lps  (Novus Biologicals)

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular vesicles derived from Enterococcus faecalis : inflammatory activation does not require internalization

doi: 10.1186/s12964-026-02926-9

Figure Lengend Snippet: Isolation and characterization of Ef -EVs. A Growth curve of E. faecalis DSM 20478. The optical density (OD 600 ) was measured from E. faecalis cultures in BHI medium grown under static conditions at 37 °C. Results are shown as mean ± SD ( N = 3, n = 3). B Representative chromatogram obtained by protein concentration analysis of eluted fractions after size exclusion chromatography. Protein concentration was quantified by the BCA assay. The upper panel indicates the protein concentration of the first forty 1-mL fractions, indicating the elution peak corresponding to impurities (black area). The zoomed lower panel shows the protein concentration of the first 20 fractions, highlighting the EV-rich zone (red area). Results are shown as mean ± SD ( n = 3). C Representative size distribution of particles in the vesicle-richest fraction by Nanoparticle Tracking Analysis. D Representative cryo-TEM image of Ef -EVs in the vesicle-richest fraction (scale bar = 200 nm)

Article Snippet: To elucidate whether Ef -EVs induce NF-κB/AP-1 activation by TLR2 engagement, dTHP1-XBlue were pre-incubated for 1 h with fresh medium containing 1 μg/mL of neutralizing monoclonal antibodies (anti-hTLR2-IgA mAb, cat. no. maba2-htlr2-2, Invivogen) or human IgA2 control (cat. no. maba2-ctrl, Invivogen) before adding either Ef -EVs (7000 EVs/cell), ultrapure LPS from E. coli K12 (LPS, 1 ng/mL, cat.no. tlrl-peklps, Invivogen), or Pam 3 CSK 4 (1 ng/mL) in antibody-containing medium.

Techniques: Isolation, Protein Concentration, Size-exclusion Chromatography, BIA-KA

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular vesicles derived from Enterococcus faecalis : inflammatory activation does not require internalization

doi: 10.1186/s12964-026-02926-9

Figure Lengend Snippet: Ef -EVs activate the NF-κB/AP-1 pathways in human reporter macrophages via TLR2 signaling. A dTHP1-XBlue cells were treated with Ef -EVs (100 − 50,000 EVs/cell). LPS (100 ng/mL) was used as a positive control. B dTHP1-XBlue cells were treated with EVs derived from clinical E. faecalis bloodstream isolates (1000-10,000 EVs/cell). LPS (100 ng/mL) and Pam 3 CSK 4 (100 ng/mL) were used as positive controls. C-E) dTHP1-XBlue cells were pretreated with increasing concentrations (from 0.1 to 5 µg/mL) of anti-hTLR2-IgA mAb (dark pink) or human IgA2 control mAb (light pink) for 1 h. Subsequently, cells were treated with either (C) EVs (7000 EVs/cell), (D) Pam 3 CSK 4 (TLR2 ligand, 1 ng/mL), or (E) LPS (TLR4 ligand, 1 ng/mL) in the presence of antibodies for 24 h. Cells treated with cell culture medium supplemented only with Ef -EVs (7000 EVs/cell) in (C, blue), Pam 3 CSK 4 (1 ng/mL) in (D, dark blue), or LPS (1 ng/mL) in (E, red) were used as positive activating controls while cells treated with cell culture medium supplemented alone ( C - E , light green) were used as negative controls. NF-κB/AP-1 activation was measured as the activity of secreted SEAP and expressed as a normalized value relative to the positive controls. Data are shown as means ± SD of three independent experiments ( N = 3, n ≥ 3) and analyzed either by Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test in (A and B) or by two-way analysis of variance (ANOVA) followed by Šídák multiple comparison post hoc test in ( C - E )

Article Snippet: To elucidate whether Ef -EVs induce NF-κB/AP-1 activation by TLR2 engagement, dTHP1-XBlue were pre-incubated for 1 h with fresh medium containing 1 μg/mL of neutralizing monoclonal antibodies (anti-hTLR2-IgA mAb, cat. no. maba2-htlr2-2, Invivogen) or human IgA2 control (cat. no. maba2-ctrl, Invivogen) before adding either Ef -EVs (7000 EVs/cell), ultrapure LPS from E. coli K12 (LPS, 1 ng/mL, cat.no. tlrl-peklps, Invivogen), or Pam 3 CSK 4 (1 ng/mL) in antibody-containing medium.

