Journal: Molecular medicine reports

Article Title: BCG stimulation promotes dendritic cell proliferation and expression of VDR and CYP27B1 in vitamin D‑deficient mice.

doi: 10.3892/mmr.2019.10780

Figure Lengend Snippet: Figure 2. Effect of BCG on BMDC differentiation and maturation. (A) Cell morphology during BMDC induction from days 1 to 6, as observed by light micros- copy. (B) BCG stimulated BMDC maturation. (C) Phenotypic changes of the BMDCs induced by BCG stimulation and (D) the levels of the surface molecules of DCs. All images were captured under identical magnification and microscopy conditions. **P<0.01 vs. 0 mg/ml in the normal control or vitamin D‑deficiency groups. ##P<0.01, vitamin D‑deficiency group vs. normal control group. Scale bar, 20 µm. BCG, Bacillus Calmette‑Guérin; BMDC, bone marrow‑derived dendritic cell; CD11c, integrin alpha‑X; CD80, T‑lymphocyte activation antigen CD80; MHC‑I, major histocompatibility complex class I; MHC‑II, major histocompatibility complex class II; CD86, T‑lymphocyte activation antigen CD86.

Article Snippet: The cell concentration was adjusted to 1x106 cells/ml and the cells were fixed with 0.1 ml polyformaldehyde for 30 min at 25 ̊C Subsequently, fluorescein isothiocyanate‐labeled integrin alpha‐X (CD11c; 1:50; 130‐110‐700; Miltenyi Biotech), T‐lymphocyte activation antigen CD86 (CD86; 1:50; 130‐102‐506; Miltenyi Biotech), T-lymphocyte activation antigen cd80 (cd80; 1:50; 130‐116‐459; Miltenyi Biotech), major histocompatibility complex class i (MHc-i; 1:50; ab95572; abcam) and MHC‐II (1:50; 130‐112‐386; Miltenyi Biotech) were added, respectively.

Techniques: Microscopy, Control, Activation Assay, Immunopeptidomics

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Fusion proteins for versatile antigen targeting to cell surface receptors reveal differential capacity to prime immune responses.

doi: 10.4049/jimmunol.0902555

Figure Lengend Snippet: FIGURE 4. Superior CD8+ T cell priming by a fusion protein containing three protein G domains and a Ub mutation. A, Proliferation of adoptively transferred, CFSE-labeled OT-I T cells recovered 3 d after Ag injection from the draining lymph nodes of injected mice. Animals were injected with CD11c-targeted fusion protein containing 50 ng OVA. Fusion proteins containing the physiological residues (KG, Lys-Gly) or substituted (RV, Arg-Val) at Ub positions 48 and 76 were tested. Experiments were per- formed twice, using seven mice per group total. Fusion protein P3UOrv primed significantly better than P2UOrv (p = 0.001), whereas the differ- ence was not statistically significant for P3UOkg versus P2UOkg (p = 0.10). B, Number of divisions per OT-I precursor induced upon priming with P3UO fusion proteins carrying the amino acids shown to the left in positions 48 and 76 (e.g., RV, Arg48Val76; KG, Lys48Gly76). Experiment was performed once, using four mice per group. Priming by 50 ng P3UOrv was significantly better than priming by all of the other proteins (p , 0.03), whereas the other differences shown were not statis- tically significant.

Article Snippet: The following Abs were used: rat anti-mouse DEC-205 (clone NLDC-145), hamster anti-mouse CD11c (clone N418), and control mAb mouse anti–HLA-A2 (clone BB7.2) were purchased from American Type Culture Collection and purified from hybridoma supernatant; rat antimouse TLR2 and rat anti-mouse CD206 (MR1) were from AbD Serotec (Cergy Saint-Christophe, France); control mAb mouse anti-human TAP1 (clone 148.3) was purified from ascites.

Techniques: Mutagenesis, Labeling, Injection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Fusion proteins for versatile antigen targeting to cell surface receptors reveal differential capacity to prime immune responses.

doi: 10.4049/jimmunol.0902555

Figure Lengend Snippet: FIGURE 5. Localization of fusion protein in draining lymph nodes. Mice were injected with fusion protein in the hind footpads. Draining lymph node cryosections were stained for OVA fusion protein (P2EUO or P3UO, green) and cell markers (red). In the top row, mice were injected with 11 mg anti-CD11c targeted (left panel) or hamster Ig complexed (right panel) P2EUO 24 h before lymph node analysis. All of the other images are from mice injected with 2.5 mg CD11c-targeted fusion protein. The times between injection and lymph node collection were 1 h (CD169), 4 h (CD3, DEC205), and 24 h (CD19, CD35). Experiments were per- formed four times. Anti-CD169–stained section, original magnification 3100; anti-DEC205–stained section, original magnification 340; all others, original magnification 310.

