Journal: Frontiers in Immunology

Article Title: Regulation of human Th9 cell differentiation by lipid modulators targeting PPAR-γ and acetyl-CoA-carboxylase 1

doi: 10.3389/fimmu.2024.1509408

Figure Lengend Snippet: IL-21 maximizes IL-9 production in the presence of IL-4 and TGF-β. Naïve human CD4 T cells were cultured for 4 days with different combinations of IL-4 (50ng/mL), TGF-β (5ng/mL) and IL-21 (25ng/mL), as indicated. (A) On day 4, cells were restimulated with PMA plus ionomycin in the presence of brefeldin A and stained for IL-9 and Foxp3. Supernatants were measured for IL-9 (B) and IL-5 (C) by ELISA. Asterisks (*) represent significant differences compared to control cultures with no cytokines. (D) Cultures incubated for 4 days with or without peptide P60 (75-100μM) were assessed for IL-9 by ELISA. (E) Gene expression of GATA3, FOXP3, PPARG and SPI1, normalized to GAPDH, measured by PCR. Fold changes relative to control cultures with no cytokines are shown. Asterisks (*) represent significant differences compared to IL-4 treatment. Data in (A-C) are combined from four experiments with n=14-17. Data in (D) are combined from two experiments with n=2-3. Data in (E) are combined from three experiments with n=3-4.

Article Snippet: Additional treatments included rosiglitazone (10μM, 40μM, Tocris Bioscience), GW 9662 (10μM, Tocris), 2-deoxy-D-glucose (2DG, 1mM, Tocris), AICAR (200-500μM, Tocris), dorsomorphin dihydrochloride (5-40μM), fatostatin A (10μM, Tocris), CBM 301940 (1μM, Tocris), 5-(Tetradecyloxy)-2-furoic acid (TOFA, 10μM, Sigma-Aldrich), oleic acid (5-20μM, Cayman Chemical), and peptide P60 (75-100μM, Abbiotec).

Techniques: Cell Culture, Staining, Enzyme-linked Immunosorbent Assay, Control, Incubation, Expressing

Journal: Journal of neuroimmunology

Article Title: TGF-β1/Smad2/3/Foxp3 signaling is required for chronic stress-induced immune suppression

doi: 10.1016/j.jneuroim.2017.11.005

Figure Lengend Snippet: Critical role of TGF-β1/Smad2/3 signaling on chronic stress-induced FoxP3 and the numbers of CD25+Foxp3+ cells. (A) After stress of the indicated time periods, spleens were harvested. The expression of Foxp3 in the nucleus was examined by Western blot analysis. (B) WT mice were administrated with SB203580 (1mg/kg body weight, i.p.), SB431542 (4.2mg/kg body weight, i.p.), ISI3(2.5mg/kg body weight, i.p.), peptide P60 (4.8mg/kg body weight, i.p.) for 1 h, 2 h, 2 h and 2 h respectively, before the initiation of stress. Foxp3 expression in spleens was determined by Western blot analysis. (C) After treated the mice as protocol (B), the mice were subjected to chronic stress for 2 days of 12 h. The spleens were harvested and isolated CD4+ cells by CD4+ isolation kit, stained the isolated cells with FICT-Foxp3 and PE-CD25 and determined the cells gated on CD25+Foxp3+ staining cells by flow cytometry. (N = 5 per group. *p < 0.05, **p < 0.01, ***p < 0.001 compared with indicated groups.

Article Snippet: P38 inhibitor SB203580 (1mg/kg body weight, Calbiochem, La Jolla, CA), TGF-β inhibitor SB431542 (4.2mg/kg body weight, Sigma-Aldrich), Smad3 inhibitor ISI3 (2.5mg/kg body weight, Cayman Chemical, Michigan), and Foxp3 inhibitor peptide P60 (50 nmol/mouse (i.p., Abbiotec, San Diego, CA) were administered via i.p.1 h (for SB203580) or 2 h (for SB431542, ISI3 and peptide P60) before the initiation of stress ( Caraci et al., 2015 ; Casares et al., 2010 ; Hu et al., 2014 ; Li et al., 2010 ; Wang et al., 2013 ).