Techniques: Positive Control, Derivative Assay, Control, Cell Culture, Activation Assay, Activity Assay, Comparison

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular vesicles derived from Enterococcus faecalis : inflammatory activation does not require internalization

doi: 10.1186/s12964-026-02926-9

Figure Lengend Snippet: TLR2 controls EV-induced immune activation but does not function as an endocytic receptor for EV uptake. dTHP1-XBlue cells were pretreated with anti-hTLR2-IgA mAb (1 µg/mL) or human IgA2 control mAb (1 µg/mL) for 1 h. Cells were then treated with DiI-labeled Ef -EVs (7000 EVs/cell) in the presence of antibodies for 4 and 24 h. Cells incubated in cell culture medium alone were used as negative controls. Cells treated with cell culture medium supplemented with DiI-labeled Ef -EVs (7000 EVs/cell) were used as positive activating controls. For EV internalization assays, mean fluorescence intensity (B and C, upper panel) and EV-positive cells (B and C, lower panel) were quantified after 4 ( A and B ) and 24 h ( A and C ) of EV treatment by measuring fluorescence intensity associated with DiI-labeled Ef -EVs on the PE channel. NF-κB/AP-1 activation in D was measured after 24 h of EV incubation as the activity of secreted SEAP and expressed normalized to the positive controls. Quantitative results are presented as mean ± SD ( N = 3, n = 3) and were analyzed by Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test

Article Snippet: To elucidate whether Ef -EVs induce NF-κB/AP-1 activation by TLR2 engagement, dTHP1-XBlue were pre-incubated for 1 h with fresh medium containing 1 μg/mL of neutralizing monoclonal antibodies (anti-hTLR2-IgA mAb, cat. no. maba2-htlr2-2, Invivogen) or human IgA2 control (cat. no. maba2-ctrl, Invivogen) before adding either Ef -EVs (7000 EVs/cell), ultrapure LPS from E. coli K12 (LPS, 1 ng/mL, cat.no. tlrl-peklps, Invivogen), or Pam 3 CSK 4 (1 ng/mL) in antibody-containing medium.

Techniques: Activation Assay, Control, Labeling, Incubation, Cell Culture, Fluorescence, Activity Assay, Comparison

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular vesicles derived from Enterococcus faecalis : inflammatory activation does not require internalization

doi: 10.1186/s12964-026-02926-9

Figure Lengend Snippet: Routes of Ef -EV uptake. A and B ) dTHP1-XBlue cells were pretreated with pharmacological endocytosis inhibitors for 30 min. Afterward, cells were incubated with DiI-labeled Ef -EVs (7000 EVs/cell) in the presence of inhibitors for 4 h. Mean fluorescence intensity (B, upper panel) and EV-positive cells (B, lower panel) were quantified by measuring fluorescence intensity associated with DiI-labeled Ef -EVs on the PE channel. Cells treated by EVs dispersed in cell culture medium or medium containing DMSO (1% v/v) were used as positive uptake controls. C ) dTHP1-XBlue cells were pretreated with dynasore at 100 µM for 30 min. Afterward, cells were incubated with Ef -EVs (7000 EVs/cell) in the presence of dynasore at 100 µM for 24 h. Cells treated with cell culture medium supplemented only with Ef -EVs (7000 EVs/cell) were used as positive activating controls. NF-κB/AP-1 activation was measured as the activity of secreted SEAP and expressed as a normalized value relative to the positive control. Quantitative results are presented as mean ± SD of three independent experiments ( N = 3, n ≥ 3) and were analyzed by Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test

Article Snippet: To elucidate whether Ef -EVs induce NF-κB/AP-1 activation by TLR2 engagement, dTHP1-XBlue were pre-incubated for 1 h with fresh medium containing 1 μg/mL of neutralizing monoclonal antibodies (anti-hTLR2-IgA mAb, cat. no. maba2-htlr2-2, Invivogen) or human IgA2 control (cat. no. maba2-ctrl, Invivogen) before adding either Ef -EVs (7000 EVs/cell), ultrapure LPS from E. coli K12 (LPS, 1 ng/mL, cat.no. tlrl-peklps, Invivogen), or Pam 3 CSK 4 (1 ng/mL) in antibody-containing medium.

