Journal: Theranostics

Article Title: Sulforaphane elicts dual therapeutic effects on Renal Inflammatory Injury and crystal deposition in Calcium Oxalate Nephrocalcinosis

doi: 10.7150/thno.44054

Figure Lengend Snippet: Nrf2 suppresses TLR4 and IRF1 levels and promotes M2Mϕ polarization in vitro . (A) BMDM and COM-TECs coculture schematic diagram. Western blot (B) and qPCR (C) analyses of Nrf2, TLR4, IRF1, iNOS, and ARG-1 levels in BMDMs co-cultured with increasing COM dose stimulated TECs. GAPDH was used as an internal control. (D, F) Western blot detection of Nrf2, TLR4, IRF1, iNOS, and ARG-1 levels following SFN treatment or Nrf2 upregulation/downregulation in BMDMs co-cultured with COM-stimulated TECs. GAPDH was used as an internal control. (E, G) The distribution of iNOS (green) and ARG-1 (red) in BMDMs according to immunofluorescence. (H, I) Flow cytometric analysis of the polarization state of BMDMs using anti-CD11c and anti-CD206 in F4/80 + and CD11b + cells. The data are shown as the mean ± standard error (SE) of three independent experiments. *P < 0.05; **P < 0.01, as determined by Student's t test (C) or one-way ANOVA (E, G, H, I).

Article Snippet: Protein extract was isolated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, and incubated overnight at 4°C with primary antibodies against Nrf2 (AF7006; 72 kDa; 1:1000; Affinity Biologicals), TLR4 (GB11519; 95 kDa; 1:1000; Servicebio), IRF1 (abs118047; 37 kDa; 1:1000; Absin), iNOS (BA0362; 130 kDa; 1:200; Boster), arginase 1 (ARG-1; GB11285; 35-40 kDa; 1:5000; Servicebio), or GAPDH (T0004; 34 kDa; 1:5000; Affinity Biologicals).

Techniques: In Vitro, Western Blot, Cell Culture, Control, Immunofluorescence

Journal: Theranostics

Article Title: Sulforaphane elicts dual therapeutic effects on Renal Inflammatory Injury and crystal deposition in Calcium Oxalate Nephrocalcinosis

doi: 10.7150/thno.44054

Figure Lengend Snippet: Nrf2 inhibits TLR4 and IRF1 expression by directly binding to miR-93 promoter. (A) miRNA-array heatmap revealed the top 30 LPS-regulated miRNAs in BMDMs. Additionally, miRNAs predicted to be transcriptionally activated by Nrf2 (based on the JASPRA database) are highlighted. (B) Venn analysis identified miRNAs that could target TLR4 and IRF1 and be transcriptionally activated by Nrf2. (C) Renal expression of mmu-miR-93-5p following treatment with an Nrf2-neutralizing antibody or SFN in mice with CaOx nephrocalcinosis was detected using FISH (200× magnification for all panels; scale bar: 20 µm). (D) qPCR analysis of the expression levels of mmu-miR-93-5p in BMDMs. U6 RNA was detected as an internal control. (E, F) ChIP assays and ChIP qPCR analysis of Nrf2 binding to the predicted miR-93 ARE in BMDMs treated with Nrf2. (G) A schematic model showing Nrf2 directly binds the promoter of miR-93 and activates its transcription. (H, I) Schematic representation of mutant and WT seed sequences of miR-93 targeting the 3' UTRs of TLR4 and IRF1. Luciferase reporters harboring putative target sites in WT and mutant 3' UTRs of TLR4 (J) or IRF1 (L) were co-transfected with miR-93 mimics (100 nM). TLR4 (K) and IRF1 (M) expression detected by qPCR in BMDMs transfected with miR-93 mimics or inhibitor. Western blot (N) and qPCR (O) detection of TLR4 and IRF1 levels, as well as the Mϕ-polarization markers iNOS and ARG-1 in BMDMs transfected with miR-93 mimics or inhibitor. GAPDH was used as an internal control. Data represent the mean ± standard error (SE) of three independent experiments. *P < 0.05; **P < 0.01, as determined by Student's t test (D, F) or one-way ANOVA (J-M, O).

Article Snippet: Protein extract was isolated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, and incubated overnight at 4°C with primary antibodies against Nrf2 (AF7006; 72 kDa; 1:1000; Affinity Biologicals), TLR4 (GB11519; 95 kDa; 1:1000; Servicebio), IRF1 (abs118047; 37 kDa; 1:1000; Absin), iNOS (BA0362; 130 kDa; 1:200; Boster), arginase 1 (ARG-1; GB11285; 35-40 kDa; 1:5000; Servicebio), or GAPDH (T0004; 34 kDa; 1:5000; Affinity Biologicals).

