Journal: PLoS Pathogens

Article Title: Attachment and Entry of Chlamydia Have Distinct Requirements for Host Protein Disulfide Isomerase

doi: 10.1371/journal.ppat.1000357

Figure Lengend Snippet: (A) Immunoblot of cells transfected with PDI-targeted siRNA or non-targeting siRNA using antibody to PDI. GAPDH expression was evaluated to ensure equivalent loading. (B) Following siRNA treatment, C. psittaci and C. trachomatis attachment were examined. PDI staining is shown in red, and bacteria are stained green. Because of the significant PDI downregulation, cells in PDI siRNA panels are marked by arrows. siRNA downregulation of PDI led to a dramatic decrease in bacterial attachment, but attachment was not affected by treatment with non-targeting siRNA. (C) The number of bacteria attached to cells following treatment with non-targeting siRNA (gray) or PDI siRNA (black) was determined by quantification of the number of cell-associated bacteria by counting eight separate fields of view containing at least ten cells. Error bars indicate standard error of the mean (SEM).

Article Snippet: Blocking solution was removed and coverslips were incubated 1 h with mouse anti- Chlamydia MOMP antibody for C. trachomatis or anti- Chlamydia LPS antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for C. psittaci and then washed with HBSS.

Techniques: Western Blot, Transfection, Expressing, Staining, Bacteria

Journal: eLife

Article Title: Site-specific glycosylation regulates the form and function of the intermediate filament cytoskeleton

doi: 10.7554/eLife.31807

Figure Lengend Snippet: Analysis of vimentin GlcNDAz crosslinks. ( A ) 293T cells transfected with vimentin-myc-6xHis were treated with DMSO vehicle or GlcNDAz for 24 hr, treated with vehicle, 100 mM 5SGlcNAc, or 50 mM Thiamet-G for an additional 24 hr, and then UV-crosslinked. Lysates were analyzed by fluorescent IB (V9 antibody). Nucleoporin-62 is a positive control for GlcNDAz crosslinking. GAPDH is a loading control. ( B ) 293T cells transfected with vimentin-myc-6xHis were treated with DMSO vehicle or GlcNDAz (48 hr) and UV light (or not) as indicated. Lysates were analyzed by IB (D21H3 antibody). Vimentin-myc-6xHis and endogenous vimentin crosslink similarly. ( C ) 293T cells were transfected with vimentin-myc-6xHis for 48 hr, treated with DMSO vehicle or GlcNDAz as in , and UV-crosslinked. Crosslinked and uncrosslinked vimentin populations were purified from lysates by tandem anti-myc IP and nickel affinity chromatography. Crosslinks were visualized by colloidal Coomassie stain (left) and IB (D21H3 antibody) (right).

Article Snippet: A mouse antibody against vimentin (clone V9, Santa Cruz Biotech sc-57678) was diluted in TBS containing 5% BSA and 0.1% Triton X-100 and incubated with the samples overnight at 4 ̊C.

Techniques: Transfection, Positive Control, Control, Purification, Affinity Chromatography, Staining

Journal: eLife

Article Title: Site-specific glycosylation regulates the form and function of the intermediate filament cytoskeleton

doi: 10.7554/eLife.31807

Figure Lengend Snippet: ( D ) Peptide coverage map from GlcNDAz-crosslinked complexes of vimentin purified in (C; see ). Found peptides are highlighted in yellow. ( E ) Top protein IDs from vimentin crosslinks. Full proteomics results are available for download as a Scaffold (.sf3) file on the eLife web site . Free software to view Scaffold files is available at http://www.proteomesoftware.com/products/scaffold/download/ . ( F ) 293T cells were GlcNDAz-crosslinked as in (A; see ) and subjected to differential extraction as in Figure 1D (left). Then, the 8M urea fractions from DMSO or GlcNDAz samples were divided, half of each sample was exchanged into non-denaturing IP buffer, and samples were analyzed by vimentin IB (V9 antibody) (right). ( G ) HeLa cells were treated with DMSO vehicle or 1% IDPN for 1 hr, fixed, stained for vimentin (D21H3 antibody), and analyzed by confocal microscopy. ( H ) 293T cells were treated with DMSO (vehicle) or GlcNDAz for 48 hr and UV light as indicated. Lysates were normalized for protein amount and analyzed by fluorescent IB (V9 antibody). n = 4.

Article Snippet: A mouse antibody against vimentin (clone V9, Santa Cruz Biotech sc-57678) was diluted in TBS containing 5% BSA and 0.1% Triton X-100 and incubated with the samples overnight at 4 ̊C.

