Journal: Molecular Medicine Reports

Article Title: Rsf-1 regulates malignant melanoma cell viability and chemoresistance via NF-κB/Bcl-2 signaling

doi: 10.3892/mmr.2019.10610

Figure Lengend Snippet: Rsf-1 expression in melanoma cell lines and Rsf-1 knockdown efficiency. (A) Western blotting and RT-qPCR analysis revealed the endogenous expression levels of Rsf-1 in three melanoma cell lines (MV3, M14 and A375). (B) Western blotting and RT-qPCR analysis demonstrated that Rsf-1 siRNA transfection significantly decreased Rsf-1 expression levels in MV3 and A375 cells, while Rsf-1 plasmid transfection upregulated the protein and mRNA expression of Rsf-1 in M14 cells. Data were presented as the mean ± standard deviation of at least three experiments. *P<0.05 vs. control. Rsf-1, remodeling and spacing factor 1; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; siRNA, small interfering RNA.

Article Snippet: The targeting sequences were as follows: Rsf-1 siRNA, 5′-GGAAAGACAUCUCUACUAU-3′; and control siRNA, 5′-GCGCGATAGCGCGAATATA-3′. pCMV6-Rsf-1 and control empty plasmids were purchased from OriGene Technologies, Inc. (Rockville, MD, USA), and M14 cells were transfected with 1 µg plasmid using Lipofectamine 3000 according to the manufacturer's protocols.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Transfection, Plasmid Preparation, Standard Deviation, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Rsf-1 regulates malignant melanoma cell viability and chemoresistance via NF-κB/Bcl-2 signaling

doi: 10.3892/mmr.2019.10610

Figure Lengend Snippet: Rsf-1 regulates melanoma cell viability and invasion. (A) An MTT assay (96-well plate) revealed that Rsf-1 depletion decreased the viability of MV3 and A375 cells; conversely, Rsf-1 overexpression increased M14 cell viability. (B) A colony formation assay (culture dish diameter, 6 cm) demonstrated that the colony number was reduced in MV3 and A375 cells transfected with Rsf-1 siRNA, while Rsf-1 overexpression promoted colony formation ability in M14 cells. (C) A Transwell invasion assay (24-well plate) revealed that the number of invading cells decreased following Rsf-1 depletion in MV3 and A375, and increased following Rsf-1 overexpression in M14 cells. Data were presented as the mean ± standard deviation of at least three experiments. *P<0.05 vs. control. Magnification, ×200. Rsf-1, remodeling and spacing factor 1; siRNA, small interfering RNA.

Article Snippet: The targeting sequences were as follows: Rsf-1 siRNA, 5′-GGAAAGACAUCUCUACUAU-3′; and control siRNA, 5′-GCGCGATAGCGCGAATATA-3′. pCMV6-Rsf-1 and control empty plasmids were purchased from OriGene Technologies, Inc. (Rockville, MD, USA), and M14 cells were transfected with 1 µg plasmid using Lipofectamine 3000 according to the manufacturer's protocols.

Techniques: MTT Assay, Over Expression, Colony Assay, Transfection, Transwell Invasion Assay, Standard Deviation, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Rsf-1 regulates malignant melanoma cell viability and chemoresistance via NF-κB/Bcl-2 signaling

doi: 10.3892/mmr.2019.10610

Figure Lengend Snippet: Rsf-1 regulates cell cycle progression of melanoma and expression of MMP2, cyclin E and p-IκB. (A) Cell cycle analysis revealed that Rsf-1 depletion increased the percentage of G1 phase cells and decreased that of S phase cells in MV3 and A375 cell groups; Rsf-1 overexpression in M14 cells exhibited opposing effects. (B) Western blotting demonstrated that Rsf-1 depletion decreased the levels of MMP2, cyclin E and p-IκB in MV3 and A375 cell lines. Rsf-1 overexpression upregulated expression of MMP2, cyclin E and p-IκB in M14 cells. Data were presented as the mean ± standard deviation of at least three experiments. *P<0.05 vs. control. MMP2, matrix metalloproteinase-2; p, phosphorylated; Rsf-1, remodeling and spacing factor 1; siRNA, small interfering RNA.

