy

Article Snippet: The abundance of mRNA was measured by quantitative PCR using a StepOnePlus PCR System (Applied Biosystems), and cycle threshold (Ct) values were calculated with StepOne Software version 2.0 (Applied Biosystems) using TaqMan Gene Expression Assays (Hs00919595_g1 for TSPAN31 , Hs00170423_m1 for E ‐ cadherin , Hs00958111_m1 for Vimentin , Hs00960489_m1 for ABCC2 ; Applied Biosystems) according to the manufacturer’s instructions.

Techniques: Expressing

Journal: Cancer Science

Article Title: Overexpression of Tetraspanin31 contributes to malignant potential and poor outcomes in gastric cancer

doi: 10.1111/cas.15342

Figure Lengend Snippet: Associations between expression of Tetraspanin31 (TSPAN31) and disease recurrence in gastric cancer patients with gastrectomy

Article Snippet: The abundance of mRNA was measured by quantitative PCR using a StepOnePlus PCR System (Applied Biosystems), and cycle threshold (Ct) values were calculated with StepOne Software version 2.0 (Applied Biosystems) using TaqMan Gene Expression Assays (Hs00919595_g1 for TSPAN31 , Hs00170423_m1 for E ‐ cadherin , Hs00958111_m1 for Vimentin , Hs00960489_m1 for ABCC2 ; Applied Biosystems) according to the manufacturer’s instructions.

Techniques: Expressing

Journal: Cancer Science

Article Title: Overexpression of Tetraspanin31 contributes to malignant potential and poor outcomes in gastric cancer

doi: 10.1111/cas.15342

Figure Lengend Snippet: Suppression of malignant activities in gastric cancer cells by knockdown of Tetraspanin31 (TSPAN31). (A) Effects of TSPAN31 knockdown on cell proliferation at the indicated times by siRNA (siRNA‐TSPAN31) compared with those of control siRNA in NUGC4 (WT TP53 ) and MKN74 (mutant TP53 ) cell lines. (B) Representative results of the population in each phase of the cell cycle in each cell line, as assessed by FACS at 72 h posttreatment with siRNA. (C) Representative results of the apoptosis assay in gastric cancer cells 72 h after treatment with siRNA. (D) TSPAN31 overexpression facilitated cell proliferation at the indicated times in MKN45 (WT TP53 ), HGC27 (mutant TP53 ), and MKN7 (mutant TP53 ) cell lines. Western blot analyses showed that overexpression of TSPAN31 induced phosphorylation activation of AKT. Expression of TSPAN31 protein was confirmed by western blotting using anti‐HaloTag Ab. (E) Reduction of tumor volumes of xenografts implanted NUGC4 cells with transfection of siRNA‐Luc ( n = 4) or siRNA‐TSPAN31 ( n = 4). Subcutaneous injection of siRNA‐Luc or siRNA‐TSPAN31 with atelocollagen around the tumor was repeated once a week for 2 weeks after tumor cell implantation. Results shown are means ± SD (bars) for quadruplicate experiments. Mann–Whitney U ‐test was used for statistical analysis. * p < 0.05

Article Snippet: The abundance of mRNA was measured by quantitative PCR using a StepOnePlus PCR System (Applied Biosystems), and cycle threshold (Ct) values were calculated with StepOne Software version 2.0 (Applied Biosystems) using TaqMan Gene Expression Assays (Hs00919595_g1 for TSPAN31 , Hs00170423_m1 for E ‐ cadherin , Hs00958111_m1 for Vimentin , Hs00960489_m1 for ABCC2 ; Applied Biosystems) according to the manufacturer’s instructions.

