Journal: iScience

Article Title: Induction of Foxp3 and activation of Tregs by HSP gp96 for treatment of autoimmune diseases

doi: 10.1016/j.isci.2021.103445

Figure Lengend Snippet: TLR binding domain is essential for gp96-induced Treg activation (A) Low- (20 μg) or high- (200 μg) dose Cy5-labelled gp96 was subcutaneously injected into the backs of Foxp3-GFP KI mice. Flow cytometry analysis of Cy5-positive Tregs (CD4 + Foxp3-GFP + ), DCs (CD11c + ), B cells (CD3 – CD19 + ), conventional CD4 + T cells (CD4 + Foxp3-GFP – ) and CD8 + T cells 6 h post treatment. (B–D) Ovalbumin (OVA)-specific IgG in serum on day 10 and day 21 post OVA treatment was measured using ELISA (B). FACS analysis of Treg cells (C) and Tfr cells (D) in spleen. (E) Flow cytometry analysis of Foxp3, Helios, CD62L, CD69, ICOS, and CD137 expression on CD4 + Foxp3 + Tregs in the spleen of saline, mutant or WT gp96-immunized mice. n = 5 mice/group. (F) Serum anti-dsDNA antibodies were determined by ELISA in Lyn –/– mice at the indicated weeks of age one week after mutant gp96 treatment. (G) FACS analysis of surface TLR1/2 expression in CD4 + Foxp3 + Tregs from C57BL/6 mice. (H) Tregs were cultured with 100 μg/mL His-gp96 for 4 h before immunoprecipitation with anti-His antibody and western blotting. (I) Luciferase assay using reporter plasmids containing the Foxp3 promoter in 293T cells. Expression plasmids of TLR1 and TLR2 were transfected as indicated. Cells were either unstimulated or stimulated with 100 μg/mL gp96 for 12 h before analysis. (J) Frequency of Tregs in the spleen from Tlr2 −/− mice immunized with 200 μg gp96 or saline (control, n = 5/group). (K) Mean Fluorescence intensity (MFI) of indicated markers in the spleen from Tlr2 −/− mice immunized with 200 μg gp96 or saline (control) (n = 5/group). (L and M) Chimeric mouse model was produced and immunized with gp96 (L). Flow cytometry analysis of ICOS, KLRG1, CD69, and CD137 expression on CD4 + Foxp3 + Tregs from CD45.1 + cells and CD45.2 + cells (M). Dots represent data from individual mice. The data are representative of two independent experiments with similar results. n = 5 mice/group. Mean ± SD is shown. The Student's t test was used for statistical analysis. P < 0.05 was considered statistically significant. ns = not significant.

Article Snippet: Mouse: Tlr2 –/– (C57BL/6) , Shanghai Model Organisms , NM-KO-18026.

Techniques: Binding Assay, Activation Assay, Injection, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Expressing, Saline, Mutagenesis, Cell Culture, Immunoprecipitation, Western Blot, Luciferase, Transfection, Control, Fluorescence, Produced

Journal: iScience

Article Title: Induction of Foxp3 and activation of Tregs by HSP gp96 for treatment of autoimmune diseases

doi: 10.1016/j.isci.2021.103445

Figure Lengend Snippet:

Article Snippet: Mouse: Tlr2 –/– (C57BL/6) , Shanghai Model Organisms , NM-KO-18026.

Techniques: Recombinant, SYBR Green Assay, Cell Isolation, Staining, Plasmid Preparation, Software

Journal: PLoS ONE

Article Title: Toll-Like Receptor 4–Mediated Nuclear Factor Kappa B Activation Is Essential for Sensing Exogenous Oxidants to Propagate and Maintain Oxidative/Nitrosative Cellular Stress

doi: 10.1371/journal.pone.0073840

Figure Lengend Snippet: Cells were stimulated with 1 µg/ml of LPS from Porphyromonas gingivalis (LPS-PG) (1 µg/ml), MPLA (57 nM), PPC (1 µM), PDO (50 µM), or PN (1 mM) for 16 h. SEAP released into the supernatant was quantified using Quanti-Blue (Fig. 4A) [n = means ± SEM from 5 independent experiments; # p ≤0.01 vs treatments with LPG-PG or PPC alone; + p ≤0.01 versus treatment with LPS-PG alone]. Fig. 4B - A representative qualitative representation of LPS-PG concentration-dependent changes in purple/blue color development (absorbance) indicative of increased SEAP release (i.e., enhanced with increasing NF-κB) with increasing LPS-PG concentration after Quanti-Blue reaction. HEK-Blue mTLR2 cells incubated with different concentrations of LPS-PG at 0.1, 1.0 and 10 µg/ml for 16 h were compared to treatments with different prooxidants assumed to be potential TLR2 activators.

Article Snippet: HEK-Blue-Null1-v, HEK-Blue mouse TLR2 (mTLR2) and HEK-Blue mTLR4 cells were purchased from InvivoGen (San Diego, CA).

Techniques: Concentration Assay, Incubation