Journal: Pharmacological Reports

Article Title: Compromised diabetic heart function is not affected by miR-378a upregulation upon hyperglycemia

doi: 10.1007/s43440-023-00535-8

Figure Lengend Snippet: miR-378a is upregulated in hyperglycemic murine heart. A – I Heart lysates and J cardiac histological sections were obtained from either control or STZ-treated miR-378a+/+ and miR-378a−/− mice. The expression of A miR-378a-3p and B miR-378a-5p is increased in heart lysates of miR-378a+/+ mice 8 weeks upon STZ treatment, LNA miRCURY qPCR ( n = 4–8). mRNA level of C Igf1r , D Mapk1 and E Pik3ca (qPCR, n = 4–8). F – I Densitometric analysis of changes of IGF-1R pathway in response to STZ. F Combined analysis of IGF-1R and downstream mediators. Relative to GAPDH reference protein. The protein level of G IGF-1R (rel. to GAPDH), H pERK1/2 (rel. to total ERK1/2), and I p-AKT (rel. to total AKT), (western blot, densitometric analysis of five independent western blot repetitions, n = 3–4) upon STZ treatment. J IGF-1R expression in different cardiac cell types (CMs, SMCs, ECs; shown with arrows) (IF; ⍺-smooth muscle actin (⍺-SMA)/IGF-1R—green, laminin—purple, Cx43—red). Representative histological sections (×63 magnification, scale bar: 10 µm). Data are presented as mean ± SEM. # p < 0.05, ## p < 0.001, ### p < 0.0001—two-way ANOVA variation; * p < 0.05, ** p < 0.01 *** p < 0.001 by two-way ANOVA with Tukey’s post hoc test; $ p < 0.05, $$ p < 0.01 by unpaired two-tailed Student’s t test. Igf1r (IGF-1R) insulin-like growth factor 1 receptor, Mapk1 (ERK2) mitogen-activated protein kinase 1, Pik3ca PI3 kinase catalytic subunit alpha, (p)AKT (phosphorylated) protein kinase B, (p)ERK1/2 (phosphorylated) extracellular signal-regulated kinase 1/2

Article Snippet: Total RNA was used for reverse transcription (RT) of either mRNA (500 ng) or miRNA (10 ng) performed with RevertAid reverse transcriptase (200 U/μL, Thermo Fisher Scientific, cat. no. EP0442) and miRCURY LNA RT kit (Qiagen, cat. no. 339340), respectively, carried out in ProFlex PCR System (ThermoFisher Scientific).

Techniques: Control, Expressing, Western Blot, Two Tailed Test

Journal: Pharmacological Reports

Article Title: Compromised diabetic heart function is not affected by miR-378a upregulation upon hyperglycemia

doi: 10.1007/s43440-023-00535-8

Figure Lengend Snippet: High glucose increases miR-378a expression and stimulates pro-hypertrophic pathways in hiPSC-CMs. miR-378a+/+ hiPSCs and miR-378a−/− hiPSCs were differentiated to CMs and treated with glucose (GLU) for 48 h. Mannitol (55 mM) was used as a vehicle. The expression of A miR-378a-3p and -5p (LNA miRCURY qPCR, n = 4) and B IGF1R mRNA level (qPCR, n = 5). C – F Densitometric analysis of changes of IGF-1R pathway in response to glucose. C Combined analysis of IGF-1R and downstream mediators. Relative to GAPDH reference protein. D Protein level of IGF-1R (rel. to GAPDH), E pERK1/2 (rel. to total ERK1/2) and F p-AKT (rel. to total AKT), (western blot, densitometric analysis, n = 2–4). G Quantitative analysis of cell area ( n = 15/27/8/43) and representative pictures of hypertrophic growth of miR-378a−/− hiPSC-CMs under control conditions and 55 mM glucose (HG) (IF; ⍺-actinin—green), representative images (×200 magnification). Data are presented as mean ± SEM. # p < 0.05—two-way ANOVA variation; * p < 0.05, *** p < 0.001 by two-way ANOVA with Tukey’s post hoc test; $ p < 0.05 by unpaired two-tailed Student’s t test. GLU glucose, HG high glucose, hiPSC-CMs human induced pluripotent stem cell-derived cardiomyocytes, IGF1R (IGF-1R) insulin-like growth factor 1 receptor, (p)AKT (phosphorylated) protein kinase B, (p)ERK1/2 (phosphorylated) extracellular signal-regulated kinase 1/2

Article Snippet: Total RNA was used for reverse transcription (RT) of either mRNA (500 ng) or miRNA (10 ng) performed with RevertAid reverse transcriptase (200 U/μL, Thermo Fisher Scientific, cat. no. EP0442) and miRCURY LNA RT kit (Qiagen, cat. no. 339340), respectively, carried out in ProFlex PCR System (ThermoFisher Scientific).

Techniques: Expressing, Western Blot, Control, Two Tailed Test, Derivative Assay

Journal: Bioactive Materials

Article Title: Apoptotic vesicles ameliorate lupus and arthritis via phosphatidylserine-mediated modulation of T cell receptor signaling

doi: 10.1016/j.bioactmat.2022.07.026

Figure Lengend Snippet: ApoVs rescue dysregulated T cell proliferation and ameliorate murine lupus. (A) Scheme of 4-week systemic apoV supplementation in MRL/lpr mice. (B) Normalized ratio of apoVs/T cells in circulation and spleen of MRL/lpr mice treated with or without apoVs for 4 weeks in comparison to age-matched MRL/wt mice analyzed by flow cytometry. N = 6 per group. (C) Numbers of CD3+ cells in spleen and mesenteric lymph node (MLN) of MRL/lpr mice treated with or without apoVs for 4 weeks in comparison to age-matched MRL/wt. N = 6 per group. (D, E) Frequency of naïve, central memory (Tcm), effector memory (Tem), and effector CD4+ T cells in spleen of MRL/lpr mice treated with or without apoVs for 4 weeks in comparison to age-matched MRL/wt. N = 6 per group. (F, G) Frequency of IFNγ+CD4+ T cells in spleen and MLN analyzed by flow cytometry, and serum levels of IFNγ measured by ELISA of MRL/lpr mice treated with or without apoVs for 4 weeks in comparison to age-matched MRL/wt. N = 6–11 per group. (H) Frequency of Foxp3+CD4+ T cells in spleen and MLN of MRL/lpr mice treated with or without apoVs for 4 weeks in comparison to age-matched MRL/wt. N = 6 per group. (I) Scheme of long-term systemic apoV supplementation in MRL/lpr mice. (J) Severe proteinuria (>0.3 g/L) onset in MRL/lpr mice treated with or without apoVs for 8 weeks in comparison to age-matched MRL/wt mice. N = 6 per group. (K) Representative hematoxylin and eosin (HE) staining and Masson's trichrome staining of kidney from MRL/lpr mice treated with or without apoVs for 12 weeks in comparison to age-matched MRL/wt mice. Green triangle indicates immune cell infiltration. Yellow triangle indicates eosinophilic material deposition. Black triangle indicates necrotic glomerulus. Black contour indicates crescent formation. Scale bar = 50 μm. (L, M) Size and weight of spleen and MLN of MRL/lpr mice treated with or without apoVs for 12 weeks in comparison to age-matched MRL/wt mice. N = 6 per group. Scale bar = 1 cm. (N) Representative immunofluorescence staining of CD3+ T cells in lung and colon of MRL/lpr mice treated with or without apoVs for 12 weeks in comparison to age-matched MRL/wt mice. Scale bar = 200 μm. (O) Serum levels of anti-dsDNA IgG measured by ELISA from MRL/lpr mice treated with or without apoVs for 4 weeks in comparison to age-matched MRL/wt. N = 5–15 per group. (P) Survival of MRL/lpr mice treated with or without apoVs starting at 8 weeks in comparison to age-matched MRL/wt mice. N = 6 per group. Kruskal-Wallis test and ANOVA was used for comparison among three groups when appropriate. Mantel-Cox test was used for proteinuria and survival analysis. Data are shown as mean ± standard deviation. ns, not significant. *, p < 0.05. **, p < 0.01. ***, p < 0.001. ****, p < 0.0001.

