Journal: American Journal of Translational Research

Article Title: The therapeutic response of CDDO-Me in the esophageal squamous cell carcinoma (ESCC) cells is mediated by CaMKIIα

doi:

Figure Lengend Snippet: Top 20 upregulated protein molecules modulated by CDDO-Me in Ec109 cells

Article Snippet: Human ESCC Ec109 cells was obtained from AddexBio Inc. (San Diego, CA, USA), KYSE70 and KYSE30 cells were obtained from Sigma-Aldrich Co (St Louis, MO, USA).

Techniques:

Journal: American Journal of Translational Research

Article Title: The therapeutic response of CDDO-Me in the esophageal squamous cell carcinoma (ESCC) cells is mediated by CaMKIIα

doi:

Figure Lengend Snippet: The top ten canonical signaling pathways regulated by CDDO-Me in Ec109 cells analyzed by ingenuity pathway analysis. Abbreviations: CDDO-Me, methyl ester of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid; eIF2, eukaryotic initiation factor 2; p70S6K, p70S6 kinase; mTOR, mammalian target of rapamycin; RAN, ras-related nuclear protein; Nrf2, nuclear factor (erythroid-derived 2)-like 2.

Article Snippet: Human ESCC Ec109 cells was obtained from AddexBio Inc. (San Diego, CA, USA), KYSE70 and KYSE30 cells were obtained from Sigma-Aldrich Co (St Louis, MO, USA).

Techniques: Protein-Protein interactions, Derivative Assay

Journal: American Journal of Translational Research

Article Title: The therapeutic response of CDDO-Me in the esophageal squamous cell carcinoma (ESCC) cells is mediated by CaMKIIα

doi:

Figure Lengend Snippet: Representative blots of protein levels of VKORC1, CaMKIIα, NPLOC4, PSME3, and Dynamin 2 in various human ESCC cell lines and normal human esophageal epithelial cell line (Het-1A) were determined using Western blot analysis. β-Actin served as loading controls. Abbreviations: VKORC1, vitamin K epoxide reductase complex subunit 1; CaMKIIα, calcium/calmodulin-dependent protein kinase type II subunit alapha; NPLOC4, nuclear protein localization protein 4 homolog; PSME3, proteasome activator complex subunit 3; ESCC, esophageal squamous cell carcinoma.

Article Snippet: Human ESCC Ec109 cells was obtained from AddexBio Inc. (San Diego, CA, USA), KYSE70 and KYSE30 cells were obtained from Sigma-Aldrich Co (St Louis, MO, USA).

Techniques: Western Blot

Journal: American Journal of Translational Research

Article Title: The therapeutic response of CDDO-Me in the esophageal squamous cell carcinoma (ESCC) cells is mediated by CaMKIIα

doi:

Figure Lengend Snippet: Mitochondrial dysfunction signaling pathway regulated by CDDO-Me in Ec109 cells. Notes: Ec109 cells were treated with 0.5 μM CDDO-Me for 24 hours and the protein samples were subject to quantitative proteomic analysis. Red indicates an upregulation; green indicates a downregulation. The intensity of green and red molecule colors indicates the degree of down- or upregulation, respectively. Solid arrows indicate direct interaction and dashed arrows indicate indirect interaction. Abbreviations: CDDO-Me, methyl ester of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid.

Article Snippet: Human ESCC Ec109 cells was obtained from AddexBio Inc. (San Diego, CA, USA), KYSE70 and KYSE30 cells were obtained from Sigma-Aldrich Co (St Louis, MO, USA).

Techniques:

Journal: American Journal of Translational Research

Article Title: The therapeutic response of CDDO-Me in the esophageal squamous cell carcinoma (ESCC) cells is mediated by CaMKIIα

doi:

Figure Lengend Snippet: mTOR signaling pathway regulated by CDDO-Me in Ec109 cells. Notes: Ec109 cells were treated with 0.5 μM CDDO-Me for 24 hours and the protein samples were subject to quantitative proteomic analysis. Red indicates an upregulation; green indicates a downregulation. The intensity of green and red molecule colors indicates the degree of down- or upregulation, respectively. Solid arrows indicate direct interaction and dashed arrows indicate indirect interaction. Abbreviation: CDDO-Me, methyl ester of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid; mTOR, mammalian target of rapamycin.