Article Snippet: The targeting sequences were as follows: Rsf-1 siRNA, 5′-GGAAAGACAUCUCUACUAU-3′; and control siRNA, 5′-GCGCGATAGCGCGAATATA-3′. pCMV6-Rsf-1 and control empty plasmids were purchased from OriGene Technologies, Inc. (Rockville, MD, USA), and M14 cells were transfected with 1 µg plasmid using Lipofectamine 3000 according to the manufacturer's protocols.

Techniques: Expressing, Cell Cycle Assay, Over Expression, Western Blot, Standard Deviation, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Rsf-1 regulates malignant melanoma cell viability and chemoresistance via NF-κB/Bcl-2 signaling

doi: 10.3892/mmr.2019.10610

Figure Lengend Snippet: Rsf-1 regulates chemoresistance and the MMP of melanoma cells. (A) An MTT assay revealed that cell viability was decreased following Rsf-1 depletion in MV3 and A375 cells treated with cisplatin. Rsf-1 overexpression promoted cell viability in M14 cells treated with cisplatin. (B) Annexin V/propidium iodide analysis revealed that the percentage of apoptotic cells was significantly increased in Rsf-1-depleted MV3 and A375 cells compared with controls. Rsf-1 overexpression downregulated cisplatin-induced apoptosis in M14 cells. (C) Rsf-1 overexpression reduced MMP depolarization in M14 cells, while Rsf-1 depletion increased depolarization in MV3 and A375 cells treated with cisplatin. Data were presented as the mean ± standard deviation of at least three experiments. *P<0.05 vs. control. FITC, fluorescein isothiocyanate; MMP, mitochondrial membrane potential, Rsf-1, remodeling and spacing factor 1; siRNA, small interfering RNA.

Article Snippet: The targeting sequences were as follows: Rsf-1 siRNA, 5′-GGAAAGACAUCUCUACUAU-3′; and control siRNA, 5′-GCGCGATAGCGCGAATATA-3′. pCMV6-Rsf-1 and control empty plasmids were purchased from OriGene Technologies, Inc. (Rockville, MD, USA), and M14 cells were transfected with 1 µg plasmid using Lipofectamine 3000 according to the manufacturer's protocols.

Techniques: MTT Assay, Over Expression, Standard Deviation, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Rsf-1 regulates malignant melanoma cell viability and chemoresistance via NF-κB/Bcl-2 signaling

doi: 10.3892/mmr.2019.10610

Figure Lengend Snippet: Rsf-1 regulates Bcl-2 expression via NF-κB signaling. (A) Western blotting revealed that Bax expression levels increased, whereas cIAP1, cIAP2 and Bcl-2 expression decreased significantly following Rsf-1 depletion in MV3 and A375 cells. Rsf-1 overexpression in M14 cells exhibited opposing effects. (B) NF-κB inhibition significantly downregulated p-IκB and NF-κB p65 protein levels in M14 cells. NF-κB inhibition also eradicated the effects of Rsf-1 overexpression on Bcl-2 upregulation. Total IκB expression was markedly altered. Data were presented as the mean ± standard deviation of at least three experiments. *P<0.05 vs. control. Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; cIAP1, cellular inhibitor of apoptosis protein 1; NF-κB, nuclear factor κ-light-chain-enhancer of activated B cells; p, phosphorylated; Rsf-1, remodeling and spacing factor 1; siRNA, small interfering RNA.

