Fig 1: Visfatin-increased AREG levels attenuate the cytotoxicity of HCT-116 CRC cells in response to 5-FU. (A) HCT-116 CRC cells were pretreated with either vehicle (PBS) or Visfatin (50 ng/mL) for 1 h. They were then either maintained as controls or treated with 5-FU (5, 10, and 20 μM) for 24 h. (B) HCT-116 CRC cells were transfected with AREG-specific siRNA for 48 h. Subsequently, the cells were pretreated with PBS or Visfatin (50 ng/mL) for 1 h and then either maintained as controls or treated with 5-FU (10 μM) for 24 h. (A,B) The viability of treated cells was assessed using the MTT assay. The data were calculated from the results of three independent experiments and are presented as the mean ± SEM. The results were considered statistically significant when p < 0.05. *, compared to the untreated control cells (CL). #, compared to cells treated with 5-FU alone. **, compared to cells treated with Visfatin and 5-FU.
Fig 2: STAT3 affects AREG upregulation and subsequent 5-FU-initiated cytotoxicity in Visfatin-stimulated HCT-116 CRC cells. (A,B) HCT-116 CRC cells were transfected with control (si-CL)- or STAT3 (si-STAT3)-specific siRNA for 48 h and then either maintained as controls or treated with Visfatin (50 ng/mL) for 8 h. The mRNA (A) and protein (B) expression of AREG was determined using real-time PCR and Western blot analysis, respectively. (C) HCT-116 CRC cells were maintained as controls (CL) or treated with Visfatin (50 ng/mL) for 1, 4, 8, and 12 h and then the phosphorylation of STAT3 was examined sing Western blot analysis. (D) HCT-116 CRC cells were transfected with control (si-CL)- or STAT3 (si-STAT3)-specific siRNA for 48 h and then pretreated with vehicle (PBS) or Visfatin (50 ng/mL) for 1 h. Subsequently, the cells were either maintained as controls or treated with 5-FU (10 μM) for 24 h. The viability of treated cells was examined using the MTT assay. The data in (A–D) were calculated from the results of three independent experiments and are presented as the mean ± SEM. The results in (B,C) were obtained from three independent experiments, all of which exhibited similar outcomes. The results were considered statistically significant when p < 0.05. *, compared to (A,B) cells treated with si-CL-CL and (C,D) the untreated control cells. #, compared to (A,B) cells treated with si-CL and Visfatin and (D) the cells treated with 5-FU alone. **, compared to (D) cells treated with si-CL, Visfatin, and 5-FU.
Fig 3: Visfatin upregulates AREG expression in HCT-116 CRC cells. HCT-116 CRC cells were either maintained as untreated controls or treated with Visfatin (1, 25, 50, and 100 ng/mL) for 1, 4, 8, and 24 h. The mRNA (A,B) and protein (C,D) expression of AREG was subsequently determined using real-time PCR and Western blot analysis, respectively. The data in (A–D) were calculated from the results of three independent experiments and are presented as the mean ± SEM. The results in (C,D) were obtained from three independent experiments, all of which exhibited similar outcomes. The results were considered statistically significant when p < 0.05. *, compared to the untreated control cells (CL).
Fig 4: CREB affects AREG upregulation and subsequent 5-FU-initiated cytotoxicity in Visfatin-stimulated HCT-116 CRC cells. (A,B) HCT-116 CRC cells were transfected with control (si-CL)- or CREB (si-CREB)-specific siRNA for 48 h and then either maintained as controls or treated with Visfatin (50 ng/mL) for 8 h. The mRNA (A) and protein (B) expression of AREG was determined using real-time PCR and Western blot analysis, respectively. (C) The HCT-116 CRC cells were maintained as controls (CL) or treated with Visfatin (50 ng/mL) for 1, 4, 8, and 12 h, and then the phosphorylation of CREB was examined using Western blot analysis. (D) The HCT-116 CRC cells were transfected with control (si-CL)- or CREB (si-CREB)-specific siRNA for 48 h and then pretreated with vehicle (PBS) or Visfatin (50 ng/mL) for 1 h. Subsequently, the cells were either maintained as controls or treated with 5-FU (10 μM) for 24 h. The viability of treated cells was examined using the MTT assay. The data in (A–D) were calculated from the results of three independent experiments and are presented as the mean ± SEM. The results in (B,C) were obtained from three independent experiments, all of which exhibited similar outcomes. The results were considered statistically significant when p < 0.05. *, compared to (A,B) cells treated with si-CL-CL and (C,D) the untreated control cells. #, compared to (A,B) cells treated with si-CL and Visfatin and (D) cells treated with 5-FU alone. **, compared to (D) cells treated with si-CL, Visfatin, and 5-FU.
Fig 5: JNK1/2 and p38 signaling regulate AREG upregulation in HCT-116 CRC cells under Visfatin stimulation. (A,B) HCT-116 CRC cells were pretreated with DMSO (vehicle), PD98059 (an ERK1/2 inhibitor at 25 μM), SP600125 (a JNK1/2 inhibitor at 20 μM), or SB203580 (a p38 inhibitor at 10 μM) for 30 min and then either maintained as controls or treated with Visfatin (50 ng/mL) for 8 h. The mRNA (A) and protein (B) expression of AREG was determined using real-time PCR and Western blot analysis, respectively. (C) HCT-116 CRC cells were maintained as controls (CL) or treated with Visfatin (50 ng/mL) for 0.5, 1, 2, and 4 h, and then the phosphorylation of JNK1/2 and p38 kinases was examined using Western blot analysis. The data in (A–C) were calculated from the results of three independent experiments and are presented as the mean ± SEM. The results in (B,C) were obtained from three independent experiments, all of which exhibited similar outcomes. The results were considered statistically significant when p < 0.05. *, compared to the untreated control cells (CL). #, compared to cells treated with DMAO.
from Cell Signaling Technology for Human Amphiregulin Recombinant Protein