Journal: BMC Oral Health

Article Title: Edaravone attenuates doxorubicin-induced oral mucosal injury via modulation of oxidative stress, inflammatory signaling, and the SIRT1/TLR4/NF-kB/ACE2 axis in rats

doi: 10.1186/s12903-025-07148-y

Figure Lengend Snippet: Tongue tissue TLR4 and proinflammatory cytokines. A TLR4 (ng/mg protein), ( B ) TNF-α (ng/L/g), ( C ) IL-6 (ng/L/g) (mean ± SEM). DOX increased TLR4/TNF-α/IL-6; EDO reduced these levels. Statistics: TLR4/IL-6—ANOVA ± Tukey; TNF-α—Kruskal–Wallis ± Mann–Whitney. Significance: * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant (adjusted p ≥ 0.05)

Article Snippet: TLR4 protein levels in tongue tissue were quantified using a rat TLR4 ELISA kit (Cusabio, Cat. No: CSB-E15822r).

Techniques: MANN-WHITNEY

Journal: Stem cells (Dayton, Ohio)

Article Title: Embryonic stem cells and mammary luminal progenitors directly sense and respond to microbial products.

doi: 10.1002/stem.75

Figure Lengend Snippet: Figure 1. TLR ligands stimulated mouse ES cell proliferation in vitro and in vivo. (A): TLR ligands stimulated the proliferation of the ES cells in vitro. ES proliferation rates were measured in the ES cell (ES-D3) culture after the treatment of LPS or Poly(I:C) for 72 hours by BrdU incor- poration. Data are presented as the mean SEM of three independent experiments (*, p < .05). (B): LPS stimulated the growth of teratoma in vivo. Teratoma growth in NOD/SCID mice 5 weeks after ES cells subcutaneously inoculation (5 105 cells/mouse) with or without intraperito- neal administration of LPS (5 lg/mouse, n ¼ 4 mice per group) once every 2 days was compared. Data are shown as mean SEM of two inde- pendent experiments (*, p < .05, two-tail p value). (C): Mouse ES cells (not treated with a TLR agonist) cultured in gelatin-coated plates with the medium supplemented with leukemia inhibitory factor (1,000 U/ml; ESGRO, Chemicon) without (a) or with (b) staining for the ES cell marker alkaline phosphatase using Chemicon’s detection kits. (D): Flow cytometric analysis of the expression of ES cell markers SSEA-1 and Oct-3/4 in the cultured ES cells. (E): The surface expression of TLR-2 and TLR-4 on SSEA-1-positive ES cells by flow cytometric analysis. (F): Expression of TLRs mRNA in ES cells by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) with the TLR1-TLR9-spe- cific primers. (G): The expression of ES cell marker genes, Oct-3/4 and Nanog, and the lack of the expression of differentiating markers GATA-1 and Brachyury transcription factors in the cultured ES cells by RT-PCR analysis. Marker: 100 bp DNA Ladder (New England BioLabs, Ipswich, MA, http://www.neb.com). Abbreviations: BrdU, bromodeoxyurdine; ES, embryonic stem; LPS, lipopolysaccharide; NS, no stimulation; RAW, RAW mouse monocyte macrophage cell line; SSEA, stage-specific embryonic antigen; TLR, Toll-like receptor.

Article Snippet: It was demonstrated that mouse ES cells were efficiently transfected with Cy3-labeled siRNA oligonucleotide duplexes by nucleofection (>80%) (supporting information Fig. 2A) and the TLR4 mRNA level was significantly downregulated by mouse TLR4-siRNA oligo (sc-40261; Santa Cruz Biotechnology) (Fig. 2A).

Techniques: In Vitro, In Vivo, Cell Culture, Staining, Marker, Expressing, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

Journal: Stem cells (Dayton, Ohio)

Article Title: Embryonic stem cells and mammary luminal progenitors directly sense and respond to microbial products.

doi: 10.1002/stem.75

Figure Lengend Snippet: Figure 2. Reduced responsiveness of TLR4-silenced ES cells to LPS stimulation. (A): Semi-quantitative reverse transcription-polymer- ase chain reaction (RT-PCR) analyses showing the selective downreg- ulation of TLR4 expression in ES cells 24 hours after nucleofection of 20 nM mouse TLR4-siRNA (sc-40261) and control siRNA oligo (Santa Cruz Biotechnology) by Nucleofector II (Lonza Amaxa, Wal- kersville, MD, http://www.amaxa.com). (B): BrdU incorporation assay of wild-type and siRNA-transfected ES cells 72 hours after LPS (20 lg/ml) stimulation or NS. LPS was added at 24 hours after siRNA transfection. Data are shown as mean SEM of three independent experiments. *, p < .05, versus NS, *, p < .05, siControl versus siTLR4 (two-tail p value). (C): MyD88 mRNA expression in ES cells by RT-PCR. RAW cells mRNA was used as positive control. Abbreviations: BrdU, bromodeoxyurdine; ES, embryonic stem; LPS, lipopolysaccharide; NS, no stimulation; RAW, RAW mouse monocyte macrophage cell line; TLR, Toll-like receptor.

