Journal: Infection and Immunity

Article Title: Enhanced Direct Major Histocompatibility Complex Class I Self-Antigen Presentation Induced by Chlamydia Infection

doi: 10.1128/IAI.01254-15

Figure Lengend Snippet: Stabilization and presentation of SCRAP-SVG in JY cells. (A) Depiction of SCRAP-SVG, including the destabilization domain (DD), the SVG peptide, and VFP. In the absence of Shield-1, SCRAP-SVG is degraded by the proteasome. The addition of Shield-1 allows SCRAP-SVG to fold and gain fluorescence. (B) Flow cytometry histograms depicting VFP fluorescence of parental JY cells (shaded), JY/SCRAP-SVG cells treated with Shield-1 (black histogram), and JY/SCRAP-SVG cells treated with ethanol alone (blue trace). RL15A-stained JY/SCRAP-SVG cells (bottom histogram, black trace) and parental JY cells (shaded histogram) are used to measure HLA-A2-SVG complexes at the cell surface. (C) Dose response of JY/SCRAP-SVG cells with various concentrations of Shield-1 or epoxomicin, measuring the mean fluorescence intensity (MFI) of VFP fluorescence. (D) JY/SCRAP-SVG cells were washed in citric acid and cultured with ethanol (EtOH), 1 μM Shield-1, BFA, or emetine and harvested at the indicated times for RL15A staining. (E) The same as in panel D except the cells were harvested at 8 h and stained in triplicate. Results for Shield-1-treated cells are statistically significantly different from results for ethanol-treated and either BFA- or emetine-treated cells (*, P < 0.05).

Article Snippet: For transient transfection, 5 × 10 5 MCF7 cells were mixed with 500 ng of the pCAGGS-SCRAP-SVG vector in 20 μl of transfection solution SF (Lonza) and transfected with the Amaxa shuttle nucleofector (program FF-120).

Techniques: Fluorescence, Flow Cytometry, Staining, Cell Culture

Journal: Infection and Immunity

Article Title: Enhanced Direct Major Histocompatibility Complex Class I Self-Antigen Presentation Induced by Chlamydia Infection

doi: 10.1128/IAI.01254-15

Figure Lengend Snippet: Chlamydia infection prevents accumulation of SCRAP-SVG while enhancing presentation of the SVG peptide. (A) JY/SCRAP-SVG cells were infected with RFP-expressing C. trachomatis and cultured for 12 h prior to the addition of Shield-1. VFP fluorescence was monitored over the next 12 h, and the MFI was plotted. (B) JY/SCRAP-SVG cells were infected with either C. trachomatis or C. caviae and cultured for 12 h before the addition of 1.0 μM Shield-1. The cells were cultured for an additional 12 h and analyzed in triplicate by flow cytometry for VFP expression. VFP fluorescence was significantly decreased (P < 0.05) for infected cells. (C) JY/SCRAP-SVG cells were mock infected or infected with either C. trachomatis or C. caviae, and 24 hpi, mRNA was extracted from cells and used to synthesize cDNA. SCRAP-SVG transcripts were quantified by qPCR. (D) JY/SCRAP-SVG cells were treated with EtOH, Shield-1, or BFA for 12 h, and the HLA-A2-SVG complexes were quantified. Results seen with Shield-1 treatment were significantly different from results for both EtOH- and BFA-treated cells (*, P < 0.05). (E) JY/SCRAP-SVG cells were infected and treated with Shield-1 or ethanol as described for panel B, and HLA-A2-SVG complexes were detected by staining with the RL15A MAb. Shield-1-treated infected cells had significantly more peptide-MHC complexes (*, P < 0.05) than uninfected cells. (F) Total MHC class I was quantified by flow cytometry in both mock-infected and infected cells. (G) JY/SCRAP-SVG cells were treated with the indicated TLR ligands (x axis) for 12 h and Shield-1 for an additional 12 h. The cells were analyzed for VFP expression (top) and HLA-A2-SVG (bottom). No statistically significant changes were noted when TLR-stimulated cells were compared to untreated cells. (H) MCF7 cells were transiently transfected with SCRAP-SVG, infected with C. trachomatis serovar D/UW-3, and treated with Shield-1 12 hpi. The cells were analyzed 15 h later by gating on VFP+ cells, and the average MFI of both the Venus fluorescence and HLA-A2 staining on VFP+ cells is depicted (*, P < 0.05).

