Journal: Ecology and evolution

Article Title: Expression of TLR2/4 in the sperm-storing oviduct of the Chinese soft-shelled turtle Pelodiscus sinensis during hibernation season.

doi: 10.1002/ece3.1726

Figure Lengend Snippet: Figure 6. The Expression of TLR4 protein in November was Similar in the Magnum, Isthmus, Uterus, and Vagina. Cross-sections of the magnum, isthmus, uterus, and vagina are shown at low magnification in A, E, I, and M, respectively; B, F, J, and n refer to the amplification of the epithelium in the magnum, isthmus, uterus, and vagina, respectively; the secretory glands in magnum, isthmus, uterus, and vagina are described in C, G, K, and o; respectively; and D, H, I, and P represent the negative controls for the magnum, isthmus, uterus, and vagina, respectively. No immunoreaction products were observed. All sections were counterstained with hematoxylin. Positive staining was observed on the ciliated cell superior surface and cilium surface (white thin arrow head), secretory cell superior surface (white thin arrow), secretory cell lateral membrane (white fat arrow head), secretory cell basal membrane (white fat arrow), secretory gland vesicles membrane (black thin arrow), longitudinal muscle (black fat arrow), circular muscle (black fat arrow head), blood vessel endothelium (black thin arrow head). Scale bars: 100 lm (E and M), 50 lm (A, D, H, I, L, and P), and 10 lm (B, C, F, G, J, K, N, and O).

Article Snippet: The sections were incubated at 4°C for 15 h with 200 lg/mL of a rabbit polyclonal antibody to human, mouse, and rat TLR4 (Boster, China).

Techniques: Expressing, Staining, Membrane

Journal: Ecology and evolution

Article Title: Expression of TLR2/4 in the sperm-storing oviduct of the Chinese soft-shelled turtle Pelodiscus sinensis during hibernation season.

doi: 10.1002/ece3.1726

Figure Lengend Snippet: Figure 7. The Location of TLR4 protein was Similar in the Magnum, Isthmus, Uterus, and Vagina. Cross-sections of the magnum, isthmus, uterus, and vagina are depicted in the pictures (A, E, I, and M) at low magnification: B, F, J, and n refer to the amplification for the epithelium in the magnum, isthmus, uterus, and vagina, respectively; the secretory glands in the magnum, isthmus, uterus, and vagina are described C, G, K, and O, respectively; and D, H, I, and p show the negative controls for the magnum, isthmus, uterus, and vagina, respectively. All sections were counterstained with hematoxylin. No immunoreaction products were observed. Positive staining was observed on the ciliated cell superior surface and cilium surface (white thin arrow head), secretory cell superior surface (white thin arrow), secretory cell lateral membrane (white fat arrow head), secretory cell basal membrane (white fat arrow), secretory gland vesicles membrane (black thin arrow), longitudinal muscle (black fat arrow), circular muscle (black fat arrow head), and blood vessel endothelium (black thin arrow head). Scale bars: 50 lm (A, D, E, H, I, L, M, and P), 20 lm (B, F, J, and N), and 10 lm (C, G, K, and O).

Article Snippet: The sections were incubated at 4°C for 15 h with 200 lg/mL of a rabbit polyclonal antibody to human, mouse, and rat TLR4 (Boster, China).

Techniques: Staining, Membrane

Journal: Ecology and evolution

Article Title: Expression of TLR2/4 in the sperm-storing oviduct of the Chinese soft-shelled turtle Pelodiscus sinensis during hibernation season.

doi: 10.1002/ece3.1726

Figure Lengend Snippet: Figure 8. The IOD of TLR2/4 Positive Reaction Parts in Oviduct during Hibernation. (A) Image shows changes in numbers of TLR2-positive reaction parts in four different parts of oviduct in two different months. (B) The IOD of TLR4- positive reaction parts in oviduct during hibernation. Data are presented as mean SE of five turtles per group. Significant differences are identified as *, P < 0.05; **, P < 0.01.

