Journal: PLOS ONE

Article Title: Enhancing the immunogenicity of Wilms tumor 1 epitope in mesothelioma cells with immunoproteasome inhibitors

doi: 10.1371/journal.pone.0308330

Figure Lengend Snippet: ( A ) Structure of the 20S proteasome core and β-ring. The 20S proteasome core particle comprises four rings of seven subunits: two outer rings of α-subunits and two inner rings of β-subunits. The poly-ubiquitylated proteins are translocated into the 20S core where proteolysis occurs to produce short peptides. Aminopeptidases trim the short peptides to generate antigenic peptides of optimal length for binding to MHC-I molecules. ( B ) Three different β-subunits form a β-ring with different specificity and catalytic properties. Standard proteasome (β1c, β2c, and β5c) and immunoproteasome (β1i, β2i, and β5i) cleave substrates differently, producing different peptides (immunopeptidome). Proteasome inhibitors (MG132, epoxomicin, and bortezomib) mainly inhibit β5c and β5i, but also β1c and β2c. ONX-0914, an immunoproteasome selective inhibitor, mainly inhibits β5i but also β1i. ( C ) MESO-4 was treated with the indicated proteasome inhibitors for 3 hours, then co-cultured with reporter T cells for 20 hours, followed by a cellular immunogenicity assay. Data are represented as the mean ± SD of three independent experiments and Dunnett’s multiple comparisons test was used. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Representative data results from two independent experiments are shown.

Article Snippet: Total cell extracts were resolved on 4–12% SDS-PAGE gels and analyzed by western blotting using antibodies against WT1 (clone D8I7F, #83535), β1c (PSMB6, clone E1K9O, #13267), β2c (PSMB7, clone E1LSH, #12197), β2i (MECL-1/PSMB10, clone E6R7O, #17579), β5c (PSMB5, clone D1H6B, #12919), and β5i (LMP-7/PSMB8, clone D1K7X, #13635) (Cell Signaling Technology), β1i (Santa Cruz Biotechnology, PSMB9/LMP2, clone G-3, #373996), and GAPDH as a loading control (BioLegend, #649204).

Techniques: Binding Assay, Cell Culture, Immunopeptidomics

Journal: PLOS ONE

Article Title: Enhancing the immunogenicity of Wilms tumor 1 epitope in mesothelioma cells with immunoproteasome inhibitors

doi: 10.1371/journal.pone.0308330

Figure Lengend Snippet: ( A ) Representative western blots of protein extracts from MESO-4 treated with 20 nM ONX-0914 for 3 hours (indicated by +). Protein expressions of β1c, β2c, β5c, β1i, β2i, and β5i were analyzed by western blotting using the indicated antibodies. ( B ) Quantification of 20S proteasomal β-subunit expression was analyzed by western blotting and values were normalized to control (GAPDH). Data are represented as the mean ± SD of three independent experiments and an unpaired t-test was used. ns, not significant; **, p<0.01.

Article Snippet: Total cell extracts were resolved on 4–12% SDS-PAGE gels and analyzed by western blotting using antibodies against WT1 (clone D8I7F, #83535), β1c (PSMB6, clone E1K9O, #13267), β2c (PSMB7, clone E1LSH, #12197), β2i (MECL-1/PSMB10, clone E6R7O, #17579), β5c (PSMB5, clone D1H6B, #12919), and β5i (LMP-7/PSMB8, clone D1K7X, #13635) (Cell Signaling Technology), β1i (Santa Cruz Biotechnology, PSMB9/LMP2, clone G-3, #373996), and GAPDH as a loading control (BioLegend, #649204).

Techniques: Western Blot, Expressing, Control