Techniques: Purification, Software, Extraction, Staining, Confocal Microscopy

Journal: eLife

Article Title: Site-specific glycosylation regulates the form and function of the intermediate filament cytoskeleton

doi: 10.7554/eLife.31807

Figure Lengend Snippet: Successful CRISPR/Cas9-mediated deletion of vimentin was verified by actin-normalized qPCR ( A ) and IB (D21H3 antibody) ( B ). mRNA from WT and vimentin knockout mouse embryonic fibroblasts (MEFs) serve as positive and negative controls for the qPCR assay in ( A ). Single vimentin −/− clones (HeLa, sgRNA 1 clone 4; 293T, sgRNA 3 clone 4) were used to construct vimentin-reconstituted cell lines. ( C ) Parental (i.e., un-manipulated) HeLa cells (control) and vimentin −/− HeLa cells reconstituted with either empty vector or WT vimentin-mEmerald were lysed and analyzed by fluorescent anti-vimentin IB (V9 antibody) (left). Quantification of the fluorescence intensities (right) demonstrates that vimentin-mEmerald expression level is indistinguishable from that of endogenous vimentin. n = 2. n.s., not significant, Student’s t-test. ( D ) Vimentin −/− HeLa cells reconstituted with empty vector (control) or WT or mutant vimentin-mEmerald constructs were sorted by flow cytometry to obtain the top one-third of mEmerald-expressing cells and then used in all subsequent experiments. ( E ) Parental HeLa cells were transfected with empty vector or WT vimentin-mEmerald for 24 hr, fixed, permeabilized, and imaged for total vimentin (D21H3 antibody immunofluorescence) and vimentin-mEmerald (mEmerald tag fluorescence).

Article Snippet: A mouse antibody against vimentin (clone V9, Santa Cruz Biotech sc-57678) was diluted in TBS containing 5% BSA and 0.1% Triton X-100 and incubated with the samples overnight at 4 ̊C.

Techniques: CRISPR, Knock-Out, Clone Assay, Construct, Control, Plasmid Preparation, Fluorescence, Expressing, Mutagenesis, Flow Cytometry, Transfection, Immunofluorescence

Journal: eLife

Article Title: Site-specific glycosylation regulates the form and function of the intermediate filament cytoskeleton

doi: 10.7554/eLife.31807

Figure Lengend Snippet: 293T cells were transfected with vimentin-myc-6xHis constructs as indicated and treated with either vehicle (DMSO) or 12 µM MG132 for 13 hr. Lysates were analyzed by fluorescent IB (V9 antibody for vimentin).

Article Snippet: A mouse antibody against vimentin (clone V9, Santa Cruz Biotech sc-57678) was diluted in TBS containing 5% BSA and 0.1% Triton X-100 and incubated with the samples overnight at 4 ̊C.

Techniques: Transfection, Construct

Journal: eLife

Article Title: Site-specific glycosylation regulates the form and function of the intermediate filament cytoskeleton

doi: 10.7554/eLife.31807

Figure Lengend Snippet:

Article Snippet: A mouse antibody against vimentin (clone V9, Santa Cruz Biotech sc-57678) was diluted in TBS containing 5% BSA and 0.1% Triton X-100 and incubated with the samples overnight at 4 ̊C.

Techniques: Stable Transfection, Expressing, Recombinant, Plasmid Preparation, Sequencing, Functional Assay, Virus, Cloning

Journal: BMC Microbiology

Article Title: Inhibitory effects of 405 nm irradiation on Chlamydia trachomatis growth and characterization of the ensuing inflammatory response in HeLa cells

doi: 10.1186/1471-2180-12-176

Figure Lengend Snippet: Effects of 405 nm irradiance on chlamydial growth in HeLa cells. ( A ) HeLa cells were infected with C. trachomatis serovar E at a MOI of 5. ( B ) Infected cells were then exposed to varying doses of 405 nm at a range of energy densities (5-20 J/cm 2 ) either promptly after infection or 24 h post-infection (24 h post). Treatments are grouped based on post-hoc comparisons for convenience. The effect of 405 nm on chlamydial growth was assessed during active and persistent stages induced with penicillin ( B and C ). Growth was determined using quantitative real-time PCR to determine the ratio of chlamydial and eukaryotic housekeeping genes (16S: GAPDH respectively) 48 h post-infection on cDNA reverse transcribed from RNA. Mean ± standard deviation are plotted for the two replicated experiments. Statistical significance was determined post-hoc using a Bonferonni adjustment comparing all groups against C. trachomatis -infected HeLa cells alone (CTE); * P < 0.05, ** P < 0.005.

Article Snippet: C. trachomatis -infected HeLa cells with or without 405 nm were fixed with ice-cold methanol for 10 min. After aspiration, culture wells were washed with PBS and then stained with rabbit anti- C. trachomatis EBs (Virostat, Portland, ME) for 1 h. Wells were washed five times with PBS and counterstained with 4’, 6-diamidino-2’-phenylindole, dihydrochloride (Dapi; Thermo Scientific, Rockford, IL) for 10 min.