Article Snippet: For transient transfection, 5 × 10 5 MCF7 cells were mixed with 500 ng of the pCAGGS-SCRAP-SVG vector in 20 μl of transfection solution SF (Lonza) and transfected with the Amaxa shuttle nucleofector (program FF-120).

Techniques: Infection, Expressing, Cell Culture, Fluorescence, Flow Cytometry, Staining, Transfection

Journal: Infection and Immunity

Article Title: Enhanced Direct Major Histocompatibility Complex Class I Self-Antigen Presentation Induced by Chlamydia Infection

doi: 10.1128/IAI.01254-15

Figure Lengend Snippet: C. trachomatis protein synthesis and LOS are necessary for skewing peptide presentation. Infected and mock-infected JY/SCRAP-SVG cells were treated with RIF (A) or CAM (B) immediately after infection, Shield-1 was added 12 hpi, and HLA-A2-SVG levels were measured 24 hpi. (C) JY/SCRAP-SVG cells were infected with C. trachomatis and treated with LPC. After 24 h, the cells were visualized using fluorescence microscopy with antibodies to LOS and bacterial Hsp60. Images of a representative cell from treated and nontreated cells are shown. (D) Cells were treated as described for panel C, and DNA was extracted from cells 24 hpi to determine the C. trachomatis genome copy number by qPCR. (E) JY/SCRAP-SVG cells were infected and treated with LPC or left untreated and then exposed to Shield-1, and HLA-A2-SVG complexes were quantified by RL15A staining at 24 hpi. Results for LPC-treated cells are significantly different from results for untreated, infected cells (*, P < 0.05).

Article Snippet: For transient transfection, 5 × 10 5 MCF7 cells were mixed with 500 ng of the pCAGGS-SCRAP-SVG vector in 20 μl of transfection solution SF (Lonza) and transfected with the Amaxa shuttle nucleofector (program FF-120).

Techniques: Infection, Fluorescence, Microscopy, Staining

Journal: Infection and Immunity

Article Title: Enhanced Direct Major Histocompatibility Complex Class I Self-Antigen Presentation Induced by Chlamydia Infection

doi: 10.1128/IAI.01254-15

Figure Lengend Snippet: Chlamydia-induced loss of SCRAP-SVG is not mediated by proteasomal degradation. (A) JY/SCRAP-SVG cells were cultured overnight in the presence of Shield-1, and the following day, the cells were washed and cultured in the absence of Shield-1 but in the presence of epoxomicin or DMSO. VFP fluorescence was monitored at the indicated times. (B) JY/SCRAP-SVG cells were cultured with Shield-1 plus epoxomicin or with Shield-1 alone, and VFP accumulation was monitored at the indicated times. (C) JY/SCRAP-SVG cells were treated with Shield-1 or EtOH overnight. Existing peptide-MHC complexes were removed by a brief acid wash, and cells were cultured for 2 h in the presence or absence of the proteasome inhibitor epoxomicin. SVG-peptide presentation after Shield-1 “loss” was significantly different than that after the loss of ethanol or in cells treated with epoxomicin (*, P < 0.05). (D and E) C. caviae-infected cells were treated with Shield-1 and epoxomicin for 2 h starting at 20 hpi and analyzed for VFP fluorescence (D) or for SCRAP-SVG accumulation by Western blotting (E).

Article Snippet: For transient transfection, 5 × 10 5 MCF7 cells were mixed with 500 ng of the pCAGGS-SCRAP-SVG vector in 20 μl of transfection solution SF (Lonza) and transfected with the Amaxa shuttle nucleofector (program FF-120).