Article Snippet: Human ESCC Ec109 cells was obtained from AddexBio Inc. (San Diego, CA, USA), KYSE70 and KYSE30 cells were obtained from Sigma-Aldrich Co (St Louis, MO, USA).

Techniques:

Journal: American Journal of Translational Research

Article Title: The therapeutic response of CDDO-Me in the esophageal squamous cell carcinoma (ESCC) cells is mediated by CaMKIIα

doi:

Figure Lengend Snippet: The effect of knockdown of CaMKIIα on the CDDO-Me induced apoptosis in human ESCC cells. A. Percentages of specific cell populations showed in dot plots and apoptotic cells showed in bar graphs for the silencing of CaMKIIα in Ec109 and KYSE30 cells with or without 0.5 μM CDDO-Me treatment for 24 hours. B. Representative blots of bcl-2, bax, and cleaved caspase-3 for the proteinlysate samples, which were prepared from Ec109 and KYSE30 cells treated with CaMKIIα siRNAs or 0.5 μM CDDO-Me for 24 hours. Abbreviation: CaMKIIα, calcium/calmodulin-dependent protein kinase type II subunit alapha; CDDO-Me, methyl ester of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid; ESCC, esophageal squamous cell carcinoma; bcl-2, B-cell lymphoma-2; bax, bcl-2 associated X protein; siRNA , small interfering RNA.

Article Snippet: Human ESCC Ec109 cells was obtained from AddexBio Inc. (San Diego, CA, USA), KYSE70 and KYSE30 cells were obtained from Sigma-Aldrich Co (St Louis, MO, USA).

Techniques: Knockdown, Small Interfering RNA

Journal: American Journal of Translational Research

Article Title: The therapeutic response of CDDO-Me in the esophageal squamous cell carcinoma (ESCC) cells is mediated by CaMKIIα

doi:

Figure Lengend Snippet: The effect of knockdown of CaMKIIα on the CDDO-Me induced autophagy in human ESCC cells. A. Percentages of specific cell populations showed in dot plots and autophagic cells showed in bar graphs for the silencing of CaMKIIα in Ec109 and KYSE30 cells with or without 0.5 μM CDDO-Me treatment for 24 hours. B. CDDO-Me-induced autophagic death in the silencing of CaMKIIα in Ec109 and KYSE30 cellsdetermined by confocal microscopy. The level of autophagy was evaluated using a lysosome-specific fluorescence dye. The confocal microscopic images of autophagic Ec109 and KYSE 30 cells (stained in green) are also shown. C. Representative blots of beclin-1 and LC3I/II for the proteinlysate samples, which were prepared from Ec109 and KYSE30 cells treated with CaMKIIα siRNAs or 0.5 μM CDDO-Me for 24 hours. Abbreviation: CaMKIIα, calcium/calmodulin-dependent protein kinase type II subunit alapha; CDDO-Me, methyl ester of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid; ESCC, esophageal squamous cell carcinoma; siRNA, small interfering RNA; LC 3, microtubule-associated protein 1A/1B-light chain 3.

Article Snippet: Human ESCC Ec109 cells was obtained from AddexBio Inc. (San Diego, CA, USA), KYSE70 and KYSE30 cells were obtained from Sigma-Aldrich Co (St Louis, MO, USA).