Article Snippet: Total CD4 + T cells and naïve CD25 − CD4 + T cells were isolated from mouse spleen using magnetic beads (130-091-041, Miltenyi Biotec, Germany).

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining, Immunofluorescence, Standard Deviation

Journal: Bioactive Materials

Article Title: Apoptotic vesicles ameliorate lupus and arthritis via phosphatidylserine-mediated modulation of T cell receptor signaling

doi: 10.1016/j.bioactmat.2022.07.026

Figure Lengend Snippet: ApoVs preferentially suppress activation of effector T cells in a dose-dependent manner. (A, B) Frequency of activated CD4+ T cells measured in vitro after 3 days with or without anti-CD3/CD28 antibody stimulation in the presence of different doses of apoVs. Representative flow cytometric analyses of CD25 expression on CD4+ T cells are shown. N = 5 per group. (C) Level of IL-2 in splenocyte culture supernatants measured by ELISA after 3 days of anti-CD3/CD28 antibody stimulation in the presence of different doses of apoVs. N = 5 per group. (D) Frequency of activated CD4+ T cells measured in vitro after 3 days with 1 μg/mL (Lo) or 10 μg/mL (Hi) anti-CD3/CD28 antibody stimulation in the presence of 0.2x apoVs. Representative flow cytometric analyses are shown. N = 3–4 per group. (E) Frequency of Th1, Th17 and Th2 of CD4+ T cells measured in vitro after 3 days with or without anti-CD3/CD28 antibody stimulation in the presence of 0.2x apoVs. N = 3 per group. (F) Levels of IFNγ, IL-17A and IL-10 in splenocyte culture supernatants measured by ELISA after 3 days of anti-CD3/CD28 antibody stimulation in the presence of 0.2x apoV. N = 3 per group. (G) Frequency of Foxp3+CD4+ T cells measured in vitro after 3 days with or without anti-CD3/CD28 antibody stimulation in the presence of 0.2x apoVs. N = 4 per group. Mann-Whitney test and student's t-test was used for comparison between two groups when appropriate. Kruskal-Wallis test and ANOVA was used for comparison among multiple groups when appropriate.Data are shown as mean ± standard deviation. ns, not significant. *, p < 0.05. **, p < 0.01. ***, p < 0.001. ****, p < 0.0001.

Article Snippet: Total CD4 + T cells and naïve CD25 − CD4 + T cells were isolated from mouse spleen using magnetic beads (130-091-041, Miltenyi Biotec, Germany).

Techniques: Activation Assay, In Vitro, Expressing, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Standard Deviation

Journal: Bioactive Materials

Article Title: Apoptotic vesicles ameliorate lupus and arthritis via phosphatidylserine-mediated modulation of T cell receptor signaling

doi: 10.1016/j.bioactmat.2022.07.026

Figure Lengend Snippet: ApoVs directly interact with T cells via phosphatidylserine to inhibit TCR signaling. (A) Representative images of flow cytometric analysis of CD11b expression on CD11b-DTR mice treated with or without diphtheria toxin (DT). (B) Frequency of CD25+CD4+ T cells in circulation and spleen of CD11b-DTR mice at 24 h after treatment with or without apoVs. N = 3 per group. (C–E) The experimental scheme, representative flow cytometric analyses and quantification of CD25 expression from naïve CD4+ T cell cultures that were unstimulated (Unstim) or anti-CD3/CD28 stimulated with or without 0.2x apoVs. N = 3 per group. (F) Activation fold change of naïve CD4+ T cells stimulated in the presence of PBS (Ctr), 0.2x apoVs (ApoVs) or 0.2x annexin V (A5) pre-treated apoVs. N = 4 per group. (G) IL-2 levels in supernatants of naïve CD4+ T cells stimulated in the presence of PBS (Ctr), 0.2 x apoVs (ApoVs) or 0.2 x annexin V (A5) pre-treated apoVs. N = 4 per group. (H, I) Representative flow cytometric analyses and quantification of CD25 expression on CD4+ T cells from splenocyte cultures that were unstimulated (Unstim), anti-CD3/CD28 stimulated in the presence of PBS (Stim), apoVs, exosome or soluble fraction of apoVs (sApoVs). N = 3–4 per group. (J) Frequency of CD25+CD4+ T cells measured in vitro after 3 days with or without anti-CD3/CD28 antibody stimulation in the presence of 1x apoV (1 apoV: 1 T cell) derived from BMMSCs or T cells from C57bl6 (WT) and MRL/lpr (LPR) mice. N = 3–6 per group. (K) Frequency of activated CD4+ T cells in healthy human peripheral blood mononuclear cells (PBMC) measured in vitro after 3 days with or without anti-CD3/CD28 antibody stimulation in the presence of different doses of apoVs derived from mouse BMMSCs. N = 3–5 per group. (L) Representative immunofluorescence staining of CD3+ T cells and PKH26-labelled apoVs with or without A5 pre-treatment. Scale bar = 10 μm. (M) Frequency of apoVs+ T cells after 15, 30 and 60 min of coculture measured by immunofluorescence staining. N = 3 per group. (N) Frequency of apoVs+ T cells after 60 min of coculture with or without A5 pre-treatment measured by immunofluorescence staining. N = 3 per group. (O) Western blotting shows expression of proximal TCR signaling proteins p-CD3, p-ZAP70, p-LCK, p-PLCγ and NFAT1 in naïve CD4+ T cells stimulated with anti-CD3/CD28 antibodies in the presence of PBS (Ctr), 0.2 x apoVs (ApoVs) or 0.2 x annexin V (A5) pre-treated apoVs for 1 h. Mann-Whitney test and student's t-test was used for comparison between two groups when appropriate. Kruskal-Wallis test and ANOVA was used for comparison among three groups when appropriate. Data are shown as mean ± standard deviation. **, p < 0.01. ***, p < 0.001. ****, p < 0.0001.

