Journal: International Journal of Oncology
Article Title: Discovery of the late autophagy inhibitor FZU-0045-053 and its anti-breast cancer and immunomodulatory effects
doi: 10.3892/ijo.2025.5823
Figure Lengend Snippet: Compound 053 modulates co-stimulatory and co-inhibitory molecules on T cell surfaces. (A) The protein levels of 4-1BB, OX40 and CD40L after 5 μ M ADM, GEM (80 μ M), 053 (5 μ M) or CQ (30 μ M) treatment were analyzed by western blotting. (B) The protein levels of TIM3, PD-1 and VISTA after 5 μ M ADM, GEM (80 μ M), 053 (5 μ M) or CQ (30 μ M) treatment were analyzed by western blotting. (C) After T cells were treated with 053 (0-10 μ M) for 24 h, ICOS expression on T cell surfaces was analyzed by flow cytometry. The bar graph shows quantitative analysis of relative fluorescence intensity. (D) T cells were co-cultured with MDA-MB-231 cells at a 1:1 ratio and treated with 053 (0-10 μ M) for 24 h, followed by flow cytometric analysis of ICOS expression on T cell surfaces. The bar graph shows quantitative analysis of relative fluorescence intensity. (E) After T cells were treated with 053 (0-10 μ M) for 24 h, OX-40 expression on T cell surfaces was analyzed by flow cytometry. The bar graph shows quantitative analysis of relative fluorescence intensity. (F) T cells were co-cultured with MDA-MB-231 cells at a 1:1 ratio and treated with 053 (0-10 μ M) for 24 h, followed by flow cytometric analysis of OX-40 expression on T cell surfaces. The bar graph shows quantitative analysis of relative fluorescence intensity. (G) After T cells were treated with 053 (0-10 μ M) for 24 h, 4-1BB expression on T cell surfaces was analyzed by flow cytometry. The bar graph shows quantitative analysis of relative fluorescence intensity. (H) T cells were co-cultured with MDA-MB-231 cells at a 1:1 ratio and treated with 053 (0-10 μ M) for 24 h, followed by flow cytometric analysis of 4-1BB expression on T cell surfaces. The bar graph shows quantitative analysis of relative fluorescence intensity. (I) After T cells were treated with 053 (0-10 μ M) for 24 h, TIM3 expression on T cell surfaces was analyzed by flow cytometry. The bar graph shows quantitative analysis of relative fluorescence intensity. (J) T cells were co-cultured with MDA-MB-231 cells at a 1:1 ratio and treated with 053 (0-10 μ M) for 24 h, followed by flow cytometric analysis of TIM3 expression on T cell surfaces. The bar graph shows quantitative analysis of relative fluorescence intensity. (K) After T cells were treated with 053 (0-10 μ M) for 24 h, PD-1 expression on T cell surfaces was analyzed by flow cytometry. The bar graph shows quantitative analysis of relative fluorescence intensity. (L) T cells were co-cultured with MDA-MB-231 cells at a 1:1 ratio and treated with 053 (0-10 μ M) for 24 h, followed by flow cytometric analysis of PD-1 expression on T cell surfaces. The bar graph shows quantitative analysis of relative fluorescence intensity. Data are presented as mean ± SEM (n=3). Statistical significance was determined by one-way ANOVA followed by Dunnett's multiple comparisons test to compare all experimental groups against a single control group. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 vs. 0 μ M 053. 053, FZU-0045-053; ADM, adriamycin; 4-1BB, tumor necrosis factor receptor superfamily member 9; OX40, tumor necrosis factor receptor superfamily member 4; CD40L, CD40 ligand; ICOS, inducible T-cell co-stimulator; PD-1, programmed cell death protein 1; TIM3, T-cell immunoglobulin and mucin-domain containing-3; VISTA, V-domain Ig suppressor of T cell activation; GEM, gemcitabine; CQ, chloroquine.
Article Snippet: The sections were then blocked with 5% BSA for 1 h at room temperature and subsequently incubated overnight at 4°C with the following primary antibodies: Anti-Ki67 antibody (cat. no. GB111499-100; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-LC3 antibody (cat. no. 81004-1-RR; 1:1,000; Proteintech Group, Inc), anti-p62 antibody (cat. no. GB11239-1-100; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-PD-L1 antibody (cat. no. GB150059-100; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-PD-L2 antibody (cat. no. 27406-1-AP; 1:4,000; Proteintech Group, Inc), anti-CD3 antibody (cat. no. GB11014-100; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-CD4 antibody (cat. no. GB11064-100; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-CD8 antibody (cat. no. GB11068; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-4-1BB/CD137 antibody (cat. no. A2025; 1:200; ABclonal Biotech Co., Ltd), anti-CD134/OX40 antibody (cat. no. 32621-1-AP; 1:500; Proteintech Group, Inc), anti-PD1 antibody (cat. no. GB153744-100; 1:400; Wuhan Servicebio Technology Co., Ltd) and anti-TIM3 antibody (cat. no. 11872-1-AP; 1:1,000; Proteintech Group, Inc).
Techniques: Western Blot, Expressing, Flow Cytometry, Fluorescence, Cell Culture, Control, Activation Assay
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