Journal: Neural Plasticity

Article Title: Toll-Like Receptor 2 Attenuates Traumatic Brain Injury-Induced Neural Stem Cell Proliferation in Dentate Gyrus of Rats

doi: 10.1155/2020/9814978

Figure Lengend Snippet: Immunofluorescence (IF) microphotographs in the dentate gyrus (DG) of the DG in sham and TBI groups at different time points. (a) BrdU-labelled NSCs (red fluor), expression of TLR2 (green fluor), cell nuclei (blue fluor), and BrdU + /TLR2 + /DAPI + cells indicated that TLR2 expression in labelled proliferating cells was possible NSCs; (b) BrdU + cells showed the proliferation of NSCs in the DG during different time points posttrauma. BrdU + cells were more in the TBI group than that in the sham group ( ∗ p < 0.05), and numbers of these cells were significantly different among different time points posttrauma ( # p < 0.05); (c) numbers of BrdU + /TLR2 + cells indicated that the expression of TLR2 was quite different in proliferating cells of the DG among different time points posttrauma. There were more BrdU + cells in the TBI group than that in the sham group ( ∗ p < 0.05), and the numbers of these cells are obviously different among different time points posttrauma ( # p < 0.05). Scale bar: 50 μ m; data is shown as mean ± SEM.

Article Snippet: Primary antibodies were used as follows: mouse anti-TLR2 antibody (1 : 500, Biorbyt, orb191498, San Francisco, CA, USA) and mouse anti- β -actin antibody (1 : 3000, TDY BIOTECH, TDY041, Beijing, China) overnight at 4°C.

Techniques: Immunofluorescence, Expressing

Journal: Neural Plasticity

Article Title: Toll-Like Receptor 2 Attenuates Traumatic Brain Injury-Induced Neural Stem Cell Proliferation in Dentate Gyrus of Rats

doi: 10.1155/2020/9814978

Figure Lengend Snippet: IF in the DG of the TBI group (mouse brain got from 3 days posttrauma). (a) BrdU (red), nestin (green), and DAPI (blue), respectively, exhibited proliferating cells, NSCs, and cell nuclei in the DG. Merged pictures of BrdU + /nestin + /DAPI + showed NSCs (the percentage of NSCs in proliferating cells was 84.30% ± 6.54%); scale bar: 50 μ m; data were expressed as mean ± SEM. (b) Nestin (green), TLR2 (red), and DAPI (blue), respectively, exhibited NSCs, TLR2 expression, and cell nuclei in the DG. Merged pictures of nestin + /TLR2 + /DAPI + showed the expression of TLR2 on NSCs. Scale bar: 50 μ m; data were expressed as mean ± SEM.

Article Snippet: Primary antibodies were used as follows: mouse anti-TLR2 antibody (1 : 500, Biorbyt, orb191498, San Francisco, CA, USA) and mouse anti- β -actin antibody (1 : 3000, TDY BIOTECH, TDY041, Beijing, China) overnight at 4°C.

Techniques: Expressing

Journal: Neural Plasticity

Article Title: Toll-Like Receptor 2 Attenuates Traumatic Brain Injury-Induced Neural Stem Cell Proliferation in Dentate Gyrus of Rats

doi: 10.1155/2020/9814978

Figure Lengend Snippet: Expression of TLR2 protein and mRNA in the DG (western blotting and PCR). (a) Western blotting: electrophoresis bands of TLR2 protein controlled with β -actin; (b) western blotting: the optical density of TLR2 electrophoresis; (c) real-time PCR: the expression of TLR2 mRNA and GAPDH was used as the endogenous reference gene. The TLR2 expression in the protein and mRNA level was significantly higher in the TBI group than that in the sham group ( ∗ p < 0.05), and the TLR2 expression was significantly different among various time points ( # p < 0.05). Data were expressed as mean ± SEM.