Article Snippet: The targeting sequences were as follows: Rsf-1 siRNA, 5′-GGAAAGACAUCUCUACUAU-3′; and control siRNA, 5′-GCGCGATAGCGCGAATATA-3′. pCMV6-Rsf-1 and control empty plasmids were purchased from OriGene Technologies, Inc. (Rockville, MD, USA), and M14 cells were transfected with 1 µg plasmid using Lipofectamine 3000 according to the manufacturer's protocols.

Techniques: Expressing, Western Blot, Over Expression, Inhibition, Standard Deviation, Small Interfering RNA

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IKK? protein is a target of BAG3 regulatory activity in human tumor growth

doi: 10.1073/pnas.0907696107

Figure Lengend Snippet: BAG3 protein levels influence HSP70 association with IKKγ and IKK activity. (A) SAOS-2 cell lysate was immunoprecipitated with an anti-BAG3 polyclonal antibody and analyzed by immunoblotting with anti-HSP70 and anti-IKKγ antibodies. An antibody recognizing annexin I was used as a negative control. (B) M14 whole lysates from untreated or PEITC-treated (5 μM for 30 min) cells were immunoprecipitated with the anti-BAG3 monoclonal antibody AC-2 and analyzed by immunoblotting with anti-BAG3 TOS-2 polyclonal, anti-HSP70, or anti-GAPDH antibody. (C) M14 cells were transfected either with a bag3 overexpressing (o.e.) vector or the void vector. After 48 h, cells were treated as described in B. Immunoprecipitation was performed using an anti-IKKγ polyclonal antibody and analyzed by immunoblotting with anti-HSP70, anti-IKKγ, or anti-GAPDH antibody. The graph depicts densitometry values of bands obtained from two separate experiments. (D) M14 cells were plated at 30% confluency and transfected with scrambled or bag3 siRNA. After 48 h, cell lysates were immunoprecipitated with anti-IKKγ polyclonal antibody and analyzed by immunoblotting with anti-HSP70, anti-IKKγ, anti-IKKα, or anti-IKKβ antibody. The amount of coimmunoprecipitated HSP70 was quantified by densitometry from three separate experiments and normalized to the amount of IKKγ (OD: HSP70/IKKγ). An antibody recognizing GAPDH was used as a negative control. (E) Whole-cell lysates were prepared, and IKK kinase activity was measured after immunoprecipitation with anti-IKKα antibody using GST-IκBα (1–54) as a substrate (34). IKK recovery was determined by immunoblotting with anti-IKKβ antibody. (F) Total RNA was extracted, and ICAM-1 mRNA levels were analyzed by quantitative RT-PCR using β-actin mRNA levels for normalization.

Article Snippet: M14 xenografts were produced on the back of 6-week-old female BALB/c nu/nu mice (Charles River Laboratories) by s.c. injection of 5 × 10 6 M14 cells in 500 μL of Hanks’ balanced salt solution.

Techniques: Activity Assay, Immunoprecipitation, Western Blot, Negative Control, Transfection, Plasmid Preparation, Quantitative RT-PCR

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IKK? protein is a target of BAG3 regulatory activity in human tumor growth

doi: 10.1073/pnas.0907696107

Figure Lengend Snippet: BAG3 protein influences the intracellular levels of IKKγ protein. (A) SAOS-2 cells (30% confluency) were transfected with scrambled or bag3 siRNA. After 96 h, cell lysates were obtained and analyzed by immunoblotting with anti-BAG3 or anti-α-tubulin antibody. (B) M14 cells were plated at 30% confluency and transfected with two different bag3-specific siRNAs (indicated as bag3 siRNA a and b) or with a control scrambled siRNA. After 96 h, lysates were analyzed by immunoblotting with anti-BAG3 or anti-α-tubulin antibody. (C) M14 cells, WT or transfected, with either a bag3 cDNA construct in pcDNA3.1 vector [bag3 overexpressing (o.e.)] or the void vector were analyzed for their content of BAG3 protein by immunoblotting with TOS-2 polyclonal antibody; furthermore, IKKγ levels were checked by immunoblotting and GAPDH was used to monitor equal loading conditions. (D) Total RNA was extracted, and IKKγ mRNA levels were analyzed by quantitative RT-PCR using 18s rRNA levels for normalization. (E) M14 cells were plated at 30% confluency and transfected with bag3 or control scrambled siRNA. After 96 h, cells were treated with MG132 (10 μM) for 2 and 4 h. Cell lysates were analyzed by immunoblotting with anti-BAG3, anti-IKKγ, or anti-α-tubulin antibody.