Techniques: Knockdown, Confocal Microscopy, Fluorescence, Staining, Small Interfering RNA

Journal: Oncology Letters

Article Title: JAK/STAT3 signaling pathway mediates endothelial-like differentiation of immature dendritic cells

doi: 10.3892/ol.2015.3752

Figure Lengend Snippet: Cell morphology of the endothelial-like differentiation of iDCs induced by KYSE450 and KYSE70 supernatant. PBMCs were incubated in rhGM-CSF/rhIL-4 and 40% supernatant of KYSE450 (highly differentiated ESCC cells) or KYSE70 (poorly differentiated ESCC cells) was added at the end of day 2. The cells of the KYSE450 and KYSE70 groups had no obvious morphological change, similar to control DCs, following 4 days of induction. The cells of the KYSE450 group exhibited a fusiform shape following 8 days of induction. The KYSE70 group of cells was arranged as strip-shaped and funicular structures, distinctly different to the control dendritic cells, following 8 days of induction with the KYSE70 supernatant (magnification, ×200). DCs; dendritic cells; PBMCs, peripheral blood mononuclear cells.

Article Snippet: The KYSE450 and KYSE70 ESCC cell lines (Nanjing KeyGEN Biotech.

Techniques: Incubation, Stripping Membranes

Journal: Oncology Letters

Article Title: JAK/STAT3 signaling pathway mediates endothelial-like differentiation of immature dendritic cells

doi: 10.3892/ol.2015.3752

Figure Lengend Snippet: Expression of endothelial cell markers vWF and CD144 increased following induction by KYSE450 and KYSE70 supernatant. (A) Immunofluoresence was used to detect the expression of vWF and CD144 in control DCs and the KYSE450 and KYSE70 groups (magnification, ×200). Data are the mean ± standard deviation (SD), n=4, ***P<0.001 vs. control DCs. (B) RT-qPCR detection of mRNA levels of vWF and CD144 in control DCs and the KYSE450 and KYSE70 groups. Data are the mean ± SD, n=3, **P<0.01, ***P<0.001 vs. control DCs. (C) Dil-Ac-LDL uptake was enhanced following induction by KYSE450 and KYSE70 supernatant (magnification, ×200). Data are the mean ± SD, n=4, ***P<0.001 vs. control DCs. DCs, dendritic cells; vWF, von Willebrand factor; CD144, cluster of differentiation 144; DiI-Ac-LDL, DiI-labeled acetylated low-density lipoprotein; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Article Snippet: The KYSE450 and KYSE70 ESCC cell lines (Nanjing KeyGEN Biotech.

Techniques: Expressing, Standard Deviation, Quantitative RT-PCR, Labeling, Real-time Polymerase Chain Reaction

Journal: Oncology Letters

Article Title: JAK/STAT3 signaling pathway mediates endothelial-like differentiation of immature dendritic cells

doi: 10.3892/ol.2015.3752

Figure Lengend Snippet: Activation of JAK/STAT3 signaling by KYSE 450 and KYSE70 supernatant. (A) RT-qPCR analysis revealed that JAK and STAT3 mRNA expression levels increased significantly following incubation with KYSE450 and KYSE70 supernatant. Additionally, the known targets of JAK/STAT3 signaling, VEGF and IL-6 had significantly increased mRNA expression. Data are the mean ± SD (n=3). *P<0.05, **P<0.01, ***P<0.001 vs. control dendtritic cells (DCs). (B) Immunofluorescence detection of the activation of JAK and STAT3 in control DCs and the KYSE450 and KYSE70 groups (magnification, ×200). Data are the mean ± SD (n=3). ***P<0.001 vs. control DCs. JAK, janus tyrosine kinase; VEGF, vascular endothelial growth factor; STAT3, signal transducer and activator of transcription 3.

Article Snippet: The KYSE450 and KYSE70 ESCC cell lines (Nanjing KeyGEN Biotech.

Techniques: Activation Assay, Quantitative RT-PCR, Expressing, Incubation, Immunofluorescence

