Journal:

Article Title: Changes in the gene expression profile of A375 human melanoma cells induced by over-expression of multifunctional pigment epithelium-derived factor

doi: 10.1097/CMR.0b013e32834495c3

Figure Lengend Snippet: Characterization of PEDF-over-expressing A375 cell lines. (a) Western blot analysis of secreted extracellular PEDF (PEDFe) protein in 48 h conditioned medium (CM) from control (A375-pCEP4 Pool) and PEDF-over-expressing (A375-pCEP4-PEDF Pool) cells. Numbers below blot show densitometry values normalized to A375-pCEP4 Pool expression. (b) Quantitative RT-PCR analysis of PEDF mRNA levels in A375-pCEP4 Pool and A375-pCEP4-PEDF Pool cells. PEDF mRNA levels are shown relative to A375-pCEP4 Pool after normalization to GAPDH. Bars represent average ± standard deviation (SD). (c) Invasion assay of A375-pCEP4 Pool and A375-pCEP4-PEDF Pool cells toward 10% FBS for 24 h. Statistical significance was determined by Student’s t-test (****, p<0.0001). Results are representative of two independent experiments.

Article Snippet: A375 human melanoma cell line, originally established in culture from a lymph node metastasis of a melanoma patient [ 28 , 29 ], was kindly provided by Dr Vidal-Vanaclocha (Universidad del Pais Vasco, Bilbao, Spain).

Techniques: Expressing, Western Blot, Control, Quantitative RT-PCR, Standard Deviation, Invasion Assay

Journal:

Article Title: Changes in the gene expression profile of A375 human melanoma cells induced by over-expression of multifunctional pigment epithelium-derived factor

doi: 10.1097/CMR.0b013e32834495c3

Figure Lengend Snippet: Distribution in functional categories of genes regulated by PEDF in A375 melanoma cells. (a) Heat map of expression of genes regulated by PEDF in two independent hybridizations (Hybr. 1 and Hybr. 2) of A375-pCEP4-PEDF Pool cells compared to control A375-pCEP4 Pool cells. Coloring represents normalized signal intensity of the ratio log2 (A375-pCEP4-PEDF Pool/A375-pCEP4 Pool): red, upregulation; black, no change; green, downregulation. (b) Diagram showing genes regulated by PEDF grouped into biological processes and/or functional categories as described in the main text. Percentage of regulated genes in each category relative to total number of regulated genes is shown. For some categories the most relevant genes are listed in the lower boxes, being shown in red when upregulated and in green when downregulated in A375-pCEP4-PEDF Pool compared to A375-pCEP4 Pool.

Article Snippet: A375 human melanoma cell line, originally established in culture from a lymph node metastasis of a melanoma patient [ 28 , 29 ], was kindly provided by Dr Vidal-Vanaclocha (Universidad del Pais Vasco, Bilbao, Spain).

Techniques: Functional Assay, Expressing, Control

Journal:

Article Title: Changes in the gene expression profile of A375 human melanoma cells induced by over-expression of multifunctional pigment epithelium-derived factor

doi: 10.1097/CMR.0b013e32834495c3

Figure Lengend Snippet: Distribution in functional categories of genes regulated by PEDF in A375 human melanoma cell line Selected relevant genes regulated by PEDFe are shown classified into functional categories described in the main text. Upregulated genes are included at the top of the list followed by downregulated genes. Shown are gene symbol, description and the linear ratio of GCRMA-normalized expression PEDF/control in the two hybridizations from independently isolated RNAs from A375-pCEP4-PEDF Pool y A375-pCEP4 Pool cells (Hybr. 1 and Hybr. 2).

Article Snippet: A375 human melanoma cell line, originally established in culture from a lymph node metastasis of a melanoma patient [ 28 , 29 ], was kindly provided by Dr Vidal-Vanaclocha (Universidad del Pais Vasco, Bilbao, Spain).

