Journal: Frontiers in Cell and Developmental Biology

Article Title: Isothiocyanate From Moringa oleifera Seeds Inhibits the Growth and Migration of Renal Cancer Cells by Regulating the PTP1B-dependent Src/Ras/Raf/ERK Signaling Pathway

doi: 10.3389/fcell.2021.790618

Figure Lengend Snippet: MIC-1 induces apoptosis and cell cycle arrest in RCC cells. 786-O cells and 769-P cells were treated with MIC-1 (0, 2, or 4 μM) for 48 h (A,B) The cell apoptosis of 786-O cells and 769-P cells were determined by flow cytometry. (C) The protein expression of Bax and Bcl-2 in 786-O cells and 769-P cells were detected by western blotting. β -actin served as a loading control. (D,E) The cell cycle distribution of 786-O cells and 769-P cells were determined by flow cytometry. (F) The protein expression of CDK2 and cyclinA2 in 786-O cells, and the protein expression of cyclinD1 in 769-P cells were detected by western blotting. β -actin served as a loading control. Values represent the means ± SEM from three independent experiments and statistical analysis was performed by one-way ANOVA; * p < 0.05, ** p < 0.01, *** p < 0.001: MIC-1 compared with the control (0 μM).

Article Snippet: Equal amounts of protein were separated by 8% or 10% SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, United States), blocked with 5% bovine serum albumin (BSA) (Solarbio) for 30 min, and then incubated overnight at 4°C with primary antibodies targeting Bax, Bcl-2, CDK2, K-Ras, and cyclinD1 (Cat. #sc-7480, sc-7382, sc-6248, sc-30, and sc-8396, respectively; 1:1,000 dilution; Santa Cruz Biotechnology, CA, United States), PTP1B and p-Src (Tyr529) (Cat. #ab244207, 32,078; 1:2000 dilution; Abcam, Cambridge, MA, United States), Src, p-Src (Tyr416), cyclinA2, Raf, p-Raf, ERK1/2, p-ERK1/2, and β -actin (Cat. #2109, 59,548, 4,656, 9422S, 9,431, 4,695, 4,370, and 3,700, respectively; 1:2000 dilution; Cell Signaling Technology, Danvers, MA, United States).

Techniques: Flow Cytometry, Expressing, Western Blot, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Isothiocyanate From Moringa oleifera Seeds Inhibits the Growth and Migration of Renal Cancer Cells by Regulating the PTP1B-dependent Src/Ras/Raf/ERK Signaling Pathway

doi: 10.3389/fcell.2021.790618

Figure Lengend Snippet: MIC-1 inhibits the growth and migration of 786-O cells by inhibiting the PTP1B-mediated activation of the Src/Ras/Raf/ERK signaling pathway. (A) 786-O cells were treated with MIC-1 (0, 2, or 4 μM) for 48 h, and the expression of Src, p-Src (Tyr416), p-Src (Tyr529), K-Ras, Raf, p-Raf, ERK1/2, and p-ERK1/2 was detected by western blotting. β -actin served as a loading control. (B) Detection of PTP1B mRNA and protein expression in 786-O cells transfected with a PTP1B cDNA expression vector by RT-PCR and western blotting, respectively. (C) Untransfected and PTP1B-overexpressing 786-O cells were treated with different concentrations of MIC-1 (0, 2, or 4 μM) for 48 h after which cell viability was analyzed by the MTT assay. (D) Clonogenic ability was analyzed by a colony formation assay. (E) Untransfected and PTP1B-overexpressing 786-O cells were treated with different concentrations of MIC-1 (0, 1, or 2 μM) for 24 h after which migratory ability was analyzed using a wound healing assay. (F) Untransfected and PTP1B-overexpressing 786-O cells were treated with MIC-1 (0, 2, or 4 μM) for 48 h, and the expression of Src, p-Src (Tyr416), p-Src (Tyr529), ERK1/2, and p-ERK1/2 was detected by western blotting. β -actin served as a loading control. Values represent the means ± SEM from three independent experiments and statistical analysis was performed by one-way ANOVA; * p < 0.05, ** p < 0.01, *** p < 0.001: MIC-1-treated cells compared with control 786-O cells (0 μM) or PTP1B-overexpressing 786-O cells (0 μM); PTP1B-overexpressing 786-O cells compared with control 786-O cells.