Article Snippet: M14 xenografts were produced on the back of 6-week-old female BALB/c nu/nu mice (Charles River Laboratories) by s.c. injection of 5 × 10 6 M14 cells in 500 μL of Hanks’ balanced salt solution.

Techniques: Transfection, Western Blot, Control, Construct, Plasmid Preparation, Quantitative RT-PCR

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IKK? protein is a target of BAG3 regulatory activity in human tumor growth

doi: 10.1073/pnas.0907696107

Figure Lengend Snippet: Knockdown of BAG3 protein results in diminishing tumor IKKγ levels, increasing tumor cell apoptosis, inhibiting tumor growth, and enhancing animal survival in M14-xenografted mice. (A) M14 cells (5 × 106) were injected s.c. onto the back of 6-week-old female BALB/c nu/nu mice. Two weeks later, animals were randomized into three groups (10 animals per group) and control PBS (100 μL), bag3siRNA-Ad, or scrRNA-Ad (108 pfu per 100 μL) was injected into the tumors twice a week. After 2 weeks of treatment, four tumors per animal group were excised and BAG3 protein content was analyzed by Western blot. (B) BAG3 and IKKγ protein were detected by immunohistochemistry, whereas apoptosis was analyzed by TUNEL assay, in tumor sections. Representative results are shown. (C) Tumor sizes in bag3siRNA-Ad, scrRNA-Ad, treated, or untreated remaining mice (6 per group) were measured every week using calipers. (D) Evaluation of animal survival was carried out according to Kaplan–Meier analysis. Differences among the treatment groups were analyzed by ANOVA.

Article Snippet: M14 xenografts were produced on the back of 6-week-old female BALB/c nu/nu mice (Charles River Laboratories) by s.c. injection of 5 × 10 6 M14 cells in 500 μL of Hanks’ balanced salt solution.

Techniques: Knockdown, Injection, Control, Western Blot, Immunohistochemistry, TUNEL Assay

Journal: Oncology Letters

Article Title: Expression of neddylation-related proteins in melanoma cell lines and the effect of neddylation on melanoma proliferation

doi: 10.3892/ol.2014.1953

Figure Lengend Snippet: Percentage of cells in GI, G2+S and apoptosis phases in transfected and non-transfected M14 cells.

Article Snippet: M14 and MV3 human melanoma cell lines were purchased from KeyGen Biotech Co. Ltd. (Nanjing, China).

Techniques: Transfection

Journal: Oncology Letters

Article Title: Expression of neddylation-related proteins in melanoma cell lines and the effect of neddylation on melanoma proliferation

doi: 10.3892/ol.2014.1953

Figure Lengend Snippet: Effect of neddylation on proteins involved in the cell cycle or apoptosis of M14 cells. The relative density of proteins were identified using β-actin as the loading control.

Article Snippet: M14 and MV3 human melanoma cell lines were purchased from KeyGen Biotech Co. Ltd. (Nanjing, China).

Techniques:

Journal: Oncology Letters

Article Title: Expression of neddylation-related proteins in melanoma cell lines and the effect of neddylation on melanoma proliferation

doi: 10.3892/ol.2014.1953

Figure Lengend Snippet: Relative density of Bax, P21, P27 and Cyclin D in transfected and non-transfected M14 cells.

Article Snippet: M14 and MV3 human melanoma cell lines were purchased from KeyGen Biotech Co. Ltd. (Nanjing, China).

Techniques: Transfection