Journal: Human reproduction (Oxford, England)

Article Title: Toll-like receptor 4-mediated growth of endometriosis by human heat-shock protein 70.

doi: 10.1093/humrep/den195

Figure Lengend Snippet: Figure 1: TLR4 protein and gene expression in macrophages. (A) The immunolocalization of Toll-like receptor 4 (TLR4) in CD68-positive macrophages (Mf) derived from the peritoneal fluid of women with or without endometriosis. (B) Western-blot analysis showing a band of TLR4 of 78 kDa molecular size. A lysate of Ramos, a B-lymphoma cell line was used to show a control band posi- tive for TLR4. For Ramos cells, Lane 1 indicates plasma lysate, Lane 2 indicates 50% diluted fraction of plasma lysate, Lane 3 indicates expression in total cell lysates (B). For Mf, Lane 1 indicates plasma lysate, Lanes 2 and 3 indicate 50 and 100% dilution of plasma lysate and Lane 4 indicates TLR4 expression in total cell lysates. We found more expression of TLR4 in total cell lysates and minimal expression in diluted fraction of plasma lysate. (C) TLR4 mRNA (406 bp) expression was detected by standard RT-PCR. Total RNA was extracted from cultured Mf derived from three women each with endometriosis (endoþ) and without endometriosis (endo2).

Article Snippet: A blocking experiment was performed with anti-TLR4 antibody (10 mg/ml) (HTA-125, HyCult Biotechnology) 20 min prior to the treatment with recombinant human Hsp70 (10 mg/ml) in order to examine any change in the secretion of cytokines and growth factors in culture media without washing the pre-incubated antibodies.

Techniques: Gene Expression, Derivative Assay, Western Blot, Control, Clinical Proteomics, Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture

Journal: Human reproduction (Oxford, England)

Article Title: Toll-like receptor 4-mediated growth of endometriosis by human heat-shock protein 70.

doi: 10.1093/humrep/den195

Figure Lengend Snippet: Figure 3: Neutralizing effect of anti-TLR4 antibody on the levels of HGF, VEGF, IL-6 and TNFa in the culture media of Mf (105 cells /well) derived from the peritoneal fluid of women with endometriosis. Mf were pre-treated with anti-TLR4 antibody (10 mg/ml) (black bar) and without antibody (white bar) for 20 min and then further treated with and without Hsp70 for a period of 24 h. Pre-treatment of cells with anti-TLR4 antibody was able to significantly decrease all these macromol- ecules when compared with non-pre-treatment group (P , 0.05 for each). All these data are expressed as mean+SEM of three separate exper- iments for each group and were normalized with same number of cells.

Article Snippet: A blocking experiment was performed with anti-TLR4 antibody (10 mg/ml) (HTA-125, HyCult Biotechnology) 20 min prior to the treatment with recombinant human Hsp70 (10 mg/ml) in order to examine any change in the secretion of cytokines and growth factors in culture media without washing the pre-incubated antibodies.

Techniques: Derivative Assay

Journal: Human reproduction (Oxford, England)

Article Title: Toll-like receptor 4-mediated growth of endometriosis by human heat-shock protein 70.

doi: 10.1093/humrep/den195

Figure Lengend Snippet: Figure 4: HGF gene expression in macrophages by human Hsp70. (A) The effect of a variable concentration of Hsp70 (0–10 mg/ml) and anti-TLR4 antibody (10 mg/ml) on the mRNA expression of HGF in per- itoneal Mf derived from women with or without endometriosis was studied by standard RT-PCR. (B) The individual mRNA band (505 bp) of HGF was normalized with the corresponding band of internal control (b-actin) and is represented by the fold increase of their corresponding control (without treatment with Hsp70). Values of each transcript after single treatment with Hsp70 or pre-treatment with anti-TLR4 antibody (10 mg/ml) were normalized to 1 (dose 0). For HGF, a significance of P , 0.05 was found at the dose of 1 and 10 mg/ml of Hsp70 (endometriosis versus non-endometriosis) and P , 0.05 was found when compared with anti-TLR4 antibody non-treated Mf. The results are expressed as mean+ SEM of three different experiments derived from three separate patients.

Article Snippet: A blocking experiment was performed with anti-TLR4 antibody (10 mg/ml) (HTA-125, HyCult Biotechnology) 20 min prior to the treatment with recombinant human Hsp70 (10 mg/ml) in order to examine any change in the secretion of cytokines and growth factors in culture media without washing the pre-incubated antibodies.

