Journal: International Journal of Molecular Sciences

Article Title: 6-Shogaol Overcomes Gefitinib Resistance via ER Stress in Ovarian Cancer Cells

doi: 10.3390/ijms24032639

Figure Lengend Snippet: 6-shogaol induces the binding of CHOP on DR5 promoter in ovarian cancer cells. ( A – D ) After A2780 and OVCAR-3 cells were transfected with CHOP siRNA (30 nM, 24 h), WST-1, intracellular Ca 2+ assay, Western blotting analyses, and ChIP assay were performed with/without 6-shogaol (30 μM, 24 h) treatment.; *, p < 0.05. Western blotting analyses were carried out to determine the expression of CHOP, DR4, DR5, and caspase-3 cleavage in 6-shogaol-treated CHOP knockdown cells. β-actin was used as a protein loading control. ( E – H ) After A2780 and OVCAR-3 cells were transfected with DR5 (30 nM, 24 h), WST-1, LDH cytotoxicity assay, intracellular Ca 2+ assay, and Western blotting analyses were performed with/without 6-shogaol (30 μM, 24 h) treatment.; *, p < 0.05. n.s = not significant. Western blotting was carried out to identify the expression of DR5, caspase-3, and caspase-8 cleavage in 6-shogaol-treated DR5 knockdown cells. β-actin was used as a protein loading control.

Article Snippet: The primary antibodies used included β-actin (Santa Cruz, 1:1000, sc-47778), eIF2α (Santa Cruz, 1:1000, sc-133132), GRP78 (Santa Cruz, 1:1000, sc-166490); CD63 (Abcam, 1:1000, ab216130); Nox4 (Proteintech, 1:1000, 14347-1-AP); and cleaved caspase-3 (Cell Signaling, 1:1000, #9664), cleaved caspase-8 (Cell Signaling, 1:1000, #9748), cleaved caspase-9 (Cell Signaling, 1:1000, #20750), p-PERK(Thr980) (Cell Signaling, 1:1000, #3179), PERK (Cell Signaling, 1:1000, #5683), p-eIF2α (Ser51) (Cell Signaling, 1:1000, #3398), ATF4 (Cell Signaling, 1:1000, #11815), CHOP (Cell Signaling, 1:1000, #2895), DR4 (Cell Signaling, 1:1000, #42533), DR5 (Cell Signaling, 1:1000, #8074), E-cadherin (Cell Signaling, 1:1000, #14472), N-cadherin (Cell Signaling, 1:1000, #13116), Slug (Cell Signaling, 1:1000, #9585), Snail (Cell Signaling, 1:1000, #3879), and vimentin (CellSignaling, 1:1000, #5741).

Techniques: Binding Assay, Transfection, Western Blot, Expressing, Knockdown, Control, LDH Cytotoxicity Assay

Journal: Oncology Reports

Article Title: Expression of DR5 and c-FLIP proteins as novel prognostic biomarkers for non-small cell lung cancer patients treated with surgical resection and chemotherapy

doi: 10.3892/or.2019.7355

Figure Lengend Snippet: Expression levels of DR5 and c-FLIP proteins in lung ADC, lung SCC and Non-CLT were detected by IHC. (A and B) Strong positive staining of DR5 was observed in the cell nucleus and cytoplasm of lung ADC and SCC. (C) Positive staining of DR5 was also found in Non-CLT. (D and E) Positive staining of c-FLIP was observed in the cytoplasm of lung ADC and SCC. (F) Negative control staining of c-FLIP was found in Non-CLT. (IHC, DAB staining; original magnification ×400 and ×50). ADC, adenocarcinoma; SCC, squamous cell carcinoma; non-CLT, non-cancerous control lung tissues; IHC, immunohistochemistry.

Article Snippet: A 1:4,000 dilution of the primary antibody to DR5: Apo2/TRAIL-R2/TRICK2/KILLER (rabbit polyclonal antibody, SKU 2019, ProSci) and a 1:300 dilution of the primary antibody to FLIP S/L (rabbit polyclonal antibody, catalog no. SC-8347; Santa Cruz Biotechnology, Inc.) were applied to assess the expression of these two proteins.

