Journal: Clinical and Translational Medicine

Article Title: NOX5 mediates the crosstalk between tumor cells and cancer‐associated fibroblasts via regulating cytokine network

doi: 10.1002/ctm2.472

Figure Lengend Snippet: The expression profile of cytokines between NFs and CAFs. ELISA of αSMA levels in cell lysates of primary NFs and paired CAFs (six pairs). (B, C) NFs (a mixture of pairs 1, 2, and 3) or adipose‐derived MSCs cultured with the CM from KYSE30, KYSE410, KYSE510, or primary ESCC cells for 3 days. After the tumor CM was removed, NFs (a mixture of pairs 1, 2, and 3) and adipose‐derived MSCs were incubated with fresh RPMI1640 medium for 2 days, and fluorescent staining of αSMA in NFs, or adipose‐derived MSCs alone or these cells incubated with the CM from indicated ESCC cells. Scale bar, 20 μm as indicated (B). ELISA of αSMA levels in cell lysates of indicated stromal cells (C). Primary CAFs (a mixture of pairs 1, 2, and 3) were used as positive control. (D) The differential secreting status of cytokines in primary NF versus CAF from the same ESCC patient (pair 2), as assayed by cytokine antibody array. (E) ELISA assay showing the secretion of IL‐6, IL‐7, IL‐8, CCL5, and TGF‐β1 from six paired primary NFs and CAFs. (F) The experimental condition of (F) was consistent with that of (B). ELISA assay showing the secretion of IL‐6, IL‐7, IL‐8, CCL5, and TGF‐β1 from NFs (a mixture of pairs 1, 2, and 3) or adipose‐derived MSCs alone or incubated with the CM from indicated ESCC cells. Primary CAFs (a mixture of pairs 1, 2, and 3) were used as positive control. (G) Transcriptional factor activity assay of NF‐κB p65 activity in the nucleus of primary NFs and paired CAFs (six pairs). (H) The experimental condition of (H) was consistent with that of (B). NFs (a mixture of pairs 1, 2, and 3) or adipose‐derived MSCs cultured with the CM from indicated ESCC cells. NF‐κB p65 activity was examined using transcriptional factor activity assay. Primary CAFs (a mixture of pairs 1, 2, and 3) were used as positive control. ** p < 0.01;*** p < 0.001; two‐tailed unpaired Student's t ‐test. Error bars represent mean ± SD of three independent experiments

Article Snippet: The culture condition of these cell lines was according to our previous reports., One case of normal esophageal epithelial cell (NEEC) isolated from the adjacent nontumorous esophageal tissue (over 5 cm from the clinical stage II ESCC tissue ), one case of primary ESCC cell (clinical stage: II), and six paired primary CAFs and NFs isolated from six cases of clinical ESCC tissues (clinical stage: II) and their adjacent nontumorous esophageal tissues (over 5 cm from the tumor tissue , ), were obtained from CHI Scientific, Inc. Human adipose‐derived mesenchymal stem cells (MSCs; isolate from normal human adipose tissue) and lymphatic capillary endothelial cells (HLECs; isolate from normal human lymph node) were purchased from Sciencell, Inc. All primary cells were cultured with RPMI1640 medium containing 10% fetal bovine serum (FBS).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Derivative Assay, Cell Culture, Incubation, Staining, Positive Control, Ab Array, Activity Assay, Two Tailed Test

Journal: Clinical and Translational Medicine

Article Title: NOX5 mediates the crosstalk between tumor cells and cancer‐associated fibroblasts via regulating cytokine network