Techniques: Incubation, Labeling, Fluorescence, Cell Culture, Activation Assay, Activity Assay, Positive Control, Comparison

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular vesicles derived from Enterococcus faecalis : inflammatory activation does not require internalization

doi: 10.1186/s12964-026-02926-9

Figure Lengend Snippet: Ef -EVs are internalized by primary human macrophages and modulate their morphology. A and B ) HMDMs were incubated with DiI-labeled Ef -EVs (30,000 EVs/cell) for 24 and 48 h. Mean fluorescence intensity (B, left panel) and EV-positive cells (B, right panel) were quantified by measuring fluorescence intensity associated with DiI-labeled Ef -EVs on the PE channel. Results are presented as mean ± SD of three individual donors ( N = 3, n = 1). C Representative images of macrophages treated with Ef -EVs (1000-10,000 EVs/cell) for 24 h (scale bar in full micrograph = 400 μm, scale bar in zoomed micrograph = 100 μm). D X-fold change of roundness compared to medium-treated cells (0 EVs/cell). Statistical comparisons of two groups in B were performed by Mann-Whitney test. Results in D were analyzed by Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test

Article Snippet: To elucidate whether Ef -EVs induce NF-κB/AP-1 activation by TLR2 engagement, dTHP1-XBlue were pre-incubated for 1 h with fresh medium containing 1 μg/mL of neutralizing monoclonal antibodies (anti-hTLR2-IgA mAb, cat. no. maba2-htlr2-2, Invivogen) or human IgA2 control (cat. no. maba2-ctrl, Invivogen) before adding either Ef -EVs (7000 EVs/cell), ultrapure LPS from E. coli K12 (LPS, 1 ng/mL, cat.no. tlrl-peklps, Invivogen), or Pam 3 CSK 4 (1 ng/mL) in antibody-containing medium.

Techniques: Incubation, Labeling, Fluorescence, MANN-WHITNEY, Comparison

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular vesicles derived from Enterococcus faecalis : inflammatory activation does not require internalization

doi: 10.1186/s12964-026-02926-9

Figure Lengend Snippet: Ef -EVs modulate gene expression and metabolic activity in primary human macrophages. A and B ) HMDMs were incubated with Ef -EVs at different concentrations (1000-10,000 EVs/cell) for 24 (A) and 48 h (B). Data are presented as frequency distributions, median, and quartiles of gene expression results from three individual donors ( N = 3, n = 3) and normalized to medium-treated cells (0 EVs/cell) as control. C and D) HMDMs were treated with Ef -EVs (10,000 and 50,000 EVs/cell) or Pam 3 CSK 4 (10 ng/mL) for 24 h. Cells incubated in cell culture medium alone served as untreated control. C Normalized ECAR values were monitored after injections of glucose, oligomycin, and 2-DG, according to the glycolysis stress test. D Glycolytic activity is shown as the x-fold change of untreated control regarding the normalized ECAR values in glycolysis. Results C and D are shown as mean ± SEM of three individual donors ( N = 3, n = 6). Results were analyzed by Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test

Article Snippet: To elucidate whether Ef -EVs induce NF-κB/AP-1 activation by TLR2 engagement, dTHP1-XBlue were pre-incubated for 1 h with fresh medium containing 1 μg/mL of neutralizing monoclonal antibodies (anti-hTLR2-IgA mAb, cat. no. maba2-htlr2-2, Invivogen) or human IgA2 control (cat. no. maba2-ctrl, Invivogen) before adding either Ef -EVs (7000 EVs/cell), ultrapure LPS from E. coli K12 (LPS, 1 ng/mL, cat.no. tlrl-peklps, Invivogen), or Pam 3 CSK 4 (1 ng/mL) in antibody-containing medium.

Techniques: Gene Expression, Activity Assay, Incubation, Control, Cell Culture, Comparison

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular vesicles derived from Enterococcus faecalis : inflammatory activation does not require internalization

doi: 10.1186/s12964-026-02926-9

Figure Lengend Snippet: Ef -EVs are internalized by primary human endothelial cells and modulate their gene expression. A and B ) HUVECs were incubated with DiI-labeled Ef -EVs (30,000 EVs/cell) for 24 and 48 h. Mean fluorescence intensity (B, left panel) and EV-positive cells (B, right panel) were quantified by measuring fluorescence intensity associated with DiI-labeled Ef -EVs on the PE channel. Results are presented as mean ± SD of three individual donors ( N = 3, n = 1). C and D ) HUVECs were incubated with Ef -EVs at different concentrations (1000-10,000 EVs/cell) for 24 (C) and 48 h (D). Data are presented as frequency distributions, median, and quartiles of gene expression results from three individual donors ( N = 3, n = 3) and normalized to medium-treated cells (0 EVs/cell) as control. Statistical comparisons of two groups in B were performed by Mann-Whitney test. Results in C and D were analyzed by Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test