Techniques: Gene Expression, Concentration Assay, Expressing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Human reproduction (Oxford, England)

Article Title: Toll-like receptor 4-mediated growth of endometriosis by human heat-shock protein 70.

doi: 10.1093/humrep/den195

Figure Lengend Snippet: Figure 5: Single and combined effect of exogenous Hsp70 and LPS on the proliferation of stromal cells derived from the eutopic endome- tria of women with endometriosis (black bar) and without endometrio- sis (white bar) as measured by 5-bromo-2-deoxyuridine incorporation. Different concentrations of recombinant human Hsp70 (0, 1, 5, 10 mg/ ml) were applied to stromal cells as shown in (A). The single and com- bined treatment of stromal cells with Hsp70 (10 mg/ml), LPS (10 ng/ ml) and pre-treatment of these cells with either polymyxin B (1 mg/ ml) or anti-TLR4 antibody (10 mg/ml) are shown in (B). The results are represented as percentage of control (without any treatment) expressed as mean+ SEM of three different experiments derived from three separate patients. A. P , 0.05 versus non-treated cells; B. P , 0.05 (Hsp70 versus control), P , 0.05 (LPS versus control), P , 0.05 (LPS alone versus LPS þ polymyxin B); P , 0.05 (com- bined Hsp70 þ LPS versus control); P , 0.05 (anti-TLR4 pre-treated cells versus without anti-TLR4 pre-treated cells).

Article Snippet: A blocking experiment was performed with anti-TLR4 antibody (10 mg/ml) (HTA-125, HyCult Biotechnology) 20 min prior to the treatment with recombinant human Hsp70 (10 mg/ml) in order to examine any change in the secretion of cytokines and growth factors in culture media without washing the pre-incubated antibodies.

Techniques: Derivative Assay, Recombinant, Control

Journal: Journal of Inflammation (London, England)

Article Title: Expression and activation of toll-like receptor 3 and toll-like receptor 4 on human corneal epithelial and conjunctival fibroblasts

doi: 10.1186/1476-9255-11-3

Figure Lengend Snippet: Forward and reverse primers of TLRs 2–4, TLR7 and TLR9

Article Snippet: We also used NT2 cells as a negative control and HPBM cells as a positive control.For flow cytometry analysis we used anti-human monoclonal antibodies to human TLR3 (TLR3.7) (Hycult Biotech) and TLR4 (HTA125) (NOVUS Biologicals).

Techniques: Sequencing

Journal: Journal of Inflammation (London, England)

Article Title: Expression and activation of toll-like receptor 3 and toll-like receptor 4 on human corneal epithelial and conjunctival fibroblasts

doi: 10.1186/1476-9255-11-3

Figure Lengend Snippet: HCE, HCF and HPBM cells expression of TLR3 and TLR4 on the cell surface, examined by flow cytometry. NT2 cells served as a negative control. Cells were immunostained with TLR3-specific antibody, TLR4-specific antibody or isotype control antibody. Data shown (mean ± SD) are representative of four independent experiments. The single asterisk (p < 0.05), the double asterisks (p < 0.01) and the three asterisks (p < 0.001) represents statistical significance for experiments. TLR: Toll-like receptors, ISO: Isotype-control, NT2: Neuron-committed teratocarcinoma, HPBM: Human peripheral blood mononuclear, HCE: Human corneal epithelial cells.

Article Snippet: We also used NT2 cells as a negative control and HPBM cells as a positive control.For flow cytometry analysis we used anti-human monoclonal antibodies to human TLR3 (TLR3.7) (Hycult Biotech) and TLR4 (HTA125) (NOVUS Biologicals).

Techniques: Expressing, Flow Cytometry, Negative Control, Control

Journal: Journal of Inflammation (London, England)

Article Title: Expression and activation of toll-like receptor 3 and toll-like receptor 4 on human corneal epithelial and conjunctival fibroblasts

doi: 10.1186/1476-9255-11-3

Figure Lengend Snippet: TLR3 and TLR4 surface expression in HCE and HCF cells decreases following activation using poly I:C or LPS, respectively. TLR3 surface expression in naïve, HCE and HCF cells following activation using poly I:C (A) . TLR4 surface expression in naïve, HCE and HCF cells following activation using LPS (B) . Data derived from three independent experiments and shown as mean ± SD. The asterisk (p < 0.05) represent statistical significance following activation. TLR: Toll-like receptors, HCE: Human corneal epithelium, LPS: Lipopolysaccharide, Poly I:C: polyinosinic:polycytidylic acid.

Article Snippet: We also used NT2 cells as a negative control and HPBM cells as a positive control.For flow cytometry analysis we used anti-human monoclonal antibodies to human TLR3 (TLR3.7) (Hycult Biotech) and TLR4 (HTA125) (NOVUS Biologicals).

