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: Scientific Reports

Article Title: Nuclear Transport Factor 2 (NTF2) suppresses WM983B metastatic melanoma by modifying cell migration, metastasis, and gene expression

doi: 10.1038/s41598-021-02803-0

Figure Lengend Snippet: NTF2 affects lamin A nuclear localization, nuclear size, and heterochromatin distribution. VGP primary melanoma refers to a primary vertical growth phase melanoma cell line (WM983A) that was derived from the same patient as NTF2 low (WM983B) and represents an earlier stage of disease. (A) The indicated cells were stained for lamin A and representative images are shown. Images were acquired with the same exposure time and brightness/contrast were identically adjusted. Line scans were acquired across the nuclear rim as indicated by the yellow line. In the top graph, lamin A intensity was quantified across at least 50 line scans per cell line for two independent experiments (see “ ”). To measure the lamin A intensity at the nuclear envelope (NE), the highest lamin A intensities from each line scan were averaged. To measure the total nuclear lamin A intensity, the total fluorescence intensity of thresholded nuclei was quantified for at least 102 nuclei per condition. To measure the NE/nucleoplasm lamin A intensity, the maximum value along the line scan was divided by the minimum value within the nucleus. Error bars are SD. One-way ANOVA followed by Dunnett’s multiple comparisons test with ***p < 0.0001 and ns not significant. We also measured a 44% reduction in lamin B1 staining intensity at the NE in NTF2 high dox + cells compared to NTF2 low cells (data not shown). (B) Nuclear cross-sectional area, which serves as a good proxy for nuclear surface area and volume , , , , was measured for at least 274 Hoechst-stained nuclei per cell line (918 nuclei on average). The normal melanocyte cell line is AG21857. Error bars are SD. One-way ANOVA followed by Dunnett’s multiple comparisons test with ***p < 0.0001. (C) The indicated cells were stained for the heterochromatin marker H3K9me3 and representative images are shown. Images were acquired with the same exposure time and brightness/contrast were identically adjusted. To quantify heterochromatin distribution, H3K9me3 intensity was quantified along line scans drawn from the nuclear rim toward the center (see “ ”). Averages from at least 150 line scans are shown for each cell line for two independent experiments. Error bars are SEM. One-way ANOVA followed by Dunnett’s multiple comparisons test with **p < 0.01, ***p < 0.0001. The statistical comparisons are for NTF2 low and VGP primary melanoma compared to NTF2 high dox + at line scan position 0.2 µm.

Article Snippet: The human melanocyte cell line (AG21857) was obtained from the NIA Cell Culture Repository through the Coriell Institute (only used in Fig. B).

Techniques: Derivative Assay, Staining, Fluorescence, Marker

Journal: Heliyon

Article Title: Identification of the role of autophagy-related TNFSF10/ hsa-let-7a-5p axis in vitiligo development and potential herbs exploring based on a bioinformatics analysis

doi: 10.1016/j.heliyon.2023.e23220

Figure Lengend Snippet: Effects of EGCG on the 3-MA-treated PIG1 melanocytes. (A) The percentages of viable cells change in PIG1 after treatment with varying concentrations of 3-MA (0–10 mM). (B) The cell viability of PIG1 treated with 3-MA and 3-MA + EGCG by the CCK-8 assay. (C) Real-time PCR of changes in mRNA levels of TNFSF10，MAPK1 and BID in PIG1. Data were presented as mean ± SEM of three independent experiments; **P < 0.01 compared with the control group; ##P < 0.01 compared with the 3-MA group.

Article Snippet: The human melanocyte line PIG1 was purchased from ScienCell Research Laboratories, Inc (California, USA).

Techniques: CCK-8 Assay, Real-time Polymerase Chain Reaction, Control

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