Techniques: Expressing, Staining, Negative Control, Control, Immunohistochemistry

Journal: Oncology Reports

Article Title: Expression of DR5 and c-FLIP proteins as novel prognostic biomarkers for non-small cell lung cancer patients treated with surgical resection and chemotherapy

doi: 10.3892/or.2019.7355

Figure Lengend Snippet: Comparison of the expression of DR5 and c-FLIP in lung SCC and lung ADC compared to the noncancerous tissues. The percentages of positive expression of DR5 in the lung SCC and lung ADC were significantly higher than these in the noncancerous tissues (both P<0.001). The percentages of positive expression of c-FLIP in lung SCC and lung ADC were significantly higher than these in the noncancerous tissues (both P<0.001). Positive expression of DR5 exhibited a higher percentage in lung ADC than SCC and positive expression of c-FLIP had a higher percentage in lung SCC compared to ADC, and the differences were statistically significant (both P<0.001). ADC, adenocarcinoma; SCC, squamous cell carcinoma.

Article Snippet: A 1:4,000 dilution of the primary antibody to DR5: Apo2/TRAIL-R2/TRICK2/KILLER (rabbit polyclonal antibody, SKU 2019, ProSci) and a 1:300 dilution of the primary antibody to FLIP S/L (rabbit polyclonal antibody, catalog no. SC-8347; Santa Cruz Biotechnology, Inc.) were applied to assess the expression of these two proteins.

Techniques: Comparison, Expressing

Journal: Oncology Reports

Article Title: Expression of DR5 and c-FLIP proteins as novel prognostic biomarkers for non-small cell lung cancer patients treated with surgical resection and chemotherapy

doi: 10.3892/or.2019.7355

Figure Lengend Snippet: Association between expression of DR5 and c-FLIP proteins and clinicopathological features of the NSCLC patients (n=227).

Article Snippet: A 1:4,000 dilution of the primary antibody to DR5: Apo2/TRAIL-R2/TRICK2/KILLER (rabbit polyclonal antibody, SKU 2019, ProSci) and a 1:300 dilution of the primary antibody to FLIP S/L (rabbit polyclonal antibody, catalog no. SC-8347; Santa Cruz Biotechnology, Inc.) were applied to assess the expression of these two proteins.

Techniques: Expressing

Journal: Oncology Reports

Article Title: Expression of DR5 and c-FLIP proteins as novel prognostic biomarkers for non-small cell lung cancer patients treated with surgical resection and chemotherapy

doi: 10.3892/or.2019.7355

Figure Lengend Snippet: Pairwise association between expression of DR5 and c-FLIP proteins in lung SCC and ADC.

Article Snippet: A 1:4,000 dilution of the primary antibody to DR5: Apo2/TRAIL-R2/TRICK2/KILLER (rabbit polyclonal antibody, SKU 2019, ProSci) and a 1:300 dilution of the primary antibody to FLIP S/L (rabbit polyclonal antibody, catalog no. SC-8347; Santa Cruz Biotechnology, Inc.) were applied to assess the expression of these two proteins.

Techniques: Expressing

Journal: Oncology Reports

Article Title: Expression of DR5 and c-FLIP proteins as novel prognostic biomarkers for non-small cell lung cancer patients treated with surgical resection and chemotherapy

doi: 10.3892/or.2019.7355

Figure Lengend Snippet: Kaplan-Meier curves for overall survival of NSCLC patients as assessed using the log-rank test (all tests were 2-sided). (A) High expression of DR5 was significantly associated with a prolonged overall survival (P=0.029). (B) Negative expression of c-FLIP was significantly associated with a more favorable prognosis (P=0.046), as well as (C) without lymph node metastasis (P=0.001), (D) well and moderate differentiation (P=0.002) and (E) early-stage disease (P<0.001). (F) Histological type was not significantly associated with overall survival (P=0.453). NSCLC, non-small cell lung cancer.

Article Snippet: A 1:4,000 dilution of the primary antibody to DR5: Apo2/TRAIL-R2/TRICK2/KILLER (rabbit polyclonal antibody, SKU 2019, ProSci) and a 1:300 dilution of the primary antibody to FLIP S/L (rabbit polyclonal antibody, catalog no. SC-8347; Santa Cruz Biotechnology, Inc.) were applied to assess the expression of these two proteins.