doi: 10.1002/ctm2.472

Figure Lengend Snippet: Intratumoral NOX5 is critically contributed to the activation of NFs into CAFs. NFs (a mixture of pairs 1, 2, and 3) were incubated with the CM from KYSE30, KYSE410, KYSE510, and primary ESCC cells harbored control or NOX5 shRNA for 3 days. Then, fibroblasts were cultured with fresh RPMI1640 medium for 2 days. The intracellular expression of αSMA was evaluated using immunoblotting (B) and confocal assay (C). The secretion of IL‐6, IL‐7, IL‐8, CCL5, and TGF‐β1 from fibroblasts was assessed using ELISA assay (D). Primary CAFs (a mixture of pairs 1, 2, and 3) were used as positive control. (B, C) Stably silencing NOX5 in the indicated ESCC cell lines and primary ESCC cells analyzed by immunoblotting. GAPDH was used as the internal control (B, upper panel). Immunoblotting (B, lower panel) or confocal (C) of αSMA levels in NFs or fibroblasts derived from NFs incubated with the CM from indicated ESCC cells harbored control shRNA or NOX5 shRNA. Scale bar, 20 μm as indicated. (D) ELISA assay showing the secretion of IL‐6, IL‐7, IL‐8, CCL5, and TGF‐β1 from NFs or fibroblasts derived from NFs incubated with the CM from indicated ESCC cells harbored control shRNA or NOX5 shRNA. # represents the statistical significance of NFs alone versus NFs treated with the CM from indicated ESCC cells harbored control shRNA. * represents the statistical significance of NFs treated with the CM from indicated ESCC cells harbored control shRNA versus NFs treated with the CM from indicated ESCC cells harbored NOX5 shRNA. ## p < 0.01; ### p < 0.001; ** p < 0.01; *** p < 0.001; two‐tailed unpaired Student's t ‐test. Error bars, mean ± SD of three independent experiments

Article Snippet: The culture condition of these cell lines was according to our previous reports., One case of normal esophageal epithelial cell (NEEC) isolated from the adjacent nontumorous esophageal tissue (over 5 cm from the clinical stage II ESCC tissue ), one case of primary ESCC cell (clinical stage: II), and six paired primary CAFs and NFs isolated from six cases of clinical ESCC tissues (clinical stage: II) and their adjacent nontumorous esophageal tissues (over 5 cm from the tumor tissue , ), were obtained from CHI Scientific, Inc. Human adipose‐derived mesenchymal stem cells (MSCs; isolate from normal human adipose tissue) and lymphatic capillary endothelial cells (HLECs; isolate from normal human lymph node) were purchased from Sciencell, Inc. All primary cells were cultured with RPMI1640 medium containing 10% fetal bovine serum (FBS).

Techniques: Activation Assay, Incubation, shRNA, Cell Culture, Expressing, Western Blot, Confocal Assay, Enzyme-linked Immunosorbent Assay, Positive Control, Stable Transfection, Derivative Assay, Two Tailed Test

Journal: Clinical and Translational Medicine

Article Title: NOX5 mediates the crosstalk between tumor cells and cancer‐associated fibroblasts via regulating cytokine network

doi: 10.1002/ctm2.472

Figure Lengend Snippet: Intratumoral NOX5 is critically contributed to the activation of adipose‐derived MSCs into CAFs. The experimental condition of this figure was consistent with that of Figure 3. (A, B) Immunoblotting (A) or confocal (B) of αSMA levels in adipose‐derived MSCs, or fibroblasts derived from adipose‐derived MSCs incubated with the CM from KYSE410, KYSE30, KYSE510, or primary ESCC cells harbored control shRNA or NOX5 shRNA. Scale bar, 20 μm as indicated. (C) ELISA assay showing the secretion of IL‐6, IL‐7, IL‐8, CCL5, and TGF‐β1 from adipose‐derived MSCs and fibroblasts derived from adipose‐derived MSCs incubated with the CM from indicated ESCC cells harbored control shRNA or NOX5 shRNA. Primary CAFs (a mixture of pairs 1, 2, and 3) were used as positive control. # represents the statistical significance of adipose‐derived MSCs alone versus adipose‐derived MSCs incubated with the CM from indicated ESCC cells harbored control shRNA. *represents the statistical significance of adipose‐derived MSCs incubated with the CM from indicated ESCC cells harbored control shRNA versus adipose‐derived MSCs incubated with the CM from indicated ESCC cells harbored NOX5 shRNA. ### p < 0.001; ** p < 0.01; *** p < 0.001; two‐tailed unpaired Student's t ‐test. Error bars, mean ± SD of three independent experiments

