Journal: Frontiers in Microbiology

Article Title: iTRAQ-Based Quantitative Proteomics Analysis of HeLa Cells Infected With Chlamydia muridarum TC0668 Mutant and Wild-Type Strains

doi: 10.3389/fmicb.2019.02553

Figure Lengend Snippet: Indirect immunofluorescence assay (IFA) of HeLa cells infected with C. muridarum TC0668 wt or TC0668 mut strains. With 2.5 × 10 5 IFU/well inoculum (MOI = 1), Chlamydia -infected HeLa cells were photographed using optical microscopy at 6, 12, 18, and 24 h p.i. Chlamydial inclusion bodies (green) are visible in both TC0668 wt - and TC0668 mut -infected cells, whereas the TC0668 protein (red) is only visible in TC0668 wt -infected HeLa cells. Magnification, ×200. TC0668 wt -infected HeLa cells were photographed by using phase contrast microscopy (A) and IFA (B) at 6, 12, 18, and 24 h p.i. TC0668 mut -infected HeLa cells were photographed by using phase contrast microscopy (C) and IFA (D) at 6, 12, 18, and 24 h p.i.

Article Snippet: HeLa cells (human cervical carcinoma epithelial cells, CCL-2; American Type Culture Collection) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM-10) at 37°C in 5% CO 2 .

Techniques: Immunofluorescence, Infection, Microscopy

Journal: Frontiers in Microbiology

Article Title: iTRAQ-Based Quantitative Proteomics Analysis of HeLa Cells Infected With Chlamydia muridarum TC0668 Mutant and Wild-Type Strains

doi: 10.3389/fmicb.2019.02553

Figure Lengend Snippet: qRT-PCR analysis of tc0668 gene copy number in C. muridarum TC0668 wt - and TC0668 mut -infected HeLa cells. With 1 × 10 6 IFU/well inoculum (MOI = 1), the copy number of the C. muridarum gene tc0668 was determined using qRT-PCR, and the C. muridarum plasmid gene pgp8 was used as the control. 16S rRNA was used to normalize tc0668 and pgp8 signals. Three biological replicates of each time point were performed, and points represent mean and standard errors. Copy number differences of tc0668 between TC0668 wt - and TC0668 mut -infected cells were statistically significant (one-way ANOVA, P < 0.05).

Article Snippet: HeLa cells (human cervical carcinoma epithelial cells, CCL-2; American Type Culture Collection) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM-10) at 37°C in 5% CO 2 .

Techniques: Quantitative RT-PCR, Infection, Plasmid Preparation, Control

Journal: Frontiers in Microbiology

Article Title: iTRAQ-Based Quantitative Proteomics Analysis of HeLa Cells Infected With Chlamydia muridarum TC0668 Mutant and Wild-Type Strains

doi: 10.3389/fmicb.2019.02553

Figure Lengend Snippet: Quantitative proteomic analysis of HeLa cells infected with C. muridarum TC0668 wt or TC0668 mut strains. (A) Basic statistics of proteome results from iTRAQ. Spectra, secondary mass spectra after quality control; Unique peptides, identified peptides that belong to only a group of proteins; and protein, identified proteins using Mascot 2.3.02 software. (B) Trends of differentially expressed proteins in TC0668 wt -infected cells at 6, 12, 18, and 24 h p.i. (C) Trends of differentially expressed proteins in TC0668 mut -infected cells at 6, 12, 18, and 24 h p.i.

Article Snippet: HeLa cells (human cervical carcinoma epithelial cells, CCL-2; American Type Culture Collection) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM-10) at 37°C in 5% CO 2 .

Techniques: Infection, Multiplex sample analysis, Control, Software

Journal: Frontiers in Microbiology

Article Title: iTRAQ-Based Quantitative Proteomics Analysis of HeLa Cells Infected With Chlamydia muridarum TC0668 Mutant and Wild-Type Strains

doi: 10.3389/fmicb.2019.02553

Figure Lengend Snippet: Four-way Venn diagram of the total number of proteins significantly differentially expressed ( P < 0.05) between HeLa cells infected with C. muridarum TC0668 wt or TC0668 mut strains at 6, 12, 18, and 24 h p.i. Numbers of shared or unique proteins are indicated at the intersections of the circles in the Venn diagram.

Article Snippet: HeLa cells (human cervical carcinoma epithelial cells, CCL-2; American Type Culture Collection) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM-10) at 37°C in 5% CO 2 .

Techniques: Infection

Journal: Frontiers in Microbiology

Article Title: iTRAQ-Based Quantitative Proteomics Analysis of HeLa Cells Infected With Chlamydia muridarum TC0668 Mutant and Wild-Type Strains

doi: 10.3389/fmicb.2019.02553

Figure Lengend Snippet: Validation of profiling data with qRT-PCR. mRNA expression levels of seven up-regulated (encoded by SRPRB, JAK1, PMM1, HLA-DQB1, BCAP31, ITPR1, and THBS1) and three down-regulated (encoded by MAPKAPK2, TRAFD1, and IFI16) proteins in C. muridarum TC0668 mut -infected HeLa cells were determined using qRT-PCR at 18 h p.i, and compared with those of C. muridarum TC0668 wt -infected group. mRNA levels from three replicates for each group are expressed as mean and the standard errors. *represents that all copy number differences between TC0668 wt and TC0668 mut were statistically significant ( t test, P < 0.05).

