Article Figures & Data
Figures
- Figure 1.
Panobinostat inhibits cell growth in vitro. Resting or proliferating (P-) PBMCs from healthy donors (HD1 and HD2) and from patients with metastatic melanoma (MD1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, and -13); PBMCs from a healthy donor or a patient with metastatic melanoma were genetically modified to express the TCR for melanoma antigen MART-1 (T-HD1). Bone marrow from patients with multiple myeloma (BM1) or breast cancer (BM2), or 12 melanoma cell lines (M202, M229, M233, M249, M263, M370, M376, M285, M395, M308, M408, and M417) were treated with 0 to 100 μmol/L LBH589. Data represent the mean of triplicate experiments performed three independent times (n = 9). Bars, error bars.
- Figure 2.
Effect of panobinostat on cell-cycle progression and apoptosis in melanoma cell lines and activated lymphocytes. A, two melanoma cell lines (M229 and M370), PBMC cultures from a healthy donor (HD1), from patients with metastatic melanoma (MD1, -2, -3, and -4), or from a healthy donor followed by genetic modification to express the TCR for melanoma antigen MART-1 (T-HD1) were treated with 0 to 1 μmol/L LBH589 or 1 μmol/L staurosporine (SSP) for 24 hours. B, quantitative analysis of the percentage of cells in G0–G1 (white), G2–M (black), or S phase (gray). C, quantitative analysis of the percentage of cells with cleaved PARP. Columns, mean values of three independent experiments (n = 3); bars, SEM.
- Figure 3.
Panobinostat induces DNA damage in melanoma cell lines and human lymphocytes. A, M229 and M370, PBMCs from a healthy donor (HD-1), from patients with metastatic melanoma (MD1, -2, -3, and -4), or from a healthy donor followed by genetic modification to express the TCR for melanoma antigen MART-1 (T-HD1) were treated with 0 to 10 μmol/L LBH589 for 24 hours, stained for pH2Ax, and analyzed by flow cytometry. Shading, fold change with respect to the vehicle control (dark gray, decreased; black, no change; light gray, increased). Numbers, magnitude of the fold change (negative, decreased; positive, increased relative to controls). B, quantitative analysis of DNA damage. n = 3; bars, SEM. Increasing concentrations from left to right side.
- Figure 4.
Single-cell phosphoprotein flow cytometry gating strategy. Cells were treated with 0 to 1 μmol/L panobinostat for 24 hours. Cell events were first identified by gating on morphology (SSC-A vs. FSC-A) and singlets (SSC-A vs. SSC-W). After that, the six barcoded cell populations with a combination of Ax-350-NHS 0, 3, or 8 μg/mL and Ax-750-NHS 0, 3, or 8 μg/mL were deconvoluted. Then, for each of the six populations, plotting CD20 versus CD3 identified B- and T-cell populations. Further plotting the CD3 population for CD4 and CD8 identified these T-cell subpopulations. Histograms of intracellular phosphorylated proteins such as pSTAT5 were then obtained. FSC-A, forward scatter area; SSC-A, side scatter area; W, width.
- Figure 5.
Quantitative analysis of the effect of panobinostat on intracellular protein phosphorylation at the single-cell level. PBMCs from a healthy donor (HD1), a patient with metastatic melanoma, or bone marrow from a patient with multiple myeloma (BM1) were treated with 0 to 1 μmol/L LBH589 for 24 hours. Cells were not stimulated (VC, vehicle control) or stimulated with IL-2 or IFN-α for 15 minutes, then fluorescently barcoded. CD20+, CD4+, or CD8+ cells were then evaluated for phosphorylated intracellular proteins. Results, fold change relative to controls (blue, decreased; black, no change; yellow, increased. Values, magnitude of fold change relative to controls).
Tables
- Table 1.
Characteristics of melanoma cell lines
Cell line BRAF or NRAS mutational status Other oncogenic events or tumor-suppressor deletions Sensitive (S) or resistant (R) to vemurafeniba M285 Wild-type (36) R M229 BRAF V600E MITF amplification S (34, 35, 45) AKT1 amplification PTEN heterozygous M233 BRAF V600E AKT1 amplification R (35) CCND1 amplification EGFR amplification CDKN2A heterozygous PTEN heterozygous M249 BRAF V600E MITF amplification S (34, 35, 45) AKT2 amplification PTEN homozygous M263 BRAF V600E CDKN2A heterozygous R (34, 35) M308 BRAF V600E MITF amplification R (35) AKT2 amplification EGFR amplification CDKN2A homozygous M370 BRAF V600E R (34) M395 BRAF V600E S (34, 45) M417 BRAF V600E ABL1 E255K, Y253H R EGFR P753S M202 NRAS Q61L EGFR amplification R (35) CDKN2A homozygous M408 NRAS Q61K R M376 BRAFV600E/NRAS Q61K R (34) -
↵aSensitive, <1 μmol/L; resistant, >1 μmol/L.
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Supplementary Data
Files in this Data Supplement:
- Supplementary Table 1 - PDF file - 26KB, Antibody Cocktails for Phosphoproteomic at single cell level.
Volume 2, Issue 5
