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HN-1 treatment induced DC activation. ( A ) PBMCs from healthy volunteers were stained for HLA-A2 and detected via flow cytometry. ( B and C ) Collected PBMCs were stimulated with IL-4 and GM-CSF for 5 days. DCs were confirmed based on ( B ) morphological features and ( C ) CD11c expression. ( D ) MDA-MB-231 cells were pretreated with HN-1 for 24 h, co-cultured with DCs for an additional 48 h, and then separated by staining with CD45. CD45 + cells were collected as DCs, and the relative expression levels of CD83 , CD86 , IFNG , IL10 , IL4 , CD274 , IL12A , and IL2 were detected via qRT‒PCR. ( E ) The expression levels of CD80, CD83, HLA-DR, and CD86 were detected via flow cytometry. An isotype antibody was used as a control. The data are presented as the median fluorescence intensity (MFI). ( F ) The culture supernatant was collected, and the concentrations of IL10, IL-2, IL-12, <t>and</t> <t>IFN-γ</t> were detected via <t>ELISA.</t> The data were acquired from three independent experiments. ** p < 0.01. NC, negative control
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HN-1 treatment induced DC activation. ( A ) PBMCs from healthy volunteers were stained for HLA-A2 and detected via flow cytometry. ( B and C ) Collected PBMCs were stimulated with IL-4 and GM-CSF for 5 days. DCs were confirmed based on ( B ) morphological features and ( C ) CD11c expression. ( D ) MDA-MB-231 cells were pretreated with HN-1 for 24 h, co-cultured with DCs for an additional 48 h, and then separated by staining with CD45. CD45 + cells were collected as DCs, and the relative expression levels of CD83 , CD86 , IFNG , IL10 , IL4 , CD274 , IL12A , and IL2 were detected via qRT‒PCR. ( E ) The expression levels of CD80, CD83, HLA-DR, and CD86 were detected via flow cytometry. An isotype antibody was used as a control. The data are presented as the median fluorescence intensity (MFI). ( F ) The culture supernatant was collected, and the concentrations of IL10, IL-2, IL-12, <t>and</t> <t>IFN-γ</t> were detected via <t>ELISA.</t> The data were acquired from three independent experiments. ** p < 0.01. NC, negative control
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HN-1 treatment induced DC activation. ( A ) PBMCs from healthy volunteers were stained for HLA-A2 and detected via flow cytometry. ( B and C ) Collected PBMCs were stimulated with IL-4 and GM-CSF for 5 days. DCs were confirmed based on ( B ) morphological features and ( C ) CD11c expression. ( D ) MDA-MB-231 cells were pretreated with HN-1 for 24 h, co-cultured with DCs for an additional 48 h, and then separated by staining with CD45. CD45 + cells were collected as DCs, and the relative expression levels of CD83 , CD86 , IFNG , IL10 , IL4 , CD274 , IL12A , and IL2 were detected via qRT‒PCR. ( E ) The expression levels of CD80, CD83, HLA-DR, and CD86 were detected via flow cytometry. An isotype antibody was used as a control. The data are presented as the median fluorescence intensity (MFI). ( F ) The culture supernatant was collected, and the concentrations of IL10, IL-2, IL-12, <t>and</t> <t>IFN-γ</t> were detected via <t>ELISA.</t> The data were acquired from three independent experiments. ** p < 0.01. NC, negative control
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HN-1 treatment induced DC activation. ( A ) PBMCs from healthy volunteers were stained for HLA-A2 and detected via flow cytometry. ( B and C ) Collected PBMCs were stimulated with IL-4 and GM-CSF for 5 days. DCs were confirmed based on ( B ) morphological features and ( C ) CD11c expression. ( D ) MDA-MB-231 cells were pretreated with HN-1 for 24 h, co-cultured with DCs for an additional 48 h, and then separated by staining with CD45. CD45 + cells were collected as DCs, and the relative expression levels of CD83 , CD86 , IFNG , IL10 , IL4 , CD274 , IL12A , and IL2 were detected via qRT‒PCR. ( E ) The expression levels of CD80, CD83, HLA-DR, and CD86 were detected via flow cytometry. An isotype antibody was used as a control. The data are presented as the median fluorescence intensity (MFI). ( F ) The culture supernatant was collected, and the concentrations of IL10, IL-2, IL-12, <t>and</t> <t>IFN-γ</t> were detected via <t>ELISA.</t> The data were acquired from three independent experiments. ** p < 0.01. NC, negative control
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HN-1 treatment induced DC activation. ( A ) PBMCs from healthy volunteers were stained for HLA-A2 and detected via flow cytometry. ( B and C ) Collected PBMCs were stimulated with IL-4 and GM-CSF for 5 days. DCs were confirmed based on ( B ) morphological features and ( C ) CD11c expression. ( D ) MDA-MB-231 cells were pretreated with HN-1 for 24 h, co-cultured with DCs for an additional 48 h, and then separated by staining with CD45. CD45 + cells were collected as DCs, and the relative expression levels of CD83 , CD86 , IFNG , IL10 , IL4 , CD274 , IL12A , and IL2 were detected via qRT‒PCR. ( E ) The expression levels of CD80, CD83, HLA-DR, and CD86 were detected via flow cytometry. An isotype antibody was used as a control. The data are presented as the median fluorescence intensity (MFI). ( F ) The culture supernatant was collected, and the concentrations of IL10, IL-2, IL-12, and IFN-γ were detected via ELISA. The data were acquired from three independent experiments. ** p < 0.01. NC, negative control

Journal: Cell Communication and Signaling : CCS

Article Title: Hainanenin-1, an oncolytic peptide, triggers immunogenic cell death via STING activation in triple-negative breast cancer

doi: 10.1186/s12964-024-01731-6

Figure Lengend Snippet: HN-1 treatment induced DC activation. ( A ) PBMCs from healthy volunteers were stained for HLA-A2 and detected via flow cytometry. ( B and C ) Collected PBMCs were stimulated with IL-4 and GM-CSF for 5 days. DCs were confirmed based on ( B ) morphological features and ( C ) CD11c expression. ( D ) MDA-MB-231 cells were pretreated with HN-1 for 24 h, co-cultured with DCs for an additional 48 h, and then separated by staining with CD45. CD45 + cells were collected as DCs, and the relative expression levels of CD83 , CD86 , IFNG , IL10 , IL4 , CD274 , IL12A , and IL2 were detected via qRT‒PCR. ( E ) The expression levels of CD80, CD83, HLA-DR, and CD86 were detected via flow cytometry. An isotype antibody was used as a control. The data are presented as the median fluorescence intensity (MFI). ( F ) The culture supernatant was collected, and the concentrations of IL10, IL-2, IL-12, and IFN-γ were detected via ELISA. The data were acquired from three independent experiments. ** p < 0.01. NC, negative control

Article Snippet: The quantification of cytokines in the supernatant was performed using a human ELISA detection kit (IL-2, D2050; IL-10, D1000B, IFN-γ, DIF50C; R&D Systems, MN, USA) in accordance with the instructions provided by the manufacturer.

Techniques: Activation Assay, Staining, Flow Cytometry, Expressing, Cell Culture, Control, Fluorescence, Enzyme-linked Immunosorbent Assay, Negative Control