Journal: Scientific Reports

Article Title: Janus-faced Acrolein prevents allergy but accelerates tumor growth by promoting immunoregulatory Foxp3+ cells: Mouse model for passive respiratory exposure

doi: 10.1038/srep45067

Figure Lengend Snippet: ( A ) Mice nasally treated with KLH (K), acrolein ( A ), PBS (P) or with KLH in combination with acrolein (KA) were exposed to increasing doses of methacholine and the degree of airway hyperreactivity was measured by whole body plethysmography according to change in area of enhanced pause (Penh). ( B ) Rectal temperature drop 20 min after ip-challenge with KLH as a parameter to determine systemic anaphylactic reactions ( C ) Summary of CD4+CD25+Foxp3+ cell numbers counted in the spleens of the different treated mice of two independently performed experiments. ( D) Whole lung sections were imaged by TissueFAX and positive stainings were quantified by Histoquest. Summary of immune-histochemical quantification % of CD3+ cells, ( D ) % of Foxp3+ cells ( E ) Foxp3/CD3 ratio of lung sections in the different treated groups. ( F ) Representative regions of lung sections stained for CD3 and Foxp3. 2 stained sections per animal were analysed by TissueFAX. Groups were compared by ANOVA following Newman-Keuls Multiple Comparison test. Mean ± SEM; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: After cooling down slides were blocked for endogenous peroxidase (3% H 2 O 2 in Methanol, 10 min), permeabilization with 0.2% Tween in PBS and for unspecific binding with 1.5% blocking serum (rabbit ABC staining system, Santa Cruz) and incubated with rabbit anti-mouse Foxp3 (1:400 in 1.5% goat serum)(1.98 mg/ml) or isotype control, rabbit IgG, overnight at 4 °C.

Techniques: Staining, Comparison

Journal: Scientific Reports

Article Title: Janus-faced Acrolein prevents allergy but accelerates tumor growth by promoting immunoregulatory Foxp3+ cells: Mouse model for passive respiratory exposure

doi: 10.1038/srep45067

Figure Lengend Snippet: ( A ) As depicted in the treatment protocol, mice were intranasally pretreated 6 times in biweekly intervals with acrolein or PBS. After one week interval, murine mammary carcinoma cell-line D2F2 derived from BALB/c mice were injected subcutaneously in the right flank and their growth was monitored. ( B ) Representative pictures of excised tumors of mice previously pre-treated intranasally with PBS (P, n = 8), or acrolein (A, n = 8). Summary of ( C ) tumor size and ( D ) body weight of mice 11 days post-tumor engraftment in the different groups. Whole tumor sections were imaged by TissueFAX and positive stainings were quantified by Histoquest. Summary of immune-histochemical quantification of ( E ) CD3+ cells, ( F ) Foxp3+ cells and ( G ) Foxp3/CD3 ratio of tumor sections in the different treated groups. ( H ) Representative regions of tumor sections stained for CD3, Foxp3 and their respective isotype controls are shown. 2 sections/animal and staining were analyzed by Tissue FAX. Groups were compared by unpaired T test. Mean ± SEM; * P < 0.05; ** P < 0.01.

Article Snippet: After cooling down slides were blocked for endogenous peroxidase (3% H 2 O 2 in Methanol, 10 min), permeabilization with 0.2% Tween in PBS and for unspecific binding with 1.5% blocking serum (rabbit ABC staining system, Santa Cruz) and incubated with rabbit anti-mouse Foxp3 (1:400 in 1.5% goat serum)(1.98 mg/ml) or isotype control, rabbit IgG, overnight at 4 °C.