Article Snippet: The culture condition of these cell lines was according to our previous reports., One case of normal esophageal epithelial cell (NEEC) isolated from the adjacent nontumorous esophageal tissue (over 5 cm from the clinical stage II ESCC tissue ), one case of primary ESCC cell (clinical stage: II), and six paired primary CAFs and NFs isolated from six cases of clinical ESCC tissues (clinical stage: II) and their adjacent nontumorous esophageal tissues (over 5 cm from the tumor tissue , ), were obtained from CHI Scientific, Inc. Human adipose‐derived mesenchymal stem cells (MSCs; isolate from normal human adipose tissue) and lymphatic capillary endothelial cells (HLECs; isolate from normal human lymph node) were purchased from Sciencell, Inc. All primary cells were cultured with RPMI1640 medium containing 10% fetal bovine serum (FBS).

Techniques: Activation Assay, Derivative Assay, Western Blot, Incubation, shRNA, Enzyme-linked Immunosorbent Assay, Positive Control, Two Tailed Test

Journal: Clinical and Translational Medicine

Article Title: NOX5 mediates the crosstalk between tumor cells and cancer‐associated fibroblasts via regulating cytokine network

doi: 10.1002/ctm2.472

Figure Lengend Snippet: NOX5 Y476/478 sites are critical for NOX5‐mediated CAFs activation. The experimental condition of this figure was consistent with that of Figure 3. (A) Transfection of NOX5 Y476/478F plasmid into KYSE30 and KYSE410 cells. The transfection efficiency was evaluated using immunoblotting. GAPDH was used as the loading control. (B) The concentration of TNF‐α and IL‐1β in CM from KYSE30 and KYSE410 cells harbored control vector or NOX5 Y476/478F plasmid, was assayed by ELISA. (C) Confocal analysis of αSMA levels in NFs (a mixture of pairs 1, 2, and 3), adipose‐derived MSCs incubated with the CM from indicated ESCC cells harbored control vector or NOX5 Y476/478F plasmid. Scale bar, 20 μm as indicated. (D) ELISA assay showing the secretion of IL‐6, IL‐7, IL‐8, CCL5, and TGF‐β1 from NFs (a mixture of pairs 1, 2, and 3) and adipose‐derived MSCs incubated with the CM from KYSE30 and KYSE410 cells harbored control vector or NOX5 Y476/478F plasmid. ** p < 0.01; *** p < 0.001; two‐tailed unpaired Student's t ‐test. Error bars, mean ± SD of three independent experiments

Article Snippet: The culture condition of these cell lines was according to our previous reports., One case of normal esophageal epithelial cell (NEEC) isolated from the adjacent nontumorous esophageal tissue (over 5 cm from the clinical stage II ESCC tissue ), one case of primary ESCC cell (clinical stage: II), and six paired primary CAFs and NFs isolated from six cases of clinical ESCC tissues (clinical stage: II) and their adjacent nontumorous esophageal tissues (over 5 cm from the tumor tissue , ), were obtained from CHI Scientific, Inc. Human adipose‐derived mesenchymal stem cells (MSCs; isolate from normal human adipose tissue) and lymphatic capillary endothelial cells (HLECs; isolate from normal human lymph node) were purchased from Sciencell, Inc. All primary cells were cultured with RPMI1640 medium containing 10% fetal bovine serum (FBS).