Techniques: Expressing

Journal: Oncology Reports

Article Title: Expression of DR5 and c-FLIP proteins as novel prognostic biomarkers for non-small cell lung cancer patients treated with surgical resection and chemotherapy

doi: 10.3892/or.2019.7355

Figure Lengend Snippet: Summary of the univariate and multivariate analyses for overall survival in the 227 NSCLC cases.

Article Snippet: A 1:4,000 dilution of the primary antibody to DR5: Apo2/TRAIL-R2/TRICK2/KILLER (rabbit polyclonal antibody, SKU 2019, ProSci) and a 1:300 dilution of the primary antibody to FLIP S/L (rabbit polyclonal antibody, catalog no. SC-8347; Santa Cruz Biotechnology, Inc.) were applied to assess the expression of these two proteins.

Techniques: Expressing

Journal: FEBS Open Bio

Article Title: Death receptor 5 promotes tumor progression in gastric cancer

doi: 10.1002/2211-5463.13725

Figure Lengend Snippet: Bioinformatic analysis of death receptor 5 (DR5) (TNFRSF10B) expression in gastric cancer. (A) Association matrix of genes mRNA expression between DR5 and malignant genes in the Cancer Genome Atlas (TCGA) dataset. (B) The volcano plot of differentiated expressed genes as the 25% top highest versus the 25% lowest expression of DR5. (C) Gene Ontology (GO) annotation of differentiated expressed genes in upregulation passage and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation of differentiated genes in downregulation passage. (D) The correlation of DR5 and tumor mutation burden (TMB) in pan‐cancer comparison of TCGA. (E) The correlation plots between DR5 and TMB score, and microsatellite instability (MSI) score. (F) The correlation plots between DR5 and MKI67, CD44, and mRNAsi (stemness index) score. (G) The heatmap of m6A‐related genes in patients' group with high (G1) or low (G2) expression of DR5, and normal group. (H) The heatmap of immune checkpoint in patients' group with high (G1) or low (G2) expression of DR5, and normal group. (I) The comparison of DR5 expression between groups of nonresponse and response in cancer patients treated with the immune checkpoint inhibitor (IMvigor210). (J) The single‐cell RNA‐Seq (scRNA‐Seq) tSNE reduction in the subcluster of epithelial or tumor cells ( GSE163558 ). (K) The scRNA‐Seq‐based comparison of DR5 expression in normal cells and primary or metastatic gastric cancer cells (LN, lymph node; NT, adjacent normal tissue). Spearman correlation analysis, Mann–Whitney U ‐test, and Kruskal–Wallis test were used. ** P ‐value < 0.01 and *** P ‐value < 0.001.

Article Snippet: The si‐Control (sc‐37007) and siRNA of DR4 (sc‐35218) or DR5 (sc‐40237) were purchased from Santa Cruz.

Techniques: Expressing, Mutagenesis, Comparison, RNA Sequencing, MANN-WHITNEY

Journal: FEBS Open Bio

Article Title: Death receptor 5 promotes tumor progression in gastric cancer

doi: 10.1002/2211-5463.13725

Figure Lengend Snippet: Immunohistochemical staining of death receptor 4 (DR4) and DR5 expression in gastric cancer. (A–C) DR4 expression in normal (A) and tumor tissue from gastric cancer patients (B,C). (D–F) DR5 expression in normal (D) and tumor tissue from gastric cancer patients (E,F). (G–I) DR5 sublocalization in cytoplasm (G), membrane (H), and nucleus (I) in gastric tissue. Scale bars, 100 μm (A–I).

Article Snippet: The si‐Control (sc‐37007) and siRNA of DR4 (sc‐35218) or DR5 (sc‐40237) were purchased from Santa Cruz.

Techniques: Immunohistochemical staining, Staining, Expressing, Membrane

Journal: FEBS Open Bio

Article Title: Death receptor 5 promotes tumor progression in gastric cancer

doi: 10.1002/2211-5463.13725

Figure Lengend Snippet: Relationship between death receptor 4 (DR4)/DR5 expression and clinicopathological features of patients with gastric cancer. The data in parentheses are the percentage of negative or positive gene expression in different subgroups of features. The data outside the parentheses were patients' number used for statistical comparison. The chi‐squared test was used to compare the expression in different clinicopathological features. P < 0.05 was statistically significant and is labeled with an asterisk.