Article Snippet: The sections were incubated at 4°C for 15 h with 200 lg/mL of a rabbit polyclonal antibody to human, mouse, and rat TLR4 (Boster, China).

Techniques:

Journal: Pharmaceutical Biology

Article Title: Berberine attenuates septic cardiomyopathy by inhibiting TLR4/NF-κB signalling in rats

doi: 10.1080/13880209.2021.1877736

Figure Lengend Snippet: Effects of berberine on key effectors of the TLR4/NF-κB signalling pathway. (A) TLR4 protein expression in the myocardial tissue and quantitation. (B) Nuclear p65 protein expression in the rat sepsis cardiomyopathy model measured by ELISA. Effects of berberine on TNFα (C) and IL 1β (D) levels in the myocardial tissue. * p < 0.001 indicates significance in comparison with the Con group. △ p < 0.001 indicates significance in comparison with the LPS group.

Article Snippet: The membranes were blocked with 5% skim milk, followed by successive incubations with rabbit anti-mouse TLR4 polyclonal antibody (1:2000, Santa Cruz, USA) at 4 °C overnight and mouse anti-rabbit IgG monoclonal secondary antibody (1:10000, Bioss, China) for 2 h. The membranes were next incubated with ECL ultra western HRP substrate (Millipore, Billerica, MA, USA) and detected on a gel imaging system (ImageQuant LAS 4000, GE Healthcare, USA).

Techniques: Expressing, Quantitation Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Parasites & Vectors

Article Title: Toxoplasma gondii -induced adverse pregnancy outcomes: insight into the inhibitory role of Trem2 on TLR4/TRAF6/JNK signaling pathway

doi: 10.1186/s13071-025-07000-w

Figure Lengend Snippet: The absence of Trem2 further promoted the T. gondii -triggered upregulation of TLR4, TRAF6, and P-JNK in mouse placentas. A The docking models of TLR4 and Trem2 were predicted using the GRAMM docking software and visualized with PyMol. The calculated binding energy between TLR4 (green) and Trem2 (pink) was −15 kcal/mol. B Endogenous immunoprecipitation was conducted to assess the interaction between Trem2 and TLR4. RAW 264.7 cell lysate was immunoprecipitated with Trem2 antibody. Expression of Trem2 and TLR4 was assayed by western blotting. C Multiplex immunofluorescence analysis was conducted to examine the colocalization of TLR4, Trem2, and TRAF6. RAW 264.7 cells were incubated with Trem2 antibody (green), TLR4 antibody (red), TRAF6 antibody (magenta), and DAPI dye (blue). Fluorescence intensity and co-localization of the three proteins were analyzed using ImageJ software. Scale bar: 10 μm. D Expression of Trem2 in the placentas from WT pregnant mice challenged with/without T. gondii , along with corresponding quantitative analysis. Placental tissue was extracted from WT pregnant mice on G17.5, and western blotting was employed to assay Trem2 expression ( n = 6). E Expression of TLR4, JNK, P-JNK, and TRAF6 in placental tissues from Trem2 −/− and WT pregnant mice challenged with/without T. gondii , along with corresponding quantitative analysis. Placental tissue was extracted from WT and Trem2 −/− pregnant mice on G17.5. Western blotting was employed to assay the level of TLR4, JNK, P-JNK, and TRAF6 ( n = 6). WT: Wild type, TI: T. gondii infection. Significant changes were determined with a two-tailed unpaired Student t -test ( D ) and two-way ANOVA with Sidak’s multiple comparisons test ( E ) at P < 0.05. * P < 0.05

