Journal: Biomedicines

Article Title: RICTOR Affects Melanoma Tumorigenesis and Its Resistance to Targeted Therapy

doi: 10.3390/biomedicines9101498

Figure Lengend Snippet: RICTOR-mediated melanoma spheroid growth induces an MIC phenotype ( A , B ) Colo829, A375, and SkMel5 cell lines were cultivated under the same condition as and treated with DMSO or an mTOR inhibitor (OSI-027 1 μM) for 10 days and counted (scale bar represents 1000 μm). Graphs represent the mean ± s.d. of an experiment in triplicate (similar results were obtained in three independent experiments). Significance was calculated using the Student’s t -test: * p < 0.05, ** p < 0.01. ( C ) The expressions of Myc-tagged RICTOR, RICTOR, phosphorylated STAT3 (Y705), total STAT3, MITF, SOX10, and AXL were assessed by Western blotting in Colo829 cells transfected with an empty vector (Control) or with a vector expressing human RICTOR cDNA (RICTOR). ( D ) Relative mRNA expressions of the different tumor-initiating cell markers (JARID, Tie1, and BMPR1), as well as RICTOR, were assessed by qPCR and normalized to GAPDH in Colo829 cells transfected with an empty vector (Control) or with a vector expressing human RICTOR cDNA (RICTOR).

Article Snippet: Membranes were probed with the following primary antibodies: Myc-Tag, RICTOR, ACTIN, p-AKT (S473), AKT, p-ERK, ERK, pSTAT3 (Y705), STAT3, SOX10, AXL (Cell Signaling Technology, Danvers, MA, USA), and MITF (OriGene Technologies Inc, Rockville, MD, USA).

Techniques: Western Blot, Transfection, Plasmid Preparation, Control, Expressing

Journal: International Journal of Medical Sciences

Article Title: Hydroxyurea regulates the development and survival of B16 Melanoma Cells by upregulating MiR-7013-3p

doi: 10.7150/ijms.52177

Figure Lengend Snippet: Primers used for RT-qPCR

Article Snippet: Next, the proteins were transferred to PVDF membranes for 1 h. After protein transfer, the PVDF membranes were blocked in Tris-buffered saline with Tween-20 (TBST) containing 5% bovine serum albumin (BSA) for 90 min and then incubated with rabbit polyclonal MITF IgG (ImmunoWay Biotechnology Company, Newark, DE, USA), rabbit polyclonal TYR IgG (Abcam, Cambridge, MA, USA) and rabbit polyclonal GAPDH (Cell Signaling Technology, USA) primary antibodies at 4 °C overnight before being washed 3 times with TBST for 15 min per wash. After being washed with TBST, the membrane were incubated with secondary antibodies for 55 min, after which the membranes were washed with TBST 3 times for 15 min each.

Techniques: Sequencing

Journal: International Journal of Medical Sciences

Article Title: Hydroxyurea regulates the development and survival of B16 Melanoma Cells by upregulating MiR-7013-3p

doi: 10.7150/ijms.52177

Figure Lengend Snippet: Identification of differentially expressed miRNAs after HU treatment that bind to MITF. After stimulation of B16 cells with HU (400 µM) for 24 h, MITF gene expression levels (A) and miRNA expression levels were altered (B). (C) Predictive maps of complementary targeting sites for miR-7013-3p and the MITF mRNA 3'UTR. (D) Luciferase activity after cotransfection with the MITF-3'UTR WT plasmid and miR-7013-3p mimic. The data summarize three independent experiments and are given as the mean±SD. * P <0.05.

Article Snippet: Next, the proteins were transferred to PVDF membranes for 1 h. After protein transfer, the PVDF membranes were blocked in Tris-buffered saline with Tween-20 (TBST) containing 5% bovine serum albumin (BSA) for 90 min and then incubated with rabbit polyclonal MITF IgG (ImmunoWay Biotechnology Company, Newark, DE, USA), rabbit polyclonal TYR IgG (Abcam, Cambridge, MA, USA) and rabbit polyclonal GAPDH (Cell Signaling Technology, USA) primary antibodies at 4 °C overnight before being washed 3 times with TBST for 15 min per wash. After being washed with TBST, the membrane were incubated with secondary antibodies for 55 min, after which the membranes were washed with TBST 3 times for 15 min each.

Techniques: Gene Expression, Expressing, Luciferase, Activity Assay, Cotransfection, Plasmid Preparation

Journal: International Journal of Medical Sciences

Article Title: Hydroxyurea regulates the development and survival of B16 Melanoma Cells by upregulating MiR-7013-3p

doi: 10.7150/ijms.52177

Figure Lengend Snippet: MiR-7013-3p inhibited pigmentation. (A) Relative MITF and pigmentation-related gene expression after transfection with the miR-7013-3p mimic. (B) Relative MITF and pigmentation-related gene expression after transfection with MITF siRNA. (C) Melanin levels in B16 melanoma cells transfected with the miR-7013-3p mimic and mimic NC. (D, E) Relative protein expression levels of MITF after transfection with the miRNA-7013-p mimic and mimic NC. The data summarize three independent experiments and are given as the mean±SD. *P<0.05.