Article Snippet: Primary antibodies were used as follows: mouse anti-TLR2 antibody (1 : 500, Biorbyt, orb191498, San Francisco, CA, USA) and mouse anti- β -actin antibody (1 : 3000, TDY BIOTECH, TDY041, Beijing, China) overnight at 4°C.

Techniques: Expressing, Western Blot, Electrophoresis, Real-time Polymerase Chain Reaction

Journal: Neural Plasticity

Article Title: Toll-Like Receptor 2 Attenuates Traumatic Brain Injury-Induced Neural Stem Cell Proliferation in Dentate Gyrus of Rats

doi: 10.1155/2020/9814978

Figure Lengend Snippet: Gene sequences for primer synthesis.

Article Snippet: Primary antibodies were used as follows: mouse anti-TLR2 antibody (1 : 500, Biorbyt, orb191498, San Francisco, CA, USA) and mouse anti- β -actin antibody (1 : 3000, TDY BIOTECH, TDY041, Beijing, China) overnight at 4°C.

Techniques: Sequencing

Journal: Nature Microbiology

Article Title: Gut–liver translocation of pathogen Klebsiella pneumoniae promotes hepatocellular carcinoma in mice

doi: 10.1038/s41564-024-01890-9

Figure Lengend Snippet: a , Representative SEM (top) and TEM (bottom) images of Hep3B and Huh7 HCC cell lines after co-culture with K. pneumoniae (MOI = 10). After co-culture of HCC cells with K. pneumoniae , cell lysate was spread on BHI agar for bacterial culture. n = 3 independent experiments with similar results. b , Effect of pasteurized K. pneumoniae (MOI = 10) on HCC cell proliferation (top) ( n = 3 biologically independent samples), colony formation (middle) ( n = 3 biologically independent samples) and growth of HCC patient-derived organoid (bottom) ( n = 20 biologically independent samples). c , Effect of pasteurized K. pneumoniae (MOI = 10) on PCNA expression in HCC cells. n = 3 (Ctrl), 6 (pasteurized KP). d , Screening of K. pneumoniae adhesins by biotin pull-down assay. e , Hep3B membrane protein was incubated with GST or GST–PBP1B together with GST magnetic beads for GST pull-down assay. Corresponding bands in GST–PBP1B and Hep3B cell membrane protein groups were subjected to mass spectrometry analysis. In d and e , n = 3 independent experiments with similar results. f , Binding affinity between PBP1B and TLR4 was detected using SPR. K d , dissociation constant. g , Representative structure of PBP1B and TLR4 after molecular docking (left) and the residues involved in the interaction between PBP1B with TLR4 (right). The structure of TLR4 is coloured pink while the PBP1B is coloured yellow. The light green dash represents hydrogen bond. h , TLR4 from Hep3B and Huh7 cell lysates was pulled down by GST–PBP1B according to the GST pull-down assay. i , Immunoprecipitation of TLR4 from Hep3B or Huh7 cells lysates validated the binding between TLR4 and GST–PBP1B. In h and i , n = 3 independent experiments with similar results. j , Effect of PBP1B (0.05 μM) on HCC cell proliferation (top) ( n = 3 biologically independent samples), colony formation (middle) ( n = 3 biologically independent samples) and growth of HCC patient-derived organoid (bottom) ( n = 20 biologically independent samples). n = 3 independent experiments with similar results. k , Effect of PBP1B (0.05 μM) on TLR4, MyD88, p-P65, P65 and PCNA protein expression in HCC cells, as determined by western blot. n = 3 biologically independent samples. l , m , TLR4i (30 μM) abolished the effect of PBP1B on HCC cell proliferation ( l ) and colony formation ( m ). n = 3 biologically independent samples. n , Effect of K. pneumoniae on TLR4 expression in liver tissues of both germ-free and SPF mice with or without DEN treatment by IHC staining. In b , c , j – m , data are presented as mean ± s.e.m. Each data point in bar plots represents one mouse. Cell proliferation was analysed using two-way ANOVA. In b , c , j , k , m , comparisons between two groups were analysed using Student’s t -test. Unless otherwise stated, statistical significance was calculated using one-way ANOVA. Adjustments were made for multiple comparisons.

