COS7 cells were transiently transfected with vector and after 96 h the fusion protein was purified by Sepharose A column from cell culture supernatants

April 24, 2017

cells repeatedly. 1:100 diluted cell lysates containing EB were transferred to uninfected cells that had been seeded into 24 wells on glass slides the day before, incubated for 24 h at 35uC HHV6 Co-Infection Induces Chlamydial Persistence and fixed in 4% formaldehyde. After permeabilization with PBS +0.2% Triton X-100 16291771 for 20 min at RT and blocking with PBS +10% FBS for 1 h at RT, cells were stained with an antibody against chlamydial Hsp60 in PBS +2% FBS at RT for 1 h. Cells were washed with PBS thrice and incubated with a Cy2labeled secondary antibody and Hoechst in PBS +2% FBS for 1 h in the dark at RT. Slides were washed twice in PBS, once in distilled water to remove PBS and were mounted on Mowiol. The number of inclusions was determined by counting ten random fields using epifluorescence microscope at 40x magnification. For confocal laser scanning microscopy, samples were stained with Draq5 instead of Hoechst/Dapi and analyzed using a Leica TCS SPE equipped with 488, 532, and 635 nm solid-state lasers for excitation. Images were taken using appropriate excitation and emission filters for the fluorescence dyes. Overlay images of the single channels were obtained using ImageJ. Infectivity was also determined by quantifying chlamydial Hsp60 by immunoblotting using antibodies against Chlamydia specific Hsp60. The values were normalized to actin after signal quantification using densitometric analysis and ImageJ software. Number of inclusion forming units per ml was determined by analyzing five random fields in an epifluorescence microscope with 400-fold magnification. measured 17804601 by an ELISA plate reader using an excitation filter of 480 nm and emission filter of 530 nm. DCF Assay for Cellular ROS Activity Study Total cellular ROS activity was measured using OxiSelectTM Intracellular ROS Assay Kit according to the manufacturer’s protocol and an ELISA plate reader with an excitation filter of 480 nm and emission filter of 530 nm. Statistical Analysis Statistical significance of acquired data was calculated using two-sided Student’s t-test. Supporting Information Immunoblotting Cell lysates were resolved by 10% sodium dodecyl sulfate poly- acrylamide gel electrophoresis. Proteins were transferred to polyvinylidene difluoride membranes and blocked with 10% skimmed milk. The membranes were then probed with respective primary antibodies and subsequently with HRPconjugated secondary antibodies. Proteins were MMAE site detected with peroxidase-coupled secondary antibody using the ECL system. Antisera and antibodies used in this study are listed under materials and methods S1. Cell Transfections and RNA Interference Transfections were performed using Lipofectamine 2000 as per manufacturer’s protocol. To silence CD46, G6PDH, GSR, Nox1 gene expression by RNA interference, 16105 cells per well were seeded into a 12-well plate on the day of transfection. Freshly seeded HeLa cells were transfected with 160 nM siRNAs using RNAiFect transfection reagent according to the manufacturer’s instructions. Efficiency of gene silencing was generally validated either by western blot analysis or qPCR at 72 h post-transfection. All the siRNAs including a control siRNA pool were purchased from Dharmacon, Thermo Fisher Scientific. 6B and C. pneumoniae or HHV6A and S. negevensis and infectivity was determined after 3 or 4 days post infection, respectively. Cell supernatants were collected 72 h p.i. and were added to freshly growing HeLa cells. At 24 h p.i., cells were fixed an