Article Snippet: Next, the proteins were transferred to PVDF membranes for 1 h. After protein transfer, the PVDF membranes were blocked in Tris-buffered saline with Tween-20 (TBST) containing 5% bovine serum albumin (BSA) for 90 min and then incubated with rabbit polyclonal MITF IgG (ImmunoWay Biotechnology Company, Newark, DE, USA), rabbit polyclonal TYR IgG (Abcam, Cambridge, MA, USA) and rabbit polyclonal GAPDH (Cell Signaling Technology, USA) primary antibodies at 4 °C overnight before being washed 3 times with TBST for 15 min per wash. After being washed with TBST, the membrane were incubated with secondary antibodies for 55 min, after which the membranes were washed with TBST 3 times for 15 min each.

Techniques: Gene Expression, Transfection, Expressing

Journal: International Journal of Medical Sciences

Article Title: Hydroxyurea regulates the development and survival of B16 Melanoma Cells by upregulating MiR-7013-3p

doi: 10.7150/ijms.52177

Figure Lengend Snippet: MiR-7013-3p inhibited proliferation in B16 melanoma cells. (A) The RTCA system was used to measure B16 melanoma cell proliferation. (B) Relative ratio of cell proliferation after transfection with the miR-7013-3p mimic compared with the mimic NC. (C) Flow cytometry was used to measure the cell cycle distribution. (D) qRT-PCR was used to analyze relative CDK2 gene expression after transfection with the miR-7013-3p mimic and MITF siRNA. The data summarize three independent experiments and are given as the mean±SD. *P<0.05.

Article Snippet: Next, the proteins were transferred to PVDF membranes for 1 h. After protein transfer, the PVDF membranes were blocked in Tris-buffered saline with Tween-20 (TBST) containing 5% bovine serum albumin (BSA) for 90 min and then incubated with rabbit polyclonal MITF IgG (ImmunoWay Biotechnology Company, Newark, DE, USA), rabbit polyclonal TYR IgG (Abcam, Cambridge, MA, USA) and rabbit polyclonal GAPDH (Cell Signaling Technology, USA) primary antibodies at 4 °C overnight before being washed 3 times with TBST for 15 min per wash. After being washed with TBST, the membrane were incubated with secondary antibodies for 55 min, after which the membranes were washed with TBST 3 times for 15 min each.

Techniques: Transfection, Flow Cytometry, Quantitative RT-PCR, Gene Expression

Journal: International Journal of Medical Sciences

Article Title: Hydroxyurea regulates the development and survival of B16 Melanoma Cells by upregulating MiR-7013-3p

doi: 10.7150/ijms.52177

Figure Lengend Snippet: miR-7013-3p promoted apoptosis in B16 melanoma cells. (A-C) Flow cytometry was used to measure apoptosis. The apoptosis rate of the cells of the miR-7013-3p overexpression group (Fig. B) was higher than that in the NC group (Fig. A). (D) The qRT-PCR results show the relative expression of BCL2 genes after transfection with the miR-7013-3p mimic and MITF siRNA. The data summarize three independent experiments and are given as the mean±SD. *P<0.05.

Article Snippet: Next, the proteins were transferred to PVDF membranes for 1 h. After protein transfer, the PVDF membranes were blocked in Tris-buffered saline with Tween-20 (TBST) containing 5% bovine serum albumin (BSA) for 90 min and then incubated with rabbit polyclonal MITF IgG (ImmunoWay Biotechnology Company, Newark, DE, USA), rabbit polyclonal TYR IgG (Abcam, Cambridge, MA, USA) and rabbit polyclonal GAPDH (Cell Signaling Technology, USA) primary antibodies at 4 °C overnight before being washed 3 times with TBST for 15 min per wash. After being washed with TBST, the membrane were incubated with secondary antibodies for 55 min, after which the membranes were washed with TBST 3 times for 15 min each.

Techniques: Flow Cytometry, Over Expression, Quantitative RT-PCR, Expressing, Transfection

Journal: International Journal of Medical Sciences

Article Title: Hydroxyurea regulates the development and survival of B16 Melanoma Cells by upregulating MiR-7013-3p

doi: 10.7150/ijms.52177

Figure Lengend Snippet: MiR-7013-3p inhibited cell migration and invasion. (A-E) The migration of B16 melanoma cells was measured by a wound-healing assay. (F) The qRT-PCR results show the relative expression of BCL2 genes after transfection with the miR-7013-3p mimic and MITF siRNA. The data summarize three independent experiments and are given as the mean±SD. *P<0.05.

Article Snippet: Next, the proteins were transferred to PVDF membranes for 1 h. After protein transfer, the PVDF membranes were blocked in Tris-buffered saline with Tween-20 (TBST) containing 5% bovine serum albumin (BSA) for 90 min and then incubated with rabbit polyclonal MITF IgG (ImmunoWay Biotechnology Company, Newark, DE, USA), rabbit polyclonal TYR IgG (Abcam, Cambridge, MA, USA) and rabbit polyclonal GAPDH (Cell Signaling Technology, USA) primary antibodies at 4 °C overnight before being washed 3 times with TBST for 15 min per wash. After being washed with TBST, the membrane were incubated with secondary antibodies for 55 min, after which the membranes were washed with TBST 3 times for 15 min each.

Techniques: Migration, Wound Healing Assay, Quantitative RT-PCR, Expressing, Transfection