Article Snippet: TLR4 human recombinant protein (HY-P73586, MCE) was immobilized on a Series S Sensor Chip CM5 (29149603, GE Healthcare) using an amine coupling kit (BR-1000-50, GE Healthcare) with a flow rate of 10 μl min −1 , resulting in a final ligand coupling of ~180 response units.

Techniques: Co-Culture Assay, Derivative Assay, Expressing, Pull Down Assay, Membrane, Incubation, Magnetic Beads, Mass Spectrometry, Binding Assay, Immunoprecipitation, Western Blot, Immunohistochemistry

Journal: Nature Microbiology

Article Title: Gut–liver translocation of pathogen Klebsiella pneumoniae promotes hepatocellular carcinoma in mice

doi: 10.1038/s41564-024-01890-9

Figure Lengend Snippet: (a) Design of K. oxytoca on K. pneumoniae induced HCC promotion in germ-free mice with DEN treatment (GFD/PBS group n=6; GFD/EC group n=6; GFD/KO group n=6; GFD/KP group n=7; GFD/KOP group n=8) and relevant results (b–e) . (b) Images of liver gross morphology (yellow circles indicate tumor area), and H&E with tumor incidence, tumor number and tumor burden. n=6 (GFD/PBS), 6 (GFD/EC), 6 (GFD/KO) 7 (GFD/KP), 8 (GFD/KOP). (c) Alcian blue staining, E-Cad, CLDN 3, CLDN 1 IHC, and Cy3-conjugated EUB338 probe detection with quantitative analysis. n=6 biologically independent samples. (d) Gut permeability assay using FITC-dextran. n=6 (GFD/PBS), 6 (GFD/EC), 6 (GFD/KO) 7 (GFD/KP), 8 (GFD/KOP). (e) Live bacteria culture of liver tissues on blood agar plates with quantitative analysis. n=6 (GFD/PBS), 6 (GFD/EC), 6 (GFD/KO) 7 (GFD/KP), 8 (GFD/KOP). (f) Design of TLR4i on K. pneumoniae induced HCC promotion in germ-free mice with DEN treatment (GFD/PBS group n=6; GFD/EC group n=6; GFD/KP group n=7; GFD/KPi group n=7) and relevant results (g-k) . (g) Images of liver gross morphology (yellow circles indicate tumor area), and H&E with tumor incidence, tumor number and tumor burden. n=6 (GFD/PBS), 6 (GFD/EC), 7 (GFD/KP), 7 (GFD/KPi). (h) E-Cad IHC and Cy3-conjugated EUB338 probe (GFD/PBS group), E. coli probe (GFD/EC), K. pneumoniae probe (GFD/KP) and GFD/KPi groups) detection of colon tissues with quantitative analysis. n=6 biologically independent samples. (i) Gut permeability assay using FITC-dextran. n=6 biologically independent samples. (j) Live bacteria culture of liver tissues on blood agar plates with quantitative analysis. n=6 (GFD/PBS), 6 (GFD/EC), 7 (GFD/KP), 7 (GFD/KPi). (k) Cy3-conjugated conjugated EUB338 probe (GFD/PBS group), E. coli probe (GFD/EC), K. pneumoniae probe (GFD/KP) and GFD/KPi groups) FISH, TLR4, PCNA, Ki-67 IHC, and Sirius red staining of liver sections. n=6 biologically independent samples. Data (excluding tumor incidence and liver with live bacteria) are presented as mean ± SEM. Each data point in bar plots represents one mouse. Tumor incidence and Liver with live bacteria were calculated using Fisher’s exact test. Unless otherwise stated, statistical significance was calculated using one-way ANOVA. Adjustments were made for multiple comparisons.

Article Snippet: TLR4 human recombinant protein (HY-P73586, MCE) was immobilized on a Series S Sensor Chip CM5 (29149603, GE Healthcare) using an amine coupling kit (BR-1000-50, GE Healthcare) with a flow rate of 10 μl min −1 , resulting in a final ligand coupling of ~180 response units.