Techniques: Activation Assay, Transfection, Plasmid Preparation, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Incubation, Two Tailed Test

Journal: Clinical and Translational Medicine

Article Title: NOX5 mediates the crosstalk between tumor cells and cancer‐associated fibroblasts via regulating cytokine network

doi: 10.1002/ctm2.472

Figure Lengend Snippet: NOX5‐induced TNF‐α or IL‐1β mediates the activation of NFs and adipose‐derived MSCs into CAFs. Control or NOX5‐overexpressing KYSE30 and KYSE410 cells were treated with inhibitors of ROS/Src/NF‐κB pathway, and the secretion of TNF‐α and IL‐1β was evaluated using ELISA assay (B). NFs (a mixture of pairs 1, 2, and 3) or adipose‐derived MSCs were incubated with recombinant human TNF‐α or IL‐1β protein, or CM from KYSE30 and KYSE410 cells alone or in the presence of TNF‐α or IL‐1β Ab for 3 days. Then, fibroblasts were cultured with fresh RPMI1640 medium for 2 days. The intracellular expression of αSMA was evaluated using immunoblotting and confocal assay (C). The secretion of IL‐6, IL‐7, IL‐8, CCL5, and TGF‐β1 from fibroblasts was assessed using ELISA assay (D). Primary CAFs (a mixture of pairs 1, 2, and 3) were used as positive control. (B) Control vector or NOX5‐overexpressing KYSE30 (black bar) and KYSE410 (gray bar) cells were pretreated with ROS scavenger NAC (2 mM, pretreated with 90 min), or treated with H 2 O 2 scavenger‐PEG‐catalase (400 units/ml), 100 nM dasatinib, 1 μM PP2, 5 μM JSH‐23, 5 μM SC75741, or control solvent. The concentration of TNF‐α and IL‐1β in CM from indicated ESCC cells was assayed by ELISA. # represents the statistical significance of indicated treatments versus control cells. * represents the statistical significance of indicated treatments versus NOX5‐overexpressing cells. # p < 0.05; ## p < 0.01; ### p < 0.001; *** p < 0.001; two‐tailed unpaired Student's t ‐test. (C, D) NFs (a mixture of pairs 1, 2, and 3) or adipose‐derived MSCs were treated with recombinant TNF‐α or IL‐1β protein (10 ng/ml), or the CM from KYSE30 or KYSE410 cells in the presence or absence of the TNF‐α or IL‐1β Ab (10 μg/ml) for 3 days. Then, culture media were removed and added fresh RPMI1640 medium to these fibroblasts for 2 days. (C) Immunoblotting (left panel) or confocal assay (right panel) evaluating the expression of αSMA in NFs (a mixture of pairs 1, 2, and 3) or adipose‐derived MSCs. Scale bar, 20 μm as indicated. (D) ELISA assay was used to detect the secretion of IL‐6, IL‐7, IL‐8, CCL5, and TGF‐β1 from indicated stromal cells. Primary CAFs (a mixture of pairs 1, 2, and 3) were used as positive control. ** p < 0.01; *** p < 0.001; two‐tailed unpaired Student's t ‐test. Error bars, mean ± SD of three independent experiments

Article Snippet: The culture condition of these cell lines was according to our previous reports., One case of normal esophageal epithelial cell (NEEC) isolated from the adjacent nontumorous esophageal tissue (over 5 cm from the clinical stage II ESCC tissue ), one case of primary ESCC cell (clinical stage: II), and six paired primary CAFs and NFs isolated from six cases of clinical ESCC tissues (clinical stage: II) and their adjacent nontumorous esophageal tissues (over 5 cm from the tumor tissue , ), were obtained from CHI Scientific, Inc. Human adipose‐derived mesenchymal stem cells (MSCs; isolate from normal human adipose tissue) and lymphatic capillary endothelial cells (HLECs; isolate from normal human lymph node) were purchased from Sciencell, Inc. All primary cells were cultured with RPMI1640 medium containing 10% fetal bovine serum (FBS).

Techniques: Activation Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay, Incubation, Recombinant, Cell Culture, Expressing, Western Blot, Confocal Assay, Positive Control, Plasmid Preparation, Concentration Assay, Two Tailed Test