Techniques: Expressing, Western Blot, Isolation, Staining, Flow Cytometry

Journal: Journal of neuroimmunology

Article Title: TGF-β1/Smad2/3/Foxp3 signaling is required for chronic stress-induced immune suppression

doi: 10.1016/j.jneuroim.2017.11.005

Figure Lengend Snippet: Inhibition of TGF-β1/Smad2/3 signal prevents splenocyte apoptosis induced by chronic stress. WT mice were administrated SB203580, SB431542, ISI3, and P60 2 h before the mice were subjected to chronic stress for 2 days of 12 h. (A) Apoptotic cells in spleens were determined by TUNEL assay. (B) Cellular lysates were isolated from mouse spleens and the levels of Bax, Bcl-2, and total and cleaved caspase-3 were determined by western blot analysis. (C) Apoptosis regulated kinase1 (ASK1) expression levels were detected in the mouse spleens by western blot analysis. N = 5 per group. *p < 0.05, **p < 0.01, ***p < 0.001 compared with indicated groups.

Article Snippet: P38 inhibitor SB203580 (1mg/kg body weight, Calbiochem, La Jolla, CA), TGF-β inhibitor SB431542 (4.2mg/kg body weight, Sigma-Aldrich), Smad3 inhibitor ISI3 (2.5mg/kg body weight, Cayman Chemical, Michigan), and Foxp3 inhibitor peptide P60 (50 nmol/mouse (i.p., Abbiotec, San Diego, CA) were administered via i.p.1 h (for SB203580) or 2 h (for SB431542, ISI3 and peptide P60) before the initiation of stress ( Caraci et al., 2015 ; Casares et al., 2010 ; Hu et al., 2014 ; Li et al., 2010 ; Wang et al., 2013 ).

Techniques: Inhibition, TUNEL Assay, Isolation, Western Blot, Expressing

Journal: Journal of neuroimmunology

Article Title: TGF-β1/Smad2/3/Foxp3 signaling is required for chronic stress-induced immune suppression

doi: 10.1016/j.jneuroim.2017.11.005

Figure Lengend Snippet: Inhibition of TGF-β1/Smad2/3 restores chronic-induced immune suppression. WT mice were pretreated with SB203580, SB431542, ISI3 and peptide P60 as Fig. 7. After 2 days of 12 h chronic stress, the mice were challenged with 10 μg of bacterial LPS (i.p.). After 6 h, blood and spleens were harvested. (A) The transcriptional levels of TNF-α, IL-17, TGF-β1, and IL-10 cytokines were determined by quantitative RT-PCR. (B) The circulating levels of TNF-α, IL-17, TGF-β1, and IL-10 cytokines were examined by ELISA. N = 5 per group. *p < 0.05, **p < 0.01, *** p < 0.001 compared with indicated groups.

Article Snippet: P38 inhibitor SB203580 (1mg/kg body weight, Calbiochem, La Jolla, CA), TGF-β inhibitor SB431542 (4.2mg/kg body weight, Sigma-Aldrich), Smad3 inhibitor ISI3 (2.5mg/kg body weight, Cayman Chemical, Michigan), and Foxp3 inhibitor peptide P60 (50 nmol/mouse (i.p., Abbiotec, San Diego, CA) were administered via i.p.1 h (for SB203580) or 2 h (for SB431542, ISI3 and peptide P60) before the initiation of stress ( Caraci et al., 2015 ; Casares et al., 2010 ; Hu et al., 2014 ; Li et al., 2010 ; Wang et al., 2013 ).