Techniques: Expressing, Activation Assay, Derivative Assay

Journal: Journal of Inflammation (London, England)

Article Title: Expression and activation of toll-like receptor 3 and toll-like receptor 4 on human corneal epithelial and conjunctival fibroblasts

doi: 10.1186/1476-9255-11-3

Figure Lengend Snippet: TLR3 and TLR4-specific mRNA levels are affected by receptor stimulation. Relative expression of TLR3-specific mRNA in naïve cells, and in cells treated with poly I:C in comparison to medium (A) . Relative expression of TLR4-specific mRNA in naïve cells and in cells treated with LPS in comparison to medium (B) . Data derived from four independent experiments. The asterisk (p < 0.05) represent statistical significance for experiments. All values are expressed as mean ± SD. TLR: Toll-like receptors, Human peripheral blood mononuclear, HCE: Human corneal epithelium, LPS: Lipopolysaccharide, Poly I:C: polyinosinic:polycytidylic acid.

Article Snippet: We also used NT2 cells as a negative control and HPBM cells as a positive control.For flow cytometry analysis we used anti-human monoclonal antibodies to human TLR3 (TLR3.7) (Hycult Biotech) and TLR4 (HTA125) (NOVUS Biologicals).

Techniques: Expressing, Comparison, Derivative Assay

Journal: Journal of Parasitology

Article Title: Toll-Like Receptor 2 andMuc2Expression on Human Intestinal Epithelial Cells by Gymnophalloides seoi Adult Antigen

doi: 10.1645/ge-2195.1

Figure Lengend Snippet: FIGURE 2. TLR4 expression on HT29 cells by RT-PCR and flow cytometry. (A) Quantification of mRNA expression. Products of RT-PCR were separated by 2% agarose gel electrophoresis (left). Marker 5 100 bp DNA ladder. (B) Values were calculated from OD/mm of each group divided by that of the housekeeping gene (GAPDH) (right). Significantly (*, P , 0.05) enhanced compared to the negative control. Gs, Gymnophalloides seoi; Nb, Nippostrongylus brasiliensis; Sp, sparganum. (C–E) Flow cytometric analysis of TLR4 protein expression on the surface of HT29 cells. (C) Non- stimulated cells. (D) Cells stimulated with G. seoi antigen. (E) Cells stimulated with G. seoi antigen plus human IFN-c. (F) Comparison of TLR4 protein expression on IECs under various culture conditions. Significantly (*, P , 0.05) enhanced compared to cells stimulated by G. seoi antigen. Gs, G. seoi.

Article Snippet: The mAbs, human TLR2-FITC (IgG2a) (Imgenex), and TLR4-FITC (IgG2a) (Imgenex) were used for single fluorescence flow cytometry to analyze the expression of TLRs on the cell surface.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Agarose Gel Electrophoresis, Marker, Negative Control, Comparison

Journal: Clinical Cancer Research

Article Title: 18F-FMISO PET Imaging Identifies Hypoxia and Immunosuppressive Tumor Microenvironments and Guides Targeted Evofosfamide Therapy in Tumors Refractory to PD-1 and CTLA-4 Inhibition

doi: 10.1158/1078-0432.ccr-21-2394

Figure Lengend Snippet: Figure 4. Normoxic tumors identified by 18F-FMISO PET have increased TH1 signaling. A, Longitudinal comparison of individual genes comparing hypoxic (blue) to normoxic (red) tumorswithIfng(5.23 0.92vs.3.75 1.22;P¼0.019),Tnfreceptor(5.42 1.25vs.3.86 0.65;P¼ 0.015),Tlr2(7.36 0.66vs.6.55 0.39;P¼ 0.019),Tlr4(7.16 1.15vs. 5.45 1.08; P ¼ 0.013), and Ccr2 (9.25 1.03 vs. 7.47 0.92; P ¼ 0.005). B, Tumor stratification for 18F-FMISO responder and 18F-FMISO nonresponder. C–F, Cytokine analysisfromthesupernatantofexcisedtumorsquantifyingthelevelofIFNg (C;2,7941,040mg/mLvs.677788mg/mL;P¼ 0.0003),TNF(F;5,5105,222mg/mLvs. 417 385mg/mL;P ¼ 0.011),HMGB1(E;267.2 126.0mg/mLvs.77.12 66.41 mg/mL;P ¼ 0.0015),andgranzymeB(GZB;D;4.420 1.935mg/mLvs.1.287 1.057mg/mL; P ¼ 0.0009). G, Depiction ofproposedbiologicalmechanism correlating thegenes thatshowdifferentialexpressionbetweenhypoxic and normoxictumorsthat received immunotherapy. All errors shown using SEM. Tumors were collected from two biological repeats with a minimum of four mice per group.