Article Snippet: To elucidate whether Ef -EVs induce NF-κB/AP-1 activation by TLR2 engagement, dTHP1-XBlue were pre-incubated for 1 h with fresh medium containing 1 μg/mL of neutralizing monoclonal antibodies (anti-hTLR2-IgA mAb, cat. no. maba2-htlr2-2, Invivogen) or human IgA2 control (cat. no. maba2-ctrl, Invivogen) before adding either Ef -EVs (7000 EVs/cell), ultrapure LPS from E. coli K12 (LPS, 1 ng/mL, cat.no. tlrl-peklps, Invivogen), or Pam 3 CSK 4 (1 ng/mL) in antibody-containing medium.

Techniques: Gene Expression, Incubation, Labeling, Fluorescence, Control, MANN-WHITNEY, Comparison

Journal: Neoplasia (New York, N.Y.)

Article Title: Microbe-associated molecular patterns differentially mediate carcinogenic alterations of the breast tissue in the context of obesity

doi: 10.1016/j.neo.2026.101284

Figure Lengend Snippet: LPS and flagellin activate the NF-κB pathway and induce the transcription of inflammatory cytokines. A,G,M. Western blotting analysis of the nuclear (N) and cytosolic (C) fractions of NF-kB (p65) after treatment of HMT-3522 S1 cells with vehicle (control), the MAMPs LPS (A), LTA (G) or flagellin (M), in the absence or presence of inhibitor for their cognate TLR. Quantification of the nuclear to cytosolic ratios of NF-kB is shown in the graphs (B, H, N). Expression of TNF-α and IL-8 was quantified by qPCR in breast acini 1 h and 72 h after treatment with LPS (C-F), LTA (I-L), and flagellin (O-R). ns, not significant; *P<0.05; **P<0.01.

Article Snippet: Paraffin-embedded mammary gland sections were stained as previously detailed [ ] using antibodies against LPS (LSBio; LS- C56569 ), LTA (Invitrogen; MA1-40134), flagellin (Abcam; Ab93713), NF-κB p65 (Cell Signaling Technology; 8242), TLR2 (Abcam; ab209216), TLR4 (Abcam; ab22048), and TLR5 (Abcam; ab13876).

Techniques: Western Blot, Control, Expressing

Journal: Neoplasia (New York, N.Y.)

Article Title: Microbe-associated molecular patterns differentially mediate carcinogenic alterations of the breast tissue in the context of obesity

doi: 10.1016/j.neo.2026.101284

Figure Lengend Snippet: LPS and flagellin induce DNA double-strand breaks in breast acini in a TLR-dependent manner. A. Representative comet images from neutral comet assays performed on 3D cultures of breast acini treated with vehicle (control), 1 μg/ml LPS, 1 μg/ml LTA, and 10 ng/ml flagellin for 72 h. Bleomycin (BLM; 20 mU/ml; 2h) was used as positive control. B. Comet assay quantification. n=300 acini from 3 biological replicates. C. Representative images for immunofluorescence staining of 53BP1 (red) in acini treated with MAMPs ± TLR inhibitors. Nuclei are counterstained with DAPI (blue). D-F. Quantification of 53BP1 foci numbers in acini treated with LPS ± TLR4 inhibitor (D), with LTA ± TLR2 inhibitor (E) or with flagellin ± TLR5 inhibitor (F). ns is not significant. *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: Paraffin-embedded mammary gland sections were stained as previously detailed [ ] using antibodies against LPS (LSBio; LS- C56569 ), LTA (Invitrogen; MA1-40134), flagellin (Abcam; Ab93713), NF-κB p65 (Cell Signaling Technology; 8242), TLR2 (Abcam; ab209216), TLR4 (Abcam; ab22048), and TLR5 (Abcam; ab13876).