Techniques: Functional Assay, Expressing, Isolation, Permeability

Journal:

Article Title: Changes in the gene expression profile of A375 human melanoma cells induced by over-expression of multifunctional pigment epithelium-derived factor

doi: 10.1097/CMR.0b013e32834495c3

Figure Lengend Snippet: Validation of candidate target genes regulated by PEDF in A375 melanoma cells. (a) Quantitative RT-PCR analysis of mRNA levels of genes related to: 1) angiogenesis/adhesion/migration/invasion: COL4A2, FN1, IL8 and TGFA; 2) melanoma progression: FGF13, IGFBP3, JAG1 and LGALS3; 3) melanoma markers: MIA and S100B; 4) melanin secretion: MLPH and RAB27A. Empty bars correspond to A375-pCEP4 Pool cells and filled bars to A375-pCEP4-PEDF Pool cells. For each gene mRNA levels are shown relative to A375-pCEP4 Pool after normalization to GAPDH. Bars represent average ± SD. Statistical significance was determined by Student’s t-test or Welch-corrected t-test (the latter for COL4A2, TGFA, LGALS3, MLPH and S100B) (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). (b) ELISA analysis of secreted IL8 protein levels in 48 h CM from A375-pCEP4 Pool and A375-pCEP4-PEDF Pool cells. Bars represent average ± SD. Statistical significance was determined by Student’s t-test (****, p<0.0001). Results are representative of two independent experiments.

Article Snippet: A375 human melanoma cell line, originally established in culture from a lymph node metastasis of a melanoma patient [ 28 , 29 ], was kindly provided by Dr Vidal-Vanaclocha (Universidad del Pais Vasco, Bilbao, Spain).

Techniques: Biomarker Discovery, Quantitative RT-PCR, Migration, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Changes in the gene expression profile of A375 human melanoma cells induced by over-expression of multifunctional pigment epithelium-derived factor

doi: 10.1097/CMR.0b013e32834495c3

Figure Lengend Snippet: Confirmation of regulation of several target genes in other PEDF-over-expressing melanoma cell lines. (a) Quantitative RT-PCR analysis of PEDF mRNA levels in A375-pCEP4 Pool, A375-pCEP4-PEDF Clone 1 and A375-pCEP4-PEDF Clone 4 cells. PEDF mRNA levels are shown relative to A375-pCEP4 Pool after GADPH normalization. Bars represent average ± SD. (b) Quantitative RT-PCR analysis of IL8 (left panel), JAG1 (middle panel) and MLPH (right panel) mRNA levels in A375-pCEP4 Pool, A375-pCEP4-PEDF Clone 1 and A375-pCEP4-PEDF Clone 4 cells. For each gene mRNA levels are shown relative to A375-pCEP4 Pool after GAPDH normalization. Bars represent average ± SD. Statistical significance was determined by one-way ANOVA test using Tukey-Kramer post-test (*, p<0.05; ***, p<0.001). Results are representative of two independent experiments. (c) Western blot analysis of PEDFe protein in 48 h CM from control (UCD-GFP or C8161-GFP) and PEDF-over-expressing (UCD-PEDF or C8161-PEDF) cells. Numbers below blots show densitometry values normalized to each control cells expression. (d) Quantitative RT-PCR analysis of IGFBP3, MIA and IL8 mRNA levels in UCD-GFP, UCD-PEDF, C8161-GFP and C8161-PEDF cells. For each gene mRNA levels are shown relative to control cells after GAPDH normalization. Bars represent average ± SD. Statistical significance was determined by Student’s t-test (***, p<0.001; ****, p<0.0001). Results are representative of two independent experiments.