Techniques: Staining, Permeability, Bacteria

Journal: Nature Microbiology

Article Title: Gut–liver translocation of pathogen Klebsiella pneumoniae promotes hepatocellular carcinoma in mice

doi: 10.1038/s41564-024-01890-9

Figure Lengend Snippet: (a) Design of TLR4i on HCC-FMT induced HCC promotion in germ-free mice with DEN treatment (GFD/PBS group n=8; GFD/HCC-FMT group n=15; GFD/HCC-FMTi group n=10) and relevant results (b–g) . TLR4i was daily gavaged in this experiment. (b) Representative images of liver gross morphology (yellow circles indicate tumor area), and H&E staining of mice liver sections with tumor incidence, tumor number, and tumor burden. n=8 (GFD/PBS), 15 (GFD/HCC-FMT), 10 (GFD/HCC-FMTi). (c) Representative bacterial culture of liver tissues under anaerobic and aerobic conditions with quantitative analysis. n=8 (GFD/PBS), 15 (GFD/HCC-FMT), 10 (GFD/HCC-FMTi). From left to right and top to bottom, the culture plates are blood agar plate, chocolate blood agar plate, MacConkey agar plate, and Columbia blood agar plate, respectively. Gut permeability assay using 500 kDa FITC-dextran. n=6 biologically independent samples. (d) Representative pictures of Alcian blue staining, E-Cad IHC staining, and Cy3-conjugated EUB338 probe FISH detection of colon tissues with quantitative analysis. n=6 biologically independent samples. (e) Cy3-conjugated EUB338 probe FISH, Cy3-conjugated K. pneumoniae probe FISH, TLR4, PCNA, Ki-67 IHC of liver sections. n=6 biologically independent samples. (f) α-SMA, Desmin IHC staining, and Sirius red staining of liver sections. n=6 biologically independent samples. (g) Mouse Cancer Pathway Finder Array and Inflammatory Response and Autoimmunity PCR Array of liver tissues. n=3 independent experiments with similar results. Data (excluding tumor incidence, liver with live bacteria and PCR array results) are presented as mean ± SEM. Each data point in bar plots represents one mouse. Tumor incidence and Liver with live bacteria were calculated using Fisher’s exact test. Unless otherwise stated, statistical significance was calculated using one-way ANOVA. Adjustments were made for multiple comparisons.

Article Snippet: TLR4 human recombinant protein (HY-P73586, MCE) was immobilized on a Series S Sensor Chip CM5 (29149603, GE Healthcare) using an amine coupling kit (BR-1000-50, GE Healthcare) with a flow rate of 10 μl min −1 , resulting in a final ligand coupling of ~180 response units.

Techniques: Staining, Chocolate, Permeability, Immunohistochemistry, Bacteria

Journal: International Journal of Molecular Sciences

Article Title: Curcumin Alleviates HMGB1-Mediated Inflammation Through the Signaling Pathway of TLR2 -NF-κB in Bovine Ovarian Granulosa Cells

doi: 10.3390/ijms26189180

Figure Lengend Snippet: Curcumin inhibited protein expression of toll-like receptors and phospho-NF-κB p65 induced by HMGB1. ( A ) Immunoblots of TLR2 , phospho-NF-κB p65, total NF-κB, TLR1 , and TLR6 in BOGCs; ( B ) Relative band intensity of TLR2 /ACTB; ( C ) Relative band intensity of phospho-NF-κB p65/total NF-κB; ( D ) Relative band intensity of TLR1 /ACTB; ( E ) Relative band intensity of TLR6 /ACTB. ACTB as an internal control. Distinct lowercase letters in the figure denote statistically significant differences ( p < 0.05).