Techniques: Control, Positive Control, Single Cell Gel Electrophoresis, Immunofluorescence, Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: Microbe-associated molecular patterns differentially mediate carcinogenic alterations of the breast tissue in the context of obesity

doi: 10.1016/j.neo.2026.101284

Figure Lengend Snippet: LPS causes nuclear oxidative stress and the formation of single-strand breaks. A-C. Analysis of nuclear H 2 O 2 with the HyPer7 probe in MCF10A cells treated with MAMPs, in the presence or absence of glutathione (GSH). Cells were treated with LPS (A), flagellin (B), or LTA (C). n≈50 nuclei from 3 biological replicates. *P<0.05, **P<0.01 compared to control. # P<0.05, ## P<0.01 compared to the GSH condition. D. Representative images of 8-OHdG staining (green) in MCF10A cells treated with LPS, LPS+GSH, and culture medium (control). Nuclei are counterstained with DAPI (blue). E-G. Quantification of nuclear 8-OHdG signals in MFC10A cells treated with control medium or with LPS (±GSH) (E), LTA (±GSH) (F), or flagellin (±GSH) (G). H. Representative images showing comets from alkaline comet assays performed on acini cultures treated with vehicle (control), LPS, LTA, flagellin or bleomycin (BLM; positive control). I. Comet assay quantification in acini treated with MAMPs. n=300 acini from 3 biological replicates. ns is not significant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: Paraffin-embedded mammary gland sections were stained as previously detailed [ ] using antibodies against LPS (LSBio; LS- C56569 ), LTA (Invitrogen; MA1-40134), flagellin (Abcam; Ab93713), NF-κB p65 (Cell Signaling Technology; 8242), TLR2 (Abcam; ab209216), TLR4 (Abcam; ab22048), and TLR5 (Abcam; ab13876).

Techniques: Control, Staining, Positive Control, Single Cell Gel Electrophoresis

Journal: Neoplasia (New York, N.Y.)

Article Title: Microbe-associated molecular patterns differentially mediate carcinogenic alterations of the breast tissue in the context of obesity

doi: 10.1016/j.neo.2026.101284

Figure Lengend Snippet: LPS and flagellin cause DNA damage and inflammation in mice mammary glands. A. Schematic of the MAMPs injection experiment. B-D. Representative images for 3, 3′-diaminobenzidine (DAB) immunohistochemistry against LPS (B), LTA (C), and flagellin (D) in mice mammary glands. Hematoxylin was used as a counterstain. E. Representative comet images from the neutral comet assay performed on cells extracted from the mammary glands of mice treated with LPS, LTA, Flagellin, or control medium as indicated in the schematic. F. Quantification of comet assay results. G. Schematic of the obesogenic diet experiment. H. Plasma LPS concentrations (pg/ml) in the dietary groups. I. Comet assay analysis showing Spearman’s correlation between DNA damage levels (tail moments) and LPS concentrations (pg/ml) in the mice dietary groups. AU is Arbitrary units and ns is not significant. *P<0.05.

Article Snippet: Paraffin-embedded mammary gland sections were stained as previously detailed [ ] using antibodies against LPS (LSBio; LS- C56569 ), LTA (Invitrogen; MA1-40134), flagellin (Abcam; Ab93713), NF-κB p65 (Cell Signaling Technology; 8242), TLR2 (Abcam; ab209216), TLR4 (Abcam; ab22048), and TLR5 (Abcam; ab13876).

Techniques: Injection, Immunohistochemistry, Neutral Comet Assay, Control, Single Cell Gel Electrophoresis, Clinical Proteomics

Journal: Neoplasia (New York, N.Y.)

Article Title: Microbe-associated molecular patterns differentially mediate carcinogenic alterations of the breast tissue in the context of obesity

doi: 10.1016/j.neo.2026.101284

Figure Lengend Snippet: Hyper-acylated (immunogenic) forms of LPS cause higher levels of DNA damage in mammary glands than hypo-acylated forms of LPS. A. Schematic of the experiment. B. Representative comet images from the neutral comet assay performed on cells extracted from the mammary glands of control diet-fed mice treated with HF LPS, LF LPS or vehicle (saline) as indicated in the schematic. C. Quantification of comet assay results. D. Pearson’s correlation between anti-LPS IgG and DNA damage levels assessed by the comet assay. E. Representative comet images from the neutral comet assay performed on cells extracted from the mammary glands of lard diet-fed mice treated with HF LPS, LF LPS or vehicle (saline) as indicated in the schematic. F. Quantification of comet assay results. G. Pearson’s correlation between anti-LPS IgA and DNA damage levels assessed by the comet assay. AU is Arbitrary units and ns is not significant. *P<0.05, ***P<0.001.

Article Snippet: Paraffin-embedded mammary gland sections were stained as previously detailed [ ] using antibodies against LPS (LSBio; LS- C56569 ), LTA (Invitrogen; MA1-40134), flagellin (Abcam; Ab93713), NF-κB p65 (Cell Signaling Technology; 8242), TLR2 (Abcam; ab209216), TLR4 (Abcam; ab22048), and TLR5 (Abcam; ab13876).