Article Snippet: A375 human melanoma cell line, originally established in culture from a lymph node metastasis of a melanoma patient [ 28 , 29 ], was kindly provided by Dr Vidal-Vanaclocha (Universidad del Pais Vasco, Bilbao, Spain).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control

Journal:

Article Title: Changes in the gene expression profile of A375 human melanoma cells induced by over-expression of multifunctional pigment epithelium-derived factor

doi: 10.1097/CMR.0b013e32834495c3

Figure Lengend Snippet: Validation of regulation of IL8 by transient transduction of A375 melanoma cells using lentivirus-PEDF. (a) Transduction efficiency of A375 melanoma cell line after infection with control (A375-lenti-GFP) or PEDF (A375-lenti-PEDF) lentivirus. Fluorescence images show more than 95% GFP-positive cells after 72 h of infection. Corresponding phase-contrast images are shown. (b) Western blot analysis of secreted extracellular PEDF (PEDFe) protein in 48 h CM from A375 melanoma cell line after 72 h of transduction with control lentivirus-GFP (A375-lenti-GFP) or lentivirus-PEDF (A375-lenti-PEDF). Numbers below blot show densitometry values relative to A375-lenti-GFP. (c) Quantitative RT-PCR analysis of PEDF mRNA levels in A375 melanoma cell line after 72 h of transduction with control lentivirus-GFP (A375-lenti-GFP) or lentivirus-PEDF (A375-lenti-PEDF). PEDF mRNA levels are shown relative to A375-lenti-GFP after normalization to GAPDH. Bars represent average ± SD. (d) Quantitative RT-PCR analysis of IL8 mRNA levels in A375 melanoma cell line after 72 h of transduction with control lentivirus-GFP (A375-lenti-GFP) or lentivirus-PEDF (A375-lenti-PEDF). IL8 mRNA levels are shown relative to A375-lenti-GFP after normalization to GAPDH. Bars represent average ± SD. Statistical significance was determined by Student’s t-test (*, p<0.01). (e) ELISA analysis of secreted IL8 protein levels in 48 h CM from A375 melanoma cell line after 72 h of transduction with control lentivirus-GFP (A375-lenti-GFP) or lentivirus-PEDF (A375-lenti- PEDF). Bars represent average ± SD. Statistical significance was determined by Student’s t-test (**, p<0.01). (f) IL8 transcriptional activity reporter assay in the A375-lenti-GFP and A375-lenti-PEDF melanoma cells mentioned above after 24 h treatment with 10% FBS or 10 ng/ml TNFα. Luciferase levels relative to A375-lenti-GFP in 10% FBS after normalization to renilla levels are shown. Bars represent average ± SD. Results are representative of two independent experiments.

Article Snippet: A375 human melanoma cell line, originally established in culture from a lymph node metastasis of a melanoma patient [ 28 , 29 ], was kindly provided by Dr Vidal-Vanaclocha (Universidad del Pais Vasco, Bilbao, Spain).

Techniques: Biomarker Discovery, Transduction, Infection, Control, Fluorescence, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Activity Assay, Reporter Assay, Luciferase

Journal: Molecular Medicine Reports

Article Title: Reprogramming A375 cells to induced-resembled neuronal cells by structured overexpression of specific transcription genes

doi: 10.3892/mmr.2016.5598

Figure Lengend Snippet: Rapid generation of induced-resembled neuronal cells from A375 cells. (A and B) A375 melanoma cells infected by the empty viral vector with enhanced green fluorescent protein. The infection rate reached >95% with an MOI=20. The designated concentrations were derived as follows: MOI=1, 1×10 8 TU/ml of lentivirus + 0.1 µ l lentivirus solution; MOI=10, 1×10 8 TU/ml of lentivirus + 1 µ l lentivirus solution; MOI=100, 1×10 8 TU/ml of lentivirus + 1 µ l lentivirus solution). The A375 cells were readily infected by the lentivirus. (A) Green fluorescence imaging was used to identify individual cells. (B) The same field was in white. (C and D) Following screening using puromycin, the percentage of cells infected by myelin transcription factor 1, which remained of the original group was ~65%, and the majority of cells exhibited axon-like changes. Magnification, ×200. MOI, multiplicity of infection. (C) The cells prior to screening. (D) The cells following screening.