Article Snippet: The following Abs were used: rat anti-mouse DEC-205 (clone NLDC-145), hamster anti-mouse CD11c (clone N418), and control mAb mouse anti–HLA-A2 (clone BB7.2) were purchased from American Type Culture Collection and purified from hybridoma supernatant; rat antimouse TLR2 and rat anti-mouse CD206 (MR1) were from AbD Serotec (Cergy Saint-Christophe, France); control mAb mouse anti-human TAP1 (clone 148.3) was purified from ascites.

Techniques: Injection, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Fusion proteins for versatile antigen targeting to cell surface receptors reveal differential capacity to prime immune responses.

doi: 10.4049/jimmunol.0902555

Figure Lengend Snippet: FIGURE 6. Effector functions of fusion protein-primed T cells. A, A total of 2 3 106 naive OT-I cells expressing the congeneic marker CD45.2 were injected i.v. in C57BL/6 mice expressing CD45.1. Twenty-four hours later, the mice were injected in the footpads with PBS, OVA, or CD11c/P3UOrv with or without adjuvants LPS, poly(I:C), and/or anti-CD40. Two weeks later, the mice were boosted s.c. with the same Ag without adjuvant. Three days later, the draining lymph nodes were taken and analyzed by flow cytometry. B, C57BL/6 mice were immunized s.c. with the indicated amounts of CD11c-targeted P3UOrv together with poly(I:C) and anti-CD40, which were injected s.c. or i.p., as indicated. One week later, the draining lymph node and spleen lymphocytes were stimulated in vitro with peptide S8L for 6 h before staining for intracellular cytokines. The insets show the aggregate percentages of cytokine-producing cells among all of the CD8+ T lymphocytes. The percentages of CD8+ cells producing one (white), two (gray), or three (black) cytokines are indicated in the pie charts. Cells stimulated with control peptide T8V did not produce cytokines (data not shown). C, C57BL/6 mice, injected 24 h earlier with 1 3 106 OT-I cells, were immunized s.c. with CD11c-targeted P3UOrv equivalent containing 130 ng OVA with or without adjuvant poly (I:C) and anti-CD40. Twelve days later, 3 3 106 CFSE-labeled syngeneic splenocytes pulsed with control peptide T8V (CFSE low) or cognate peptide S8L (CFSE high) were injected, followed 16 h later by flow cytometric analysis of draining lymph nodes and spleen. D, C57BL/6 mice were immunized s.c. with different amounts of Ab/fusion protein complexes (DEC205-P3UOrv, 1.3 mg; MR-P3UOrv, 1.3 mg; TLR2-P3UOrv, 0.225 mg; CD11c-P3UOrv, 0.13 mg; P3UOrv, 1 mg) and adjuvant poly(I:C) s.c. and anti-CD40 i.p. and boosted 5 wk later s.c. with the same Ags without adjuvant. One week later, the presence of OVA-specific serum Abs with different isotypes was tested by ELISA. Experiments were performed twice.

Article Snippet: The following Abs were used: rat anti-mouse DEC-205 (clone NLDC-145), hamster anti-mouse CD11c (clone N418), and control mAb mouse anti–HLA-A2 (clone BB7.2) were purchased from American Type Culture Collection and purified from hybridoma supernatant; rat antimouse TLR2 and rat anti-mouse CD206 (MR1) were from AbD Serotec (Cergy Saint-Christophe, France); control mAb mouse anti-human TAP1 (clone 148.3) was purified from ascites.

Techniques: Expressing, Marker, Injection, Adjuvant, Cytometry, In Vitro, Staining, Control, Labeling, Enzyme-linked Immunosorbent Assay

Journal: Journal of nanobiotechnology

Article Title: Lactiplantibacillus plantarum -derived extracellular vesicles alleviate acute lung injury by inhibiting ferroptosis of macrophages.