Article Snippet: Equal amounts of protein were separated by 8% or 10% SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, United States), blocked with 5% bovine serum albumin (BSA) (Solarbio) for 30 min, and then incubated overnight at 4°C with primary antibodies targeting Bax, Bcl-2, CDK2, K-Ras, and cyclinD1 (Cat. #sc-7480, sc-7382, sc-6248, sc-30, and sc-8396, respectively; 1:1,000 dilution; Santa Cruz Biotechnology, CA, United States), PTP1B and p-Src (Tyr529) (Cat. #ab244207, 32,078; 1:2000 dilution; Abcam, Cambridge, MA, United States), Src, p-Src (Tyr416), cyclinA2, Raf, p-Raf, ERK1/2, p-ERK1/2, and β -actin (Cat. #2109, 59,548, 4,656, 9422S, 9,431, 4,695, 4,370, and 3,700, respectively; 1:2000 dilution; Cell Signaling Technology, Danvers, MA, United States).

Techniques: Migration, Activation Assay, Expressing, Western Blot, Control, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, MTT Assay, Colony Assay, Wound Healing Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Isothiocyanate From Moringa oleifera Seeds Inhibits the Growth and Migration of Renal Cancer Cells by Regulating the PTP1B-dependent Src/Ras/Raf/ERK Signaling Pathway

doi: 10.3389/fcell.2021.790618

Figure Lengend Snippet: MIC-1 inhibits the growth of subcutaneous xenograft tumors in vivo . 786-O cells were injected subcutaneously into nude mice to evaluate the in vivo effects of MIC-1. (A) Representative images of the tumors are shown. Changes in tumor volume (B) , tumor weight (C) , body weight (D) , water intake (E) , and food intake (F) in the different groups of nude mice. (G) Western blotting analysis was used to determine the protein levels of Bax and Bcl-2, with β -actin serving as a loading control. The results are expressed as the means ± SEM from three independent experiments and statistical analysis was performed by one-way ANOVA or two-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001: MIC-1 compared with the control.

Article Snippet: Equal amounts of protein were separated by 8% or 10% SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, United States), blocked with 5% bovine serum albumin (BSA) (Solarbio) for 30 min, and then incubated overnight at 4°C with primary antibodies targeting Bax, Bcl-2, CDK2, K-Ras, and cyclinD1 (Cat. #sc-7480, sc-7382, sc-6248, sc-30, and sc-8396, respectively; 1:1,000 dilution; Santa Cruz Biotechnology, CA, United States), PTP1B and p-Src (Tyr529) (Cat. #ab244207, 32,078; 1:2000 dilution; Abcam, Cambridge, MA, United States), Src, p-Src (Tyr416), cyclinA2, Raf, p-Raf, ERK1/2, p-ERK1/2, and β -actin (Cat. #2109, 59,548, 4,656, 9422S, 9,431, 4,695, 4,370, and 3,700, respectively; 1:2000 dilution; Cell Signaling Technology, Danvers, MA, United States).

Techniques: In Vivo, Injection, Western Blot, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Isothiocyanate From Moringa oleifera Seeds Inhibits the Growth and Migration of Renal Cancer Cells by Regulating the PTP1B-dependent Src/Ras/Raf/ERK Signaling Pathway

doi: 10.3389/fcell.2021.790618

Figure Lengend Snippet: Effect of MIC-1 on PTP1B-related Src/Ras/Raf/ERK signaling pathway in non-renal cancer cells. (A) HCT-116 cells, Hep-G2 cells, and A431 cells were treated with MIC-1 (0 or 4 μM) for 48 h, and the expression of Src, p-Src (Tyr416), K-Ras, ERK1/2, and p-ERK1/2 was detected by western blotting. β -actin served as a loading control. Quantification of the relative levels of p-Src (Tyr416) (B) , K-Ras (C) , and p-ERK1/2 (D) ; each value was normalized to that of Src, β -actin, and ERK1/2, respectively. Values represent the means ± SEM from three independent experiments and statistical analysis was performed by unpaired two-tailed Student’s t tests.

Article Snippet: Equal amounts of protein were separated by 8% or 10% SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, United States), blocked with 5% bovine serum albumin (BSA) (Solarbio) for 30 min, and then incubated overnight at 4°C with primary antibodies targeting Bax, Bcl-2, CDK2, K-Ras, and cyclinD1 (Cat. #sc-7480, sc-7382, sc-6248, sc-30, and sc-8396, respectively; 1:1,000 dilution; Santa Cruz Biotechnology, CA, United States), PTP1B and p-Src (Tyr529) (Cat. #ab244207, 32,078; 1:2000 dilution; Abcam, Cambridge, MA, United States), Src, p-Src (Tyr416), cyclinA2, Raf, p-Raf, ERK1/2, p-ERK1/2, and β -actin (Cat. #2109, 59,548, 4,656, 9422S, 9,431, 4,695, 4,370, and 3,700, respectively; 1:2000 dilution; Cell Signaling Technology, Danvers, MA, United States).

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