Journal: Clinical and Translational Medicine

Article Title: NOX5 mediates the crosstalk between tumor cells and cancer‐associated fibroblasts via regulating cytokine network

doi: 10.1002/ctm2.472

Figure Lengend Snippet: Activated CAFs assist ESCC malignant progression in vitro . NFs (a mixture of pairs 1, 2, and 3) were incubated with the CM from KYSE30 or KYSE410 cells for 3 days. Then, the tumor CM was removed, the fresh RPMI1640 medium was added to fibroblasts for 2 days to generate CM for MTS assay, and the activated CAFs were applied to Transwell invasion assay. KYSE30 or KYSE410 harbored control shRNA or NOX5 shRNA cells were incubated with the CM from corresponding parental cells‐activated CAFs. The growth ability was evaluated using MTS assay (B). CAFs regulated the invasion of ESCC cells was assessed by Transwell invasion assay. The activated CAFs primed by KYSE30 or KYSE410 cells were plated in the lower chamber. KYSE30 or KYSE410 harbored control shRNA or NOX5 shRNA cells (corresponding to their respective parental cells‐activated CAFs) were placed in the upper chamber (C). For evaluation of CAFs‐induced HLECs migration, NFs (a mixture of pairs 1, 2, and 3) were incubated with the CM from KYSE30 or KYSE410 harbored control shRNA or NOX5 shRNA cells for 3 days. Then, the tumor CM was removed, the fresh RPMI1640 medium was added to fibroblasts for 2 days, and these fibroblasts were used for subsequent assay. Migration of HLECs was evaluated using Transwell migration assay. Fibroblasts primed by KYSE30 or KYSE410 harbored control shRNA or NOX5 shRNA cells were plated in the lower chamber. HLECs were seeded in the upper chamber (D). (B) Growth rates of KYSE30 or KYSE410 cells harbored control shRNA incubated with the CM from corresponding parental cells‐activated CAFs alone or in the presence of IL‐6, IL‐7, IL‐8, CCL5, or TGF‐β1 Ab (10 μg/ml), or KYSE30 or KYSE410 cells harbored NOX5 shRNA alone or incubated with CM from their corresponding parental ESCC cells for 4 days. Cell growth was assayed by MTS assay. (C) Boyden chamber assay for KYSE30 or KYSE410 cells harbored control shRNA plated on the upper cell culture inserts with their corresponding parental ESCC cells‐activated CAFs in lower chambers alone or in the presence of IL‐6, IL‐7, IL‐8, CCL5, or TGF‐β1 Ab (10 μg/ml), or KYSE30 or KYSE410 cells harbored NOX5 shRNA plated on the upper cell culture inserts with or without their corresponding parental ESCC cells‐primed CAFs in lower chambers. (D) CytoSelect 96‐well cell migration assay for HLECs plated on upper cell culture inserts with NFs (a mixture of pairs 1, 2, and 3), NFs (a mixture of pairs 1, 2, and 3)‐activated CAFs (primed by KYSE30 or KYSE410 cells harbored control shRNA) alone or in the presence of IL‐6, IL‐7, IL‐8, CCL5, or TGF‐β1 Ab (10 μg/ml), or NFs (a mixture of pairs 1, 2, and 3) primed by the CM from KYSE30 or KYSE410 cells harbored NOX5 shRNA in lower chambers. n.s. no significant difference; * p < 0.05; *** p < 0.001; two‐tailed unpaired Student's t ‐test. Error bars, mean ± SD of five independent experiments

Article Snippet: The culture condition of these cell lines was according to our previous reports., One case of normal esophageal epithelial cell (NEEC) isolated from the adjacent nontumorous esophageal tissue (over 5 cm from the clinical stage II ESCC tissue ), one case of primary ESCC cell (clinical stage: II), and six paired primary CAFs and NFs isolated from six cases of clinical ESCC tissues (clinical stage: II) and their adjacent nontumorous esophageal tissues (over 5 cm from the tumor tissue , ), were obtained from CHI Scientific, Inc. Human adipose‐derived mesenchymal stem cells (MSCs; isolate from normal human adipose tissue) and lymphatic capillary endothelial cells (HLECs; isolate from normal human lymph node) were purchased from Sciencell, Inc. All primary cells were cultured with RPMI1640 medium containing 10% fetal bovine serum (FBS).

