braf v600e mutant human melanoma cell line a375  (ATCC)

Journal: Journal of experimental & clinical cancer research : CR

Article Title: miR-126-3p down-regulation contributes to dabrafenib acquired resistance in melanoma by up-regulating ADAM9 and VEGF-A.

doi: 10.1186/s13046-019-1238-4

Figure Lengend Snippet: Fig. 1 miRNAs differentially expressed between A375R and A375 cells and KEGG pathway analysis of putative targets. a Heatmap showing miRNA up-regulated (red = expression above the mean) and down-regulated (blue = expression below the mean) in dabrafenib-resistant A375R cells as compared with dabrafenib-sensitive A375 cells (SAM analysis; FC ≥2, FDR = 0%). miRNAs are indicated according to annotation provided by Affymetrix. b Putative target genes of differentially expressed miRNAs were obtained from TargetScan and used for KEGG pathway enrichment analysis. Only pathways with an adjusted P value <0.01 were considered and listed according to a decreasing value of the combined score. c, d Expression of miRNAs down-regulated (c) or up-regulated (d) in A375R cells according to microarray results was validated using specific TaqMan® MicroRNA Assays. The data were normalized to the level of RNU44 in each sample and expressed as 2-ΔCtx105 values. Each value represents the arithmetic mean of at least three independent experiments performed with duplicate samples. Bars, standard error of the mean (SEM). **P<0.01 and *P<0.05, A375R versus (vs) A375.

Article Snippet: The BRAF-mutant human melanoma cell lines A375 and SK-Mel28 were purchased from the European Collection of Cell Cultures (Salisbury, UK) and American Type Culture Collection (ATCC, Manassas, VA), respectively.

Techniques: Expressing, Microarray

Journal: Journal of experimental & clinical cancer research : CR

Article Title: miR-126-3p down-regulation contributes to dabrafenib acquired resistance in melanoma by up-regulating ADAM9 and VEGF-A.

doi: 10.1186/s13046-019-1238-4

Figure Lengend Snippet: Fig. 5 miR-126-3p is up-regulated by dabrafenib only in drug-sensitive cells and inhibits their proliferation. a, b Melanoma cells were incubated with 100 nM dabrafenib (DAB) or with DMSO alone and after 48 h of culture miR-126-3p expression was evaluated by qRT-PCR. 2-ΔCtx105 values calculated relative to RNU44 as the internal reference are shown. Each value represents the arithmetic mean of three independent experiments performed with duplicate samples. Bars, SEM. **P<0.01 and *P<0.05, DAB vs matched DMSO; ##P<0.01 and #P<0.05, resistant cells/DMSO vs parental cells/DMSO. c A375 and SK-Mel28 were transiently transfected with 50 nM pre-miR-126-3p or pre-miR-CTRL, cultured for six days and then assayed for proliferation by the MTT assay. Data are expressed in terms of absorbance at 595 nM. Each value represents the arithmetic mean of three (A375) or four (SK-Mel28) independent experiments performed with triplicate samples. Bars, SEM. *P<0.05, pre-miR-126-3p vs pre-miR-CTRL.

Article Snippet: The BRAF-mutant human melanoma cell lines A375 and SK-Mel28 were purchased from the European Collection of Cell Cultures (Salisbury, UK) and American Type Culture Collection (ATCC, Manassas, VA), respectively.

Techniques: Incubation, Expressing, Quantitative RT-PCR, Transfection, Cell Culture, MTT Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: miR-126-3p down-regulation contributes to dabrafenib acquired resistance in melanoma by up-regulating ADAM9 and VEGF-A.

doi: 10.1186/s13046-019-1238-4

Figure Lengend Snippet: Fig. 9 ADAM9 silencing delays the development of resistance to dabrafenib. A375 cells were seeded into 96-well plates and every eight days transfected with 50 nM siADAM9 or siCTRL and treated with 100 nM dabrafenib (DAB) or DMSO. Cell cultures were photographed and processed for quantitative analysis of proliferation on day 0 (i.e after the first transfection), 8, 16 and 24. Images from a representative experiment are shown. b Quantitative analysis of proliferation of cell cultures described in (a). Crystal violet was solubilized and absorbance was read at 595 nm. Each value represents the arithmetic mean of three independent experiments performed with triplicate cultures. Bars, SEM. **P<0.01, siADAM9 vs matched siCTRL; §§P<0.01, siCTRL/DAB/Day 8 vs siCTRL/DMSO/Day 8; ††P<0.01, siADAM9/DAB/Day 8 vs siADAM9/DMSO/Day8; ##P<0.01, siCTRL/ DAB/Day 16 vs siCTRL/DAB/Day 8; ⁋⁋P<0.01, siCTRL/DAB/Day 24 vs siCTRL/DAB/Day 16.

Article Snippet: The BRAF-mutant human melanoma cell lines A375 and SK-Mel28 were purchased from the European Collection of Cell Cultures (Salisbury, UK) and American Type Culture Collection (ATCC, Manassas, VA), respectively.

Techniques: Transfection