Techniques: Cell Culture, Fluorescence, Infection, Western Blot

Journal: Infection and Immunity

Article Title: Enhanced Direct Major Histocompatibility Complex Class I Self-Antigen Presentation Induced by Chlamydia Infection

doi: 10.1128/IAI.01254-15

Figure Lengend Snippet: Quantitative Western blot analysis can be used to calculate loss of SCRAP-SVG induced by Chlamydia infection. (A) Representative quantitative Western blot of JY/SCRAP-SVG total cell lysates from cells treated with Shield-1 or ethanol that were either infected or uninfected. Recombinant GFP (rGFP) standards, prepared in lysates of JY cells that do not express SCRAP-SVG, are included on the blot. (B) Standard curves for quantification of GFP (left) and antibody staining (right) are shown. Signal detected by Western blotting is plotted as a function of GFP concentration. For antibody staining, latex beads with known molar equivalents of fluorescent dye (MEF) were analyzed by flow cytometry, and their corresponding MFI is plotted. The shaded region in each plot represents the range of signals detected in each experiment.

Article Snippet: For transient transfection, 5 × 10 5 MCF7 cells were mixed with 500 ng of the pCAGGS-SCRAP-SVG vector in 20 μl of transfection solution SF (Lonza) and transfected with the Amaxa shuttle nucleofector (program FF-120).

Techniques: Western Blot, Infection, Recombinant, Staining, Concentration Assay, Flow Cytometry

Journal: Infection and Immunity

Article Title: Enhanced Direct Major Histocompatibility Complex Class I Self-Antigen Presentation Induced by Chlamydia Infection

doi: 10.1128/IAI.01254-15

Figure Lengend Snippet: Efficiency of SVG-peptide presentation from SCRAP-SVG precursors

Article Snippet: For transient transfection, 5 × 10 5 MCF7 cells were mixed with 500 ng of the pCAGGS-SCRAP-SVG vector in 20 μl of transfection solution SF (Lonza) and transfected with the Amaxa shuttle nucleofector (program FF-120).

Techniques: Infection

Journal: Nature Communications

Article Title: LDLR is used as a cell entry receptor by multiple alphaviruses

doi: 10.1038/s41467-024-44872-5

Figure Lengend Snippet: a , b HEK 293T cells expressing mouse LDLR, LDLRAD3 or TIMD4 or transfected with pCAGGS vector (negative control) were infected with the rGETV-EGFP. At 24 h post infection viral RNA (vRNA) levels in infected cells were measured via qRT-PCR (normalized relative to WT cells) ( a ) and viral titers in supernatant ( b ) were determined on BHK-21 cells. c – k HEK 293T cells stably overexpressing hamster LDLRAD3, MXRA8 or LDLR were infected with GETV ( c ), SFV ( d ), BEBV ( e ), MIDV ( f ), VEEV ( g ), RRV ( h ), CHIKV ( i ), MAYV ( j ) or VSV ( k ) EGFP-expressing pseudo-viruses. EGFP-positive cells were counted using flow cytometry. Data are presented as mean values ± SD ( n = 3 independent experiments) and two-tailed P -values are calculated by unpaired Student’s t test. Unless otherwise labeled, the displayed P -values are the significance between the experimental group and the control group (Vector or WT). Source data are provided as a Source Data file.

Article Snippet: To generate the sequences for expression of receptor proteins with C-terminal Flag-tag, their coding sequences were PCR amplified with the designed specific pairs of primers (Supplementary Table ), and then cloned into the pCAGGS vector (NovoPro #V008798) for transient transfection or pCDH-CMV-MCS-EF1-Puro vector (SBI Systems Biosciences #CD510B-1) for packaging into lentivirus particles.

Techniques: Expressing, Transfection, Plasmid Preparation, Negative Control, Infection, Quantitative RT-PCR, Stable Transfection, Flow Cytometry, Two Tailed Test, Labeling, Control