Techniques: In Vitro, Incubation, MTS Assay, Transwell Invasion Assay, shRNA, Migration, Subsequent Assay, Transwell Migration Assay, Boyden Chamber Assay, Cell Culture, Cell Migration Assay, Two Tailed Test

Journal: Clinical and Translational Medicine

Article Title: NOX5 mediates the crosstalk between tumor cells and cancer‐associated fibroblasts via regulating cytokine network

doi: 10.1002/ctm2.472

Figure Lengend Snippet: CAFs promote the malignancy of ESCC tumors in vivo . Tumor volume was measured at day 33. Mice‐bearing control or NOX5 shRNA KYSE30 tumors with NFs (a mixture of pairs 1, 2, and 3)‐activated CAFs (primed by KYSE30 cells) were treated with control solvent or several Abs, including IL‐6, IL‐7, IL‐8, or TGF‐β1 Ab (10 μg/mouse twice times per week, i.v.). (B) The expression of Ki‐67, CD31, or LYVE1 in indicated KYSE30 tumor tissues was evaluated using IHC assay. n.s. no significant difference; ** p < 0.01; *** p < 0.001; two‐tailed unpaired Student's t ‐test. Error bars, mean ± SD of five independent experiments

Article Snippet: The culture condition of these cell lines was according to our previous reports., One case of normal esophageal epithelial cell (NEEC) isolated from the adjacent nontumorous esophageal tissue (over 5 cm from the clinical stage II ESCC tissue ), one case of primary ESCC cell (clinical stage: II), and six paired primary CAFs and NFs isolated from six cases of clinical ESCC tissues (clinical stage: II) and their adjacent nontumorous esophageal tissues (over 5 cm from the tumor tissue , ), were obtained from CHI Scientific, Inc. Human adipose‐derived mesenchymal stem cells (MSCs; isolate from normal human adipose tissue) and lymphatic capillary endothelial cells (HLECs; isolate from normal human lymph node) were purchased from Sciencell, Inc. All primary cells were cultured with RPMI1640 medium containing 10% fetal bovine serum (FBS).

Techniques: In Vivo, shRNA, Expressing, Two Tailed Test

Journal: Clinical and Translational Medicine

Article Title: NOX5 mediates the crosstalk between tumor cells and cancer‐associated fibroblasts via regulating cytokine network

doi: 10.1002/ctm2.472

Figure Lengend Snippet: CAFs promote the malignancy of ESCC tumors in vivo . A popliteal lymph node metastasis model was established in mice ( n = 5 biologically independent mouse/group) by inoculating the foot pads with KYSE30 cells and NFs (a mixture of pairs 1, 2, and 3)‐activated CAFs (primed by KYSE30 cells). The popliteal lymph nodes were enucleated and analyzed 5 weeks after inoculation, and the volumes of popliteal lymph nodes were shown. * p < 0.05; ** p < 0.01; *** p < 0.001; two‐tailed unpaired Student's t ‐test. Error bars represent mean ± SD of five independent experiments

Article Snippet: The culture condition of these cell lines was according to our previous reports., One case of normal esophageal epithelial cell (NEEC) isolated from the adjacent nontumorous esophageal tissue (over 5 cm from the clinical stage II ESCC tissue ), one case of primary ESCC cell (clinical stage: II), and six paired primary CAFs and NFs isolated from six cases of clinical ESCC tissues (clinical stage: II) and their adjacent nontumorous esophageal tissues (over 5 cm from the tumor tissue , ), were obtained from CHI Scientific, Inc. Human adipose‐derived mesenchymal stem cells (MSCs; isolate from normal human adipose tissue) and lymphatic capillary endothelial cells (HLECs; isolate from normal human lymph node) were purchased from Sciencell, Inc. All primary cells were cultured with RPMI1640 medium containing 10% fetal bovine serum (FBS).