Article Snippet: Total CD4 + T cells and naïve CD25 − CD4 + T cells were isolated from mouse spleen using magnetic beads (130-091-041, Miltenyi Biotec, Germany).

Techniques: Expressing, Activation Assay, In Vitro, Derivative Assay, Immunofluorescence, Staining, Western Blot, MANN-WHITNEY, Standard Deviation

Journal: Bioactive Materials

Article Title: Apoptotic vesicles ameliorate lupus and arthritis via phosphatidylserine-mediated modulation of T cell receptor signaling

doi: 10.1016/j.bioactmat.2022.07.026

Figure Lengend Snippet: Blocking phosphatidylserine on apoVs stunts their immunoregulatory effect in murine lupus. (A) Scheme of 4-week systemic infusion of apoVs or A5 pre-treated apoVs in MRL/lpr mice. (B) Normalized ratios of apoVs/T cells in circulation and spleen of MRL/lpr mice treated with apoVs or A5 pre-treated apoVs for 4 weeks in comparison with age-matched MRL/lpr mice analyzed by flow cytometry. N = 6 per group. (C, D) Frequency of IFNγ+CD4+ T cells in circulation and spleen analyzed by flow cytometry, and serum levels of IFNγ measured by ELISA of MRL/lpr mice treated with apoVs or A5 pre-treated apoVs for 4 weeks in comparison with age-matched MRL/lpr mice analyzed by flow cytometry. N = 6 per group. (E, F) Frequency of naïve, central memory (Tcm), effector memory (Tem), and effector CD4+ T cells in spleen of MRL/lpr mice treated with apoVs or A5 pre-treated apoVs for 4 weeks in comparison with age-matched MRL/lpr mice. N = 4–6 per group. (G) Serum levels of anti-dsDNA IgG measured by ELISA from MRL/lpr mice treated with apoVs or A5 pre-treated apoVs for 4 weeks in comparison with age-matched MRL/lpr mice. N = 6 per group. Mann-Whitney test and student's t-test was used for comparison between two groups when appropriate. Kruskal-Wallis test and ANOVA was used for comparison among three groups when appropriate. Data are shown as mean ± standard deviation. ns, not significant. *, p < 0.05. **, p < 0.01. ***, p < 0.001. ****, p < 0.0001.

Article Snippet: Total CD4 + T cells and naïve CD25 − CD4 + T cells were isolated from mouse spleen using magnetic beads (130-091-041, Miltenyi Biotec, Germany).

Techniques: Blocking Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Standard Deviation

Journal: Bioactive Materials

Article Title: Apoptotic vesicles ameliorate lupus and arthritis via phosphatidylserine-mediated modulation of T cell receptor signaling

doi: 10.1016/j.bioactmat.2022.07.026

Figure Lengend Snippet: ApoVs prevent T cell activation in murine arthritis via phosphatidylserine. (A) Activation fold change of naïve CD4+ T cells stimulated in the presence of PBS (Ctr), 0.2 x apoVs (ApoVs) or 0.2 x phosphatidylserine (PS) liposomes. N = 3 per group. (B) Scheme of D0 systemic apoV injection in collagen-induced arthritic DBA/1 (CIA) mice. (C, D) Comparison of clinical scores in mice receiving D0 PBS, apoVs, A5 pre-treated apoVs, or PS liposomes measured every 3 days from D21 until the endpoint. N = 5–6 per group. (E) Representative images of arthritic front and hind paws of CIA mice receiving D0 PBS, apoVs, A5 pre-treated apoVs, or PS liposomes at the time of harvest. (F) Frequency of Tem CD4+ T cells in circulation of CIA mice receiving D0 PBS, apoVs, A5 pre-treated apoVs, or PS liposomes at D14 and D42. N = 3–4 per group. (G) Frequency of Th17 cells from draining lymph nodes (DLN) and spleen of CIA mice receiving D0 PBS, apoVs, A5 pre-treated apoVs, or PS liposomes. N = 3–5 per group. Kruskal-Wallis test and ANOVA was used for comparison among three groups when appropriate. Data are shown as mean ± standard deviation. ns, not significant. *, p < 0.05. **, p < 0.01. ***, p < 0.001. ****, p < 0.0001.

Article Snippet: Total CD4 + T cells and naïve CD25 − CD4 + T cells were isolated from mouse spleen using magnetic beads (130-091-041, Miltenyi Biotec, Germany).

Techniques: Activation Assay, Injection, Standard Deviation

Journal: Frontiers in Physiology

Article Title: Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation

doi: 10.3389/fphys.2021.807747

Figure Lengend Snippet: Expression of TIMP-3 in vitreous fluid samples. Equal volumes (15 μl) of vitreous fluid samples from patients with proliferative diabetic retinopathy (PDR) ( n = 12) and non-diabetic patients with rhegmatogenous retinal detachment (RD) ( n = 12) were subjected to gel electrophoresis and the presence of TIMP-3 was detected by Western blot analysis. A representative set of samples is shown.

Article Snippet: The antibodies used for Western blot analysis in the present study were as follows: rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1,500, MAB1018, R&D Systems, Minneapolis, MA, United States), mouse monoclonal anti-phospho-p65 subunit of nuclear factor-kappa B (NF-κB) antibody (1:500, sc-136548, Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States), rabbit polyclonal anti-phospho vascular endothelial growth factor receptor (VEGFR-2) antibody (1:1,000, ab194806, Abcam, Cambridge, United Kingdom), mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:1,000, sc-8439, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-vascular cell adhesion protein-1 (VCAM-1) antibody (1:1,000, sc-13160, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-VEGF antibody (1:1,500, MAB293, R&D Systems), rabbit polyclonal caspase-3 antibody (1:1,000, sc-7148, Santa Cruz Biotechnology Inc.), rabbit polyclonal ADAM-17 antibody (1:1,000, ab39163, Abcam), and rabbit monoclonal anti-TIMP-3 antibody (1:1,000, ab277794, Abcam).

Techniques: Expressing, Nucleic Acid Electrophoresis, Western Blot

Journal: Frontiers in Physiology

Article Title: Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation

doi: 10.3389/fphys.2021.807747

Figure Lengend Snippet: Tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) attenuates retinal inflammation and prevents diabetes-induced blood-retinal barrier (BRB) breakdown. The BRB breakdown [panel (A) ] was quantified with the fluorescein isothiocyanate-conjugated dextran technique after treatment with intravitreal injection of 350 μM TIMP-3 in 5 μl in one eye and the same volume of phosphate-buffered saline (PBS) in the contralateral eye of rats 10 weeks after induction of diabetes. Results are expressed as mean ± standard deviation of five rats in each group. Evaluation of inflammatory mediators was evaluated shortly after diabetes induction (see section “Materials and Methods”). Western blot analysis of rat retinas was performed to evaluate protein expression levels of phospho-ERK1/2 [panel (B) ], the p65 subunit of NF-κB [panel (C) ], intercellular adhesion molecule-1 (ICAM-1) [panel (D) ], and vascular endothelial growth factor (VEGF) [panel (E) ]. Results are expressed as mean ± standard deviation of 12 rats in each group. One-way ANOVA and independent t -test were used for comparisons between the three and two groups, respectively, panels (A–E) . * p < 0.05 compared with non-diabetic controls. # p < 0.05 compared with PBS-treated diabetic rats.