Article Snippet: HeLa cells (human cervical carcinoma epithelial cells, CCL-2; American Type Culture Collection) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM-10) at 37°C in 5% CO 2 .

Techniques: Biomarker Discovery, Quantitative RT-PCR, Expressing, Infection

Journal: Frontiers in Microbiology

Article Title: iTRAQ-Based Quantitative Proteomics Analysis of HeLa Cells Infected With Chlamydia muridarum TC0668 Mutant and Wild-Type Strains

doi: 10.3389/fmicb.2019.02553

Figure Lengend Snippet: Activation of NF-κB and PI3K/Akt signal pathways as determined using western blotting and IFA. With 1 × 10 6 IFUs/well inoculum (six-well plate) or 2.5 × 10 5 IFUs/well inoculum (24-well plate), expression of PI3K, p-Akt, p53, and NF-κB (p65) representing activation of PI3K/Akt and NF-κB signal pathways were determined using western blotting or IFA, respectively. Gel quantification software was used to calculate the relative intensity of the corresponding signals. The relative value (target protein/GAPDH) differences of PI3K, p53, and NF-κB (p65) molecules and the relative expression level of p-Akt (p-Akt/Akt) between TC0668 wt - and TC0668 mut -infected cells were statistically significant (one-way ANOVA, P < 0.05). (A) Expression of PI3K molecules in TC0668 mut - and TC0668 wt -infected cells were determined using western blotting at 6, 12, 18, and 24 h post-infection. (B) Expression of p-Akt and total Akt in TC0668 mut - and TC0668 wt -infected cells were determined using western blotting at 6, 12, 18, and 24 h post-infection. (C) Expression of p53 molecules in the TC0668 mut - and TC0668 wt -infected cells at 6, 12, 18, and 24 h post-infection. (D) Expression of p65 molecules in the TC0668 mut - and TC0668 wt -infected cells at 6, 12, 18, and 24 h post-infection. (E) NF-κB molecules in the cytoplasm and nuclei of TC0668 mut - and TC0668 wt -infected HeLa cells at 6, 12, 18, and 24 h post-infection. DAPI dye core (blue), NF-κB fluorescence secondary antibody is 488 dye (green).

Article Snippet: HeLa cells (human cervical carcinoma epithelial cells, CCL-2; American Type Culture Collection) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM-10) at 37°C in 5% CO 2 .

Techniques: Activation Assay, Western Blot, Expressing, Software, Infection, Fluorescence

Journal: Frontiers in Microbiology

Article Title: iTRAQ-Based Quantitative Proteomics Analysis of HeLa Cells Infected With Chlamydia muridarum TC0668 Mutant and Wild-Type Strains

doi: 10.3389/fmicb.2019.02553

Figure Lengend Snippet: Differentially expressed proteins related with inflammation in TC0668 mut - vs. TC0668 wt -infected HeLa cells at 18 h p.i.

Article Snippet: HeLa cells (human cervical carcinoma epithelial cells, CCL-2; American Type Culture Collection) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM-10) at 37°C in 5% CO 2 .

Techniques: Infection, Multiplex sample analysis, Ubiquitin Proteomics, Histone Deacetylase Assay, Membrane, Variant Assay

Journal: Frontiers in Microbiology

Article Title: iTRAQ-Based Quantitative Proteomics Analysis of HeLa Cells Infected With Chlamydia muridarum TC0668 Mutant and Wild-Type Strains

doi: 10.3389/fmicb.2019.02553

Figure Lengend Snippet: Differentially expressed proteins related with fibrosis in TC0668 mut - vs. TC0668 wt -infected HeLa cells at 18 h p.i.

Article Snippet: HeLa cells (human cervical carcinoma epithelial cells, CCL-2; American Type Culture Collection) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM-10) at 37°C in 5% CO 2 .

Techniques: Infection, Multiplex sample analysis, Ubiquitin Proteomics

Journal: Frontiers in Microbiology

Article Title: iTRAQ-Based Quantitative Proteomics Analysis of HeLa Cells Infected With Chlamydia muridarum TC0668 Mutant and Wild-Type Strains

doi: 10.3389/fmicb.2019.02553

Figure Lengend Snippet: Protein-protein interaction network of statistically differentially expressed proteins ( P < 0.05) associated with inflammation and fibrosis at 18 h p.i. The proteins interconnectivity of two categories (inflammation and fibrosis) are shown. The yellow circles marked with symbols combining numbers and letters, represent the protein ID of differentially expressed proteins screened by comparison of TC0668 mut -infected and TC0668 wt -infected HeLa cells. The corresponding information of proteins can be analyzed via UniProt ( https://www.uniprot.org/ ), each protein ID corresponds to a protein. For example, the protein ID “P01023” corresponds to the protein “pha-2-macroglobulin”.

Article Snippet: HeLa cells (human cervical carcinoma epithelial cells, CCL-2; American Type Culture Collection) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM-10) at 37°C in 5% CO 2 .

Techniques: Comparison, Infection