Journal: EMBO Molecular Medicine
Article Title: MHC-I upregulation by macbecin II in the solid tumors potentiates the effect of active immunotherapy
doi: 10.1038/s44321-025-00213-7
Figure Lengend Snippet: ( A ) Flow cytometry analysis for INF-γ-CD8 + T cells in the four groups. ( B ) Granzyme B expression was examined by immunohistochemistry (left) and quantified (right) in the four groups. The staining intensity was analyzed by the unpaired two-tailed Student’s t-test ( n = 4/group, biological replicates). ( C ) LAMP1 expression was examined through immunohistochemistry (left) and quantified (right) in the four groups. The staining intensity was measured and analyzed by the one-way ANOVA with a Tukey post-hoc test ( n = 4/group, biological replicates). ( D , E ) MHC-I expression was examined in EPCAM+ tumor cells and CD11c+ dendritic cells in the dissociated tumor by FACS. Statistical analysis was performed using the one-way ANOVA with a Tukey post-hoc test ( n = 3/group, biological replicates). ( F , G ) Animal weight ( F ) and AST activity ( G ) in the blood of mice were measured at the end-point. Data are represented as mean +/− SEM ( n = 5/group, biological replicates). ( H ) B16-Ova cells (500 cells/well, n = 3/group, biological replicates, were seeded in a 96-well plate and treated with a combination of macbecin II (0.1 µM) and OT-1 T cells (E:T ratio 5:1) with or without ectopic MHC-I expression for 48 h. The T cells were activated with IL2-ep13nsEV that were isolated from B16-Ova lysate-pulsed BMDCs (E:T ratio 5:1). After the incubation, cells were washed with ice-cold PBS to remove dead cells. The live cells were fixed with methanol at room temperature for 15 min and stained with crystal violet. The dye was dissolved in 10% acetic acid and measured at 590 nm. ( I ) E0771 cells (500 cells/well, n = 3/group, biological replicates) were seeded in a 96-well plate and treated with a combination of T cells (E:T ratio 5:1) and macbecin II (0.1 µM) with or without ectopic MHC-I expression for 48 h. The T cells were isolated from the spleen of E0771-bearing syngeneic mouse and they were activated with IL2-ep13nsEV that were isolated from E0771 lysate-pulsed BMDCs. After the incubation, cells were processed as described in ( H ). Statistical significance was determined by the one-way ANOVA with a Tukey post-hoc test. ( J ) MHC-I was ectopically expressed in B16-F1 cells, and the MHC-I expression was confirmed by FACS. The result was analyzed by the unpaired two-tailed Student’s t-test ( n = 3/group, biological replicates). ( K ) Cell viability was examined by MTS assay for B16-F1 with or without MHC-I over expression (OE) ( n = 3/group, biological replicates). ( L , M ) Animal weight ( L ) and AST activity ( M ) were measured at the end-point ( n = 5/group, biological replicates). Data are presented as mean +/− SEM.
Article Snippet: LAMP1 antibody , Bioss , BS-1970R.
Techniques: Flow Cytometry, Expressing, Immunohistochemistry, Staining, Two Tailed Test, Activity Assay, Isolation, Incubation, MTS Assay, Over Expression
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