Article Snippet: The antibodies used for Western blot analysis in the present study were as follows: rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1,500, MAB1018, R&D Systems, Minneapolis, MA, United States), mouse monoclonal anti-phospho-p65 subunit of nuclear factor-kappa B (NF-κB) antibody (1:500, sc-136548, Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States), rabbit polyclonal anti-phospho vascular endothelial growth factor receptor (VEGFR-2) antibody (1:1,000, ab194806, Abcam, Cambridge, United Kingdom), mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:1,000, sc-8439, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-vascular cell adhesion protein-1 (VCAM-1) antibody (1:1,000, sc-13160, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-VEGF antibody (1:1,500, MAB293, R&D Systems), rabbit polyclonal caspase-3 antibody (1:1,000, sc-7148, Santa Cruz Biotechnology Inc.), rabbit polyclonal ADAM-17 antibody (1:1,000, ab39163, Abcam), and rabbit monoclonal anti-TIMP-3 antibody (1:1,000, ab277794, Abcam).

Techniques: Injection, Standard Deviation, Western Blot, Expressing

Journal: Frontiers in Physiology

Article Title: Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation

doi: 10.3389/fphys.2021.807747

Figure Lengend Snippet: Müller cells were left untreated or treated with high-glucose (HG, 25 mM) [panel (A) ] cobalt chloride (CoCl 2 ) (300 μM) [panel (B) ] or tumor necrosis factor -α (TNF-α) (50 ng/ml) [panel (C) ] for 24 h or TIMP-3 (100 ng/ml) for 1 h followed by HG, CoCl 2 , or TNF-α. For HG treatment, cultures containing 25 mM mannitol were used as a control. Levels of vascular endothelial growth factor (VEGF) and monocyte chemotactic protein-1 (MCP-1) were quantified in the culture media by ELISA. Results are expressed as median (interquartile range) from three different experiments performed in triplicate. Kruskal-Wallis test and Mann-Whitney test were used for comparison between three groups and two groups, respectively. * p < 0.05 compared with values obtained from control cells. # p < 0.05 compared with values obtained from cells treated with HG, CoCl 2 , or TNF-α.

Article Snippet: The antibodies used for Western blot analysis in the present study were as follows: rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1,500, MAB1018, R&D Systems, Minneapolis, MA, United States), mouse monoclonal anti-phospho-p65 subunit of nuclear factor-kappa B (NF-κB) antibody (1:500, sc-136548, Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States), rabbit polyclonal anti-phospho vascular endothelial growth factor receptor (VEGFR-2) antibody (1:1,000, ab194806, Abcam, Cambridge, United Kingdom), mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:1,000, sc-8439, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-vascular cell adhesion protein-1 (VCAM-1) antibody (1:1,000, sc-13160, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-VEGF antibody (1:1,500, MAB293, R&D Systems), rabbit polyclonal caspase-3 antibody (1:1,000, sc-7148, Santa Cruz Biotechnology Inc.), rabbit polyclonal ADAM-17 antibody (1:1,000, ab39163, Abcam), and rabbit monoclonal anti-TIMP-3 antibody (1:1,000, ab277794, Abcam).

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Frontiers in Physiology

Article Title: Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation

doi: 10.3389/fphys.2021.807747

Figure Lengend Snippet: Müller cells were pre-incubated with TIMP-3 (100 ng/ml) or dilution medium for 1 h before increasing the sugar content of the cultures [25 mM of mannitol as control or 25 mM of glucose (HG)]. After 24 h levels of the p65 subunit of NF-κB [panel (A) ], phospho-ERK1/2 [panel (B) ], caspase-3 [panel (C) ], and ADAM17 [panel (D) ] in cell lysates was determined by Western blot analysis. Results are expressed as median (interquartile range) from three different experiments performed in triplicate. Kruskal-Wallis test and Mann-Whitney test were used for comparisons between three groups and two groups, respectively. * p < 0.05 compared with values obtained from cells treated with mannitol. # p < 0.05 compared with values obtained from cells treated with HG.

Article Snippet: The antibodies used for Western blot analysis in the present study were as follows: rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1,500, MAB1018, R&D Systems, Minneapolis, MA, United States), mouse monoclonal anti-phospho-p65 subunit of nuclear factor-kappa B (NF-κB) antibody (1:500, sc-136548, Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States), rabbit polyclonal anti-phospho vascular endothelial growth factor receptor (VEGFR-2) antibody (1:1,000, ab194806, Abcam, Cambridge, United Kingdom), mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:1,000, sc-8439, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-vascular cell adhesion protein-1 (VCAM-1) antibody (1:1,000, sc-13160, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-VEGF antibody (1:1,500, MAB293, R&D Systems), rabbit polyclonal caspase-3 antibody (1:1,000, sc-7148, Santa Cruz Biotechnology Inc.), rabbit polyclonal ADAM-17 antibody (1:1,000, ab39163, Abcam), and rabbit monoclonal anti-TIMP-3 antibody (1:1,000, ab277794, Abcam).

Techniques: Incubation, Western Blot, MANN-WHITNEY

Journal: Frontiers in Physiology

Article Title: Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation

doi: 10.3389/fphys.2021.807747

Figure Lengend Snippet: THP-1 monocytes were left untreated or treated with TIMP-3 (100 ng/ml) for 24 h. Subsequently, the THP-1 cells were fluorescently labeled and adhesion to a human retinal microvascular endothelial cell (HRMEC) monolayer was assessed [panel (A) ]. Results are expressed as median (interquartile range) from two independent experiments (each treatment condition: 6 wells) (* p < 0.05; Mann-Whitney test). Alternatively, HRMECs were pre-incubated with TIMP-3 (100 ng/ml) or dilution medium for 1 h before stimulation with dilution medium, tumor necrosis factor-α (TNF-α) (25 ng/ml) [panel (B) ] or vascular endothelial growth factor (VEGF) (50 ng/ml) [panel (C) ] for 24 h. Adhesion of fluorescently labeled monocytic cells to the HRMEC monolayer was assessed. Results are expressed as median (interquartile range) from three independent experiments (each treatment condition: 6 wells). Kruskal-Wallis test and Mann-Whitney test were used for comparisons between three groups and two groups, respectively (RFU = relative fluorescence unit). HRMECs were left untreated or were stimulated with TNF-α (50 ng/ml) for 24 h with/without a 1-h pre-incubation with TIMP-3 (100 ng/ml). Protein expression of vascular cell adhesion molecule-1 (VCAM-1) [panel (D) ] and intercellular adhesion molecule-1 (ICAM-1) [panel (E) ] was determined by Western blot analysis. Results are expressed as mean ± standard deviation from three independent experiments (each treatment condition: 8 wells). One-way ANOVA and independent t -test were used for comparisons between three groups and two groups, respectively. * p < 0.05 compared with values obtained from untreated cells. # p < 0.05 compared with values obtained from cells treated with TNF-α or VEGF.