Techniques: Expressing, Binding Assay, Control, ChIP-qPCR, Mutagenesis, Luciferase, Transfection, Western Blot

Journal: Theranostics

Article Title: Sulforaphane elicts dual therapeutic effects on Renal Inflammatory Injury and crystal deposition in Calcium Oxalate Nephrocalcinosis

doi: 10.7150/thno.44054

Figure Lengend Snippet: SFN dependeds on the Nrf2-miR-93-TLR4/IRF1 axis to suppress TLR4 and IRF1 expression and promote M2Mϕ polarization in vitro . Western blot (A) and qPCR (B) analyses of Nrf2, TLR4, IRF1, iNOS, and ARG-1 levels in BMDMs treated with SFN and/or miR-93 inhibitor. GAPDH was used as an internal control. (C) The distribution of iNOS (green) and ARG-1 (red) in BMDMs treated with SFN and/or miR-93 inhibitor according to immunofluorescence (200× magnification; scale bar: 20 µm). (D) Fluorescence microscopy analysis of BMDM phagocytic ability. COM crystals were labeled with an Alexa Fluor 488-conjugated IgG and directly cultured with treated BMDMs (200× magnification; scale bar: 20 µm). Flow cytometric analysis of BMDM polarization (E) and TECs necrosis (F) in co-cultured cells treated with SFN and/or miR-93 inhibitor. Data represent the mean ± standard error (SE) of three independent experiments. *P < 0.05; **P < 0.01, as determined by one-way ANOVA (B-G).

Article Snippet: Protein extract was isolated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, and incubated overnight at 4°C with primary antibodies against Nrf2 (AF7006; 72 kDa; 1:1000; Affinity Biologicals), TLR4 (GB11519; 95 kDa; 1:1000; Servicebio), IRF1 (abs118047; 37 kDa; 1:1000; Absin), iNOS (BA0362; 130 kDa; 1:200; Boster), arginase 1 (ARG-1; GB11285; 35-40 kDa; 1:5000; Servicebio), or GAPDH (T0004; 34 kDa; 1:5000; Affinity Biologicals).

Techniques: Expressing, In Vitro, Western Blot, Control, Immunofluorescence, Fluorescence, Microscopy, Labeling, Cell Culture

Journal: Theranostics

Article Title: Sulforaphane elicts dual therapeutic effects on Renal Inflammatory Injury and crystal deposition in Calcium Oxalate Nephrocalcinosis

doi: 10.7150/thno.44054

Figure Lengend Snippet: SFN dependeds on the Nrf2-miR-93-TLR4/IRF1 axis to suppress CaOx crystal deposition and kidney injury in vivo . (A) Diagram of the experimental design. (B) Polarized-light optical microscopy detection of renal CaOx crystal deposition in mice treated with SFN and/or antagomiR-93 (20× magnification; scale bar: 500 µm). Pizzolato staining to detect corticomedullary CaOx crystal deposition. PAS and TUNEL staining to detect renal tubular epithelial cell injury (200× magnification; scale bar: 20 µm). (C) 18 F-FDG PET-CT scanning to detect kidney inflammation in CaOx nephrocalcinosis mice treated with SFN and/or antagomiR-93. (D) IHC detection of Nrf2, TLR4, and IRF1 levels (400× magnification; scale bar: 40 µm) and FISH detection of miR-93 expression in kidney tissue (200× magnification; scale bar: 20 µm). (E) The distributions of iNOS (red) and ARG-1 (green) in kidney tissues according to immunofluorescence. (F) Serum levels of the proinflammatory cytokines IL-1β, TNF-α, and IL-6 and the anti-inflammatory cytokine IL-10 according to ELISA on days 3, 4, and 10. *P < 0.05; **P < 0.01, as determined by one-way ANOVA (B-F).

Article Snippet: Protein extract was isolated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, and incubated overnight at 4°C with primary antibodies against Nrf2 (AF7006; 72 kDa; 1:1000; Affinity Biologicals), TLR4 (GB11519; 95 kDa; 1:1000; Servicebio), IRF1 (abs118047; 37 kDa; 1:1000; Absin), iNOS (BA0362; 130 kDa; 1:200; Boster), arginase 1 (ARG-1; GB11285; 35-40 kDa; 1:5000; Servicebio), or GAPDH (T0004; 34 kDa; 1:5000; Affinity Biologicals).

Techniques: In Vivo, Microscopy, Staining, TUNEL Assay, Positron Emission Tomography-Computed Tomography, Expressing, Immunofluorescence, Enzyme-linked Immunosorbent Assay

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

Article Title: Tuberculosis is associated with a down-modulatory lung immune response that impairs Th1-type immunity.

doi: 10.4049/jimmunol.0801212

Figure Lengend Snippet: FIGURE 8. Expression of human NOS2 by lung cells. A, Comparisons of NOS2 levels between those in TB patients and healthy volunteers (VOL) are illustrated as the mean (SEM) percentage of positive cells (alveolar macrophages) obtained at diagnosis (time (T) 0) and at day 15 of anti-TB treatment. Statistically significant differences in NOS2 levels were noted between those TB patients and the healthy volunteers (, p 0.05; Mann- Whitney U test). B, Mean NOS2 levels quantified from TB cases at time of diagnosis to 180 days are illustrated. Of the 15 TB cases studies, the num- ber of patients with positive NOS2 lung cells at time of diagnosis (T0) and during anti-TB treatment (days 15, 30, 60, and 180), respectively, were 13, 10, 5, 6, and 3. All of the nine healthy volunteers had NOS2 detected. Statistical differences in the expressions of NOS2 at different times during anti-TB treatment are shown (, p 0.0; Student’s t test for repeated measures).