Article Snippet: The following antibodies were utilized in the experiments: anti-TRAF6 antibody (1:2000, rabbit monoclonal, ab40675, Abcam), anti-TLR4 antibody (1:1000, mouse monoclonal, 66350-1-Ig, Proteintech), anti-Phospho-SAPK/JNK antibody (Thr183/Tyr185; 1:1000, rabbit monoclonal, 4668, Cell Signaling Technology), anti-GAPDH (1:50,000, mouse monoclonal, 60004-1-Ig, Proteintech), anti-mouse HRP-IgG (1:5000, SA00001-1, Proteintech), anti-rabbit HRP-IgG (1:5000, SA00001-2, Proteintech), anti-Trem2 antibody (1:3000, sheep monoclonal, AF1729, R&D Systems), and anti-sheep HRP-IgG (1:5000; sc2473, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Software, Binding Assay, Immunoprecipitation, Expressing, Western Blot, Multiplex Assay, Immunofluorescence, Incubation, Fluorescence, Infection, Two Tailed Test

Journal: Parasites & Vectors

Article Title: Toxoplasma gondii -induced adverse pregnancy outcomes: insight into the inhibitory role of Trem2 on TLR4/TRAF6/JNK signaling pathway

doi: 10.1186/s13071-025-07000-w

Figure Lengend Snippet: Toxoplasma gondii antigens suppressed Trem2 expression but activated the downstream TLR4 signaling pathway in RAW 264.7 cells. A Immunoblot analysis of RAW 264.7 cells stimulated with/without Tg Ag. Western blotting was conducted to measure the expression of TLR4, P-JNK, JNK, TRAF6, and Trem2 in RAW 264.7 cells that were treated with/without Tg Ag (5 µg/ml) for 24 h ( n = 3). B – E Representative immunofluorescence photograph and quantitative analysis of the expression of TLR4, TRAF6, and P-JNK in RAW 264.7 cells stimulated with/without Tg Ag. Immunofluorescence was used to assess the fluorescence intensity of TLR4 (green), TRAF6 (magenta), P-JNK (red), and Trem2 (green) in RAW 264.7 cells stimulated with/without Tg Ag (5 μg/ml) for 24 h. Scale bar: 20 μm. Fluorescence intensity was quantified by using ImageJ software ( n = 3). F Endogenous immunoprecipitation was employed to measure the interaction between TLR4 and Trem2. RAW 264.7 cells were stimulated with Tg Ag (5 μg/ml) for 24 h, which was followed by immunoprecipitation with Trem2 antibody. TLR4 expression was measured by western blotting ( n = 3). Tg Ag: T. gondii antigens, Con: untreated group. Significant changes were determined with a two-tailed unpaired Student t -test ( A – F ) at P < 0.05. * P < 0.05

Article Snippet: The following antibodies were utilized in the experiments: anti-TRAF6 antibody (1:2000, rabbit monoclonal, ab40675, Abcam), anti-TLR4 antibody (1:1000, mouse monoclonal, 66350-1-Ig, Proteintech), anti-Phospho-SAPK/JNK antibody (Thr183/Tyr185; 1:1000, rabbit monoclonal, 4668, Cell Signaling Technology), anti-GAPDH (1:50,000, mouse monoclonal, 60004-1-Ig, Proteintech), anti-mouse HRP-IgG (1:5000, SA00001-1, Proteintech), anti-rabbit HRP-IgG (1:5000, SA00001-2, Proteintech), anti-Trem2 antibody (1:3000, sheep monoclonal, AF1729, R&D Systems), and anti-sheep HRP-IgG (1:5000; sc2473, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Immunofluorescence, Fluorescence, Software, Immunoprecipitation, Two Tailed Test

Journal: Parasites & Vectors

Article Title: Toxoplasma gondii -induced adverse pregnancy outcomes: insight into the inhibitory role of Trem2 on TLR4/TRAF6/JNK signaling pathway