Article Snippet: It was demonstrated that mouse ES cells were efficiently transfected with Cy3-labeled siRNA oligonucleotide duplexes by nucleofection (>80%) (supporting information Fig. 2A) and the TLR4 mRNA level was significantly downregulated by mouse TLR4-siRNA oligo (sc-40261; Santa Cruz Biotechnology) (Fig. 2A).

Techniques: Reverse Transcription, Polymer, Reverse Transcription Polymerase Chain Reaction, Expressing, Control, BrdU Incorporation Assay, Transfection, Positive Control

Journal: Stem cells (Dayton, Ohio)

Article Title: Embryonic stem cells and mammary luminal progenitors directly sense and respond to microbial products.

doi: 10.1002/stem.75

Figure Lengend Snippet: Figure 3. Toll-like receptor (TLR) ligands activated TLR-mediated signaling pathways in embryonic stem (ES) cells and promote hematopoi- etic differentiation. (A): Quantitative RT-PCR analysis of mRNA levels of representative genes in mouse ES cells at different time points after stimulation with LPS (10 lg/ml). (B): Proteasome-dependent transient IkBa degradation in ES cells induced by LPS. Total cell extracts of ES cells that were treated with MG132 (10 lM; Sigma-Aldrich), lactacystin (10 lM; Sigma-Aldrich), and Resveratrol (50 lM; Sigma-Aldrich) at dif- ferent time points after LPS stimulation (1 lg/ml) were subjected to Western blotting with an anti-IkBa antibody. The same blots were reprobed with an anti-b-actin antibody. (C, D): LPS stimulated the hematopoietic differentiation of EBs in vitro. In vitro hematopoietic differentiation assays of ES-D3 cells in the presence or absence of LPS (10 lg/ml) were performed using the two-step differentiation procedure. The percentages of each colony populations (C) in total colonies numbers of each group (n ¼ 5 plates/per group) and histograms of myeloid cell markers (F4/80 and Gr-1) from the pooled differentiated cells (D) after 14 days of in vitro culture are presented from one representative of three independent experiments. Abbreviations: BFU-E, burst forming unit-erythroid; CFU-GM, colony-forming unit-granulocyte-macrophage; CFU-M, colony-form- ing unit macrophage; LPS, lipopolysaccharide; NS, nonspecific protein band as an additional sample loading control.

Article Snippet: It was demonstrated that mouse ES cells were efficiently transfected with Cy3-labeled siRNA oligonucleotide duplexes by nucleofection (>80%) (supporting information Fig. 2A) and the TLR4 mRNA level was significantly downregulated by mouse TLR4-siRNA oligo (sc-40261; Santa Cruz Biotechnology) (Fig. 2A).

Techniques: Protein-Protein interactions, Quantitative RT-PCR, Western Blot, In Vitro, Control

Journal: Stem cells (Dayton, Ohio)

Article Title: Embryonic stem cells and mammary luminal progenitors directly sense and respond to microbial products.

doi: 10.1002/stem.75

Figure Lengend Snippet: Figure 5. TLR4-MD2 expression on mammary CD61þ progenitor cells. (A): (a) Gating strategy used to select Lin

Article Snippet: It was demonstrated that mouse ES cells were efficiently transfected with Cy3-labeled siRNA oligonucleotide duplexes by nucleofection (>80%) (supporting information Fig. 2A) and the TLR4 mRNA level was significantly downregulated by mouse TLR4-siRNA oligo (sc-40261; Santa Cruz Biotechnology) (Fig. 2A).

Techniques: Expressing

Journal: Scientific Reports

Article Title: CD36 regulates lipopolysaccharide-induced signaling pathways and mediates the internalization of Escherichia coli in cooperation with TLR4 in goat mammary gland epithelial cells

doi: 10.1038/srep23132

Figure Lengend Snippet: ( A ) Relative distribution of CD36 and TLR4 mRNA expression in different goat tissues. The relative expression values are shown on the top of each bar. ( B , C ) Histology of the dairy goat mammary gland. In healthy dairy goats, epithelial tight junctions, intact acinar structures, and no infiltrated inflammatory cells are observed in the mammary tissue of the uninfected udder ( B , left). Acinar epithelial cells and inflammatory cells infiltrated the acinar lumina, causing interstitial edema, and the acinar structures disintegrated in E. coli -induced mastitis ( C , right). ( D ) Relative to the healthy dairy goat, the mRNA expression of CD36, TLR4, and MyD88 increased significantly in E. coli -infected goats (** P < 0.01). ( E ) NF-κB, c-JUN, p38-MAPK, and TRAF6 protein levels were enhanced in mastitis tissues compared to health tissues. The values are the means ± SEM for three individuals. Quantitative PCR data were normalized to GAPDH, UXT, and MRPL39.