Techniques: Knockdown, Control, Mutagenesis, Apoptosis Assay, Over Expression, Western Blot, Phospho-proteomics, Activation Assay, Expressing, Transfection, Injection, MANN-WHITNEY

Journal: Cancer Science

Article Title: Overexpression of Tetraspanin31 contributes to malignant potential and poor outcomes in gastric cancer

doi: 10.1111/cas.15342

Figure Lengend Snippet: Molecular mechanisms by which the overexpression of Tetraspanin31 (TSPAN31) contributes to malignant potential in gastric cancer (GC) cells. (A) Knockdown of TSPAN31 by transfection with siRNA‐TSPAN31 suppressed the phosphorylation activation of AKT, induced the production of p21, suppressed the production of cyclin D1, and induced phosphorylation inactivation of retinoblastoma (Rb) in NUGC4 (WT TP53 ) and MKN74 (mutant TP5 3) cells. Moreover, knockdown of TSPAN31 also induced the cleavage of caspase 3 and poly(ADP‐ribose) polymerase (PARP) in GC cells. (B) A hypothetical model of the overexpression or activation of TSPAN31 in GC cells

Article Snippet: The abundance of mRNA was measured by quantitative PCR using a StepOnePlus PCR System (Applied Biosystems), and cycle threshold (Ct) values were calculated with StepOne Software version 2.0 (Applied Biosystems) using TaqMan Gene Expression Assays (Hs00919595_g1 for TSPAN31 , Hs00170423_m1 for E ‐ cadherin , Hs00958111_m1 for Vimentin , Hs00960489_m1 for ABCC2 ; Applied Biosystems) according to the manufacturer’s instructions.

Techniques: Over Expression, Knockdown, Transfection, Phospho-proteomics, Activation Assay, Mutagenesis

Journal: Cancer Science

Article Title: Overexpression of Tetraspanin31 contributes to malignant potential and poor outcomes in gastric cancer

doi: 10.1111/cas.15342

Figure Lengend Snippet: Suppression of migration, invasion, and epithelial–mesenchymal transition (EMT) in gastric cancer (GC) cells by knockdown of Tetraspanin31 (TSPAN31). (A) Knockdown TSPAN31 by siRNA‐TSPAN31 suppressed migration and invasion in NUGC4 (WT TP53 ) and MKN74 (mutant TP53 ) cell lines. Bottom graphs show means ± SD (bars; n = 4). Mann–Whitney U ‐test test was used for statistical analysis. p < 0.05 was considered statistically significant. (B) Identification of appropriate GC cell lines for an investigation into whether knockdown of TSPAN31 can suppress EMT. The panel mRNA expression levels of E ‐ cadherin and Vimentin were investigated, and the Vimentin / E ‐ cadherin ratio was calculated. In MKN7 cells, Vimentin mRNA expression was higher, and E ‐ cadherin mRNA expression was lower compared to other TSPAN31‐overexpressing cell lines. (C) Knockdown of TSPAN31 induced significant morphological change, increased the expression of E‐cadherin protein, and reduced the expression of Vimentin and Snail proteins

Article Snippet: The abundance of mRNA was measured by quantitative PCR using a StepOnePlus PCR System (Applied Biosystems), and cycle threshold (Ct) values were calculated with StepOne Software version 2.0 (Applied Biosystems) using TaqMan Gene Expression Assays (Hs00919595_g1 for TSPAN31 , Hs00170423_m1 for E ‐ cadherin , Hs00958111_m1 for Vimentin , Hs00960489_m1 for ABCC2 ; Applied Biosystems) according to the manufacturer’s instructions.

Techniques: Migration, Knockdown, Mutagenesis, MANN-WHITNEY, Expressing

Journal: Cancer Science

Article Title: Overexpression of Tetraspanin31 contributes to malignant potential and poor outcomes in gastric cancer

doi: 10.1111/cas.15342

Figure Lengend Snippet: Improvement in chemosensitivity by Tetraspanin31 (TSPAN31) knockdown in gastric cancer (GC) cells. (A) When treated with cisplatin (CDDP), transfection of TP53 WT NUGC4 and TP53 mutant MKN74 cells with siRNA‐TSPAN31 increased early apoptosis 48 h posttransfection compared to treatment with CDDP or transfection with control siRNA alone. (B) Knockdown of TSPAN31 suppressed ABCC2 expression in both NUGC4 (WT TP53 ) and MKN74 (mutant TP53 ) GC cells. Moreover, the distribution of TSPAN31 in GC cells was similar to that of ABCC2. (C) Knockdown of TSPAN31 induced a significant decrease in the mRNA expression level of ABCC2 . (D) Co‐immunoprecipitation (IP) showed TSPAN31 complexed with ABCC2 in NUGC4

