DR4 Antibody - #AF0304

Fig. 3. DR5 upregulation is critical for NaF-induced caspase-8 activation in BEAS-2B cells. (A–C) BEAS-2B cells were stimulated with 2 mM NaF for the indicated time. The mRNA and protein levels of DR4 and DR5 were tested using qRT-PCR and Western blot, respectively. (D) Cells were treated with or without 2 mM NaF for 9 h and cell surface expression of DR5 was determined by flow cytometry using an FITC-labeled anti-DR5 antibody. (E) The BEAS-2B cells transfected with scramble siRNA or DR5 siRNA were further stimulated with 2 mM NaF for 9 h, Western blotting was performed to detect the expression of DR5. BEAS-2B cells were transfected with scramble or DR5 siRNA (48 h), treated with 2 mM NaF (24 h). Enzymatic activity assays for caspase-8 (F) and caspase-3 (G). (H) Percentages of cells that underwent apoptosis for BEAS-2B cells treated with NaF. (I) Sum of Annexin V+PI− (Q4 quadrant) and Annexin V+PI+ (Q2 quadrant) populations is represented as the percentage of Annexin V+ cells. Results are expressed as means ± S.D and are representative of three independent experiments. **p < 0.01.

Figure 5. | Amuc_1434* mediated the activation of the apoptosis pathway in LS174T cells. (A) The expression of death receptor 4 (DR4), death receptor 5 (DR5), cysteinyl aspartate specific proteinase 8(caspase 8) and cysteinyl aspartate specific proteinase 3 (caspase 3) induced by Amuc_1434* in LS174T cells was dependent on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). (a) LS174T cells were treated with 8 and 64 µg/mL Amuc_1434* for 24 h, respectively. The cell lysates were analyzed by Western blot.

Figure 5 C20/C22 increased the sensitivity of HepG2 cells to TRAIL-induced apoptosis. (A,B) WB analysis of the expression of DR4 and 5 in HepG2 cells that were treated with 2 μM C20/C22 for 0, 6, 12, and 24 h. ImageJ software was used to calculate the gray value (n = 3). (C,D) The immunofluorescence assays used to determine the intracellular and cell surface distribution of DR4/5. (E,F) Annexin V/PI double staining was used to detect the apoptotic cells after the indicated treatments (n = 3). (G,H) WB analysis of the expression of Fas in HepG2 cells treated with C20/C22 for 0, 6, 12, and 24 h. (I) The immunofluorescence assays used to determine the intracellular and cell surface distribution of Fas. (J,K) Flow cytometry analysis of the apoptotic cells after the indicated treatments (n = 3). Scale bars, 10 µm. Data shown correspond to the mean ± SD of three independent experiments. The p-value for all datasets was analyzed by one-way ANOVA followed by Tukey’s test using GraphPad Prism version 8.00, except the data in (B) whose p-value was analyzed by nonparametric Dunnett’s test using GraphPad Prism version 8.00. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns nonsignificant vs. 0 h/control group.

Figure 5 C20/C22 increased the sensitivity of HepG2 cells to TRAIL-induced apoptosis. (A,B) WB analysis of the expression of DR4 and 5 in HepG2 cells that were treated with 2 μM C20/C22 for 0, 6, 12, and 24 h. ImageJ software was used to calculate the gray value (n = 3). (C,D) The immunofluorescence assays used to determine the intracellular and cell surface distribution of DR4/5. (E,F) Annexin V/PI double staining was used to detect the apoptotic cells after the indicated treatments (n = 3). (G,H) WB analysis of the expression of Fas in HepG2 cells treated with C20/C22 for 0, 6, 12, and 24 h. (I) The immunofluorescence assays used to determine the intracellular and cell surface distribution of Fas. (J,K) Flow cytometry analysis of the apoptotic cells after the indicated treatments (n = 3). Scale bars, 10 µm. Data shown correspond to the mean ± SD of three independent experiments. The p-value for all datasets was analyzed by one-way ANOVA followed by Tukey’s test using GraphPad Prism version 8.00, except the data in (B) whose p-value was analyzed by nonparametric Dunnett’s test using GraphPad Prism version 8.00. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns nonsignificant vs. 0 h/control group.

Figure 2.| Expression of DR4, DR5, cleaved caspase‑3 and c‑Myc in HepG2 cells. (A) The protein expression of DR4, DR5, cleaved caspase‑3 and c‑Myc was investigated using western blotting. Quantification and statistical analysis was performed on (B) DR4, (C) DR5, (D) cleaved caspase‑3 and (E) c‑Myc. n=3, *P<0.05, **P<0.001 vs. control group; #P<0.05, ##P<0.001 vs. control + DDP group. DR. death receptor; DDP, cisplatin; SMS2, sphyngomyelin synthase 2; c‑Myc, avian myelocytomatosis viral oncogene homolog.