Article Snippet: The antibodies used for Western blot analysis in the present study were as follows: rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1,500, MAB1018, R&D Systems, Minneapolis, MA, United States), mouse monoclonal anti-phospho-p65 subunit of nuclear factor-kappa B (NF-κB) antibody (1:500, sc-136548, Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States), rabbit polyclonal anti-phospho vascular endothelial growth factor receptor (VEGFR-2) antibody (1:1,000, ab194806, Abcam, Cambridge, United Kingdom), mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:1,000, sc-8439, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-vascular cell adhesion protein-1 (VCAM-1) antibody (1:1,000, sc-13160, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-VEGF antibody (1:1,500, MAB293, R&D Systems), rabbit polyclonal caspase-3 antibody (1:1,000, sc-7148, Santa Cruz Biotechnology Inc.), rabbit polyclonal ADAM-17 antibody (1:1,000, ab39163, Abcam), and rabbit monoclonal anti-TIMP-3 antibody (1:1,000, ab277794, Abcam).

Techniques: Labeling, MANN-WHITNEY, Incubation, Fluorescence, Expressing, Western Blot, Standard Deviation

Journal: Frontiers in Physiology

Article Title: Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation

doi: 10.3389/fphys.2021.807747

Figure Lengend Snippet: Tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) inhibits vascular endothelial growth factor (VEGF)-mediated human retinal microvascular endothelial cell (HRMEC) migration, chemotaxis and proliferation. A scratch was made in confluent monolayers of overnight starved HRMECs with a micropipette tip. Subsequently, the cultures were pre-incubated with TIMP-3 (100 ng/ml) or dilution medium for 1 h before stimulation with dilution medium or vascular endothelial growth factor (VEGF) (50 ng/ml) for 24 h. Cells were visualized using an inverted microscope. Three independent experiments were performed. Each experiment was done in triplicate and 6–8 independent field images were taken for the migration analysis which was done by using image J software. In the figure one representative image is shown [panel (A) ]. Results are expressed as median (interquartile range). Kruskal-Wallis test and Mann-Whitney test were used for comparisons between three groups and two groups, respectively. * p < 0.05 compared with untreated cells. # p < 0.05 compared with VEGF-treated cells. Chemotaxis and proliferation of HRMECs stimulated with 10 ng/ml VEGF was modulated by a 30-min pre-incubation with TIMP-3 (10 or 100 ng/ml). Cell migration was monitored using the xCELLigence RTCA-DP system. The median (interquartile range) percentage of inhibition of VEGF-induced chemotaxis [panel (B) ] or proliferation [panel (C) ] is shown. In total five chemotaxis experiments were performed and conditions were tested in duplicate or triplicate within 1 experiment. For proliferation, 6 experiments were performed and conditions were tested at least in triplicate within 1 experiment. * p < 0.05; Mann-Whitney test (compared with VEGF).

Article Snippet: The antibodies used for Western blot analysis in the present study were as follows: rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1,500, MAB1018, R&D Systems, Minneapolis, MA, United States), mouse monoclonal anti-phospho-p65 subunit of nuclear factor-kappa B (NF-κB) antibody (1:500, sc-136548, Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States), rabbit polyclonal anti-phospho vascular endothelial growth factor receptor (VEGFR-2) antibody (1:1,000, ab194806, Abcam, Cambridge, United Kingdom), mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:1,000, sc-8439, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-vascular cell adhesion protein-1 (VCAM-1) antibody (1:1,000, sc-13160, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-VEGF antibody (1:1,500, MAB293, R&D Systems), rabbit polyclonal caspase-3 antibody (1:1,000, sc-7148, Santa Cruz Biotechnology Inc.), rabbit polyclonal ADAM-17 antibody (1:1,000, ab39163, Abcam), and rabbit monoclonal anti-TIMP-3 antibody (1:1,000, ab277794, Abcam).

Techniques: Migration, Chemotaxis Assay, Incubation, Inverted Microscopy, Software, MANN-WHITNEY, Inhibition

Journal: European Journal of Medical Research

Article Title: Ferrostatin-1 facilitated neurological functional rehabilitation of spinal cord injury mice by inhibiting ferroptosis

doi: 10.1186/s40001-023-01264-7

Figure Lengend Snippet: Ferrostatin-1 promotes GPX4 to disturbe Nrf2/HO-1 signaling pathway during ferroptosis. A Representative results of western blot of Nrf2/HO-1 signaling pathway change with Ferrostatin-1 and Ferrostatin-1 + Spinosin administration. B The quantification of Fe 2+ concentration in injured spinal cord

Article Snippet: After conventional electrophoretic separation, membrane transfer, and blocking, primary antibodies against Glutathione Peroxidase 4 (GPX4) (ab252833, abcam), HO-1 (ab305290, abcam), Nrf2 (ab92946, abcam) and secondary antibodies were sequentially applied.

Techniques: Western Blot, Concentration Assay

Journal: Immunity, Inflammation and Disease

Article Title: Transcription factor Krüppel‐like factor 4 upregulated G protein‐coupled receptor 30 alleviates intestinal inflammation and apoptosis, and protects intestinal integrity from intestinal ischemia–reperfusion injury

doi: 10.1002/iid3.940

Figure Lengend Snippet: KLF4/GPR30 regulates NLRP3 inflammasome and pyroptosis in OGD/R‐exposed Caco‐2 cells. (A) Untransfected and transfected Caco‐2 cells were exposed to OGD/R, and protein expression of NLRP3, cleaved‐caspase1, caspase1, and GSDMD‐N was measured using western blot. (B) Immunofluorescence was conducted to examine GSDMD‐N expression. OGD/R, oxygen‐glucose deprivation/reoxygenation. *** p < .001 versus control; ### p < .001 versus OGD/R; @@@ p < .001 versus OGD/R+oe‐GPR30+sh‐NC.

Article Snippet: After being blocked with skimmed milk, the membranes were probed with primary antibodies against GPR30 (ab39742, Abcam), p‐p65 (#3031, Cell Signaling Technology), p65 (#8242, Cell Signaling Technology), Cox2 (ab179800, Abcam), iNOS (ab178945, Abcam), Bcl‐2 (ab32124, Abcam), Bax (ab32503, Abcam), cleaved‐PARP (ab32064, Abcam), PARP (ab32138, Abcam), Claudin‐1 (ab211737, Abcam), Occludin‐1 (ab216327, Abcam), ZO‐1 (ab216880, Abcam), KLF4 (ab215036, Abcam), nod‐like receptor pyrin 3 (NLRP3; ab263899, Abcam), Cleaved‐caspase1 (#4199, Cell Signaling Technology), caspase1 (#3866, Cell Signaling Technology), N‐terminal Gasdermin D (GSDMD‐N; ab215203, Abcam), and GAPDH (ab9485, Abcam) at 4°C overnight, after which was the cultivation with horse radish peroxidase (HRP)‐conjugated secondary antibody (ab6721, Abcam) at room temperature for 2 h. The visualization and analysis of protein blots were operated employing enhanced chemiluminescence and Image Lab™ software 5.2.1 (Bio‐Rad Laboratories, Inc), respectively.