Article Snippet: Primary antibodies used include CD8 (Thermo Fisher Scientific Cat. No. MA5–18153, RRID:AB_2539527), GZMB (Biorbyt Cat. No. orb10738-CF647, RRID:AB_2893361), Texas Red Tomato Lectin preinjected 1minute before tumor extraction (Vector Laboratories Cat. No. TL-1176, RRID:AB_2336563), pimonidazole preinjected 1 hour prior to tumor extraction (Hypoxyprobe Cat. No. HP1–1000, RRID: AB_2811309), and secondary Hypoxyprobe Red PE (Hypoxyprobe HP11–100kit, RRID:AB_2893362), TLR4 (Novus Biologicals NB100– 56723AF647, RRID:AB_2893363), PD-1 (Thermo Fisher Proteintech Cat. No. CL647–66220, RRID:AB_2883732), F4/80 (Abcam Cat. No. ab204467, RRID:AB_2810932), TNFa (Abcam ab237353, RRID: AB_2893364), Arginase (Thermo Fisher Proteintech Cat. No. CL488–66129, RRID:AB_2883268), and CD11c (Thermo Fisher Proteintech Cat. No. CL488–60258, RRID:AB_2883128).

Techniques: Comparison

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

Article Title: The PPE18 of Mycobacterium tuberculosis interacts with TLR2 and activates IL-10 induction in macrophage.

doi: 10.4049/jimmunol.0901367

Figure Lengend Snippet: FIGURE 2. The recombinant PPE18 protein specifically binds to TLR2 receptor on macrophage to stimulate IL-10 production. A, PMA-differentiated THP-1 macrophages were either left untreated or incubated for 10–15 min with increasing concentrations of either rPPE18-biotin or rPPE18-biotin preincubated with F(ab)2 of either anti-PPE18 Ab or NMS. Cells were washed and incubated with streptavidin-FITC. The amount of bound rPPE18 was measured by flow cytometry. B, Binding efficiency of rPPE18 at a concentration of 3 g/ml was compared between undifferentiated and PMA-differentiated THP-1 macrophages by flow cytometry. C, IL-10 induction by rPPE18 at concentration of 3 g/ml was compared between undifferentiated and PMA- differentiated THP-1 macrophages. D, IL-10 production in PMA-differentiated THP-1 macrophages stimulated with rPPE18 (3 g/ml) in the presence of titrating concentrations of anti-TLR2 or anti-TLR4 or anti-CD14 or isotype (IgG2a) control Ab. Results are mean SD of three different experiments.

Article Snippet: For TLR2 and TLR4 staining, cells were treated (30 min at 4°C) with anti-human TLR2 mAb (Imgenex) or anti-human TLR4 mAb HTA125 (mouse IgG2a) (Imgenex) or isotype control IgG2a and then incubated with FITC-conjugated goat anti-mouse IgG (Sigma-Aldrich) at 4°C for a further 30 min. To examine PPE18 binding, cells (0.5–1 106) were incubated at room temperature with increasing concentrations of rPPE18 conjugated to biotin (PPE18-biotin) for 10–15 min. For competition assay for binding of rPPE18, cells were preincubated with 100 and 10-fold excess of Pam3CSK4 (Calbiochem) for 15 min on ice followed by incubation with rPPE18-biotin.

Techniques: Recombinant, Incubation, Cytometry, Binding Assay, Concentration Assay, Control

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

Article Title: The PPE18 of Mycobacterium tuberculosis interacts with TLR2 and activates IL-10 induction in macrophage.