Article Snippet: The abundance of mRNA was measured by quantitative PCR using a StepOnePlus PCR System (Applied Biosystems), and cycle threshold (Ct) values were calculated with StepOne Software version 2.0 (Applied Biosystems) using TaqMan Gene Expression Assays (Hs00919595_g1 for TSPAN31 , Hs00170423_m1 for E ‐ cadherin , Hs00958111_m1 for Vimentin , Hs00960489_m1 for ABCC2 ; Applied Biosystems) according to the manufacturer’s instructions.

Techniques: Knockdown, Transfection, Mutagenesis, Control, Expressing, Immunoprecipitation

Journal: Cell stress & chaperones

Article Title: Salidroside protects against bleomycin-induced pulmonary fibrosis: activation of Nrf2-antioxidant signaling, and inhibition of NF-κB and TGF-β1/Smad-2/-3 pathways.

doi: 10.1007/s12192-015-0654-4

Figure Lengend Snippet: Fig. 6 Salidroside blocks recombinant TGF-β1-induced EMT-like shift in AECs in vitro. Recombinant TGF-β1 (5 or 10 ng/mL) and salidroside (50 μM) were utilized to co-treat the primarily cultured rat AECs or human A549 cells for 48 h. a–d Then, these cells were subjected to immunohistochemical analysis using antibodies against E- cadherin (green) and fibronectin (red). Cell nuclei were stained with DAPI (blue). e, f Protein expression levels of E-cadherin and fibronectin were analyzed with Western blot assay. β-Actin was used as the endogenous control

Article Snippet: Both cell and tissue samples were blocked with goat serum and then incubated with rabbit polyclonal antibodies for E-cadherin (1:100 diluted for tissues and 1:200 diluted for cells; Boster, Wuhan, China) and fibronectin (1:200 diluted for cells; Boster) overnight (4 °C).

Techniques: Recombinant, In Vitro, Cell Culture, Immunohistochemical staining, Staining, Expressing, Western Blot, Control

Journal: Cell Death & Disease

Article Title: Stat3 modulates chloride channel accessory protein expression in normal and neoplastic mammary tissue

doi: 10.1038/cddis.2016.302

Figure Lengend Snippet: KIM-2 mammary epithelial cells exhibit upregulation of mCLCA2 and mCLCA5 expression upon differentiation, which persists during hormone withdrawal. ( a ) Upon stimulation with prolactin, KIM-2 cells form dome-like epithelial islands (d), which exhibit robust E-cadherin expression, surrounded by more elongate, loosely arranged cells (l). Immunofluorescence staining for E-cadherin (green) and DNA (Hoechst; blue). Right panel shows merged images. Scale bar=100 μ m. ( b ) RT-PCR analysis for mCLCA2, mCLCA5, β -casein and cyclophilin A (housekeeping gene), in KIM-2 cells that are undifferentiated (undiff), KIM-2 cells that have received lactogenic hormones for 14 days (14d Diff), or KIM-2 cells that have received lactogenic hormones for 11–14 days and have then undergone hormone withdrawal for 72 (72 h HW) or 96 h (96 h HW). Two independent biological repeats of each condition are shown. ( c ) Expression of mCLCA2 and mCLCA5 in undifferentiated and differentiated KIM-2 cells, measured by qRT-PCR relative to expression of cyclophilin A (housekeeping gene); values are mean±S.D. from at least three biological repeats; * P <0.05, as determined by Student's t -test. NTC, no template control

Article Snippet: Immunofluoresence staining for E-cadherin (1:500; 610182, BD Biosciences, Oxford Science Park, Oxford, UK), mCLCA5 (1:100; sc99224; Santa Cruz Biotechnology), vimentin (1:200; #5741) and murine total Stat3 (1:100; #9139) (both Cell Signaling Technology, Danvers, MA, USA) was carried out following de-paraffinization and antigen retrieval for 20 min at 90 °C using Dako Envision Flex Target Antigen Retrieval Solution High pH in a PT Link and Pre-Treatment Module for Tissue Specimens (both Dako).