doi: 10.1186/s12951-025-03405-y

Figure Lengend Snippet: Fig. 2 LpEVs alleviated lung inflammation and injury in mice. A. Schematic depiction of the experiment. B. Representative images showing histopatho logical alterations in lungs of different treatment groups. ICR mice (n = 5) were sitimulated with LPS (2.5 mg/kg, intratracheally) for 3 h, then administered PBS or LpEVs (100 µg) via the tail vein injection method. Mice were euthanized 24 h post-treatment. Scale bar = 200 μm. C. Representative F4/80 and MPO IHC staining images in different lungs. Scale bars represent 50 μm. D. Protein concentration in BALF. E. Cell counts in BALF. F. The concentration of cytokines/chemokines in the BALF was assessed using ELISA. G. Flow cytometry analysis of neutrophils (CD11b+ Ly6G+) in the BALF. H. Flow cytometry analysis of macrophages (CD11b+ F4/80+) in the BALF. I. Flow cytometry analysis of alveolar macrophages (CD11c+ Siglec F+) in the BALF

Article Snippet: The population of alveolar macrophages (anti-CD11c, anti-Siglec F) (E-AB-F0991E, Elabscience, Wuhan, China) (12-1702- 82, Thermo Fisher, USA), macrophages (anti-F4/80, anti-CD11b) (E-AB-F0995D, E-AB-F1081E, Elabscience, Wuhan, China), and neutrophils (anti-CD11b, anti-Ly6G) (E-AB-F1081E, E-AB-F1108C, Wuhan, China) were assessed using flow cytometry with specific antibodies.

Techniques: Injection, Immunohistochemistry, Protein Concentration, Concentration Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Journal of nanobiotechnology

Article Title: Lactiplantibacillus plantarum -derived extracellular vesicles alleviate acute lung injury by inhibiting ferroptosis of macrophages.

doi: 10.1186/s12951-025-03405-y

Figure Lengend Snippet: Fig. 4 LpEVs treatment inhibited LPS-induced ferroptosis in vivo. A. The levels of reactive oxygen species (ROS) in lung BALF cells were evaluated using DCFH-DA. The left panel displays a flow cytometry diagram, while the right panel shows the mean fluorescence intensity of ROS. B. Relative MDA content in lungs. C. Relative Fe2+ content in lungs. D. Relative GSH content in lungs. E. Relative mRNA expression of ferroptotic-related genes were analyzed by qRT-PCR. F. The expression levels of ferroptotic-related proteins in lungs were analyzed by western blotting. ACTIN is adopted as the loading control. G. Representative IHC staining images for GPX4, SLC7A11 and ACSL4 were obtained from different lung samples. Scale bars represent 50 μm. H. The statisti cal graph of the expression of GPX4, SLC7A11 and ACSL4 in IHC staining. I. Representative IHC staining images for macrophages M1 marker CD86 and M1 marker CD206 in lungs. Scale bars represent 50 μm. J. The statistical graph of the expression of CD86 and CD206 in IHC staining. Quantification of proteins expresion levels in IHC staining were determined by image J software. The values for the LPS-PBS and LPS-EV were expressed as percentages of the values for the Sham group. *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001 and ns, no significant difference

Article Snippet: The population of alveolar macrophages (anti-CD11c, anti-Siglec F) (E-AB-F0991E, Elabscience, Wuhan, China) (12-1702- 82, Thermo Fisher, USA), macrophages (anti-F4/80, anti-CD11b) (E-AB-F0995D, E-AB-F1081E, Elabscience, Wuhan, China), and neutrophils (anti-CD11b, anti-Ly6G) (E-AB-F1081E, E-AB-F1108C, Wuhan, China) were assessed using flow cytometry with specific antibodies.

Techniques: In Vivo, Flow Cytometry, Fluorescence, Expressing, Quantitative RT-PCR, Western Blot, Control, Immunohistochemistry, Marker, Software

Journal: Journal of nanobiotechnology

Article Title: Lactiplantibacillus plantarum -derived extracellular vesicles alleviate acute lung injury by inhibiting ferroptosis of macrophages.

doi: 10.1186/s12951-025-03405-y

Figure Lengend Snippet: Fig. 5 LpEVs facilitated the transition of macrophages towards an anti-inflammatory phenotype. A. Confocal laser scanning microscopy images dis played the presence of Dio-labeled LpEVs in MH-S cells. Green: Dio labeled LpEVs; Blue: Hoechst. Scale bar = 20 μm. B. The Dio-positive MH-S cells were determined by flow cytometry. C. Relative mRNA expression levels of inflammatory factors were assessed by qRT-PCR. M1 proinflammatory genes: Il1β, Il6, Tnfα, and iNos, M2 anti-inflammatory genes: Arg-1, and Cd206. The M1 macrophages were induced by LPS treatment, and M2 macrophages were induced by IL-4 treatment. D. The levels of pro-inflammatory and anti-inflammatory cytokines in the medium supernatants were quantified using ELISA. E. The expression of CD86 (an M1 marker) on MH-S cells 12 h after exposure to LPS was assessed using flow cytometry, with representative histograms and average relative mean fluorescence intensity (MFI) provided. F. Flow cytometry was used to detect the expression of CD206, an M2 marker, on MH-S cells 12 h after exposure to IL4