Techniques: Neutral Comet Assay, Control, Saline, Single Cell Gel Electrophoresis

Journal: Neoplasia (New York, N.Y.)

Article Title: Microbe-associated molecular patterns differentially mediate carcinogenic alterations of the breast tissue in the context of obesity

doi: 10.1016/j.neo.2026.101284

Figure Lengend Snippet: Proteobacteria is associated with higher DNA damage levels in breast tissues. A. 16S rRNA sequencing results showing relative abundance of LPS-containing bacteria, LTA-containing bacteria, and bacteria with unknown status in each of the study subjects. B. Bar graph showing the relative abundance of bacterial phyla in breast tissue samples from each subject. C-D. Spearman’s correlation between BMI and the relative abundance of LPS-containing bacteria (C) or Proteobacteria (D). E. Spearman’s correlation between BMI and breast tissue levels of anti-MAMPs IgA, IgM, and IgG. F-G. Spearman’s correlation between DNA damage and the relative abundance of LPS-containing bacteria (F) or Proteobacteria (G). H. Representative neutral comet assay images from breast tissues samples. I-J. Spearman’s correlation between the relative abundance of LPS-containing bacteria (I) or Proteobacteria (J) and 8-OHdG concentrations in breast tissue.

Article Snippet: Paraffin-embedded mammary gland sections were stained as previously detailed [ ] using antibodies against LPS (LSBio; LS- C56569 ), LTA (Invitrogen; MA1-40134), flagellin (Abcam; Ab93713), NF-κB p65 (Cell Signaling Technology; 8242), TLR2 (Abcam; ab209216), TLR4 (Abcam; ab22048), and TLR5 (Abcam; ab13876).

Techniques: Sequencing, Bacteria, Neutral Comet Assay

Journal: Inflammation

Article Title: Leonurine Mitigates Experimental Autoimmune Prostatitis by Modulating Macrophage M1 Polarization Through the TLR4/NF-κB Signaling Pathway

doi: 10.1007/s10753-026-02468-9

Figure Lengend Snippet: Leonurine inhibits the TLR4/NF-κB axis. ( A - D ) Representative Western blots and quantitative analysis of TLR4, MYD88, p-p65, and p65 expression across the indicated groups. ( E , F ) Immunohistochemical analysis of TLR4 and MYD88 levels in the Control, EAP, EAP + Leo (Low), and EAP + Leo (High) groups. ( G ) Immunofluorescence analysis of p65 in the Control, EAP, EAP + Leo (Low), and EAP + Leo (High) groups. ( H - K ) Western blot analysis of TLR4, MYD88, p-p65, and p65 levels in the Control, LPS, LPS + Leo (10 µM), and LPS + Leo (20 µM) groups

Article Snippet: Additionally, LPS and the TLR4 agonist RS09 were procured from MedChemExpress (China).

Techniques: Western Blot, Expressing, Immunohistochemical staining, Control, Immunofluorescence

Journal: Inflammation

Article Title: Leonurine Mitigates Experimental Autoimmune Prostatitis by Modulating Macrophage M1 Polarization Through the TLR4/NF-κB Signaling Pathway

doi: 10.1007/s10753-026-02468-9

Figure Lengend Snippet: The TLR4 agonist RS09 reactivates the TLR4/NF-κB pathway and counteracts the anti-inflammatory effects of leonurine in vitro. ( A ) The levels of iNOS mRNA in the Control, LPS, LPS + Leo (20 µM), and LPS + Leo (20 µM) + RS09 groups were detected by Real-time PCR. ( B ) Western blot analysis and quantification of iNOS levels in the prostate tissues of mice from each group. ( C , D ) Flow cytometry analysis and quantitative assessment of M1 macrophage proportions in iBMDMs from the Control, LPS, LPS + Leo (20 µM), and LPS + Leo (20 µM) + RS09 groups. ( E , G ) Real-time PCR analysis of TNF-α, IL-1β, and IL-6 mRNA levels in the iBMDMs of mice from the Control, LPS, LPS + Leo (20 µM), and LPS + Leo (20 µM) + RS09 groups. ( H - K ) Western blot analysis of TLR4, MYD88, p-p65, and p65 levels in the Control, LPS, LPS + Leo (20 µM), and LPS + Leo (20 µM) + RS09 groups

Article Snippet: Additionally, LPS and the TLR4 agonist RS09 were procured from MedChemExpress (China).

Techniques: In Vitro, Control, Real-time Polymerase Chain Reaction, Western Blot, Flow Cytometry