Article Snippet: Anti-Myc (Proteintech, China) and anti-Flag (Proteintech) antibodies were used to detect Myc-TLR4 and Flag-CD36 protein levels in the pGMECs (see ).

Techniques: Expressing, Infection, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: CD36 regulates lipopolysaccharide-induced signaling pathways and mediates the internalization of Escherichia coli in cooperation with TLR4 in goat mammary gland epithelial cells

doi: 10.1038/srep23132

Figure Lengend Snippet: ( A , B ) Changes in CD36 and TLR4 mRNA levels are shown. Cells were pretreated with NC or si-CD36 for 24 h and then treated with LPS for 12 h. CD36 and TLR4 mRNA levels increased following the addition of various concentrations of LPS. ( C ) Bright field and fluorescence images of pGMECs infected with Ad-GFP (left, upper) and Ad-CD36 (left, lower) adenovirus for 24 h (MOI = 100). The changes in CD36 mRNA levels compared with the control group, Ad-GFP group, and Ad-CD36 group are shown. ( D , E ) CD36 and TLR4 mRNA changes in pGMECs pretreated with siRNA or adenovirus and then exposed to 10 μg/ml LPS for 12 h. ( F ) The variation in NF-κB mRNA levels following the manipulation of CD36 in pGMECs. NF-κB mRNA levels were influenced by knockdown (left) or overexpression (right) of CD36 in pGMECs, which were then stimulated with 10 μg/ml LPS. ( G ) MyD88 mRNA levels and TRAF6 protein levels were influenced by CD36 expression in LPS-stimulated pGMECs. The values are the mean ± SEM for three individuals. Quantitative PCR data were normalized to GAPDH, UXT, and MRPL39. All data are presented as the mean ± SEM from three experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, and not significant (NS).

Article Snippet: Anti-Myc (Proteintech, China) and anti-Flag (Proteintech) antibodies were used to detect Myc-TLR4 and Flag-CD36 protein levels in the pGMECs (see ).

Techniques: Fluorescence, Infection, Over Expression, Expressing, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: CD36 regulates lipopolysaccharide-induced signaling pathways and mediates the internalization of Escherichia coli in cooperation with TLR4 in goat mammary gland epithelial cells

doi: 10.1038/srep23132

Figure Lengend Snippet: ( A , B ) Cells were transfected with pBiFC-VC155-TLR4 ( A , left upper) or pBiFC-VN155-CD36 ( B , right upper) alone, and then Hoechst 33342-labeled E. coli was added for 2 h at 37 °C. ( C ) Hoechst 33342-stained E. coli bacteria were used to stimulate cotransfection of VC-155-TLR4 and VN-155-CD36 cells for 2 h. ( D ) pGMECs were cotransfected with pBiFC-VC155-TLR4 and pBiFC-VN155-CD36 plasmids, and the cells were stained with Hoechst 33342 to visualize the nuclei. ( E ) pGMECs were cotransfected with pBiFC-VC155-TLR4 and pBiFC-VN155-CD36 plasmids, and then the cells were stimulated with LPS (10 μg/ml) and Hoechst 33342 for 2 h. The images are representative of multiple fields from three experiments. Scale bar: 10 μm. ( F ) Cells were transiently cotransfected with pef-NEO-Flag-CD36 and pef-NEO-Myc-TLR4 and then incubated with LPS for 12 h or with E. coli for 2 h. The cell lysates (input) were probed for Flag-CD36, Myc-TLR4, and β-actin. ( G ) Flag-CD36 was immunoprecipitated from the cell lysates using mouse anti-Flag antibody. ( H ) The cells were lysed and plated on agar after incubation with E. coli (10 7 cfu/ml) in pGMEC manipulated with mock, Ad-CD36, or si-CD36 for 2 h. Equal protein amounts were immunoprecipitated (IP: Flag-CD36) using anti-Flag antibody. All data are presented as the mean ± SEM from three experiments. * P < 0.05, ** P < 0.01, and not significant (NS).

Article Snippet: Anti-Myc (Proteintech, China) and anti-Flag (Proteintech) antibodies were used to detect Myc-TLR4 and Flag-CD36 protein levels in the pGMECs (see ).

Techniques: Transfection, Labeling, Staining, Cotransfection, Incubation, Immunoprecipitation