Techniques: Derivative Assay, Injection, Staining

Journal: Scientific Reports

Article Title: Janus-faced Acrolein prevents allergy but accelerates tumor growth by promoting immunoregulatory Foxp3+ cells: Mouse model for passive respiratory exposure

doi: 10.1038/srep45067

Figure Lengend Snippet: ( A ) NF-κB inhibition was measured with monocytic THP1-XBlue cells, which were treated with LPS and increasing concentrations of acrolein or cinnamaldehyde for 18 hours. Bars represent summary from two independent experiments normalized to cells stimulated with LPS alone. ( B ) AZ-AhR cells were treated with different concentrations of acrolein or cinnamaldehyde crotonaldehyde, propanal and methacrolein in increasing concentrations (0.018/0.18/1.8/9/18/45/90/180 μM), with ( C ) acrolein alone or in combination with the antagonists resveratrol or 3′-methoxy-4′-nitroflavone for 18 h, before luciferase-activity was measured in the supernatant. Bars represent data from three independent experiments normalized to medium alone for B and two independent experiments for C. Data presented as mean ± SD, Statistical analyses was made using 2way RM ANOVA following Sidak’s multiple comparisons test, respectively. Significances to controls are depicted. ( D ) PBMCs were stimulated for 72 h with cinnamaldehyde, acrolein and or resveratrol. Gating Strategy and summary of % of CD4+CD25+Foxp3+ cells as determined by FACS of 5 independent experiments (n = 15). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001.

Article Snippet: After cooling down slides were blocked for endogenous peroxidase (3% H 2 O 2 in Methanol, 10 min), permeabilization with 0.2% Tween in PBS and for unspecific binding with 1.5% blocking serum (rabbit ABC staining system, Santa Cruz) and incubated with rabbit anti-mouse Foxp3 (1:400 in 1.5% goat serum)(1.98 mg/ml) or isotype control, rabbit IgG, overnight at 4 °C.

Techniques: Inhibition, Luciferase, Activity Assay

Journal: Cancers

Article Title: CD11c-CD8 Spatial Cross Presentation: A Novel Approach to Link Immune Surveillance and Patient Survival in Soft Tissue Sarcoma

doi: 10.3390/cancers13051175

Figure Lengend Snippet: Foxp3+ cells reside in TA-TLSs. ( a ) Representative image of CD11c+ cells and Foxp3+ cells residing primarily within a TA-TLS (intra-TLS) and not in the peri-TLS region. ( b ) Quantification of Foxp3+ cell numbers in relation to total cell numbers and TA-TLS formation (+/−SD). Each dot in the graph represents the QuPath image quantification result originating from one out of three middle-sized TA-TLSs. Statistical comparisons were performed with One-way Anova comparing intra-TLS regions with regions outside the TLS (close and distant). * p < 0.05, ** p < 0.01, *** p < 0.001; p < 0.05 is considered significant.

Article Snippet: Serum block was performed before applying the primary antibodies directed against CD11c (NCL-TL-TCD11c-T563, Novocastra, 1:75), CD8 (M7103, DAKO, 1:75), and Foxp3 (98377, Cell Signaling, 1:150).

Techniques:

Journal: Cancers

Article Title: CD11c-CD8 Spatial Cross Presentation: A Novel Approach to Link Immune Surveillance and Patient Survival in Soft Tissue Sarcoma

doi: 10.3390/cancers13051175

Figure Lengend Snippet: High gene expression levels of ITGAX together with CD8A are prognostic by transcriptomics, irrespective of FOXP3 levels, in human STS. ( a ) Contrast group analysis of gene expression levels of ITGAX (CD11c) and CD8A (CD8) in the TCGA sarcoma cohort; ( b ) Contrast group analysis of gene expression levels of ITGAX (CD11c) and CD8A (CD8) in the TCGA sarcoma cohort where FOXP3 low and FOXP3 high indicate subgroupings according to total FOXP3 levels in the TME. * p < 0.05, ** p < 0.01; p < 0.05 is considered significant.

Article Snippet: Serum block was performed before applying the primary antibodies directed against CD11c (NCL-TL-TCD11c-T563, Novocastra, 1:75), CD8 (M7103, DAKO, 1:75), and Foxp3 (98377, Cell Signaling, 1:150).

