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: Pathogens

Article Title: Bartonella henselae Detected in Malignant Melanoma, a Preliminary Study

doi: 10.3390/pathogens10030326

Figure Lengend Snippet: Melanoma A375 cells co-cultured with B. henselae . ( a ) Melanoma A375 cells were cultured without and ( b ) with B. henselae organisms (green). After 24 h, cultured cells were fixed and immunostained with anti- Bartonella spp. (green). The length of dendritic processes of the Melanoma A375 cells were shorter when cultured with B. henselae (t 2 = 4.18, p < 0.001 ( n = 10). ( c ) At 24 h, immuno-reactive B. henselae (green) appeared to be within as well as outside of melanoma A375 cells. Z-projection of merged Bartonella spp.-immunoreactivity (green), DNA stain TOPRO3 (red), and differential interference contrast (DIC) imaging. Single optical sections were captured with confocal microscopy at 10× UPLSAPO NA: 0.40. Scale bar = 50 microns.

Article Snippet: The cell line A375 was authenticated by IDEXX BioAnalytics (Columbia, MO, USA) using short tandem repeat (STR) analysis in February 2020.

Techniques: Cell Culture, Staining, Imaging, Confocal Microscopy

Journal: Pathogens

Article Title: Bartonella henselae Detected in Malignant Melanoma, a Preliminary Study

doi: 10.3390/pathogens10030326

Figure Lengend Snippet: A video documenting the intracellular location of B. henselae in melanoma cells. Melanoma A375 cells were cultured with B. henselae organisms and at 24 h co-cultures were stained with the nuclear dye Syto9. Z-projection total thickness is 19.3-microns, constructed from 64 optical sections each 0.3 microns thick (Nikon Ti-E Motorized Microscope. Plan Fluor 100× Oil DIC H, Nikon. Scale bar = 5 microns). Data were deconvoluted using Imaris microscopy software with a 5-micron size filter was applied to detect bacteria and only bacteria within the melanoma cells are shown. AVI movie file of this Z-stack is shown in the Video S1 movie file.

Article Snippet: The cell line A375 was authenticated by IDEXX BioAnalytics (Columbia, MO, USA) using short tandem repeat (STR) analysis in February 2020.

Techniques: Cell Culture, Staining, Construct, Microscopy, Software, Bacteria

Journal: Pathogens

Article Title: Bartonella henselae Detected in Malignant Melanoma, a Preliminary Study

doi: 10.3390/pathogens10030326

Figure Lengend Snippet: Bartonella henselae triggers cytokine release during co-culture with melanoma cells. ( a ) An ELISA documented a significant increase in VEGFC expression at 24 h** after inoculation of melanoma cells with B. henselae (Student’s t -test: t 2 = 7, p < 0.01, n = 6). Significance was determined by Student’s t -test. ( b ) VEGFC expression was greater in co-cultures and the increase in VEGFC-expression occurred earlier, namely 24 h, when B. henselae was present as compared to melanoma cells alone. The rate of VEGFC expression when B. henselae was present was significantly greater than that for melanoma cells alone at 24 h (t 4 = 7, p < 0.05), but did not differ from that of melanoma alone after 48 (t 4 = 0.71, p = 0.54) and 72 h (t 4 = 0.25, p = 0.82). n = 6. ( c ) After inoculation of A375 melanoma cells with B. henselae , media was collected and analyzed for interleukin 8 (IL-8) expression at 24, 48, and 72 h. IL-8 expression was significantly increased in response to the presence of Bartonella at 24 and 48 h time points ** (Student’s t -tests: t 4 = 27.08, p < 0.001, and t 4 = 14.84, p < 0.001 for 24 and 48 h, respectively). IL-8 expression was higher in the presence of Bartonella at 72 h, but the difference was not significant (t 4 = 1.72, p = 0.16). n = 6.

Article Snippet: The cell line A375 was authenticated by IDEXX BioAnalytics (Columbia, MO, USA) using short tandem repeat (STR) analysis in February 2020.

Techniques: Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Expressing

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