Techniques: In Vivo, Two Tailed Test

Journal: British Journal of Nutrition

Article Title: Mitochondrial dysfunction in the liver of type 2 diabetic Goto–Kakizaki rats: improvement by a combination of nutrients

doi: 10.1017/s0007114511000493

Figure Lengend Snippet: Fig. 5. Apoptosis-related factors p53 and p21 in liver tissue: (a) Western blotting images of p21, p53 and a-tubulin, respectively and (b) quantitative results of p53 and p21. Quantitative values were computed as the ratios of the density of the targeted protein to a-tubulin. Results are expressed as fold change of control. Values are means, with their standard errors represented by vertical bars (n 10). Mean value was significantly different from that of the Wistar group: * P,0·05, ** P,0·01. , Wistar; , GK; , GK with nutrients; , GK with pioglitazone.

Article Snippet: Liver tissue proteins were subjected to 10 % SDS-PAGE and detected with primary antibodies against p53 (1:1000, Mouse Ab no. 12 506; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p21 (1:1000, Mouse Ab no. 2946; Cell Signaling Technology, Danvers, MA, USA) or a-tubulin (1:5000).

Techniques: Western Blot, Control

Journal: British Journal of Nutrition

Article Title: Chronic ingestion of deoxynivalenol and fumonisin, alone or in interaction, induces morphological and immunological changes in the intestine of piglets

doi: 10.1017/s0007114511004946

Figure Lengend Snippet: Fig. 5. Effect of individual and combined deoxynivalenol (DON) and fumonisins (FB) exposure on the intestinal expression of E-cadherin. Pigs received a control diet ( ), or a diet contaminated with DON ( ), FB ( ), or both DON and FB ( ). (A) Jejunum of a control piglet showing a strong and (B) homogeneous immunor- eactivity to E-cadherin. Immunoperoxidase, 20£ . (C) Percentage of animals showing strong immunoreactivity to E-cadherin. a,b Mean values with unlike letters were significantly different (P,0·05).

Article Snippet: The antibodies used in the present study were E-cadherin (24E10) rabbit mAb (diluted 1:500; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-occludin (672381A, diluted 1:500; Invitrogen, Cergy-Pontoise, France) and b-actin mouse mAb (8H10D10; Cell Signaling).

Techniques: Expressing, Control

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Expression of Signal Transducer and Activator of Transcription 3 and Suppressor of Cytokine Signaling 3 in Urothelial Carcinoma

doi: 10.1016/s1607-551x(09)70569-8

Figure Lengend Snippet: Figure 1. Immunohistochemical staining of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) Tyr705 and cytokine signaling 3 (SOCS3) in urothelial carcinomas. Nuclear staining of p-STAT3 (Tyr705): (A) low intensity grade (insert in A is normal urothelium); (B) high intensity grade. Cytoplasmic staining of SOCS3: (C) low intensity grade; (D) high intensity grade. Original magnification, 400×.

Article Snippet: The membranes were blocked with 5% non-fat dried milk in Tris-buffered Saline (pH 7.4) with Tween-20, and then incubated with either anti-phospho-STAT3 (Tyr705) (1:1,000 dilution; Cell Signaling Technology) or SOCS3 rabbit polyclonal primary antibody (Santa Cruz Biotechnology Inc.).

Techniques: Immunohistochemical staining, Staining

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Expression of Signal Transducer and Activator of Transcription 3 and Suppressor of Cytokine Signaling 3 in Urothelial Carcinoma

doi: 10.1016/s1607-551x(09)70569-8

Figure Lengend Snippet: Figure 2. Western blotting of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) Tyr705 and cytokine signaling 3 (SOCS3) protein in low-grade and high-grade UCs. p-STAT3 (Tyr705) expression was low and high in low- and high-grade UCs, respectively. Lane 1 = low-grade UC; lane 2=high-grade UC. However, the expression of SOCS3 was simi- lar in low- and high-grade UCs. GADPH protein expression was used as an internal control.

Article Snippet: The membranes were blocked with 5% non-fat dried milk in Tris-buffered Saline (pH 7.4) with Tween-20, and then incubated with either anti-phospho-STAT3 (Tyr705) (1:1,000 dilution; Cell Signaling Technology) or SOCS3 rabbit polyclonal primary antibody (Santa Cruz Biotechnology Inc.).

Techniques: Western Blot, Expressing, Control