Journal: Oncology Letters

Article Title: JAK/STAT3 signaling pathway mediates endothelial-like differentiation of immature dendritic cells

doi: 10.3892/ol.2015.3752

Figure Lengend Snippet: Blocking the activation of JAK and STAT3 inhibited endothelial-like differentiation of iDCs. (A) Addition of 10 µmol AG490 at the end of day 2, decreased the expression of vWF and CD144 in iDCs induced by KYSE450 and KYSE70 supernatant. RT-qPCR results shown are the mean ± SD (n=3). **P<0.01, ***P<0.001 vs. KYSE450 group. **P<0.01, ***P<0.001 vs.KYSE70 group. (B) Immunofluorescence detection of the protein expression of the EC markers CD144 and vWF in the KYSE450, KYSE450 + AG, KYSE70, and KYSE70 + AG groups (magnification, ×200). Data are the mean ± SD (n=3). ***P<0.001 vs. KYSE450 group. ***P<0.001 vs. KYSE70 group. (C) Dil-Ac-LDL uptake decreased after induction by KYSE450 + AG490 and KYSE70 + AG490. Original magnification, ×200. Results shown are the mean ± SD (n=4). ***P<0.001 vs. KYSE450 group. ***P<0.001 vs. KYSE70 group. AG, AG490; iDCs, immature dendritic cells; vWF, von Willebrand factor; CD144, cluster of differentiation 144; DiI-Ac-LDL, DiI-labeled acetylated low-density lipoprotein; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Article Snippet: The KYSE450 and KYSE70 ESCC cell lines (Nanjing KeyGEN Biotech.

Techniques: Blocking Assay, Activation Assay, Expressing, Quantitative RT-PCR, Immunofluorescence, Labeling, Real-time Polymerase Chain Reaction

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 human ESCC cell lines were generously provided by Dr. Yutaka Shimada of Kyoto University, and characteristics of ESCC cell lines are listed in Tables and .

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 human ESCC cell lines were generously provided by Dr. Yutaka Shimada of Kyoto University, and characteristics of ESCC cell lines are listed in Tables and .

Techniques: Activation Assay, Incubation, Control, 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 human ESCC cell lines were generously provided by Dr. Yutaka Shimada of Kyoto University, and characteristics of ESCC cell lines are listed in Tables and .

Techniques: Activation Assay, Derivative Assay, Western Blot, Incubation, Control, 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 human ESCC cell lines were generously provided by Dr. Yutaka Shimada of Kyoto University, and characteristics of ESCC cell lines are listed in Tables and .

Techniques: Activation Assay, Transfection, Plasmid Preparation, Western Blot, Control, 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 human ESCC cell lines were generously provided by Dr. Yutaka Shimada of Kyoto University, and characteristics of ESCC cell lines are listed in Tables and .

Techniques: Activation Assay, Derivative Assay, Control, Enzyme-linked Immunosorbent Assay, Incubation, Recombinant, Cell Culture, Expressing, Western Blot, Confocal Assay, Positive Control, Plasmid Preparation, Solvent, 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 human ESCC cell lines were generously provided by Dr. Yutaka Shimada of Kyoto University, and characteristics of ESCC cell lines are listed in Tables and .

Techniques: In Vitro, Incubation, MTS Assay, Transwell Invasion Assay, Control, 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 human ESCC cell lines were generously provided by Dr. Yutaka Shimada of Kyoto University, and characteristics of ESCC cell lines are listed in Tables and .

Techniques: In Vivo, Control, shRNA, Solvent, 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 human ESCC cell lines were generously provided by Dr. Yutaka Shimada of Kyoto University, and characteristics of ESCC cell lines are listed in Tables and .

Techniques: In Vivo, Two Tailed Test

Journal: Molecular Oncology

Article Title: Regulation of MRE11A by UBQLN4 leads to cisplatin resistance in patients with esophageal squamous cell carcinoma