doi: 10.1186/s13071-025-07000-w

Figure Lengend Snippet: Toxoplasma gondii antigens inhibited Trem2 to activate the TLR4/TRAF6/JNK axis. A Schematic representation of the treatment of TLR4 blocking antibody on RAW 264.7 cells. The cells, primed with TLR4 blocking antibody (20 μg/ml) or isotype control for 30 min, were stimulated with/without Tg Ag (5 μg/ml) for 24 h. Expression of P-JNK, JNK, Trem2, and TRAF6 was assayed by western blotting ( n = 3). B Schematic diagram of Trem2 knockdown in RAW 264.7 cells. Cells that were transfected with si- Trem2 or negative control (si-NC) for 6 h were stimulated with/without Tg Ag (5 μg/ml) for 24 h. Expression of P-JNK, JNK, TLR4, and TRAF6 was measured by western blotting ( n = 3). C Schematic diagram of Trem2 overexpression in RAW 264.7 cells. Cells were stimulated with or without Tg Ag for an additional 24 h. Expression of P-JNK, JNK, TLR4, Trem2, and TRAF6 was assayed by western blotting ( n = 3). D Schematic illustration of the treatment with the TRAF6 inhibitor on RAW 264.7 cells. Cells, primed with 20 µM C25-140 (TRAF6 inhibitor) for 2 h, were exposed to Tg Ag (5 μg/ml) for 24 h. Expression of P-JNK, JNK, TLR4, Trem2, and TRAF6 was assayed by western blotting ( n = 3). Created in BioRender. Cao, Y. (2025) https://BioRender.com/mwqhkc7 . Tg Ag: T. gondii antigens, Con: untreated group. Significant changes were determined with two-way ANOVA with Sidak’s multiple comparisons test ( A – D ) at P < 0.05. * P < 0.05

Article Snippet: The following antibodies were utilized in the experiments: anti-TRAF6 antibody (1:2000, rabbit monoclonal, ab40675, Abcam), anti-TLR4 antibody (1:1000, mouse monoclonal, 66350-1-Ig, Proteintech), anti-Phospho-SAPK/JNK antibody (Thr183/Tyr185; 1:1000, rabbit monoclonal, 4668, Cell Signaling Technology), anti-GAPDH (1:50,000, mouse monoclonal, 60004-1-Ig, Proteintech), anti-mouse HRP-IgG (1:5000, SA00001-1, Proteintech), anti-rabbit HRP-IgG (1:5000, SA00001-2, Proteintech), anti-Trem2 antibody (1:3000, sheep monoclonal, AF1729, R&D Systems), and anti-sheep HRP-IgG (1:5000; sc2473, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Blocking Assay, Control, Expressing, Western Blot, Knockdown, Transfection, Negative Control, Over Expression

Journal: Parasites & Vectors

Article Title: Toxoplasma gondii -induced adverse pregnancy outcomes: insight into the inhibitory role of Trem2 on TLR4/TRAF6/JNK signaling pathway

doi: 10.1186/s13071-025-07000-w

Figure Lengend Snippet: Trem2 deficiency in macrophages further activated the TLR4/TRAF6/JNK signaling cascade. A Flowchart depicting the extraction process of BMDMs derived from WT and Trem2 −/− mice. Created in BioRender. Cao, Y. (2025) https://BioRender.com/mwqhkc7 . B Expression of P-JNK, JNK, TLR4, and TRAF6 in BMDMs was assayed by western blotting. BMDMs derived from either WT or Trem2 −/− mice were stimulated with/without Tg Ag (5 μg/ml) for 24 h ( n = 3). C Expression of P-JNK, JNK, Trem2, and TRAF6 in BMDMs was assayed by western blotting. BMDMs isolated from WT mice, primed with TLR4 blocking antibody (20 μg/ml) or isotype control for 30 min, were stimulated with/without Tg Ag (5 μg/ml) for 24 h ( n = 3). D The messenger RNA (mRNA) levels of inflammatory factors in WT or Trem2 −/− mouse BMDMs. BMDMs isolated from either WT or Trem2 −/− mice were exposed to Tg Ag (5 μg/ml) for 24 h. Then, the mRNA levels of TNF-α , IL-10 , IFN-γ , and TGF-β were assayed by real-time PCR ( n = 5). Tg Ag: T. gondii antigens, Con: untreated group; anti-TLR4: TLR4/MD-2 Complex Antibody. Significant changes were determined with two-way ANOVA with Sidak’s multiple comparisons test ( B – D ) at P < 0.05. * P < 0.05