Techniques: Inhibition, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Journal of neuroimmunology

Article Title: TGF-β1/Smad2/3/Foxp3 signaling is required for chronic stress-induced immune suppression

doi: 10.1016/j.jneuroim.2017.11.005

Figure Lengend Snippet: Critical role of TGF-β1/Smad2/3 signaling on chronic stress-induced FoxP3 and the numbers of CD25+Foxp3+ cells. (A) After stress of the indicated time periods, spleens were harvested. The expression of Foxp3 in the nucleus was examined by Western blot analysis. (B) WT mice were administrated with SB203580 (1mg/kg body weight, i.p.), SB431542 (4.2mg/kg body weight, i.p.), ISI3(2.5mg/kg body weight, i.p.), peptide P60 (4.8mg/kg body weight, i.p.) for 1 h, 2 h, 2 h and 2 h respectively, before the initiation of stress. Foxp3 expression in spleens was determined by Western blot analysis. (C) After treated the mice as protocol (B), the mice were subjected to chronic stress for 2 days of 12 h. The spleens were harvested and isolated CD4+ cells by CD4+ isolation kit, stained the isolated cells with FICT-Foxp3 and PE-CD25 and determined the cells gated on CD25+Foxp3+ staining cells by flow cytometry. (N = 5 per group. *p < 0.05, **p < 0.01, ***p < 0.001 compared with indicated groups.

Article Snippet: Animals treatment protocol P38 inhibitor SB203580 (1mg/kg body weight, Calbiochem, La Jolla, CA), TGF-β inhibitor SB431542 (4.2mg/kg body weight, Sigma-Aldrich), Smad3 inhibitor ISI3 (2.5mg/kg body weight, Cayman Chemical, Michigan), and Foxp3 inhibitor peptide P60 (50 nmol/mouse (i.p., Abbiotec, San Diego, CA) were administered via i.p.1 h (for SB203580) or 2 h (for SB431542, ISI3 and peptide P60) before the initiation of stress ( Caraci et al., 2015 ; Casares et al., 2010 ; Hu et al., 2014 ; Li et al., 2010 ; Wang et al., 2013 ).

Techniques: Expressing, Western Blot, Isolation, Staining, Flow Cytometry

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Immunization against HIF-1α inhibits the growth of basal mammary tumors and targets mammary stem cells in vivo

doi: 10.1158/1078-0432.CCR-16-1678

Figure Lengend Snippet: HIF-1α-specific antibodies can be detected in the sera of breast cancer patients with the highest incidence and levels in those with the triple negative subtype. (A) Incidence of positive HIF-1α-specific antibodies in each group. *p<0.05, **p<0.01, ***p<0.001 compared to the control group. (B) HIF-1α-specific IgG (μg/ml ± SEM) for the experimental groups. ***p<0.001 between groups. TNBC: triple negative breast cancer; ER+: estrogen receptor positive breast cancer; HER2+: human epidermal growth factor receptor 2 positive breast cancer.

Article Snippet: Each mouse was injected with 50 µl of a HIF-1α peptide pool (p38-53, p60-82, and p93-117; 50 μg each) as a mixture in complete Freund’s adjuvant/incomplete Freund’s adjuvant (Sigma).

Techniques:

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Immunization against HIF-1α inhibits the growth of basal mammary tumors and targets mammary stem cells in vivo

doi: 10.1158/1078-0432.CCR-16-1678

Figure Lengend Snippet: Th1-inducing epitopes derived from the HIF-1α protein sequence can be identified. Mean percent responding donors to HIF-1α epitopes with IFN-γ (A) or IL-10 (B). CSPW for the indicated epitope presented as interquartile box plots with Tukey whiskers for IFN-γ (C) and IL-10 (D). Median CSPW is indicated by the horizontal bar.

Article Snippet: Each mouse was injected with 50 µl of a HIF-1α peptide pool (p38-53, p60-82, and p93-117; 50 μg each) as a mixture in complete Freund’s adjuvant/incomplete Freund’s adjuvant (Sigma).