doi: 10.1002/1878-0261.12929

Figure Lengend Snippet: Loss of MRE11A leads to chemotherapy resistance. (A) Scheme of the study design. (B) Pie chart showing the proportion of ESCC cases with high or low MRE11A in the TCGA ESCA database. ESCC patients were divided according to the z ‐score values into 1) ≥ 1.5, 2) ≤ −1.5, or 3) < 1.5 and > −1.5 for MRE11A mRNA expression levels. (C) MRE11A mRNA expression levels in normal adjacent esophageal epithelia ( n = 11) and primary ESCC tumors ( n = 95) in the TCGA ESCA database (* P = 0.05). (D) Representative images of normal adjacent esophageal epithelia and primary ESCC tumors showing MRE11A protein levels stained for MRE11A using IHC. Scale bars = 50 µm. Right top insets on each picture show a magnification of MRE11A staining. Scale bars = 10 µm. (E) Comparison of H ‐scores for MRE11A protein levels for normal adjacent esophageal epithelia ( n = 7) and primary ESCC tumor tissues ( n = 59) (*** P < 0.001). (F) Kaplan–Meier curves comparing OS in ESCC patients with low ( n = 34) versus high ( n = 25) MRE11A protein levels ( P < 0.01). (G) Representative images of core biopsy tissues from nonresponders (Patients 1 and 2) and responders (Patients 1 and 2) ESCC patients to NAC that were stained for MRE11A using IHC. Scale bars = 50 µm. Right top insets on each picture show a magnification of MRE11A staining. Scale bars = 10 µm. (H) Comparison of H ‐scores for MRE11A protein levels in core biopsy tissues from nonresponders ( n = 53) or responders ( n = 8) ESCC patients to NAC (*** P < 0.001). Error bars represent the mean ± SD. Statistical differences were tested using Mann–Whitney test (C), an unpaired two‐tailed t ‐test with Welch’s correction (E and H) and log‐rank test (F).

Article Snippet: Established human ESCC cell lines TE‐4 (CVCL_3337), TE‐8 (CVCL_1766), and TE‐10 (CVCL_1760) were obtained from Keio University.

Techniques: Expressing, Staining, Comparison, MANN-WHITNEY, Two Tailed Test

Journal: Molecular Oncology

Article Title: Regulation of MRE11A by UBQLN4 leads to cisplatin resistance in patients with esophageal squamous cell carcinoma

doi: 10.1002/1878-0261.12929

Figure Lengend Snippet: Clinicopathological features for primary ESCC patients ( n = 60). NA, not applicable.

Article Snippet: Established human ESCC cell lines TE‐4 (CVCL_3337), TE‐8 (CVCL_1766), and TE‐10 (CVCL_1760) were obtained from Keio University.

Techniques:

Journal: Molecular Oncology

Article Title: Regulation of MRE11A by UBQLN4 leads to cisplatin resistance in patients with esophageal squamous cell carcinoma

doi: 10.1002/1878-0261.12929

Figure Lengend Snippet: Univariable and multivariable analysis for clinicopathological features in relation to OS in ESCC patients ( n = 60).

Article Snippet: Established human ESCC cell lines TE‐4 (CVCL_3337), TE‐8 (CVCL_1766), and TE‐10 (CVCL_1760) were obtained from Keio University.

Techniques:

Journal: Molecular Oncology

Article Title: Regulation of MRE11A by UBQLN4 leads to cisplatin resistance in patients with esophageal squamous cell carcinoma

doi: 10.1002/1878-0261.12929

Figure Lengend Snippet: MRE11A expression determines cisplatin resistance in ESCC cell lines. (A, B) Western blot analysis for MRE11A, UBQLN4, and β‐actin (loading control) comparing si‐Ctrl and si‐MRE11A in TE‐10 (A) and TE‐8 (B) parental cell lines. (C, D) Drug sensitivity assays comparing si‐Ctrl or si‐MRE11A in TE‐10 (C) and TE‐8 (D) parental cell lines treated with different cisplatin concentrations (** P < 0.01, *** P < 0.001). (E, F) MRE11A, UBQLN4, and β‐actin (loading control) comparing EV and MRE11A‐OV in TE‐10 (E) or TE‐8 (F) parental cell lines. (G, H) Drug sensitivity assays for TE‐10 (G) and TE‐8 (H), EV or MRE11A‐OV parental cell lines treated with different cisplatin concentrations (** P < 0.01, *** P < 0.001). (I, J) Drug sensitivity assays for TE‐10 (I) and TE‐8 (J) parental or established cisplatin‐resistant (Cis‐Res) cell lines treated with different cisplatin concentrations (* P < 0.05, *** P < 0.001). (K, L) Western blot analysis for MRE11A, UBQLN4, and β‐actin (loading control) comparing parental and cisplatin‐resistant (Cis‐Res) TE‐10 (K) or TE‐8 (L) ESCC cell lines. (M, N) Quantification of MRE11A protein levels analyzed by western blot comparing parental and cisplatin‐resistant (Cis‐Res) TE‐10 (M) or TE‐8 (N) ESCC cell lines (** P < 0.01, *** P < 0.001). Error bars represent the mean ± SD from n = 3 replicates. Statistical differences were tested using two‐way ANOVA test and post hoc Bonferroni test (C, D, G, H, I, and J) and unpaired two‐tailed t ‐test (M and N).