Article Snippet: The A375 human melanoma cell line was obtained from GeneChem Co., Ltd. (Shanghai, China) and maintained in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.).

Techniques: Infection, Plasmid Preparation, Derivative Assay, Fluorescence, Imaging

Journal: Molecular Medicine Reports

Article Title: Reprogramming A375 cells to induced-resembled neuronal cells by structured overexpression of specific transcription genes

doi: 10.3892/mmr.2016.5598

Figure Lengend Snippet: Importance of transduced factors in the reprogramming procedure. Quantification of Tuj1-positive cells with the indicated factor combinations of the brain protein 2 (B), achaete-scute homolog 1 (A), myelin transcription factor 1 (M) and neuronal differentiation factor 1 (N) genes 7 days following infection. Data are presented as the mean ± standard deviation of three randomly selected images (AxioCam), captured with a 200× objective on a Zeiss Axio Observer microscope. The total quantity of virus was constant between the different factor combinations, and the same numbers of A375 cells were seeded in each condition prior to infection. * P<0.05. Tuj1, tubulin β3.

Article Snippet: The A375 human melanoma cell line was obtained from GeneChem Co., Ltd. (Shanghai, China) and maintained in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.).

Techniques: Infection, Standard Deviation, Microscopy, Virus

Journal: Molecular Medicine Reports

Article Title: Reprogramming A375 cells to induced-resembled neuronal cells by structured overexpression of specific transcription genes

doi: 10.3892/mmr.2016.5598

Figure Lengend Snippet: Results of reverse transcription-quantitative polymerase chain reaction analysis of A375 irNCs. Statistical analyses of the expression levels of neuron-specific markers in the empty-virus, four-factor and five-factor groups at week (A) 1, (B) 2, (C) 3 and (D) 4. The levels of MAP2, NEUN and SYN1 from the four-factor and five-factor group were higher, compared with the negative control in the 4 weeks. Data are expressed as the mean ± standard deviation. ##** P<0.01 and #* P<0.05. irNCs, induced resembled neuronal cells; MAP2, microtubule-associated protein 2; NEUN, neuronal nuclei; SYN1, synapsin 1.

Article Snippet: The A375 human melanoma cell line was obtained from GeneChem Co., Ltd. (Shanghai, China) and maintained in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.).

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Virus, Negative Control, Standard Deviation

Journal: Genes

Article Title: Integrative Transcriptomic Analysis Identify Potential m6A Pathway-Related Drugs That Inhibit Cancer Cell Proliferation

doi: 10.3390/genes13112011

Figure Lengend Snippet: Effect of R428 on human melanoma cell line cell line A375. ( A , B ) The proliferation of the human breast cancer cell line A375 was significantly inhibited by R428 treatment. The results from the cell count assay ( A ) and the MTT assay ( B ) are shown. Data represented the mean ± SEM from five independent experiments, t -test, * p < 0.05, ** p < 0.01. Some SEM values in the drug-treated experimental group that are too small to be shown are specified here: ( A ) 24 h. 0.009; 48 h, 0.012; 72 h, 0.006; 96 h, 0.000; (B) 24 h. 0.004; 48 h, 0.002; 72 h, 0.006; 96 h, 0.002. ( C , D ) The protein expression of METTL3 in the A375 cell line was significantly reduced by drug treatment, t -test, ** p < 0.01. The summary of Western blot grey values as the mean ± SEM from three independent experiments ( C ) and the representative Western blot result ( D ) are shown. ( E ) The RNA m6A modification level in the A375 cell line was significantly decreased after R428 treatment. Data represented the mean ± SEM from five independent experiments, t -test, * p < 0.05. ( F , G ) Representative images of treated and control cells observed under an inverted 10× microscopy.

Article Snippet: The breast cancer cell line MCF7 and the human melanoma cell line A375 were provided by Biomol Inc. (Hamburg, Germany).

Techniques: Cell Counting, MTT Assay, Expressing, Western Blot, Modification, Control, Microscopy