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Supplemental Figures 1 - 8 from BRAFV600E Co-opts a Conserved MHC Class I Internalization Pathway to Diminish Antigen Presentation and CD8+ T-cell Recognition of Melanoma

Authors :
Gregory Lizée
Patrick Hwu
Michael A. Davies
Laszlo G. Radvanyi
Chantale Bernatchez
Fenge Li
Wanleng Deng
Mayra Whittington
Shujuan Liu
Tania Rodriguez-Cruz
Jahan S. Khalili
Amjad Talukder
Brenda Melendez
Zeming Chen
Sherille D. Bradley
Publication Year :
2023
Publisher :
American Association for Cancer Research (AACR), 2023.

Abstract

Supplemental Figure 1. Three melanoma cell lines (CHL1, Mel888, and WM793) were treated with DMSO (0), 10uM, or 50uM of dabrafenib (BRAFi) for 3 hours. Whole cell lysates were prepared, and levels of phospho-ERK and total ERK were analyzed by immunoblotting. Supplemental Figure 2. Mel888 cells were treated with vehicle (DMSO) or 50 uM of BRAFi for 3 hours. Following treatment, cells were stained with fluorescently-labeled antibodies specific for MHC-I (HLA-A,B,C), MHC-II (HLA-DR), PD-L1, or melanoma-associated chondroitin sulfate proteoglycan (MCSP), and analyzed by flow cytometry. Data shown are representative of at least three replicate experiments with similar results. * indicates p < 0.05; ns, not significant. Supplemental Figure 3. HLA-A2-transduced Mel888 melanoma cell lines were treated with vehicle DMSO (0), 10uM, or 50uM of BRAFi for 3 hours. Whole cell lysates were prepared and levels of phospho-ERK and total ERK were analyzed by immunoblotting. Supplemental Figure 4. HLA-A2-transduced Mel888 or WM793 cells were treated with DMSO or MEKi for 3 hours. Following treatment, cells were stained with a fluorescently-labeled HLA-A2-specific antibody and analyzed by flow cytometry. These experiments were repeated at least four times with similar results. *** indicates p < 0.005; ns, not significant. Supplemental Figure 5. T2 cells were pulsed with titrated amounts of MART-1(27-35) peptide, washed, and then used as stimulator cells for MART1-specific CD8+ TILs. Supernatants were collected after 8 hours of co-culture and analyzed by ELISA to measure interferon-gamma (IFNgamma) release. Supplemental Figure 6. Percentage of intracellular IFNgamma-positive TILs following 3 hours of co-culture with DMSO- or BRAFi-treated HLA-A*0201-transduced Mel888 cells or MART-1(27-35) peptide-pulsed WM793 cells, as measured by flow cytometry. All data are representative of experiments performed at least 3 times with similar results. *, p

Details

Database :
OpenAIRE
Accession number :
edsair.doi.dedup.....788bf3b568ad9e09fe2ab3424cef6b35