Techniques: Expressing, Immunofluorescence, Staining, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: Stat3 modulates chloride channel accessory protein expression in normal and neoplastic mammary tissue

doi: 10.1038/cddis.2016.302

Figure Lengend Snippet: mCLCA5 protein is expressed during mammary gland involution and is subjectively more abundant in the absence of epithelial Stat3 signalling. Immunofluorescence staining for mCLCA5 (red) and E-cadherin (green), and DNA (Hoechst; blue) on control and Stat3 KO tissue at 96 h of involution. Scale bar=50 μ m ( a ) and 10 μ m ( b ). Staining of residual milk (*) was considered non-specific. Some, but not all shed cells exhibited positive staining for mCLCA5 (arrowhead). Representative images are displayed. Sections from five mice were examined (control n =2; Stat3 KO n =3)

Article Snippet: Immunofluoresence staining for E-cadherin (1:500; 610182, BD Biosciences, Oxford Science Park, Oxford, UK), mCLCA5 (1:100; sc99224; Santa Cruz Biotechnology), vimentin (1:200; #5741) and murine total Stat3 (1:100; #9139) (both Cell Signaling Technology, Danvers, MA, USA) was carried out following de-paraffinization and antigen retrieval for 20 min at 90 °C using Dako Envision Flex Target Antigen Retrieval Solution High pH in a PT Link and Pre-Treatment Module for Tissue Specimens (both Dako).

Techniques: Immunofluorescence, Staining

Journal: Cell Death & Disease

Article Title: Stat3 modulates chloride channel accessory protein expression in normal and neoplastic mammary tissue

doi: 10.1038/cddis.2016.302

Figure Lengend Snippet: Although both mCLCA5 and pStat3 are expressed predominantly at the invasive edge of the tumour, minimal co-localization of nuclear Stat3 and cytoplasmic mCLCA5 is observed. Representative images showing the edge of orthotopic tumours derived from implantation of 4T1 cells. ( a ) Immunofluorescence staining for vimentin (red), E-cadherin (green) and DNA (Hoechst; blue). Scale bar=10 μ m. ( b ) Immunofluorescence staining for mCLCA5 (red), E-cadherin (green) and DNA (Hoechst; blue). Groupings of E-cadherin-positive cells express mCLCA5 (arrowhead). ( c ) Immunofluorescence staining for Stat3 (red), mCLCA5 (green) and DNA (Hoechst; blue). In each case, images are representative of results from three separate mice and are captured at the same magnification. ( d ) Graphical summary of hypothesis that subpopulations of tumour cells may express high levels of the oncogene Stat3 or mCLCA5, and that mCLCA5 may be secreted

Article Snippet: Immunofluoresence staining for E-cadherin (1:500; 610182, BD Biosciences, Oxford Science Park, Oxford, UK), mCLCA5 (1:100; sc99224; Santa Cruz Biotechnology), vimentin (1:200; #5741) and murine total Stat3 (1:100; #9139) (both Cell Signaling Technology, Danvers, MA, USA) was carried out following de-paraffinization and antigen retrieval for 20 min at 90 °C using Dako Envision Flex Target Antigen Retrieval Solution High pH in a PT Link and Pre-Treatment Module for Tissue Specimens (both Dako).