Techniques: Gene Expression

Journal: Materials today. Bio

Article Title: Contribution of Tregs to the promotion of constructive remodeling after decellularized extracellular matrix material implantation.

doi: 10.1016/j.mtbio.2024.101151

Figure Lengend Snippet: Fig. 2. Histological analysis and cell recruitment after implantation of the dECM material. (A) Representative images of dECM stained with HE, Masson, and EVG staining on days 3, 7, 14, and 28 after implantation in the mouse muscle traumatic model. Fibroblasts are indicated by green arrows, neutrophils by yellow arrows, monocytes by red arrows, capillaries by blue arrows, multinucleated giant cells by white arrows, and fibrous connective tissue by black arrows. The dECM boundaries are framed with black wire. (B, C) Representative FACS plots (B) and quantitative analysis (C) of the proportion of CD45+ cells between the dECM and sham groups on days 3, 7, 14, and 28 after implantation. (D, E) Proportion of Tregs in the dECM and sham groups was determined on days 3, 7, 14, and 28 after implantation via flow cytometry (D) and quantitative analysis (E). (F, G) Representative images (F) and statistical plots (G) of Foxp3+ cells in the dECM material, detected via immunofluorescence on days 3, 7, 14, and 28 after implantation. ns = not significant, *p ≤0.05, **p ≤0.01, ***p ≤0.001, ****p ≤0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: To assess Treg infiltration into the dECM material, the sections were sealed with 10 % donkey serum (AntGene, China), and then incubated with forkhead box protein P3 (Foxp3) (1:200, 12653, CST, USA) antibody working fluid.

Techniques: Staining, Flow Cytometry, Immunofluorescence

Journal: Materials today. Bio

Article Title: Contribution of Tregs to the promotion of constructive remodeling after decellularized extracellular matrix material implantation.

doi: 10.1016/j.mtbio.2024.101151

Figure Lengend Snippet: Fig. 5. Treg recruitment increases after injection of a CD28-SA antibody. (A) Schematic of the protocol for establishing a mouse model with increased Treg recruitment. (B) Flow cytometry results of the Treg proportion in the peripheral blood of mice indicate a persistently high proportion of Tregs throughout the study period, relative to the control group. The results were normalized relative to those of the control group. (C, D) Representative FACS plots of Treg proportion in dECM, peripheral blood, and lymph nodes on day 14 after dECM material implantation (C), with the accompanying statistical graph (D) indicating an increase in Treg proportion. (E, F) Immunofluorescence image of Foxp3+ cells in dECM material on day 14 after implantation (E), with the accompanying statistical results (F) indicating an increase in Tregs infiltrating the dECM interior. **p ≤0.01, ****p ≤0.0001.

Article Snippet: To assess Treg infiltration into the dECM material, the sections were sealed with 10 % donkey serum (AntGene, China), and then incubated with forkhead box protein P3 (Foxp3) (1:200, 12653, CST, USA) antibody working fluid.

Techniques: Injection, Flow Cytometry, Control, Immunofluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Sirtuin 6 mediates the therapeutic effect of endometrial regenerative cell-derived exosomes in alleviation of acute transplant rejection by weakening c-myc-dependent glutaminolysis

doi: 10.3389/fcell.2025.1564382

Figure Lengend Snippet: SIRT6 mediated ERC-Exos to regulate CD4 + T cell differentiation in the graft, alleviating the inflammatory infiltration. Representative graft sections for infiltration of different subsets of CD4 + T cells and immunohistochemical staining of cytokines in the graft (magnification 200×). IFN-γ (A) and IL-17A (B) were used to stain Th1 and Th17 cells and their corresponding cytokines in the graft, respectively. Foxp3 (C) was used to stain the Treg infiltration in the graft. The positive staining areas were shown by arrows. Quantitative analysis of IFN-γ (D) , IL-17A (E) , and Foxp3 (F) positive cell proportion in IHC via ImageJ software.

Article Snippet: After being blocked with 10% goat serum, the sections were incubated with rabbit anti-mouse CD4 (dilution at 1:1,000, Abcam), IFN-γ (dilution at 1:100, ABclonal), IL-17A (dilution at 1:100, ABclonal), and Foxp3 (dilution at 1:100, affinity) antibody overnight at 4 °C and then with 100 μL of enhanced enzyme-labeled goat anti-rabbit IgG polymer (DAB kit, ZSGB-BIO, Beijing, China) for 20 min the next day at room temperature.