Article Snippet: The population of alveolar macrophages (anti-CD11c, anti-Siglec F) (E-AB-F0991E, Elabscience, Wuhan, China) (12-1702- 82, Thermo Fisher, USA), macrophages (anti-F4/80, anti-CD11b) (E-AB-F0995D, E-AB-F1081E, Elabscience, Wuhan, China), and neutrophils (anti-CD11b, anti-Ly6G) (E-AB-F1081E, E-AB-F1108C, Wuhan, China) were assessed using flow cytometry with specific antibodies.

Techniques: Confocal Laser Scanning Microscopy, Labeling, Flow Cytometry, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Marker, Fluorescence

Journal: Journal of nanobiotechnology

Article Title: Lactiplantibacillus plantarum -derived extracellular vesicles alleviate acute lung injury by inhibiting ferroptosis of macrophages.

doi: 10.1186/s12951-025-03405-y

Figure Lengend Snippet: Fig. 6 LpEVs attenuated ferroptosis in macrophages. A. The ROS level in MH-S was assessed by flow cytometry. B. Relative MDA content in MH-S. C. Rela tive GSH content in MH-S. D. The intercellular Fe2+ was detected by FerroOrange (a specific probe of Fe2+). Scale bar = 100 μm. E. The expression levels of ferroptotic-related proteins and NRF2-related proteins in MH-S were detected by western blot. ACTIN is adopted as the loading control. F. SLC7A11, GPX4 and ACSL4 immunofluorescent staining results of differently treated MH-S. Scale bar = 20 μm. G. NRF2 and HO-1 immunofluorescent staining results of differently treated MH-S. Scale bar = 20 μm

Article Snippet: The population of alveolar macrophages (anti-CD11c, anti-Siglec F) (E-AB-F0991E, Elabscience, Wuhan, China) (12-1702- 82, Thermo Fisher, USA), macrophages (anti-F4/80, anti-CD11b) (E-AB-F0995D, E-AB-F1081E, Elabscience, Wuhan, China), and neutrophils (anti-CD11b, anti-Ly6G) (E-AB-F1081E, E-AB-F1108C, Wuhan, China) were assessed using flow cytometry with specific antibodies.

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

Journal: Journal of nanobiotechnology

Article Title: Lactiplantibacillus plantarum -derived extracellular vesicles alleviate acute lung injury by inhibiting ferroptosis of macrophages.

doi: 10.1186/s12951-025-03405-y

Figure Lengend Snippet: Fig. 7 LpEVs regulated ferroptosis in macrophages via delivery of cbn-let-7. A. miRNA microarray analyze the miRNA expression profile of LpEVs. B. The 4 miRNAs (cel-mir-4937, cbn-let-7, ngi-miR-92a and gsa-mir-9394b) expression level in LpEVs. C. cbn-let-7 expression in PBS and LpEVs treated MH-S cells. D, E. Structure and luciferase result of dual luciferase reporter gene. F. The relative expression of cbn-let-7 in NC, cbn-let-7 mimic and inhibitor treated MH-S cells. G. The protein expression of of ACSL4 and GPX4 in NC, cbn-let-7 mimic and inhibitor treated MH-S cells. H. The expression of cbn-let-7 in different treated cells. I. The protein expression of of ACSL4 and GPX4 in different treated cells. ACTIN is adopted as the loading control. J. The intercellular Fe2+ in different treated cells was detected by FerroOrange (a specific probe of Fe2+)

Article Snippet: The population of alveolar macrophages (anti-CD11c, anti-Siglec F) (E-AB-F0991E, Elabscience, Wuhan, China) (12-1702- 82, Thermo Fisher, USA), macrophages (anti-F4/80, anti-CD11b) (E-AB-F0995D, E-AB-F1081E, Elabscience, Wuhan, China), and neutrophils (anti-CD11b, anti-Ly6G) (E-AB-F1081E, E-AB-F1108C, Wuhan, China) were assessed using flow cytometry with specific antibodies.

Techniques: Microarray, Expressing, Luciferase, Control