Curcumin analog GO-Y078 enhanced tumor immunity cooperate with anti-OX40 antibody (Raw data)

<strong>Fig. 1 GO-Y078 treatment</strong> <strong>induced</strong> <strong>apoptotic cell death in B16-F10 melanoma cells</strong>
(<strong>A</strong>) Live cell counting assay. B16-F10 melanoma cells were cultured with 0.007 μM DMSO (control) and 12.5–1.5625 μM GO-Y078 as indicated for 20 h, followed by the addition of the Cell Counting Kit 8 reagent (4 h). The circles indicate independent experiments. The horizontal bars represent the mean. (<strong>B, C</strong>) Flow cytometry analysis using live and dead staining (Annexin V and propidium iodide). B16-F10 melanoma cells were cultured in the presence of 0.007 μM DMSO or 12.5–1.5625 μM GO-Y078 for 24 h. The data show representative density plots (<strong>B</strong>). Statistical analyses of apoptotic dead cells (Annexin V+propidium iodide+) and early apoptotic dead cells (Annexin V+propidium iodide+) compared with 0.007 μM DMSO-treated cultured B16-F10 melanoma cells. Data show triplicate and are representative of three independent experiments (<strong>C</strong>). (<strong>D</strong>) The real-time quantitative analysis results of 0.007 μM DMSO- or 1.5625 μM GO-Y078-treated B16-F10 melanoma cells for 24 h. Data show triplicate and are representative of three independent experiments. (<strong>E</strong>) l-lactate production of cultured B16-F10 melanoma cells between DMSO and GO-Y078 treatments for 24 h. Data show triplicate and are representative of three independent experiments. (<strong>F, G</strong>) Flow cytometry analysis using live and dead staining (Annexin V and propidium iodide). B16-F10 melanoma cells were cultured in the presence of 0.007 μM DMSO or 1.5625 μM GO-Y078 for 2 h. The data show representative density plots (<strong>F</strong>). Statistical analyses of apoptotic dead cells (Annexin V+propidium iodide+) compared with 0.007 μM DMSO-treated cultured B16-F10 melanoma cells. Data show triplicate and are representative of three independent experiments (<strong>G</strong>). (<strong>H</strong>) Glycolysis measured using the glycolytic rate test. Glycolysis was calculated after Rotenon/Antimycin A treatment based on the rate of OCR in gastric tumor SH-10-TC cells. White bar indicates 0.007 μM DMSO treatment; black bar indicates 1.5625 μM GO-Y022 treatment for 2 h. Student’s t-test (<strong>D, E, G, H</strong>) or one-way ANOVA with post hoc Dunnett’s test for multiple comparison (<strong>A</strong> and <strong>C</strong>) was used. Statistical significance was set at p &lt; 0.05; *p &lt; 0.05, **p &lt; 0.01, and ***p &lt; 0.001.

<strong>Fig. 2 GO-Y078 treatment prevented tumor growth</strong><em><strong> in vivo</strong></em>
(<strong>A</strong>) Representative picture of tumor size at the end of experiment (Day 13). (<strong>B</strong>) Calculation of the tumor volume (mm3) for each day beginning 7 days after tumor injection (DMSO = 7, GO-Y078 = 8, mean ± standard deviation). (<strong>C</strong>, <strong>D</strong>) Representative intracellular staining of Foxp3+ Tregs (TCRβ+ CD4+ CD8α- Zombie-Foxp3+ population) in tumor-infiltrating lymphocytes. (<strong>E</strong>, <strong>F</strong>) Representative staining of exhausted CD8+ T cells (TCRβ+ CD4- CD8α+ Zombie- PD-1+ Tim3+ population) in tumor-infiltrating lymphocytes. Student’s t-test (<strong>B,</strong> <strong>D, F</strong>) was used. Statistical significance was set at p &lt; 0.05; *p &lt; 0.05, and **p &lt; 0.01.