Article Snippet: The following antibodies were utilized in the experiments: anti-TRAF6 antibody (1:2000, rabbit monoclonal, ab40675, Abcam), anti-TLR4 antibody (1:1000, mouse monoclonal, 66350-1-Ig, Proteintech), anti-Phospho-SAPK/JNK antibody (Thr183/Tyr185; 1:1000, rabbit monoclonal, 4668, Cell Signaling Technology), anti-GAPDH (1:50,000, mouse monoclonal, 60004-1-Ig, Proteintech), anti-mouse HRP-IgG (1:5000, SA00001-1, Proteintech), anti-rabbit HRP-IgG (1:5000, SA00001-2, Proteintech), anti-Trem2 antibody (1:3000, sheep monoclonal, AF1729, R&D Systems), and anti-sheep HRP-IgG (1:5000; sc2473, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Extraction, Derivative Assay, Expressing, Western Blot, Isolation, Blocking Assay, Control, Real-time Polymerase Chain Reaction

Journal: mBio

Article Title: Candida albicans Induces Cross-Kingdom miRNA Trafficking in Human Monocytes To Promote Fungal Growth

doi: 10.1128/mbio.03563-21

Figure Lengend Snippet: Generation of hsa-miR-24-3p is dependent on CR3 and TLR4. (a) The level of hsa-miR-24-3p 1 h after infection was higher in MEV Ca than in MEV. Blocking of CR3 or TLR4 on blood monocytes had a minor effect on hsa-miR-24-3p content in vesicles. However, when both CR3 and TLR4 were blocked, hsa-miR-24-3p was absent from vesicles. Data represent mean values ± SD, P = 0.0025, unpaired two-tailed t test, n = 3 different donors. EVs were isolated from 10 6 human monocytes, and hsa-miR-24-3p levels were determined using qPCR. (b) Mmu-miR-24-3p content was significantly higher in REV Ca (vesicles from opsonized C. albicans -induced RAW 264.7 cells) than in REV (vesicles from untreated RAW 264.7 cells). The level of mmu-miR-24-3p was not elevated in CR3- or TLR4 -silenced RAW 264.7 cells. Data represent mean values ± SD, P = 0.0245 and P = 0.0166, unpaired two-tailed t test, n = 3 or 4 different experiments. (c) Candida -treated CD11b- and TLR4 -silenced RAW 264.7 cells generated the same amount of REV as nonsilenced cells. EVs isolated from the same numbers of cells were counted by DLSM. (d) CD11b and TLR4 colocalized on blood monocytes upon incubation with soluble β-glucan (sβG) and mannan but not on untreated cells, as observed by CLSM. Green, CD11b; red, TLR4. Bars, 10 μm. Data are representative of n = 3 independent experiments. (e) Colocalization of CD11b and TLR4 in the presence of soluble β-glucan (sβG) and mannan on monocytes, as confirmed by PLA. Red, CD11b–TLR4 complexes; blue, DNA. Bars, 10 μm. Data are representative of n = 3 experiments. (f) EVs isolated from sβG and mannan-treated human monocytes for 1 h (MEV sβG+mannan ) contained significantly more hsa-miR-24-3p than MEV. Data represent mean values ± SD, P = 0.0025, unpaired two-tailed t test, n = 3 different donors. REV Ca but not REV Ca(TLR4 and CD11b silenced) induced significant growth (g) and hyphal filamentation (h) in C. albicans . EVs were isolated from opsonized C. albicans -infected or control RAW 264.7 cells (REV and REV Ca , respectively). EVs were also isolated from opsonized C. albicans - infected or control TLR4- and CD11b-silenced RAW 264.7 cells [REV (TLR4 and CD11b silenced) and REV Ca(TLR4 and CD11b silenced) , respectively]. Isolated EVs were counted by DLSM, and C. albicans was incubated with identical amounts of vesicles obtained from the indicated treatments.

