Fig 1: Olaparib activates STAT3 in fibroblasts and immune cells, suppressing immune responses (A–D) Western blot showing activated (P-Y705) and total STAT3 levels after Olaparib in freshly isolated splenocytes, CD19+ B cells, and bone marrow-derived macrophages from naive mice, as well as in 3T3 and MEF fibroblasts after treatment with DMSO or 10 µM Olaparib for 24 h. Numbers on the blot show relative activated (P-Y705) vs. total STAT3 ratios as determined by ImageJ. Shown are representative blots of three (A, D) or two (B, C) independent experiments. GAPDH and a-Tubulin staining served as a loading control. (E) Western blotting showing activated (P-Y705) in freshly isolated human PBMCs from five healthy donors treated either with DMSO or 10 µM Olaparib for 48 h. Relative p-STAT3/STAT3 ratios were determined by ImageJ and are shown. a-Tubulin staining served as a loading control. (F) Real-time PCR measuring immunostimulatory IFNG, GZMB and immune-suppressive IL10 mRNA levels in human PBMCs from three different donors treated either with DMSO or 10 µM Olaparib for 48 h. Gene expression was normalized to the housekeeper gene ACTB and relative expressions against vehicle controls are shown. Unpaired two-tailed Student t-test of Olaparib treatment performed against DMSO controls. Shown are means ± SD (n = 3). *p<0.05, **p<0.01, and ***p<0.001. (G) ELISA assessing IL-10 production in the indicated donor PBMC cultures treated either with DMSO or 10 µM Olaparib for 48 h. Unpaired two-tailed Student t-test of Olaparib treatment performed against DMSO controls, shown are means ± SD (n = 3). *p<0.05, **p<0.01, and ***p<0.001 (H). Diagram showing the tumor-conditioned medium (TCM) transfer system (right). Parental or Olaparib-resistant OVCAR8 cells were plated in a 10-cm dish and incubated for 48 h. The supernatant was transferred to a 50-ml centrifuge tube and centrifuged for 5 min. Fresh PBMCs were serum-starved for 24 h, incubated with TCM from parental (TCM Par) or Olaparib-resistant (TCM Res) OVCAR8 cells for 2 h and whole cell lysates were analyzed by Western blotting showing activated (P-Y705) in human PBMCs following TCM incubation (left). For the control, full-serum media was added to PBMCs for 2 h. Activated (P-Y705) vs. total STAT3 ratios were determined by ImageJ. Data are representative of at least three independent experiments. GAPDH served as a loading control. (I) ELISA assessing IL-6 production in TCM from parental or Olaparib-resistant OVCAR8 cells. Unpaired two-tailed Student t-test of resistant TCM performed against parental controls, shown are means ± SD (n = 3). ***p<0.001, n.s., not significant.
Fig 2: Resveratrol-ßcd switch tumor-associated macrophage M2-type polarization. (A,C) Typical flow cytometry diagram of TAM (CD11b+F4/80+) or M2 macrophages (CD11b+F4/80+ CD206+). The cells were CD45+ gated. (B,D) The proportions of TAM or M2-like macrophages in the treatment and control groups. Error bar = mean ± S.D. ***p < 0.001. ****p < 0.0001. (E) The numbers of M2-like and (F) M1-like (CD206-F4/80+CD11b+) macrophages per tumor gram. **p < 0.01. (G) THP1 as the control group and THP1-derived M1 macrophages treated with resveratrol-ßcd at the indicated concentrations. TNF and (H) IL6 transcript levels and (I) IL-6 levels in the medium 48 h after treatment were measured to identify M1 polarization. Error bar = mean ± S.D. (J) MRC1, (K) IL10, and (L) IL-10 levels 48 h after treatment with the indicated concentration of resveratrol-ßCd. Error bar = mean ± S.D.
Fig 3: Ketogenic diet enhances human T-cell immune capacity in vivo Healthy volunteers conducted a 3-week KD with a limited carbohydrate consumption of < 30 g/day. Blood was taken and analyzed prior to (T0) and post-KD (T1). PBMCs were isolated. T-cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. CD4+/CD8+ T cells were separated via magnetic cell labeling. IL2, IL4, and CTLA4 mRNA expression in stimulated CD4+ T cells, n = 17/18/17 individual human subjects.Tbet and GATA3 mRNA expression in CD4+ T cells, n = 7 individual human subjects.Flow cytometric quantification of Th1/Th2 cells and respective ratio of Th1/Th2 cells, n = 7 individual human subjects.IL10 mRNA expression and flow cytometric quantification of CD4+CD25+Foxp3+ regulatory T cells (Treg), n = 19/9 individual human subjects.IFN? GZMB, PRF1, and CTLA4 mRNA expression in CD8+ T cells, n = 16/18/17/17 individual human subjects.Relative CD8+ cell lysis activity as measured by calcein fluorescence of isolated T cells, n = 4 individual experiments. Data information: Data depicted as box plots with median, 25th and 75th percentiles and range. Dots indicating individual values. *P < 0.05, paired t-test/Wilcoxon matched-pairs signed rank test, as appropriate.
Fig 4: Beta-hydroxybutyrate enhances human T-cell immune capacity in vitro Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48 h in RPMI containing 80 mg/dl glucose (NC) and supplemented with 10 mM beta-hydroxybutyrate (BHB). T-cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Human pan T-cell RNA was isolated, and cell culture supernatant was sampled. mRNA expression of CD4+ cytokines IL2, IL4, IL8, and IL22 relative to endogenous controls, n = 13/11/10/8 biological replicates.Protein expression of IL2, IL4, IL6, and IL8, analyzed in the supernatant of stimulated PBMCs, n = 9/12/11/11 biological replicates.Th1/Th2 cell transcription factors Tbet and GATA3 quantified via RT–qPCR and flow cytometric ratio of Th1/Th2 cells following differentiation, n = 6/6/4 biological replicates.mRNA expression of CD8+ cytokines IFN?, PRF1, TNFa, GZMB, and CTLA4 in stimulated human T cells relative to internal controls, n = 15/14/10/14/9 biological replicates.Protein expression of IFN?/TNFa in the supernatant of stimulated PBMCs and cell lysis-dependent calcein fluorescence of isolated T cells, n = 8/12/9 biological replicates.Foxp3 mRNA relative to internal control in human PBMCs and quantification of CD4+CD25+Foxp3+ regulatory T cells (Treg) following 5 days of Treg differentiation with a representative Foxp3 histogram plot (right side), n = 7/11 biological replicates.IL10 and TGFß1 mRNA and protein expression of human Treg cells, n = 8/9 (IL10), 5/9 (TGFß1) biological replicates. Data information: Data depicted as mean ± SEM (protein data) and box plots with median, 25th and 75th percentiles and range (all other). Dots indicating individual values. *P < 0.05, **P < 0.01, ***P < 0.001, paired t-test/Wilcoxon matched-pairs signed rank test, as appropriate.
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