Journal: Scientific Reports

Article Title: A tool kit for rapid cloning and expression of recombinant antibodies

doi: 10.1038/srep05885

Figure Lengend Snippet: (A–B) Flow cytometric histograms showing fluorescent intensity of cells incubated with purified IgG/IgE antibodies plus secondary FITC-labelled goat anti-human IgG/IgE (open) and cells with secondary antibody only (filled) against the number of cells. (A) Flow cytometric assessment of anti-CSPG4 IgG 1 /IgE antibodies shows specific binding to native CSPG4 antigen present on the cell surface of A375 melanoma cells and no binding above background to primary melanocytes. (B) Fc regions of anti-CSPG4 IgG 1 and 102.1F10 IgG 4 isotypes demonstrate effector-binding to U937 monocytic cell line, expressing human Fcγ receptors. The IgE antibody isotypes also bind similarly to RBL SX38 mast cells, expressing human FcεR1 receptor. (C) Immunofluorescence staining of A375 cells confirms specific binding of anti-CSPG4 IgG 1 /IgE and no background binding with isotype control hapten specific NIP-IgG 1 /IgE detected by goat anti-human IgG/IgE-FITC. (D) Grass pollen allergen specificity is confirmed by anti-human sandwich ELISA, showing specific binding of recombinant 102.1F10 IgG 4 /IgE and original patient serum to the ELISA plate-bound Phl p 7 allergen and no binding above background with unspecific human myeloma IgG 4 and MOv18 IgE isotype controls.

Article Snippet: Primary human epidermal melanocytes (PCS-200-012, ATCC) were grownin Dermal Cell Basal Medium (PCS-200-030, ATCC) and supplemented with the Melanocyte Growth Kit (PCS-200-041, ATCC).

Techniques: Incubation, Purification, Binding Assay, Expressing, Immunofluorescence, Staining, Control, Sandwich ELISA, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Human Mutation

Article Title: A Macrophage/Monocyte‐Related Four‐Gene Signature for Prognostic Assessment of Uveal Melanoma: BTBD6 , C2CD4B , CCL24 , and S100A4

doi: 10.1155/humu/4978880

Figure Lengend Snippet: Role of four prognostically relevant genes. (A) Quantitative reverse transcription PCR for the calculated levels of four prognostically relevant genes in human epidermal melanocytes. (B) Validation of the knockdown efficiency of C2CD4B. (C, D) Scratch and Transwell assays exploring the migration and invasion of A375 cells in vitro. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, p > 0.05.

Article Snippet: Melanoma cell line A375 (CRL‐1619) and human epidermal melanocytes (PCS‐200‐013) were purchased from American Type Culture Collection (ATCC, Manassas, Virginia) and cultured as follows.

Techniques: Reverse Transcription, Biomarker Discovery, Knockdown, Migration, In Vitro

Journal: Oncogenesis

Article Title: Participation of xCT in melanoma cell proliferation in vitro and tumorigenesis in vivo

doi: 10.1038/s41389-018-0098-7

Figure Lengend Snippet: a Western immunoblots were conducted to assess xCT protein expression from one normal human melanocytes and a panel of human melanoma cell lines. Two out of nine melanoma cell lines demonstrated relatively low levels of endogenous xCT protein (SK-mel2 and SK-mel187). Relative expression of xCT was presented as a fold change compared to normal melanocytes. b Transient transfection of empty vector and exogenous xCT cDNA in HEK293T cells was performed, followed by western analysis at 48 h post-transfection. GAPDH was used as loading control. c An example of xCT expression in human melanoma tumor specimens at various stages. Quantitative RT-PCR was performed to evaluate mRNA expression of xCT in tumor samples. In the bar graph (top panel), the values were presented as the mean ± SEM of three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001. Normalization of xCT mRNA to GAPDH levels showed an increase of xCT as the disease progressed. N.S., normal skin, C.N., congenital nevi, P.M., primary melanoma, IT.M., in-transit melanoma, N.M., nodal metastatic melanoma, L.M., lymph node metastatic melanoma. Bottom panel: western immunoblots were conducted to assess levels of xCT protein expression. Relative expression of xCT was presented as a fold change compared to normal skin. N.S., normal skin, C.N., congenital nevi, P.M., primary melanoma, IT.M., in-transit melanoma, N.M., nodal metastatic melanoma, L.M., lymph node metastatic melanoma. d Immunohistostaining of xCT on normal skin and tumor sections from stages II and III melanoma patients

Article Snippet: Human primary melanocytes were purchased from Coriell Institute for Medical Research (Camden, NJ, USA) and cultured in MelM medium containing melanocyte growth supplement (ScienCell Research Laboratories, Carlsbad, CA, USA) with 5% FBS.

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

Journal: The Journal of Biological Chemistry

Article Title: 67-kDa Laminin Receptor-dependent Protein Phosphatase 2A (PP2A) Activation Elicits Melanoma-specific Antitumor Activity Overcoming Drug Resistance *

doi: 10.1074/jbc.M114.604983

Figure Lengend Snippet: EGCG-induced PP2A activation regained the sensitivity of BRAF inhibitor (PLX4720). A, phosphorylation level and expression of P70S6k and S6 were analyzed by Western blot analysis. B, melanoma cells were treated with PLX4720 of for 48 h, and the phosphorylation level of P70S6k, S6 and ERK1/2 were analyzed by Western blot analysis. C, Hs294T cells were treated with EGCG for 24 h, and the activity of PP2A was measured. D, Hs294T cells were treated with EGCG for 96 h in the presence or absence of OA. E, Hs294T cells were treated with EGCG (5 μm) of and the phosphorylation levels of P70S6k and S6 were analyzed by Western blot analysis. F, Hs294T cells were treated with EGCG for 48 h in the presence or absence of OA, and the phosphorylation levels of P70S6k and S6 were measured. G, Hs294T cells were treated with EGCG, PLX4720, or EGCG/PLX4720 in combination for 96 h. H, NHEM were treated with EGCG or rapamycin (Rapa.) alone or combined with PLX4720 for 96h. I, Hs294T cells were treated with EGCG or rapamycin for 24 h. J, Hs294T cells were treated with EGCG, rapamycin, or PLX4720 alone, or in combination for 14 days for long term colony formation assay. K–N, effect of PLX4720 and EGCG combination on the tumor growth (K), tumor weight (L), p70S6k and ERK phosphorylation (n = 4) (M), aspartate transaminase (AST) and alanine aminotransferase (ALT) (N) of mice on day 33 after inoculation. O, signaling pathway schematic. Error bars, S.D. in vitro or S.E. in vivo (n = 3 per group in vitro or n = 7 per group in vivo). *, p < 0.05; **, p < 0.01; *** or ###, p < 0.001. DMSO, dimethyl sulfoxide; n.s., not significant.

Article Snippet: Primary normal human melanocyte (NHEM) cells in CSF-4HM-500D culture medium supplemented with human melanocyte growth supplements were obtained from DS Pharma Biomedical (Osaka, Japan).

Techniques: Activation Assay, Phospho-proteomics, Expressing, Western Blot, Activity Assay, Colony Assay, In Vitro, In Vivo