Techniques: Transfection, Expressing, Western Blot, Immunofluorescence

Journal: Immunity, Inflammation and Disease

Article Title: Transcription factor Krüppel‐like factor 4 upregulated G protein‐coupled receptor 30 alleviates intestinal inflammation and apoptosis, and protects intestinal integrity from intestinal ischemia–reperfusion injury

doi: 10.1002/iid3.940

Figure Lengend Snippet: Schematic diagram of KLF4‐upregulated GPR30 might exert a protective role against intestine I/R injury by inhibiting inflammation and apoptosis, and maintaining intestinal integrity, thereby leading to the reduction of the NLRP3‐mediated pyroptosis. I/R, ischemia/reperfusion.

Article Snippet: After being blocked with skimmed milk, the membranes were probed with primary antibodies against GPR30 (ab39742, Abcam), p‐p65 (#3031, Cell Signaling Technology), p65 (#8242, Cell Signaling Technology), Cox2 (ab179800, Abcam), iNOS (ab178945, Abcam), Bcl‐2 (ab32124, Abcam), Bax (ab32503, Abcam), cleaved‐PARP (ab32064, Abcam), PARP (ab32138, Abcam), Claudin‐1 (ab211737, Abcam), Occludin‐1 (ab216327, Abcam), ZO‐1 (ab216880, Abcam), KLF4 (ab215036, Abcam), nod‐like receptor pyrin 3 (NLRP3; ab263899, Abcam), Cleaved‐caspase1 (#4199, Cell Signaling Technology), caspase1 (#3866, Cell Signaling Technology), N‐terminal Gasdermin D (GSDMD‐N; ab215203, Abcam), and GAPDH (ab9485, Abcam) at 4°C overnight, after which was the cultivation with horse radish peroxidase (HRP)‐conjugated secondary antibody (ab6721, Abcam) at room temperature for 2 h. The visualization and analysis of protein blots were operated employing enhanced chemiluminescence and Image Lab™ software 5.2.1 (Bio‐Rad Laboratories, Inc), respectively.

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: The Laminin Receptors Basal Cell Adhesion Molecule/Lutheran and Integrin α7β1 on Human Hematopoietic Stem Cells

doi: 10.3389/fcell.2021.675240

Figure Lengend Snippet: Immunostaining of lineage negative HSPC with the anti-α7 integrin antibody. (A) The lineage-negative CBMNC fraction fixed by cytospin centrifugation was labeled with a mouse monoclonal anti-human integrin α7 chain antibody (red) and a rabbit monoclonal anti-human CD34 antibody (green). The cells were counterstained with DAPI (blue). The merged picture shows that all CD34 + HSPC express integrin α7. Representative of three independent experiments. (B) Lin- CD34+ cells were labeled with the unconjugated integrin antibody followed by an incubation with FITC-conjugated secondary antibody, and measured by flow cytometry. Representative plot of three independent experiments. (C) Two different preparations of lin- CD34+ cell extracts were analyzed by immunoblotting. An extract from HUVEC cells was used as a positive control. In all extracts a band of 117 kb can be detected.

Article Snippet: To obtain lineage-negative (lin-) stem and progenitor cells, the CBMNC fraction was magnetically labeled with the human lineage cell depletion kit (Miltenyi Biotec) containing biotin-conjugated antibodies against CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a followed by an incubation with anti-biotin microbeads followed by magnetic separation to obtain the unlabeled lineage-negative fraction.

Techniques: Immunostaining, Centrifugation, Labeling, Incubation, Flow Cytometry, Western Blot, Positive Control

Journal: Experimental and Therapeutic Medicine

Article Title: Downregulation of microRNA-185 expression in diabetic patients increases the expression of NOS2 and results in vascular injury

doi: 10.3892/etm.2021.10893

Figure Lengend Snippet: Levels of NOS2 in the blood from patients with diabetes compared with healthy subjects. (A) Relative expression of NOS2 mRNA in the blood determined by reverse transcription-quantitative PCR. (B) Content of NOS2 protein in the blood determined by enzyme-linked immunosorbent assay. * P<0.05 vs. diabetes group. N=33. NOS2, nitric oxide synthase 2.

Article Snippet: The concentrations of human NOS2 (cat. no. ab253217; Abcam) and rat NOS2 (cat. no. SBJ-R0012; SenBeiJia Biological Technology) were measured by ELISA according to the protocols provided by the kit manufacturers.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Downregulation of microRNA-185 expression in diabetic patients increases the expression of NOS2 and results in vascular injury

doi: 10.3892/etm.2021.10893

Figure Lengend Snippet: Level of miR-185 in the blood. (A) Possible binding sites between miR-185 and NOS2 gene, as predicted by bioinformatics. (B) Relative expression of miR-185 in blood from patients with diabetes in contrast to control group. ** P<0.01 vs. control group. N=33. miR, microRNA; NOS2, nitric oxide synthase 2.

Article Snippet: The concentrations of human NOS2 (cat. no. ab253217; Abcam) and rat NOS2 (cat. no. SBJ-R0012; SenBeiJia Biological Technology) were measured by ELISA according to the protocols provided by the kit manufacturers.

Techniques: Binding Assay, Expressing

Journal: Experimental and Therapeutic Medicine

Article Title: Downregulation of microRNA-185 expression in diabetic patients increases the expression of NOS2 and results in vascular injury

doi: 10.3892/etm.2021.10893

Figure Lengend Snippet: mRNA and protein expression of NOS2 in vascular tissues and blood from diabetic rats. (A) mRNA and (B) protein levels of NOS2 in vascular tissues from normal rats and diabetic rats, as determined by RT-qPCR and western blotting, respectively. (C) mRNA and (D) protein levels of NOS2 in the blood from normal rats and diabetic rats, as determined by RT-qPCR and enzyme-linked immunosorbent assay, respectively. * P<0.05 and ** P<0.01 vs. control group. N=20. NOS2, nitric oxide synthase 2; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: The concentrations of human NOS2 (cat. no. ab253217; Abcam) and rat NOS2 (cat. no. SBJ-R0012; SenBeiJia Biological Technology) were measured by ELISA according to the protocols provided by the kit manufacturers.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Experimental and Therapeutic Medicine

Article Title: Downregulation of microRNA-185 expression in diabetic patients increases the expression of NOS2 and results in vascular injury

doi: 10.3892/etm.2021.10893

Figure Lengend Snippet: Effect of miR-185 expression on the expression of NOS2. (A) Expression of miR-185 in HMEC-1 cells after transfection with agomiR-NC and agomiR-185. (B) Expression of NOS2 mRNA in HMEC-1 cells after transfection with agomiR-NC and agomiR-185. (C) Expression of NOS2 protein in HMEC-1 cells after transfection with agomiR-NC and agomiR-185. * P<0.05 and ** P<0.01 vs. agomiR-NC group. N=3. miR, microRNA; NOS2, nitric oxide synthase 2; NC, negative control.