Article Snippet: Established human ESCC cell lines TE‐4 (CVCL_3337), TE‐8 (CVCL_1766), and TE‐10 (CVCL_1760) were obtained from Keio University.

Techniques: Expressing, Western Blot, Control, Two Tailed Test

Journal: Molecular Oncology

Article Title: Regulation of MRE11A by UBQLN4 leads to cisplatin resistance in patients with esophageal squamous cell carcinoma

doi: 10.1002/1878-0261.12929

Figure Lengend Snippet: High UBQLN4 leads to worse postoperative survival in ESCC patients. (A) Pie chart showing the proportion ESCC cases with high and low UBQLN4 mRNA expression levels in the TCGA ESCA database. ESCC patients were divided according to the z ‐score values into 1) ≥ 1.5, 2) ≤ −1.5, or 3) < 1.5 and > −1.5 for UBQLN4 mRNA expression levels. (B) Comparison of UBQLN4 mRNA expression levels in normal adjacent esophageal epithelia ( n = 11) and primary ESCC tumors ( n = 95) in the TCGA ESCA database (* P < 0.05). (C) The relationship between CNV and UBQLN4 mRNA expression levels was assessed in primary ESCC tumors using the TCGA ESCA database ( n = 95) (NS, not significant, * P < 0.05, *** P < 0.001). (D) Correlation between linear CNV and UBQLN4 mRNA expression levels (Spearman r = 0.579, P < 0.001). (E) Representative images of UBQLN4 protein levels in normal adjacent esophageal epithelia and primary ESCC tumors stained for UBQLN4 by IHC. Scale bars = 50 µm. Right top insets on each picture show a magnification of MRE11A staining. Scale bars = 10 µm. (F) Comparison of H ‐scores for UBQLN4 protein levels in normal adjacent esophageal epithelia ( n = 10) and primary ESCC tumors tissues ( n = 59) (*** P < 0.001). (G) H ‐scores for UBQLN4 protein levels in normal adjacent esophageal epithelia tissue ( n = 10), nonrecurrent primary ESCC ( n = 36), and recurrent primary ESCC tumors ( n = 23) (NS, not significant, *** P < 0.001). (H) Kaplan–Meier curves comparing OS in ESCC patients with low ( n = 39) versus high ( n = 20) UBQLN4 protein levels ( P < 0.01). (I) Kaplan–Meier curves comparing OS in ESCC patients with concurrent low UBQLN4 and high MRE11A ( n = 14) versus high UBQLN4 and low MRE11A ( n = 10) protein levels ( P < 0.001). (J) Representative images of core biopsy tissues from nonresponders (Patients 1 and 2) or responders (Patients 1 and 2) ESCC patients to NAC that were stained for UBQLN4 using IHC. Scale bars = 50 µm. Right top insets on each picture show a magnification of MRE11A staining Scale bars = 10 µm. (K) Comparison of H ‐scores for UBQLN4 protein levels in core biopsy tissues from nonresponders ( n = 53) and responders ( n = 8) ESCC patients to NAC (*** P < 0.001). Error bars represent the mean ± SD. Statistical differences were tested using Mann–Whitney test (B), an unpaired two‐tailed t ‐test with Welch’s correction (F and K), ordinary one‐way ANOVA test and Bonferroni post hoc test (C and G), spearman correlation (D), and log‐rank test (H and I).

