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ed in suppression of p53 expression73 and p21, a p53 target gene. After washing, coverslips were mounted by using DAPI Vectashield mounting medium and examined by differential interference contrast and fluorescence microscopy by using a Zeiss Axioplan 2 I-BET-762 microscope. Images were captured having a digital CCD camera. Analysis of co localization of the fluorescent labels was performed by using OpenLab software with or without having three dimensional reconstruction and deconvolution as indicated. For quantitative analyses, the percentage of cells with a single or a lot more internalized B. burgdorferi particles were counted by examining sequential fields from minimum three independent experiments. Cells containing any internalized B. burgdorferi particles or cells containing internalized/intact B.
burgdorferi were counted and expressed as a percent of the total number cells examined. The mean percent of minimum three independent experiments were plotted over time and the statistical significance amongst groups was analyzed by using the nonparametric Mann Whitney U test. Quantitative I-BET-762 reverse transcriptase PCR After incubation with B. burgdorferi, cells were washed with phosphate buffered saline and RNA extracted by using Trizol as per the companies directions. very first strand synthesis of cDNA from total RNA was performed by using Improm II as per the companies directions. Quantification of cDNA was performed by quantitative PCR by using Sybrgreen technology. Cycling parameters were 60 C for 5 min and 95 C for 15 min, followed by 40 cycles of 95 C for 30 sec and 60 C for 1 min.
The specificity of each and every reaction was checked by melt curve analysis and by agarose gel electrophoresis of PCR items. Expression of target genes was referenced to expression of B actin. Calculations of expression were normalized by using the Ct approach where the amount of target, normalized to an endogenous reference and relative to a calibrator, is given by 2−Ct, where Ct would be the cycle quantity of the detection threshold. Transient transfection of MyD88 dominant damaging plasmid Raw 264. 7 cells were transiently transfected having a dominant damaging mutant of MyD88 or pCDNA3 GFP plasmid, by using a 4:1 lipid/DNA ratio of Lipofectamine 2000 transfection reagent in line with the companies protocol. The transfection mix was added to cells in DMEM serum absolutely free media and incubated at 37 C.
After 6 hours, the media was replaced with 10% FBS added DMEM, and 24 hours later, we performed phagocytosis assay as described. We estimated transfection efficiency of Raw 264. 7 cells by randomly deciding on 10 fields and counting both total cells and cells expressing GFP right after transient transfection of cells with pCDNA3 GFP plasmid. Estimated transfection efficiency for all experiments was around 70 80%. Western blotting Cellular lysates of mouse macrophages were prepared by lysis buffer after which separated by SDS Page on 4 12% acrylamide gels and transferred to a polyvinyldifluoride membrane. The membrane was incubated in blocking buffer for 1 hour at room temperature and washed three occasions for 5 minutes each and every with 15ml of TBS/T. Membranes were incubated with the main antibody overnight at 4 C.
Phospho Akt antibody and total Akt antibody were purchased from Cell Signaling. After washing three occasions with TBS/T, the membranes were incubated with anti rabbit IgG HRP conjugated secondary antibody for a single hour at 25 C. After washing three occasions with TBS/T, the membrane was incubated with LumiGlo substrate and exposed towards the film. Statistical analysis Experiments were repeated three occasions as indicated. The statistical significance amongst groups was analyzed by using the nonparametric Mann Whitney U test. Differences were regarded statistically substantial when the p values were equal to or less than 0. 05. Results Deficiencies in MyD88 mediated phagocytosis of B. burgdorferi might be complemented by activation of TLR3 dependent signaling We previously reported that MyD88 is required for uptake of B.
burgdorferi, but not for E. coli. Among the differences amongst innate immune recognition of B. burgdorferi and E. coli would be the reality that B. burgdorferi lipoproteins are recognized by TLR2, although E. coli lipopolysaccaride is recognized via TLR4. A single possible implication of this difference is that TLR4, additionally to utilizing MyD88 for activation of signaling pathways, may also activate MyD88 independent pathways via the use of TRIF adaptor pathway. To be able to determine whether or not signaling via TRIF can complement the loss of MyD88 and restore phagocytosis of B. burgdorferi in MyD88 deficient cells, we stimulated both WT and MyD88 BMDMs with the TLR3 ligand, poly I:C. Among TLRs, TLR3 is distinctive in that it can be the only identified TLR that does not make use of MyD88 and activates pathways solely via recruitment and activation of TRIF. We very first confirmed the effect of poly I:C on activation of MyD88 cells by evaluating mRNA expression of sort I interferon and tum