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: Clinical Cancer Research

Article Title: Anti-EGFR Antibody–Drug Conjugate Carrying an Inhibitor Targeting CDK Restricts Triple-Negative Breast Cancer Growth

doi: 10.1158/1078-0432.CCR-23-3110

Figure Lengend Snippet: Stochastic conjugation of cetuximab to CDK inhibitor and ADC internalization in live breast cancer cells. A, Flow cytometric evaluation of surface EGFR expression (TNBC: MDA-MB-468, HCC1143, HCC1806, MDA-MB-231, HCC1937, SUM149, and CAL51; HER2+: SKBR3; ER+: MCF7, T47D; nontumorigenic epithelial cell model: MCF10A; immune cell model: human B lymphocytes RPMI8866, RPMI8226, and monocytic cell line U937; human primary melanocyte: melanocyte). B, EGFR mRNA expression from the Cancer Cell Line Encyclopedia database showed a positive correlation with surface EGFR measured by flow cytometry in A (Spearman’s rank coefficient, r = 0.723). A high level of correlation was found between EGFR and cyclin E ( r = 0.738), but not with cyclin A or CDK2. Nonsignificant P values are marked as NS. C, Top, Schematic diagram of stochastic ADC conjugation by antibody reduction with TCEP and then conjugation to SNS-032 via MC-Val–Ala-PAB. Middle, HIC analysis confirmed an average DAR of 4.4. Bottom, SEC trace indicates negligible ADC aggregation and minimal free linker–payload (less than 0.8%). D, Surface plasmon resonance analysis demonstrated similar binding affinity ( K D ) for cetuximab (0.73 nmol/L) and ADC (1.28 nmol/L). Isotype IgG1 and isotype ADC showed no measurable binding. E, Monitoring internalization of Fabfluor-pH-labeled cetuximab, ADC, or isotype control (10 nmol/L) by Incucyte live-cell imaging. Phase and red fluorescence time-course images were captured for 24 hours. Images of internalized antibody display in cytosolic, low pH lysosomal vesicle-associated red fluorescence in cells. Scale bar, 0.2 mm. F, Cells were seeded in Matrigel for 5 days, allowing the formation of spheroids. Fabfluor-pH-labeled antibodies or ADC (10 nmol/L) were introduced in the Matrigel and showed rapid internalization in EGFR-high MDA-MB-468 and MDA-MB-231, whereas EGFR-low CAL51 displayed little red fluorescence signals. A low level of internalization was observed for isotype or isotype-ADC controls. Scale bar, 0.5 mm. P values determined by two-tailed unpaired t test of three independent experiments compared with isotype control.

Article Snippet: Human primary epidermal melanocytes were cultured in Melanocyte Growth Medium (Cell Applications Inc.).

Techniques: Conjugation Assay, Expressing, Flow Cytometry, SPR Assay, Binding Assay, Labeling, Control, Live Cell Imaging, Fluorescence, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Rewiring melanoma cell fate: TRPM8 modulators trigger apoptosis and boost NK cell cytotoxicity

doi: 10.1038/s41419-026-08469-8

Figure Lengend Snippet: A Representative Western blot showing TRPM8 protein expression in the indicated cell lines. Tubulin was used as loading control. Viability of human melanocytes ( B ) and human dermal fibroblasts ( C ) untreated or treated with compounds 4 and 9 at the concentrations indicated in the legends on the right. Absorbance values from WST-1 assays at 24, 48, and 72 h are shown. Data are presented as mean ± SD of three independent experiments. n.s . indicates not significant. Representative Live/Dead assay images of human melanocytes ( D ) and human dermal fibroblasts ( E ) treated for 24 h with compounds 4 and 9 (1 or 10 μM). Viable cells are shown in green (acridine orange; total cells), while dead cells are shown in red (propidium iodide; dead cells). Overlay images are shown. Scale bar, 100 μm. Quantification of cell death is displayed to the right of each overlay image. The percentage of dead cells was calculated as: (red-stained dead cells/green-stained total cells) × 100.

Article Snippet: The human epidermal Melanocytes ( P10853 , Innoprot, Bizkaia, Spain) were cultured in Melanocyte Medium (MeIM; Innoprot) supplemented with 5% FBS (Gibco), 1% Melanocyte Growth Supplement (MelSG; Innoprot) and 1% penicillin-streptomycin (Gibco).

Techniques: Western Blot, Expressing, Control, Live Dead Assay, Staining