Article Snippet: Immunolocalization was performed with immune alkaline phosphatase using the primary mAb, anti-human NOS2 (purified mouse monoclonal IgG1, clone 2D2-B2, 100 g; R&D Systems), diluted to 1/40 in TBS containing BSA.

Techniques: Expressing, Biomarker Discovery, MANN-WHITNEY

Journal: International immunopharmacology

Article Title: KLF15 regulates macrophage polarization patterns in deep vein thrombosis.

doi: 10.1016/j.intimp.2025.114632

Figure Lengend Snippet: Fig. 1. Macrophage polarization patterns and KLF15 expression in DVT. A-B: ELISA kits were used to measure plasma levels of P-selectin (A) and vWF (B) in Sham and DVT group mice (N = 3). C-D: western blotting (C) and IF staining (D) were used to examine the expression and localization of M1-like macrophage (iNOS) and M2-like macrophage (CD206) markers in the inferior vena cava (IVC) tissues of mice in the Sham and DVT groups (N = 3). Scale = 100 μm. E: serum levels of pro-inflammatory factors (IL-1β and IL-6) and anti-inflammatory factors (IL-10 and TGF-β) released by M1/M2-like macrophages in mice from Sham and DVT groups were detected using ELISA kits (N = 3). F: Representative images of KLF15 IHC staining of IVC tissues from Sham and DVT group mice (N = 3). Scale = 500 μm. G-H: Western blotting (G) and RT-qPCR (H) assays were performed to detect the levels of KLF15 protein and mRNA in IVC tissues (N = 3).

Article Snippet: PVDF membranes were incubated in hybridization bags with antibodies, including rabbit KLF15 pAb, rabbit CD206 pAb (18704-1- AP; Proteintech; 1:5000), mouse iNOS mAb, rabbit NEK2 pAb (24171-1- AP; Proteintech; 1:1000), rabbit recombinant P65 (80979-1-RR; Proteintech; 1:40,000), mouse β-actin mAb (TA-09; ZSGB, China; 1:4000), rabbit p –P65 pAb (bs-5661R; Bioss, China; 1:2000), goat anti-mouse/ rabbit IgG-HRP (M21001L/M21002L; Abmart; 1:4000).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Western Blot, Staining, Immunohistochemistry, Quantitative RT-PCR

Journal: International immunopharmacology

Article Title: KLF15 regulates macrophage polarization patterns in deep vein thrombosis.

doi: 10.1016/j.intimp.2025.114632

Figure Lengend Snippet: Fig. 2. KLF15 overexpression promotes M1 polarization in macrophages. A-B: the expression differences of KLF15 protein and mRNA in macrophages of PMA, M1, M1-NC and M1-KLF15 groups were detected using western blotting (A) and RT-qPCR (B) assays (N = 3). C: Effect of KLF15 overexpression on the proportion of CD86+ macrophages detected by flow cytometry (N = 3). D-E: Effect of KLF15 overexpression on the expression of M1 macrophage marker (iNOS) protein (D) and mRNA (E) (N = 3). F: representative images of iNOS IF staining in macrophages (N = 3). Scale = 100 μm. G: levels of pro-inflammatory factors (IL-1β and IL-6) in the supernatants of macrophages from the PMA, M1, M1-NC and M1-KLF15 groups were detected by ELISA kits (N = 3). H: Effect of KLF15 overexpression on IL-12 and TNF-α levels in M1 macrophages (N = 3).

Article Snippet: PVDF membranes were incubated in hybridization bags with antibodies, including rabbit KLF15 pAb, rabbit CD206 pAb (18704-1- AP; Proteintech; 1:5000), mouse iNOS mAb, rabbit NEK2 pAb (24171-1- AP; Proteintech; 1:1000), rabbit recombinant P65 (80979-1-RR; Proteintech; 1:40,000), mouse β-actin mAb (TA-09; ZSGB, China; 1:4000), rabbit p –P65 pAb (bs-5661R; Bioss, China; 1:2000), goat anti-mouse/ rabbit IgG-HRP (M21001L/M21002L; Abmart; 1:4000).

Techniques: Over Expression, Expressing, Western Blot, Quantitative RT-PCR, Flow Cytometry, Marker, Staining, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: Type I interferon governs immunometabolic checkpoints that coordinate inflammation during Staphylococcal infection

doi: 10.1016/j.celrep.2024.114607

Figure Lengend Snippet:

Article Snippet: Anti-iNOS Rabbit IgG , Cell Signaling , Cat#13120S; RRID:AB_2687529.

Techniques: Virus, Recombinant, Lactate Assay, SYBR Green Assay, cDNA Synthesis, Staining, Western Blot, Enzyme-linked Immunosorbent Assay, Bradford Assay, Sample Prep, Infection, Derivative Assay, Transgenic Assay, Control, Mutagenesis, Software, Targeted Proteomics