Article Snippet: TLR1 (orb48968) and TLR6 (orb357127) antibodies were acquired from Biorbyt, Cambridge, UK. β-actin monoclonal antibody (66009-1-Ig), HMGB1 monoclonal antibody (66525-1-Ig) and CoraLite488-conjugated Goat Anti-Rabbit IgG (SA00013-2) were acquired from Proteintech, Rosemont, IL, USA.

Techniques: Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Curcumin Alleviates HMGB1-Mediated Inflammation Through the Signaling Pathway of TLR2 -NF-κB in Bovine Ovarian Granulosa Cells

doi: 10.3390/ijms26189180

Figure Lengend Snippet: Curcumin attenuates the binding of HMGB1 to TLR1 , TLR2 , TLR6 , and phospho-NF-κB p65. ( A , C , E , G ) Co-IP analysis of HMGB1 with TLR2 , TLR1 , TLR6 and phospho-NF-κB p65 interaction. ( B , D , F , H ) Relative band intensity of TLR2 , TLR1 , TLR6 , and phospho-NF-κB p65. Distinct lowercase letters in the figure denote statistically significant differences ( p < 0.05).

Article Snippet: TLR1 (orb48968) and TLR6 (orb357127) antibodies were acquired from Biorbyt, Cambridge, UK. β-actin monoclonal antibody (66009-1-Ig), HMGB1 monoclonal antibody (66525-1-Ig) and CoraLite488-conjugated Goat Anti-Rabbit IgG (SA00013-2) were acquired from Proteintech, Rosemont, IL, USA.

Techniques: Binding Assay, Co-Immunoprecipitation Assay

Journal: International Journal of Molecular Sciences

Article Title: Curcumin Alleviates HMGB1-Mediated Inflammation Through the Signaling Pathway of TLR2 -NF-κB in Bovine Ovarian Granulosa Cells

doi: 10.3390/ijms26189180

Figure Lengend Snippet: Curcumin inhibited protein expression of toll-like receptors and phospho-NF-κB p65 induced by HMGB1. ( A ) Immunoblots of TLR2 , phospho-NF-κB p65, total NF-κB, TLR1 , and TLR6 in BOGCs; ( B ) Relative band intensity of TLR2 /ACTB; ( C ) Relative band intensity of phospho-NF-κB p65/total NF-κB; ( D ) Relative band intensity of TLR1 /ACTB; ( E ) Relative band intensity of TLR6 /ACTB. ACTB as an internal control. Distinct lowercase letters in the figure denote statistically significant differences ( p < 0.05).

Article Snippet: TLR1 (orb48968) and TLR6 (orb357127) antibodies were acquired from Biorbyt, Cambridge, UK. β-actin monoclonal antibody (66009-1-Ig), HMGB1 monoclonal antibody (66525-1-Ig) and CoraLite488-conjugated Goat Anti-Rabbit IgG (SA00013-2) were acquired from Proteintech, Rosemont, IL, USA.

Techniques: Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Curcumin Alleviates HMGB1-Mediated Inflammation Through the Signaling Pathway of TLR2 -NF-κB in Bovine Ovarian Granulosa Cells

doi: 10.3390/ijms26189180

Figure Lengend Snippet: Curcumin attenuates the binding of HMGB1 to TLR1 , TLR2 , TLR6 , and phospho-NF-κB p65. ( A , C , E , G ) Co-IP analysis of HMGB1 with TLR2 , TLR1 , TLR6 and phospho-NF-κB p65 interaction. ( B , D , F , H ) Relative band intensity of TLR2 , TLR1 , TLR6 , and phospho-NF-κB p65. Distinct lowercase letters in the figure denote statistically significant differences ( p < 0.05).

Article Snippet: TLR1 (orb48968) and TLR6 (orb357127) antibodies were acquired from Biorbyt, Cambridge, UK. β-actin monoclonal antibody (66009-1-Ig), HMGB1 monoclonal antibody (66525-1-Ig) and CoraLite488-conjugated Goat Anti-Rabbit IgG (SA00013-2) were acquired from Proteintech, Rosemont, IL, USA.

Techniques: Binding Assay, Co-Immunoprecipitation Assay