doi: 10.4049/jimmunol.0901367

Figure Lengend Snippet: FIGURE 3. PPE18 interacts with TLR2 but not with other TLRs. A, Cell surface expression of TLR2 (i) and TLR4 (ii) in HEK293 cells transfected with either WT-TLR2 or WT-TLR4 as measured by flow cytometry. In parallel experiment, binding of rPPE18 to TLR2- (iii) or TLR4- (iv) transfected HEK293 cells was assessed by flow cytometry. B, Lysates from HEK293 cells transfected with either the WT-TLR2 or the backbone vector (pcDNA3.1) were incubated with rPPE18 immobilized with TALON resin. The bound protein was extracted and loaded along with the whole cell extracts from HEK293 cells transfected with either WT-TLR2 or pcDNA3.1 vector alone and immunoblotted with 1–2 g/ml anti-TLR2 Ab. C, Cell lysates from PMA-differentiated THP-1 macrophages were incubated with rPPE18 immobilized with TALON. The bound protein (lane 1) was extracted and loaded along with the whole cell extracts from THP-1 macrophages as positive control (lane 2) and immunoblotted with 1–2 g/ml Ab to either TLR1 or TLR2 or TLR4 or TLR6. The membranes were incubated with anti-rabbit IgG-HRP and the blots were visualized by chemiluminescence. D, Human MDM were pretreated with either anti-TLR2 blocking Ab (20 g/ml) or isotype control Ab (20 g/ml) followed by incubation (10–15 min) with biotinlyated rPPE18 (3 g/ml). The cells were washed and incubated with steptavidin-FITC. The amount of bound rPPE18 was measured by flow cytometry. Data are representative of three to four independent experiments.

Article Snippet: For TLR2 and TLR4 staining, cells were treated (30 min at 4°C) with anti-human TLR2 mAb (Imgenex) or anti-human TLR4 mAb HTA125 (mouse IgG2a) (Imgenex) or isotype control IgG2a and then incubated with FITC-conjugated goat anti-mouse IgG (Sigma-Aldrich) at 4°C for a further 30 min. To examine PPE18 binding, cells (0.5–1 106) were incubated at room temperature with increasing concentrations of rPPE18 conjugated to biotin (PPE18-biotin) for 10–15 min. For competition assay for binding of rPPE18, cells were preincubated with 100 and 10-fold excess of Pam3CSK4 (Calbiochem) for 15 min on ice followed by incubation with rPPE18-biotin.

Techniques: Expressing, Transfection, Cytometry, Binding Assay, Plasmid Preparation, Incubation, Positive Control, Blocking Assay, Control

Journal: EMBO Molecular Medicine

Article Title: The HSP40 chaperone Ydj1 drives amyloid beta 42 toxicity

doi: 10.15252/emmm.202113952

Figure Lengend Snippet: A Pearson’s correlation coefficient and Manders’ co‐localization Coefficients of endoplasmatic reticulum protein KDEL and Abeta analyzed on confocal images of Kenyon cells in 15‐day‐old male fly brains immunostained with Abeta‐specific antibody (Abeta) 6E10 and KDEL‐specific antibody in Droj2 knockdown flies ( Droj2 +/− ) and corresponding isogenic w 1118 wild‐type flies ( Droj2 +/+ ) with expression of human Abeta42 (UAS‐A42). Dot plots show all data points along with the mean (bar) ± SD n = 10. Unpaired, two‐tailed t ‐test. B Representative confocal and gSTED deconvolved (decon) images of Kenyon cells in 15‐day‐old male fly brains immunostained with Abeta‐specific antibody (Abeta) 6E10 (magenta) and endoplasmic reticulum protein KDEL‐specific antibody (KDEL, green) of Droj2 knockdown flies ( Droj2 +/− ) and corresponding isogenic w 1118 wild‐type flies ( Droj2 +/+ ) expressing human Abeta42 (UAS‐A42). C Representative confocal and gSTED deconvolved (decon) microscopy of Kenyon cells in 15‐day‐old male fly brains immunostained with Abeta‐specific antibody (Abeta) 6E10 (magenta) and mitochondrial marker ATP5A‐specific antibody (ATP5A, green) of w 1118 wild‐type flies ( Droj2 +/+ ) expressing human Abeta42 (UAS‐A42). D–H Counts of mitochondria (D), mitochondria average size (E), mitochondria coverage area (F), Feret diameter (G), and min Feret diameter (H) of ATP5A‐stained mitochondria from fly brain gSTED deconvolved images representatively shown in Fig from 9 to 10 brains of w 1118 wild‐type ( Droj2 +/+ ) and knockdown ( Droj2 +/− ) male flies expressing human Abeta42 (UAS‐A42). Dot plots show all data points along with the mean (bar) ± SD n = 9–10. Unpaired, two‐tailed t ‐test or Mann–Whitney test.

Article Snippet: The following primary antibodies were used: mouse monoclonal IgG1 κ anti‐Aß antibody (clone 6E10; Biolegend, 803001; 1:200 for confocal, 1:400 for gSTED), mouse IgG2b anti‐ATP5A (clone 15H4C4, Abcam, ab14748, 1:100 for gSTED), and mouse IgG2a anti‐KDEL (clone 10C3, Enzo Lifesciences, ADI‐SPA‐827, 1:100 for gSTED).

Techniques: Expressing, Two Tailed Test, Microscopy, Marker, Staining, MANN-WHITNEY