<strong>Fig. 3 GO-Y078 treatment augmented CD8+ T cell-induced tumor cytotoxicity </strong><em><strong>in vitro</strong></em>
(<strong>A</strong>) Diagram of co-culture experiment. OVA-carrying B16-F10 melanoma cells (5 × 105 cells) labeled with CellTrace VioletTM and cultured with CD8+ T cells (1 × 105 cells) from OT-I mice. 25% culture supernatants from OVA-carrying B16-F10 melanoma cells were used and cultured in the presence of 0.007 μM DMSO or 0.25 μM GO-Y078 for 48 h at 37°C and 5% CO2. (<strong>B</strong>, <strong>C</strong>) Representative Annexin V and propidium iodide staining to check the percentage of apoptotic cell death (Annexin V+ propidium iodide+) in tumor cells (CellTrace Violet+ CD8-) or CD8+ T cells (CellTrace Violet- CD8+). (<strong>D</strong>) Absolute number of CD8+ T cells (CellTrace Violet- CD8+ Annexin V- propidium iodide-) in the co-culture experiment. (<strong>E</strong>) Scheme of tumor models using OVA-carried B16-F10 melanoma cells. 0.75 × 106 OVA-carried B16-F10 melanoma cells were subcutaneously injected at day 0. Seven days later, DMSO- or GO-Y078-PBS injected intraperitoneal in each days. In the end of experiments (day 13), collect the tumors and analyze. (<strong>F</strong>) Representative picture of tumor size at Day 13. (<strong>G</strong>) Tumors’ weight at Day 13. (<strong>H</strong>) Absolute numbers of CD8+ T cells per tumor volumes. (<strong>I</strong>, <strong>J</strong>) Representative OVA-tetramer and CD8α staining in tumor microenvironments. (<strong>K</strong>) Scheme of tumor models using Rag-1-deficient mice. Rag-1-deficient mice received B16-F10 melanoma cells (0.25 x105 cells: S.C. injection) or CD8+ T cells (1x106 cells: I.V. injection) at day 0. Seven days later, DMSO- or GO-Y078-PBS injected intraperitoneal in each days. In the end of experiments (day 13), collect the tumors and analyze. (<strong>L</strong>) Representative picture of tumor size at Day 13. (<strong>M</strong>) Calculation of the tumor volume (mm3) for each day beginning 7 days after tumor injection (n = 10, mean ± standard deviation). Student’s t-test (<strong>C,</strong> <strong>D, G, H, J</strong>) or One-way ANOVA with post hoc Dunnett’s test for multiple comparison (<strong>M</strong>) were used. Statistical significance was set at <em>p </em>&lt; 0.05; *p &lt; 0.05., **p &lt; 0.01.

<strong>Fig. 4 GO-Y078 treatment induced apoptotic cell death in CD8+ T cells even in the presence of the supernatant of tumor cells </strong>
(<strong>A</strong>) Representative picture of tumor size at the end of experiment (Day 13). (<strong>B</strong>) Calculation of the tumor volume (mm3) for each day beginning 7 days after tumor injection (n = 8, mean ± standard deviation). (<strong>A, B</strong>) Flow cytometry analysis using live and dead staining (Annexin V and propidium iodide). Naïve CD8+ T cells were cultured in the presence of 0.007 μM DMSO or 0.1–0.25 μM GO-Y078 for 48 h. The data show representative density plots (<strong>A</strong>). Statistical analyses of apoptotic dead cells (Annexin V+propidium iodide+) and early apoptotic dead cells (Annexin V+propidium iodide+) compared with 0.007 μM DMSO-treated. Data show triplicate and are representative of three independent experiments (<strong>B</strong>). (<strong>C</strong>) Live cell counting assay. Naïve CD8+ T cells were cultured with 0.007 μM DMSO (control), 0.1–0.25 μM GO-Y078 as indicated for 44 h, followed by the addition of the Cell Counting Kit 8 reagent (4 h). The circles indicate independent experiments. The horizontal bars represent the mean. (<strong>D</strong>) Absolute number of cultured naïve CD8+ T cells (48 h). 0.007 μM DMSO or 0.1–0.25 μM GO-Y078 treatment for 48 h. Data show triplicate and are representative of three independent experiments. (<strong>E, F</strong>) Flow cytometry analysis using live and dead staining (Annexin V and propidium iodide). Naïve CD8+ T cells were cultured in the presence of 0.007 μM DMSO or 0.25 μM GO-Y078 for 48 h in the presence or absence of the tumor supernatant. Data show representative density plots (<strong>E</strong>). Statistical analyses of apoptotic dead cells (Annexin V+propidium iodide+) and early apoptotic dead cells (Annexin V+propidium iodide+) compared with 0.007 μM DMSO treatment. Data show triplicate and are representative of three independent experiments (<strong>F</strong>). Student’s t-test (<strong>B</strong>, <strong>C</strong>,<strong> D</strong>, <strong>F</strong>) was used. Statistical significance was set at p &lt; 0.05; *p &lt; 0.05. Statistical significance was set at p &lt; 0.05; *p &lt; 0.05, **p &lt; 0.01, and ***p &lt; 0.001.

<strong>Fig. 5 GO-Y078 treatment in cooperation with anti-OX40 induced strong antitumor effects </strong><em><strong>in vivo</strong></em>
(<strong>A</strong>) Representative picture of tumor size at the end of experiment (Day 13). (<strong>B</strong>) Calculation of the tumor volume (mm3) for each day beginning 7 days after tumor injection (n = 8, mean ± standard deviation). (<strong>C</strong>) Representative picture of tumor-infiltrating CD8+ cells (green) and DAPI (blue). The original magnification is 20×.  (<strong>D</strong>)Absolute number of CD8+ T cells in tumor area.  (<strong>E</strong>) IFN-γ production from CD8+ T cells in TIL. (<strong>F</strong>) The real-time quantitative analysis results of Control antibody with DMSO-treated or anti-OX-40 antibody with GO-Y078-treated B16-F10 melanoma tissue at 13 days (n = 8, mean). One-way ANOVA with post hoc Dunnett’s test for multiple comparison (<strong>D</strong>) or Student’s t-test (<strong>E</strong>) was used. Statistical significance was set at p &lt; 0.05; *p &lt; 0.05.