Ytes have been purified and stained for intact (strong line) and permeabilized cells (dotted line). CD14-MACS bead-purified peripheral blood monocytes were suspended in FACS buffer with or with out PermWash buffer followed by incubation with PE-conjugated isotype control IgG2a (gray histogram) or anti-TLR5 mAb. b Cells were plated and incubated with medium alone, flagellin (b, c), profilin (d, e) or LPS (f, g) in PBS (PAMP), PAMP predigested with proteinase K (PAMP + proteinase K) or anti-huTLR5 mAb (PAMP + anti-TLR5; 1.1 g/ ml). Soon after incubation for 24 h, supernatants had been harvested and assayed for IL-6 (b, d, f) or IL-12p70 (c, e, g) by ELISA. Information shown are representative of three independently performed experiments. Fig. 4. siRNA-mediated silencing of human TLR5 inhibits profilinand flagellin-mediated proinflammatory cytokine production by human peripheral blood-derived monocytes. Peripheral blood-derived monocytes have been isolated and electroporated inside the presence of medium alone, manage siRNA or human TLR5 siRNA oligos. Cells were then stained for TLR5 as described in figure 3. a Transfected cells have been gated and TLR5 expression was analyzed. A histogram overlay of isotype control-stained cells (gray histogram), control siRNA-transfected cells (dotted line) or TLR5 siRNAtransfected cells (strong line) is shown. b, c Cells were plated and incubated inside the presence of medium alone, flagellin, profilin or LPS for 24 h. Culture supernatants were then harvested and assayed for IL-6 (b) and IL-12p70 (c) by ELISA. Information shown are representative of three independently performed experiments.a250 200 IL-6 (pg/ml) 150 100Percentage of totalTLRb0Medium Flagellin Profilin LPSIL-12p70 (pg/ml)c0 Control vector TLR5 siRNAJ Innate Immun 2014;six:68594 DOI: 10.1159/Salazar Gonzalez et al.ten,000 Percentage of maximum IL-6 (pg/ml) 1,000 100 ten 1 CTDC C14 CTDC C42 IL-12p40 (pg/ml) 100 TLR5 (MFI) 80 60 40 20 0 100 101 102 103 104 FL2-MFI 400 IL-6 (pg/ml) 300 200 one hundred 0 60 40 20LPS Flagellin Profilin1 CTDC C14 CTDC CCTDC CCTDC Cabcd1,500 IL-8 (pg/ml) Isotype manage Anti-huTLR5 mAb 1,IL-12p40 (pg/ml)CTDC C14 CTDC CCTDC C14 CTDC C300 200 100*10 01 01 1 0 0 1*01 1 10 01 0 0 1 1 0. 00 00 ten 0. 00 0. 0.Tachyzoites:monocyte (m.o.i.)*0* * *01 ten ten 00 0. 0.Tachyzoites:monocyte (m.o.i.)0.0.1,0.1,1,ef0.Tachyzoites:monocyte (m.o.i.)gFig. five. TLR5 (R392X) mutation abolishes monocyte cytokine pro-duction soon after T. gondii profilin stimulation. Peripheral blood CD14+ monocytes have been purified and stained as described in figure 3. a Histogram overlays of isotype control versus TLR5 staining of CD14+ monocytes obtained from two donors (CTDC C42, gray histogram; CTDC C14, black histogram).Rosiglitazone Empty histograms represent isotype manage Ab staining (black line for CTDC C14 and gray line for CTDC C42).Rilpivirine (hydrochloride) b Imply fluorescence intensity (MFI) in the samples.PMID:23903683 c, d Cell suspensions in the similar donors were then cultured inside the presence of medium alone, LPS, flagellin or profilin (all at 1 g/ml) for 24 h. Supernatants had been harvested and assayed forIL-6 (c) and IL-12p40 (d) by ELISA. e, f Peripheral blood monocytes from donors CTDC C14 and CTDC C42 have been incubated within the presence of reside T. gondii Rh strain tachyzoites (m.o.i. ranging from 1,000 to 0.0001); 24 h later supernatants had been harvested and assayed for IL-6 (e) and IL-12p40 (f). g HEK293 cells were plated and incubated inside the presence of isotype control Ab or anti-huTLR5 mAb and live tachyzoites as indicated in e and f. Culture supernatants had been har.