Article Snippet: CD14 was stained with Alexa Fluor 488 anti-human CD14 antibody (no. 367130; BioLegend) (1:100), CD9 was stained with Alexa Fluor 647 anti-human CD9 antibody (no. MCA469A647T; Bio-Rad) (1:1,000), TLR4 was stained with mouse anti-TLR4 antibody (no. NBP1-51697; R&D Systems) (3 μg/ml) and Alexa Fluor 647 goat anti-mouse IgG (H+L) secondary antibody (no. A-21235; Thermo Fisher) (1:500), and CD11b was stained with rabbit anti-CD11b antibody (no. 133357; Abcam) and Alexa Fluor 488 goat anti-rabbit IgG (H+L) secondary antibody.

Techniques: Infection, Blocking Assay, Two Tailed Test, Isolation, Generated, Incubation

Journal: mBio

Article Title: Candida albicans Induces Cross-Kingdom miRNA Trafficking in Human Monocytes To Promote Fungal Growth

doi: 10.1128/mbio.03563-21

Figure Lengend Snippet: hsa-miRNA-24-3p acts across species boundaries (cross-kingdom) as it regulates C. albicans gene expression. C. albicans induces the release of miRNA-containing vesicles in human monocytes upon binding of mannan and soluble β glucan to TLR4 and CR3, respectively, followed by receptor colocalization. Subsequently, EVs are released from multivesicular bodies transporting miRNAs. EVs attach via CR1 on the vesicle to opsonized C. albicans . hsa-miRNA-24-3p but not hsa-miRNA-21-5p inhibits sol1 translation in C. albicans , leading to enhanced growth and filamentation of the fungus. Graphic created with BioRender.com.

Article Snippet: CD14 was stained with Alexa Fluor 488 anti-human CD14 antibody (no. 367130; BioLegend) (1:100), CD9 was stained with Alexa Fluor 647 anti-human CD9 antibody (no. MCA469A647T; Bio-Rad) (1:1,000), TLR4 was stained with mouse anti-TLR4 antibody (no. NBP1-51697; R&D Systems) (3 μg/ml) and Alexa Fluor 647 goat anti-mouse IgG (H+L) secondary antibody (no. A-21235; Thermo Fisher) (1:500), and CD11b was stained with rabbit anti-CD11b antibody (no. 133357; Abcam) and Alexa Fluor 488 goat anti-rabbit IgG (H+L) secondary antibody.

Techniques: Expressing, Binding Assay

Journal: Biochemical and biophysical research communications

Article Title: Synergistic anti-tumorigenic effect of diosmetin in combination with 5-fluorouracil on human colon cancer xenografts in nude mice.

doi: 10.1016/j.bbrc.2024.150677

Figure Lengend Snippet: Fig. 7. IHC assessment of Ki67 and TLR4 expression level in HCT-116 xenograft tumor in different groups. Control (untreated), diosmetin (100 mg/kg), 5-FU (30 mg/kg), combination high dose (5-FU + diosmetin: 30 + 100 mg/kg), and combination low dose (5-FU + diosmetin: 15 + 50 mg/kg). Magnification: 10×; scale bar: 200 μm. A & C: microscopic images of Ki67 & TLR4, respectively, brown color indicates Ki67 & TLR4 protein expression. B & D: quantification of the brown color of Ki67 and TLR4 expression, respectively. **P < 0.01 signifies statistically significant variances compared to the control group. #P < 0.05 indicates a statistically significant difference in comparison to 5-FU. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Briefly, antigen retrieval was carried out by incubating the slides in a chamber containing antigen unmasking solution and citric acid-based buffer (Vector Laboratories, Inc. CA, 94010, US) at 120 ◦C for 30 min. Primary antibodies (Ki67 from Abcam, ab16667, US (RRIC: AB_302459); and TLR4 from Novus Biological, NB-10056566SS, US) were diluted in blocking serum (5 μL: 1000 μL) and applied to the tissue sections and incubated overnight at 4 ◦C.

Techniques: Expressing, Control, Comparison