Journal: PLoS ONE

Article Title: Direct Quantification of mRNA and miRNA from Cell Lysates Using Reverse Transcription Real Time PCR: A Multidimensional Analysis of the Performance of Reagents and Workflows

doi: 10.1371/journal.pone.0072463

Figure Lengend Snippet: Extraction efficiency in the detection of mRNA and miRNA. a

Article Snippet: The NCI-60 cell lines from 7 tissues of origin- A549 (lung, ATCC CCL185), HCT116 (colon, ATCC CCL247, gift from Prof. Sim Kim Ping), OVCAR8 (ovarian, gift from Prof. Jean Paul Thiery ), 786–0 (renal, ATCC CRL1932, gift from Dr. Deng Lih Wen), M14 (melanoma, gift from Prof. Marie-Véronique Clément ), PC3 (prostate, ATCC CRL1435), U251 (central nervous system, ATCC 09063001) were grown in Dulbecco's minimum essential medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (PS, Sigma).

Techniques: Extraction

Journal: PLoS ONE

Article Title: Direct Quantification of mRNA and miRNA from Cell Lysates Using Reverse Transcription Real Time PCR: A Multidimensional Analysis of the Performance of Reagents and Workflows

doi: 10.1371/journal.pone.0072463

Figure Lengend Snippet: M14 cell line (10 4 cells) was cultured and lysed with the Trizol reagent, in-house and Bio-Rad cell-to-Ct reagents. After reverse transcription, the cDNA samples were amplified by RPS8, ZNF706, RNF167, hsa-miR-29a, hsa-miR-197, hsa-miR-297 PCR assays. Graph bars represent (A) CVs and (B) detected Ct values of the biological quadruplicates. The error bars referred to the S.E.M and the significant differences in CVs between the Trizol reagent and cell-to-Ct reagents were calculated using unpaired, two tailed student's t-test. *, p<0.05; **, p<0.005.

Article Snippet: The NCI-60 cell lines from 7 tissues of origin- A549 (lung, ATCC CCL185), HCT116 (colon, ATCC CCL247, gift from Prof. Sim Kim Ping), OVCAR8 (ovarian, gift from Prof. Jean Paul Thiery ), 786–0 (renal, ATCC CRL1932, gift from Dr. Deng Lih Wen), M14 (melanoma, gift from Prof. Marie-Véronique Clément ), PC3 (prostate, ATCC CRL1435), U251 (central nervous system, ATCC 09063001) were grown in Dulbecco's minimum essential medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (PS, Sigma).

Techniques: Cell Culture, Reverse Transcription, Amplification, Two Tailed Test

Journal: PLoS ONE

Article Title: Direct Quantification of mRNA and miRNA from Cell Lysates Using Reverse Transcription Real Time PCR: A Multidimensional Analysis of the Performance of Reagents and Workflows

doi: 10.1371/journal.pone.0072463

Figure Lengend Snippet: M14 cell line cultured in 96-wells at various densities (10, 100, 1000, 10,000 and 100,000 cells per well) were lysed directly by (A) in-house or (B) Bio-Rad lysis buffer. The cDNA samples generated from cell lysates were amplified by PCR assays from set 2. Standard curves for isolated RNAs were plotted as Ct versus Log (cells per RT). The experiments were conducted with biological duplicates.

Article Snippet: The NCI-60 cell lines from 7 tissues of origin- A549 (lung, ATCC CCL185), HCT116 (colon, ATCC CCL247, gift from Prof. Sim Kim Ping), OVCAR8 (ovarian, gift from Prof. Jean Paul Thiery ), 786–0 (renal, ATCC CRL1932, gift from Dr. Deng Lih Wen), M14 (melanoma, gift from Prof. Marie-Véronique Clément ), PC3 (prostate, ATCC CRL1435), U251 (central nervous system, ATCC 09063001) were grown in Dulbecco's minimum essential medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (PS, Sigma).

Techniques: Cell Culture, Lysis, Generated, Amplification, Isolation

Journal: PLoS ONE

Article Title: Direct Quantification of mRNA and miRNA from Cell Lysates Using Reverse Transcription Real Time PCR: A Multidimensional Analysis of the Performance of Reagents and Workflows

doi: 10.1371/journal.pone.0072463

Figure Lengend Snippet: M14 cultured in 96-wells at 10 4 cells per well (biological triplicates) were lysed directly by (A) in-house or (B) Bio-Rad lysis buffers and incubated at room temperature across various durations (5, 10, 30, 60, 120, 240 min). The cDNA samples generated from cell lysates were amplified by PCR assays from set 2. Ct was plotted against duration of incubation to examine the stability of RNA in room temperature. The experiments were conducted with biological duplicates.

Article Snippet: The NCI-60 cell lines from 7 tissues of origin- A549 (lung, ATCC CCL185), HCT116 (colon, ATCC CCL247, gift from Prof. Sim Kim Ping), OVCAR8 (ovarian, gift from Prof. Jean Paul Thiery ), 786–0 (renal, ATCC CRL1932, gift from Dr. Deng Lih Wen), M14 (melanoma, gift from Prof. Marie-Véronique Clément ), PC3 (prostate, ATCC CRL1435), U251 (central nervous system, ATCC 09063001) were grown in Dulbecco's minimum essential medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (PS, Sigma).

Techniques: Cell Culture, Lysis, Incubation, Generated, Amplification

Journal: PLoS ONE

Article Title: Direct Quantification of mRNA and miRNA from Cell Lysates Using Reverse Transcription Real Time PCR: A Multidimensional Analysis of the Performance of Reagents and Workflows

doi: 10.1371/journal.pone.0072463

Figure Lengend Snippet: M14 cultured in 96-wells at 10 4 cells per well (biological triplicates) were directly lysed by (A) in-house or (B) Bio-Rad lysis buffers. Cell lysates were collected and aliquoted. The lysates were then frozen at −70°C, thawed in a temperature-controlled water bath at 25°C, and the freeze-thaw cycles repeated. The miRNA and mRNA expressions were quantified following 1, 3, 5, 10 freeze-thaw cycles. The qPCR assays from set 2 were used for quantifications. Ct was plotted against freeze-thaw cycles to examine the stability of RNA. The experiments were conducted in biological duplicates.

Article Snippet: The NCI-60 cell lines from 7 tissues of origin- A549 (lung, ATCC CCL185), HCT116 (colon, ATCC CCL247, gift from Prof. Sim Kim Ping), OVCAR8 (ovarian, gift from Prof. Jean Paul Thiery ), 786–0 (renal, ATCC CRL1932, gift from Dr. Deng Lih Wen), M14 (melanoma, gift from Prof. Marie-Véronique Clément ), PC3 (prostate, ATCC CRL1435), U251 (central nervous system, ATCC 09063001) were grown in Dulbecco's minimum essential medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (PS, Sigma).

Techniques: Cell Culture, Lysis

Journal: PLoS ONE

Article Title: Neuronal differentiation of hair-follicle-bulge-derived stem cells co-cultured with mouse cochlear modiolus explants

doi: 10.1371/journal.pone.0187183

Figure Lengend Snippet: Primary antibodies used for immunohistochemistry (in alphabetical order).

Article Snippet: SOX9 , 1:500 , Millipore , AB5535 , M14 melanoma cells , neural crest stem cells.

Techniques: Immunohistochemistry, Positive Control, Marker

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