Techniques: Infection, Real-time Polymerase Chain Reaction, Reverse Transcription, Standard Deviation

Journal: BMC Microbiology

Article Title: Inhibitory effects of 405 nm irradiation on Chlamydia trachomatis growth and characterization of the ensuing inflammatory response in HeLa cells

doi: 10.1186/1471-2180-12-176

Figure Lengend Snippet: Anti-chlamydial properties of 405 nm irradiance. ( A - C ) HeLa cells were infected with C. trachomatis serovar E at a MOI of 5 without exposure to photodiodes. ( D - F ) Infected cells were exposed to 405 nm LEDs at 20 J/cm 2 promptly after infection to evaluate anti-chlamydial effects during an acute chlamydial infection. Cells were fixed and stained with dapi (blue) ( B and E ) and anti-chlamydial (green) ( C and F ) antibody 48 hours post-infection. Bar = 10μm.

Article Snippet: C. trachomatis -infected HeLa cells with or without 405 nm were fixed with ice-cold methanol for 10 min. After aspiration, culture wells were washed with PBS and then stained with rabbit anti- C. trachomatis EBs (Virostat, Portland, ME) for 1 h. Wells were washed five times with PBS and counterstained with 4’, 6-diamidino-2’-phenylindole, dihydrochloride (Dapi; Thermo Scientific, Rockford, IL) for 10 min.

Techniques: Infection, Staining

Journal: BMC Microbiology

Article Title: Inhibitory effects of 405 nm irradiation on Chlamydia trachomatis growth and characterization of the ensuing inflammatory response in HeLa cells

doi: 10.1186/1471-2180-12-176

Figure Lengend Snippet: Effect of 405 nm on IL-6 production in C. trachomatis -infected epithelial cells. ( A ) HeLa cells were infected with C. trachomatis serovar E at a MOI of 5 (CTE5). ( B ) Infected cells were then exposed to varying doses of 405 nm at a range of energy densities (5-20 J/cm 2 ) either promptly after infection or 24 h post-infection (post-24 h). The effect of 405 nm on IL-6 production was assessed during active ( A and B ) and penicillin-induced persistent stages ( C ). Supernatants were collected and measured for IL-6 production using an ELISA. Treatments are grouped based on post-hoc comparisons for convenience. Mean ± SEM are plotted for the two replicated experiments. Statistical differences were determined post-hoc using a Bonferonni adjustment comparing all groups to C. trachomatis infected cells (CTE); *, P < 0.05; ** P < 0.005.

Article Snippet: C. trachomatis -infected HeLa cells with or without 405 nm were fixed with ice-cold methanol for 10 min. After aspiration, culture wells were washed with PBS and then stained with rabbit anti- C. trachomatis EBs (Virostat, Portland, ME) for 1 h. Wells were washed five times with PBS and counterstained with 4’, 6-diamidino-2’-phenylindole, dihydrochloride (Dapi; Thermo Scientific, Rockford, IL) for 10 min.

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal: BMC Microbiology

Article Title: Inhibitory effects of 405 nm irradiation on Chlamydia trachomatis growth and characterization of the ensuing inflammatory response in HeLa cells

doi: 10.1186/1471-2180-12-176

Figure Lengend Snippet: Effect of 405 nm on CCL2 production in C. trachomatis -infected epithelial cells. ( A ) HeLa cells were infected with C. trachomatis serovar E at a MOI of 5 (CTE5). ( B ) Infected cells were then exposed to varying doses of 405 nm at a range of energy densities (5-20 J/cm 2 ) either promptly after infection or 24 h post-infection (post-24 h). The effect of 405 nm on CCL2 was assessed during active ( B ) and persistent stages induced with penicillin ( C ). Supernatants were collected and measured for CCL2 production using an ELISA. Treatments are grouped based on post-hoc comparisons for convenience. Mean ± SEM are plotted for the two replicated experiments. Statistical differences were determined post-hoc using a Bonferonni adjustment comparing all groups to C. trachomatis infected cells (CTE); *, P < 0.001.

Article Snippet: C. trachomatis -infected HeLa cells with or without 405 nm were fixed with ice-cold methanol for 10 min. After aspiration, culture wells were washed with PBS and then stained with rabbit anti- C. trachomatis EBs (Virostat, Portland, ME) for 1 h. Wells were washed five times with PBS and counterstained with 4’, 6-diamidino-2’-phenylindole, dihydrochloride (Dapi; Thermo Scientific, Rockford, IL) for 10 min.

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Measurement of IgG antibodies to Chlamydia trachomatis by commercial enzyme immunoassays and immunofluorescence in sera from pregnant women and patients with infertility, pelvic inflammatory disease, ectopic pregnancy, and laboratory diagnosed Chlamydia psittaci / Chlamydia pneumoniae infection

doi:

Figure Lengend Snippet: Specificity of the commercial enzyme immunoassays (EIAs) for detecting Chlamydia trachomatis antibody when tested against a panel of Chlamydia psittaci/Chlamydia pneumoniae antibody positive sera as diagnosed by whole cell immunofluorescence assay

Article Snippet: Sera for testing were diluted 1/64 in DELFIA assay buffer (Wallac Oy, Turku, Finland) and 100 μl was loaded on to Genzyme Virotech C trachomatis coated microtitre plates (Genzyme Virotech).

Techniques: Enzyme Immunoassay, Immunofluorescence