Techniques: Derivative Assay, Sequencing

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Immunization against HIF-1α inhibits the growth of basal mammary tumors and targets mammary stem cells in vivo

doi: 10.1158/1078-0432.CCR-16-1678

Figure Lengend Snippet: HIF-1α poly-epitope vaccines are significantly more effective in inhibiting growth of basal than luminal mammary tumors. (A) IFN-γ and IL-4 ELISPOT in splenocytes after completion of three immunizations. Antigens include a pool of the HIF-1α vaccinating peptides (●), HIV p52 (○) as a negative control and ConA (▲) as a positive control. The data are presented as corrected spots per well (CSPW). The horizontal bar indicates the mean CSPW ± SEM. n=5 mice/group; ***p<0.001 compared to HIV. Mean tumor volume (mm3 ± SEM) from mice injected with adjuvant alone (●) or HIF-1α poly-epitope vaccine (▲) in TgMMTV-neu mice (B) or C3(1)Tag mice (C), n=5 mice/group; ****p<0.0001. (D) Mean tumor volume (mm3 ± SEM) from C3(1)Tag mice injected with adjuvant alone (●), or HIF-1α poly-epitope vaccine treated with mouse IgG (●), anti-CD4 (○) or anti-CD8 (■) n=5 mice/group; ****p<0.0001 compared to HIF-1α poly-epitope vaccine+IgG.

Article Snippet: Each mouse was injected with 50 µl of a HIF-1α peptide pool (p38-53, p60-82, and p93-117; 50 μg each) as a mixture in complete Freund’s adjuvant/incomplete Freund’s adjuvant (Sigma).

Techniques: Enzyme-linked Immunospot, Negative Control, Positive Control, Injection

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Immunization against HIF-1α inhibits the growth of basal mammary tumors and targets mammary stem cells in vivo

doi: 10.1158/1078-0432.CCR-16-1678

Figure Lengend Snippet: Basal-like breast cancer contains greater levels of cancer stem cells than luminal/neu breast cancer. (A) Representative Western blot of cell lysate for HIF-1α (I) and Tubulin (II). Relative pixel density ± SD as measured by densitometry from 3 independent experiments (III), **p<0.05. (B) Percent of Sca-1 expressing cells in the indicated cell line. ****p<0.0001; n=4 independent experiments. (C) Representative Western blot of Sca-1positive and Sca-1negative cell lysate for HIF-1α (I) and Tubulin (II). Relative pixel density ± SD as measured by densitometry from 3 independent experiments (III), **p<0.01.

Article Snippet: Each mouse was injected with 50 µl of a HIF-1α peptide pool (p38-53, p60-82, and p93-117; 50 μg each) as a mixture in complete Freund’s adjuvant/incomplete Freund’s adjuvant (Sigma).

Techniques: Western Blot, Expressing

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Immunization against HIF-1α inhibits the growth of basal mammary tumors and targets mammary stem cells in vivo

doi: 10.1158/1078-0432.CCR-16-1678

Figure Lengend Snippet: HIF-1α immunization results in a significant reduction of cancer stem cells in mammary tumors. (A) Percent CD4+ or CD8+ T-cells in the tumor from mice immunized with adjuvant alone (white bar) or HIF-1α peptides (gray bar) presented as box and whisker plot with Tukey outliers, n=5 mice/group; ****p<0.0001. (B) Representative Western blot of tumor cell lysate from mice immunized with adjuvant alone (control) or HIF-1α peptides for HIF-1α (I) and Tubulin (II). Relative pixel density ± SD as measured by densitometry from 3 independent experiments (III), *p<0.05. (C) Percent of Sca-1 expressing cells in tumors from mice immunized with adjuvant alone (control) or HIF-1α peptides. *p<0.05; n=9 mice/group.

Article Snippet: Each mouse was injected with 50 µl of a HIF-1α peptide pool (p38-53, p60-82, and p93-117; 50 μg each) as a mixture in complete Freund’s adjuvant/incomplete Freund’s adjuvant (Sigma).

Techniques: Whisker Assay, Western Blot, Expressing