Article Snippet: The concentrations of human NOS2 (cat. no. ab253217; Abcam) and rat NOS2 (cat. no. SBJ-R0012; SenBeiJia Biological Technology) were measured by ELISA according to the protocols provided by the kit manufacturers.

Techniques: Expressing, Transfection, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: Downregulation of microRNA-185 expression in diabetic patients increases the expression of NOS2 and results in vascular injury

doi: 10.3892/etm.2021.10893

Figure Lengend Snippet: Direct interaction between miR-185 and NOS2 mRNA. (A) Design of mutation sequence in NOS2 seed region. (B) Fluorescence intensity of 293T cells co-transfected with miR-185 and its wild-type or mutant seed regions in the 3'-untranslated region of NOS2 gene, as examined by dual-luciferase reporter assay. One-way ANOVA was performed, followed by Least Significant Difference test. * P<0.05 vs. NC group. N=3. miR, microRNA; NOS2, nitric oxide synthase 2; NC, negative control.

Article Snippet: The concentrations of human NOS2 (cat. no. ab253217; Abcam) and rat NOS2 (cat. no. SBJ-R0012; SenBeiJia Biological Technology) were measured by ELISA according to the protocols provided by the kit manufacturers.

Techniques: Mutagenesis, Sequencing, Fluorescence, Transfection, Luciferase, Reporter Assay, Negative Control

Journal: Frontiers in Immunology

Article Title: The prognostic and immune significance of C15orf48 in pan-cancer and its relationship with proliferation and apoptosis of thyroid carcinoma

doi: 10.3389/fimmu.2023.1131870

Figure Lengend Snippet: Effect of C15orf48 knockdown on THCA cell line BHT101, all experiments were performed in triplicate. (A) Expression levels of C15orf48 in different THCA cell lines; (B) RT-PCR verification of the knockout efficiency of C15orf48 in BHT101 cells; (C) The knockout efficiency of C15orf48 in BHT101 cells was verified by Western blot, and the figure below shows the statistical difference analysis of three repeated experiments; (D) CCK8 assay to analyze the effect of knocking out C15orf48 on cell proliferation; (E) Analysis of the effect of knocking out C15orf48 on cell healing ability by cell scratch test; (F) Transwell assay to analyze the effect of knocking out C15orf48 on cell migration; (G) Analysis of cell apoptosis changes by flow cytometry; (H) The percentage of apoptotic cells in the two groups. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Thereafter, the cells were stained with Annexin V-FITC/PI Apoptosis Detection kit (Vazyme, Nanjing, China) according to the manufacturer’s instructions.

Techniques: Knockdown, Expressing, Reverse Transcription Polymerase Chain Reaction, Knock-Out, Western Blot, CCK-8 Assay, Transwell Assay, Migration, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Pyrolyzed deketene curcumin controls regulatory T cell generation and gastric cancer metabolism cooperate with 2-deoxy-d-glucose

doi: 10.3389/fimmu.2023.1049713

Figure Lengend Snippet: GO-Y022 inhibits TGF-β-induced generation of Tregs. (A) Live cell-counting assay. Naïve splenic CD4 + T cells were cultured with plate-bound anti-CD3 and soluble anti-CD28 in the presence or absence of 2 ng/mL human TGF-β1 and 0.007 μM DMSO (control), 0.25 μM curcumin or 0.25 μM GO-Y022 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. One-way analysis of variance (ANOVA) with post-hoc Tukey’s multiple comparison test was employed. (B, C) Flow cytometry analysis using live and dead staining (Annexin V and Propidium iodide). Purified naïve CD4 + T cells were cultured with plate-bound anti-CD3 and soluble anti-CD28 in the presence of 2 ng/mL human TGF-β1 and 0.007 μM DMSO or 0.25 μM GO-Y022 for 24 h and the CD4 + cell population was gated. The data show representative density plots (B) . Statistical analyses of apoptotic dead cells (Annexin V + Propidium Iodide + ), early apoptotic dead cells (Annexin V + Propidium Iodide + ) or necroptosis dead cells (Annexin V - Propidium Iodide + ) were compared between 0.25 μM GO-Y022-treated and 0.007 μM DMSO-treated cultured CD4 + T cells (plate-bound anti-CD3 and soluble anti-CD28 in the presence of 2 ng/mL human TGF-β1). Data were pooled from four independent experiments (C) . (D) The real-time quantitative analysis results of 0.007 μM DMSO-, 0.25 μM curcumin-, or 0.25 μM GO-Y022 treated naïve CD4 + T cells for 24 h plate-bound anti-CD3 and soluble anti-CD28 in the presence or absence of 2 ng/mL human TGF-β1. The circles indicate independent experiments. The horizontal bars represent the mean. (E, F) Frequency of Foxp3 + Tregs in the entire CD4 + cell population. Naïve splenic CD4 + T cells were cultured with plate-bound anti-CD3 and soluble anti-CD28 in the presence or absence of 2 ng/mL human TGF-β1 or 0.007 μM DMSO or 0.25 μM GO-Y022 as indicated for 3 days. The data are representative histogram (E) . Statistical analyses of the percentages of Foxp3 + Tregs were compared between 0.25 μM GO-Y022-treated and 0.007 μM DMSO-treated cultured CD4 + T cells with plate-bound anti-CD3 and soluble anti-CD28 in the presence or absence of 2 ng/mL human TGF-β1. Data were pooled from four independent experiments (F) . The horizontal bars represent the mean. Student’s t-test (C) or one-way ANOVA with post-hoc Tukey’s multiple comparison test (A, D, F) was used. Statistical significance was set at p < 0.05; **p < 0.01, and ***p < 0.001.

Article Snippet: Naïve CD4 + T cells were isolated from mouse spleens using a mouse CD4 + CD62L hi T Cell Isolation Kit according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Cell Counting, Cell Culture, Control, Comparison, Flow Cytometry, Staining, Purification

Journal: Frontiers in Immunology

Article Title: Pyrolyzed deketene curcumin controls regulatory T cell generation and gastric cancer metabolism cooperate with 2-deoxy-d-glucose