Techniques: Derivative Assay, Immunofluorescence, Staining

Journal: International Journal of Molecular Medicine

Article Title: Neutrophil extracellular traps promote gastric cancer metastasis by inducing epithelial-mesenchymal transition

doi: 10.3892/ijmm.2021.4960

Figure Lengend Snippet: NETs promote tumor cell migration without altering cell cycle and proliferation. (A) Representative immunofluorescence microphotographs showing NETs (Hoechst-44432, blue; NE, green). Scale bar, 50 µ m; magnification, ×200. (B) MTT assay indicated that there was no significant change in the proliferation rate of AGS cells for 48 h in each group. Data are presented as the means ± SEM from n=3 separate experiments. (C) The effects of NETs on cell cycle was assessed by flow cytometry, AGS cells were stimulated by NETs or inhibiting NET CM for 24 h. (D) The percentage of cell population at each phase in different group. (E) The effects of NETs on the migration ability of GC cells was assessed by Transwell assay; scale bar, 20 µ m; magnification, ×400. (F) Statistical graphs are presented as the means ± SEM, # P<0.001; n=5. (G) The effects of NETs on the EMT markers in AGS cells and GAPDH were examined by western blot analysis. (H and I) Statistical analysis of the expression of E-cadherin and vimentin was showed. Data were normalized to the GAPDH protein level and are expressed as the mean ± SEM, ** P<0.01 vs. PMA group; n=3. NET, neutrophil extracellular trap; GC, gastric cancer.

Article Snippet: The tissue preparations were incubated with the primary antibodies for E-cadherin (#20874-1-AP, ProteinTech Group, Inc.,) at a 1:500 dilution, vimentin (#HPA001762, Sigma-Aldrich; Merck KGaA) at a 1:250 dilution at 4°C overnight, followed by incubation with the secondary antibody EnVisionTM+/HRP rabbit polymer (#P0448, Dako; Agilent Technologies, Inc.) at a 1:200 dilution at room temperature for 30 min.

Techniques: Migration, Immunofluorescence, MTT Assay, Flow Cytometry, Transwell Assay, Western Blot, Expressing

Journal: International Journal of Molecular Medicine

Article Title: Neutrophil extracellular traps promote gastric cancer metastasis by inducing epithelial-mesenchymal transition

doi: 10.3892/ijmm.2021.4960

Figure Lengend Snippet: Effects of NET inhibitors on solid tumor growth in nude mice. (A) Images of AGS residual tumor xenografts treated with Dnase I or GSK-484; scale bar, 1 cm. (B-D) The graphs of mouse weight, tumor growth curve, average tumor weight. AGS cells induced NETs deposition in the tumors. (E) Images showing representative immunostaining for neutrophil elastase (green), citrullinated histone H3 (red), and DAPI (blue) in the tumors of mice treated as indicated; scale bar, 50 µ m; magnification, ×400. (F) Representative IHC images of murine tumor sections stained for the EMT-related markers; n=5. scale bar, 50 µ m; magnification, ×400. (G) The effects of NETs inhibitors on EMT process in murine tumor tissues, protein levels of E-cadherin, vimentin and citrullinated histone H3 were detected by western blot analysis. (H-J) Statistical analysis of the expression of E-cadherin, vimentin and citrullinated histone H3. Data were normalized to the GAPDH protein level and expressed as the means ± SEM, ** P<0.01 vs. NC group, n=5. NET, neutrophil extracellular trap; GC, gastric cancer; cit-h3, citrullinated histone H3.

Article Snippet: The tissue preparations were incubated with the primary antibodies for E-cadherin (#20874-1-AP, ProteinTech Group, Inc.,) at a 1:500 dilution, vimentin (#HPA001762, Sigma-Aldrich; Merck KGaA) at a 1:250 dilution at 4°C overnight, followed by incubation with the secondary antibody EnVisionTM+/HRP rabbit polymer (#P0448, Dako; Agilent Technologies, Inc.) at a 1:200 dilution at room temperature for 30 min.

Techniques: Immunostaining, Staining, Western Blot, Expressing