Article Snippet: Established human ESCC cell lines TE‐4 (CVCL_3337), TE‐8 (CVCL_1766), and TE‐10 (CVCL_1760) were obtained from Keio University.

Techniques: Expressing, Comparison, Staining, MANN-WHITNEY, Two Tailed Test

Journal: Molecular Oncology

Article Title: Regulation of MRE11A by UBQLN4 leads to cisplatin resistance in patients with esophageal squamous cell carcinoma

doi: 10.1002/1878-0261.12929

Figure Lengend Snippet: UBQLN4 expression determines the sensitivity to cisplatin in ESCC cell lines. (A) Western blot analysis for UBQLN4 and β‐actin (loading control) in TE‐4 cell lines treated with si‐Ctrl or si‐UBQLN4 (pool siRNA). (B) TE‐4 cell lines were treated with si‐Ctrl or si‐UBQLN4 and cell proliferation was analyzed at indicated time points (*** P < 0.001). (C) TE‐4 cell lines were treated with si‐Ctrl or si‐UBQLN4 and analyzed for colony formation. The bar graph showed the quantification of colonies after 12 days of incubation (*** P < 0.001). (D) Drug sensitivity assays comparing si‐Ctrl and si‐UBQLN4 in TE‐4 cell lines treated with different cisplatin concentrations (* P < 0.05). (E, F) Cell proliferation assays were performed at indicated time points in TE‐8 (E) and TE‐10 (F) cell lines with EV or UBQLN4‐OV (* P < 0.05, *** P < 0.001). (G, H) Drug sensitivity assays comparing EV and UBQLN4‐OV in TE‐8 (G) and TE‐10 (H) cell lines treated with different cisplatin concentrations (*** P < 0.001). Error bars represent the mean ± SD from n = 3 replicates. Statistical differences were tested using two‐way ANOVA test and post hoc Bonferroni test (B, D, E, F, G, and H) and unpaired two‐tailed t ‐test (C).

Article Snippet: Established human ESCC cell lines TE‐4 (CVCL_3337), TE‐8 (CVCL_1766), and TE‐10 (CVCL_1760) were obtained from Keio University.

Techniques: Expressing, Western Blot, Control, Incubation, Two Tailed Test

Journal: Molecular Oncology

Article Title: Regulation of MRE11A by UBQLN4 leads to cisplatin resistance in patients with esophageal squamous cell carcinoma

doi: 10.1002/1878-0261.12929

Figure Lengend Snippet: UBQLN4‐OV alleviated DNA damage induced by cisplatin in ESCC cell lines. (A–D) IF staining for 53BP1 was performed in cisplatin‐treated (5 μ m , 12 h) or untreated TE‐10 EV (A), TE‐10 UBQLN4‐OV (B), TE‐8 EV (C), and TE‐8 UBQLN4‐OV (D) cell lines. Shown are 53BP1 (red), DAPI (blue), and the merged images. Scale bars: 10 µm. (E, F) Quantification of the number (#) of 53BP1 foci per cell for TE‐10 (E) and TE‐8 (F) cell lines (NS, not significant, *** P < 0.001). (G‐J) IF staining for γ‐H2AX was performed in cisplatin‐treated (5 μ m , 12 h) or untreated TE‐10 EV (G), TE‐10 UBQLN4‐OV (H), TE‐8 EV (I), and TE‐8 UBQLN4‐OV (J) cell lines. Shown are γ‐H2AX (red), DAPI (blue), and the merged images. Scale bars = 10 µm. (K‐L) Quantification of γ‐H2AX fluorescence intensity per cell for TE‐10 (K) and TE‐8 (L) cell lines (NS, not significant, ** P < 0.01, *** P < 0.001). Error bars represent the mean ± SD from n = 3 replicates. Statistical differences were tested using ordinary one‐way ANOVA test and Bonferroni post hoc test (E, F, K, and L).

Article Snippet: Established human ESCC cell lines TE‐4 (CVCL_3337), TE‐8 (CVCL_1766), and TE‐10 (CVCL_1760) were obtained from Keio University.

Techniques: Staining, Fluorescence