Article Snippet: The si‐Control (sc‐37007) and siRNA of DR4 (sc‐35218) or DR5 (sc‐40237) were purchased from Santa Cruz.

Techniques: Expressing, Gene Expression, Comparison, Labeling, Significance Assay

Journal: FEBS Open Bio

Article Title: Death receptor 5 promotes tumor progression in gastric cancer

doi: 10.1002/2211-5463.13725

Figure Lengend Snippet: Kaplan–Meier curves estimated the influence of death receptor 4 (DR4)/DR5 expression in 240 gastric cancer patients' tissues on overall survival (OS). (A) OS curves stratified by DR4 expression. (B) OS curves stratified by DR5 expression. (C) OS curves stratified by DR4 expression in a subpopulation with DR5‐positive expression. (D) OS curves stratified by combined expression of DR4 and DR5. The log‐rank test and Benjamini–Hochberg methods were performed for pairwise comparison in multiple survival curves. (E) OS curves stratified by DR5 nucleus expression.

Article Snippet: The si‐Control (sc‐37007) and siRNA of DR4 (sc‐35218) or DR5 (sc‐40237) were purchased from Santa Cruz.

Techniques: Expressing, Comparison

Journal: FEBS Open Bio

Article Title: Death receptor 5 promotes tumor progression in gastric cancer

doi: 10.1002/2211-5463.13725

Figure Lengend Snippet: Univariate and multivariate Cox's models for overall survival of GC patients. P < 0.05 was statistically significant and is labeled with an asterisk.

Article Snippet: The si‐Control (sc‐37007) and siRNA of DR4 (sc‐35218) or DR5 (sc‐40237) were purchased from Santa Cruz.

Techniques: Labeling

Journal: FEBS Open Bio

Article Title: Death receptor 5 promotes tumor progression in gastric cancer

doi: 10.1002/2211-5463.13725

Figure Lengend Snippet: Interference in death receptor 5 (DR5) expression impacted the gastric cancer cells malignance. (A,B) DR5 (TNFRSF10B) transcriptomic and proteomic expression in cell lines. (C) Extraction of nucleus proteins for detection of endogenous DR5 expression by western blot, and nuclear membrane structural components ‘Lamin B1’ as a nucleus biomarker. (D,E) Cell viability assay using CCK‐8 and cell migration assay using transwell chamber in SGC7901 and BGC823 transfected with siRNA or negative control (si‐Control). The independent experiments were performed ( n = 3). The Mann–Whitney U ‐test was used. ** P ‐value < 0.01 and *** P ‐value < 0.001. Error bars represent the standard deviation. Scale bars, 100 μm (E).

Article Snippet: The si‐Control (sc‐37007) and siRNA of DR4 (sc‐35218) or DR5 (sc‐40237) were purchased from Santa Cruz.

Techniques: Expressing, Extraction, Western Blot, Membrane, Biomarker Discovery, Viability Assay, CCK-8 Assay, Cell Migration Assay, Transfection, Negative Control, Control, MANN-WHITNEY, Standard Deviation

Journal: FEBS Open Bio

Article Title: Death receptor 5 promotes tumor progression in gastric cancer

doi: 10.1002/2211-5463.13725

Figure Lengend Snippet: Knockdown of death receptor 5 (DR5) inhibited lung metastasis of gastric cancer cells in vivo xenograft mouse model ( n = 7 each). (A) Fluorescence imaging in vivo of mice injected with NC (negative control) or KD (knockdown)‐DR5 BGC823 cells. (B) The comparison of fluorescence intensity in total ( P = 0.046) and lung ( P = 0.042) area of mice with potential metastasis between NC and KD of DR5. (C) The weight curves of mice injected with NC or KD‐DR5 cells ( P = 0.165). (D) The lung tissues dissected from injected mice, the percentage of metastasis to lung, and the average number of metastatic foci in mice ( P = 0.403). The Student's t ‐test was performed. Error bars represent the standard deviation. P < 0.05 was statistically significant and is labeled with an asterisk.

Article Snippet: The si‐Control (sc‐37007) and siRNA of DR4 (sc‐35218) or DR5 (sc‐40237) were purchased from Santa Cruz.

Techniques: Knockdown, In Vivo, Fluorescence, Imaging, Injection, Negative Control, Comparison, Standard Deviation, Labeling