doi: 10.3389/fimmu.2023.1049713

Figure Lengend Snippet: GO-Y022 prevents NFATc1 binding to Foxp3 gene regulatory elements. (A, B) TGF-β/SMAD signaling pathway. Representative fluorescence microscopic images (Leica SP8; Leica, Wetzler, Germany) of SMAD3 (Green), phospho-SMAD3 (Green) and Nuclei (stained with DAPI; Blue) of three independent experiments. Naïve CD4 + T cells were stimulated with plate-bound anti-CD3, soluble anti-CD28 and 2 ng/mL human TGF-β1 in the presence or absence of 0.25 μM GO-Y022 for 2h (A) . Statistical analyses of phospho-SMAD3 (p-SMAD3)/SMAD3 expression (n = 6, mean with standard error of the mean) (B) . (C, D) Phosphorylation levels of SMAD3 in nuclear. Representative Western blotting image of SMAD3, p-SMAD3, and α-tubulin of three independent experiments. Naïve CD4 + T cells were stimulated with plate-bound anti-CD3, soluble anti-CD28 and 2 ng/mL human TGF-β1 in the presence or absence of 0.25 μM GO-Y022 for 2h (C) . Relative p-SMAD3/SMAD3 expression in nuclear fraction. Without GO-Y022 (DMSO-treatment) of p-SMAD3/SMAD3 was set as “1.”. The horizontal bars represent the mean and standard deviation. Data represent at least three independent experiments (D) . (E) RT-PCR documenting the relative amount of chromatin immunoprecipitation (NFATc1/IgG) at the Foxp3 locus (n = 3) using cultured naïve CD4 + T cells (24 h) with plate-bound anti-CD3, soluble anti-CD28 and 2 ng/mL human TGF-β1 with or without GO-Y022 and 0.007 μM DMSO. White bar represents DMSO; black bar represents 0.25 µM GO-Y022. The horizontal bars represent the mean and standard deviation. Data represent at least three independent experiments. (F) Western blotting by using EL4/LAF lymphoma cells which is transfected control or NFATc1 shRNA particle after 72h. Control of shRNA (NFATc1/GAPDH) was set as “1.”. The horizontal bars represent the mean and standard deviation (n=3). Data represent at three independent experiments (G) . (H) Real time PCR of Foxp3 gene expression. The horizontal bars represent the mean and standard deviation (triplicate samples). Data represent at least three independent experiments. (I) A Foxp3 promoter assay was performed in EL4/LAF T lymphoma cells. Eighteen hours after plasmid transfection, the cells were incubated with 1 µM GO-Y022 or 0.007 μM DMSO in the presence or absence of 250 nM ionomycin. Without ionomycin and GO-Y022 stimulation was set as “1.” Data are pooled from three independent experiments (n = 3); the mean and standard deviation are shown. (J) The real-time quantitative analysis results of 0.007 μM DMSO- or 0.25 μM GO-Y022 treated naïve CD4 + T cells for 24 h plate-bound anti-CD3 and soluble anti-CD28 in the presence or absence of 2 ng/mL human TGF-β1, 12.5 ng/ml Cyclosporine A (CSA). The circles indicate independent experiments. The horizontal bars represent the mean. Student’s t-test (D, E, G, H) or one-way ANOVA with post-hoc Tukey’s multiple comparison test (B, I, J) was employed. Statistical significance was set at p < 0.05; *p < 0.05; **P<0.01 and ***P<0.001.

Article Snippet: Naïve CD4 + T cells were isolated from mouse spleens using a mouse CD4 + CD62L hi T Cell Isolation Kit according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Binding Assay, Fluorescence, Staining, Expressing, Phospho-proteomics, Western Blot, Standard Deviation, Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation, Cell Culture, Transfection, Control, shRNA, Real-time Polymerase Chain Reaction, Gene Expression, Promoter Assay, Plasmid Preparation, Incubation, Comparison

Journal: Frontiers in Immunology

Article Title: Pyrolyzed deketene curcumin controls regulatory T cell generation and gastric cancer metabolism cooperate with 2-deoxy-d-glucose

doi: 10.3389/fimmu.2023.1049713

Figure Lengend Snippet: GO-Y022 has little effect on the immunosuppressive ability of Tregs. (A) Live cell-counting assay. Splenic CD4 + CD25 + Treg cells were cultured in plate-bound anti-CD3, with soluble anti-CD28 and 10 ng/mL hIL-2 in the presence of 0.25 μM GO-Y022 or 0.007 μM DMSO as indicated for 68 h, followed by the addition of the Cell-Counting Kit 8 reagent (4 h). (B, C) The frequency of Foxp3 + Tregs in the entire CD4 + cell population. Splenic CD4 + CD25 + Treg cells were cultured in plate-bound anti-CD3, with soluble anti-CD28 and 10 ng/mL hIL-2 in the presence of 0.25 μM GO-Y022 or 0.007 μM DMSO as indicated for 3 days. Data represent at least three independent experiments (B) . Statistical analyses of the percentage of Foxp3 + Tregs were compared between 0.25 μM GO-Y022-treated and or 0.007 μM DMSO-treated cultured CD4 + CD25 + Tregs (stimulated with plate-bound anti-CD3, soluble anti-CD28 and 10 ng/mL hIL-2). The circles indicate independent experiments and the horizontal bars represent the mean. Data were pooled from three independent experiments (C) . (D, E) The proliferation ratio of CellTrace™ Violet-labeled CD8 + T cells isolated from CD45.1 mice and cultured with or without CD4 + CD25 + Tregs for 72 (H) Tregs were treated with 0.25 µM GO-Y022 (black fill) or 0.007 μM DMSO control (white fill) for 3 days before co-culture. The data showed a representative histogram gated on the CD8 + CD45.1 + Zombie - population (D) . The relative suppressive ability of Tregs; the percentage of non-proliferative CD8 + T cells without Tregs is set to 0 (Leftmost figure in D ). Data were pooled from three independent experiments. The mean and standard deviation is shown (E) . (F, G) Stability of Tregs. Foxp3 expression in CD4 + CD25 + Tregs, which were co-cultured with CD8 + T cells isolated from CD45.1 mice for 72 (H) The data show representative density plots (F) . Statistical analyses of the percentage of Foxp3 + Tregs were performed between 0.25 μM GO-Y022-treated and 0.007 μM DMSO-treated cultured CD4 + CD25 + Tregs in the co-cultured systems. Data were pooled from three independent experiments. The circles indicate independent experiments and the horizontal bars represent the mean (G) . The graph shows the mean and standard deviation. Student’s t-test (A, C, E, G) was employed.

Article Snippet: Naïve CD4 + T cells were isolated from mouse spleens using a mouse CD4 + CD62L hi T Cell Isolation Kit according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Cell Counting, Cell Culture, Labeling, Isolation, Control, Co-Culture Assay, Standard Deviation, Expressing

Journal: Frontiers in Immunology

Article Title: Pyrolyzed deketene curcumin controls regulatory T cell generation and gastric cancer metabolism cooperate with 2-deoxy-d-glucose

doi: 10.3389/fimmu.2023.1049713

Figure Lengend Snippet: GO-Y022 treatment of human CD4+ T cells together with gastric tumor cells. (A, B) The frequency of Foxp3 + Tregs in the entire CD4 + cell population. Naïve CD4 + T cells from human PBMCs (1 × 10 5 cells) and SH-10-TC (3 × 10 4 cells) were co-cultured in the presence of CD3/28 Dynabeads with or without 5mM 2DG for 48 (h) The data show representative density plots (A) . Statistical analyses of the percentage of Foxp3 + Tregs were compared between 0.25 μM GO-Y022-treated and 0.007 μM DMSO-treated cultured CD4 + T cells with or without 2DG. Data were pooled from three independent experiments (B) . One-way ANOVA with post-hoc Tukey’s multiple comparison test was applied. Statistical significance was set at p < 0.05; *p < 0.05, and ***p < 0.001.

Article Snippet: Naïve CD4 + T cells were isolated from mouse spleens using a mouse CD4 + CD62L hi T Cell Isolation Kit according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Cell Culture, Comparison