Techniques: Cell Differentiation, Immunohistochemical staining, Staining, Software

Journal: Frontiers in Cell and Developmental Biology

Article Title: Sirtuin 6 mediates the therapeutic effect of endometrial regenerative cell-derived exosomes in alleviation of acute transplant rejection by weakening c-myc-dependent glutaminolysis

doi: 10.3389/fcell.2025.1564382

Figure Lengend Snippet: SIRT6 mediated ERC-Exos to remodel CD4 + T cell differentiation and serum cytokine secretion level in recipient mice. Single-cell suspensions of splenocytes obtained from the untreated, ERC-Exo-treated, and SIRT6-KD-ERC-Exo-treated groups were analyzed for the frequency of CD4 + IFN-γ + , and CD4 + IL-17A + cells by flow cytometry, gated on live cells. The representative pseudocolor plots (A,C) and statistical graphs (B,D) were depicted (n = 5 per group). In addition, the frequency of CD25 + Foxp3 + cells obtained from the three groups were also analyzed by flow cytometry, gated on live cells and CD4 + cells. The representative pseudocolor plots (E) and statistical graphs (F) were depicted (n = 5 per group). (G) The representative CD4 + T cell serum cytokine levels in the recipient mice across the three groups (n = 5 per group). Specifically, compared with untreated group, ERC-Exo treatment could significantly decrease serum pro-inflammatory cytokine (IFN-γ and IL-17) level and increase IL-10 secretion, while knocking down SIRT6 in ERC-Exos slightly reversed these trends. Statistics by one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, ns = non-significant. For all panels, the bar graphs represent mean ± SD.

Article Snippet: After being blocked with 10% goat serum, the sections were incubated with rabbit anti-mouse CD4 (dilution at 1:1,000, Abcam), IFN-γ (dilution at 1:100, ABclonal), IL-17A (dilution at 1:100, ABclonal), and Foxp3 (dilution at 1:100, affinity) antibody overnight at 4 °C and then with 100 μL of enhanced enzyme-labeled goat anti-rabbit IgG polymer (DAB kit, ZSGB-BIO, Beijing, China) for 20 min the next day at room temperature.

Techniques: Cell Differentiation, Flow Cytometry

Journal: Frontiers in Cell and Developmental Biology

Article Title: Sirtuin 6 mediates the therapeutic effect of endometrial regenerative cell-derived exosomes in alleviation of acute transplant rejection by weakening c-myc-dependent glutaminolysis

doi: 10.3389/fcell.2025.1564382

Figure Lengend Snippet: SIRT6 mediated ERC-Exos to modulate CD4 + T cell differentiation and cytokine secretion level in vitro . Naïve CD4 + T cells were extracted from C57BL/6 mice. In the medium added with CD3/CD28, they were co-cultured with/withou ERC-Exos and SIRT6-KD-ERC-Exo for 72 h. Single-cell suspensions were obtained from the T-cell, T-cell + ERC-Exo, and T-cell + SIRT6-KD-ERC-Exo groups and then were analyzed for the frequency of CD4 + IFN-γ + , and CD4 + IL-17A + cells by flow cytometry, gated on live cells. The representative pseudocolor plots (A,C) and statistical graphs (B,D) were depicted (Repeat three independent holes). Similarly, the frequency of Foxp3 + cells obtained from the three groups were also analyzed by flow cytometry, gated on live cells and CD4 + cells. The representative pseudocolor plots (E) and statistical graphs (F) were depicted (Repeat three independent holes). (G) The representative cytokine levels of CD4 + T cell supernatant in the three groups (Repeat three independent holes). Compared with untreated group, ERC-Exo treatment could significantly decrease serum pro-inflammatory cytokine (IFN-γ and IL-17) level and increase IL-10 secretion, while absence of SIRT6 in ERC-Exos slightly reversed these trends. Statistics by one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, ns = non-significant. For all panels, the bar graphs represent mean ± SD.

Article Snippet: After being blocked with 10% goat serum, the sections were incubated with rabbit anti-mouse CD4 (dilution at 1:1,000, Abcam), IFN-γ (dilution at 1:100, ABclonal), IL-17A (dilution at 1:100, ABclonal), and Foxp3 (dilution at 1:100, affinity) antibody overnight at 4 °C and then with 100 μL of enhanced enzyme-labeled goat anti-rabbit IgG polymer (DAB kit, ZSGB-BIO, Beijing, China) for 20 min the next day at room temperature.

Techniques: Cell Differentiation, In Vitro, Cell Culture, Flow Cytometry