Journal: The Journal of infectious diseases

Article Title: Osteopontin impairs host defense during pneumococcal pneumonia.

doi: 10.1093/infdis/jir185

Figure Lengend Snippet: Figure 1. Pulmonary and plasma osteopontin concentrations are elevated during pneumococcal pneumonia. Osteopontin concentrations in (A) lung and (B) plasma before and 6, 24, and 48 h after infection with 104 colony-forming units of Streptococcus pneumoniae. Data are expressed as mean 6 standard error of the mean (SEM); n 5 8 mice per group. Asterisk, P , .05; double asterisk, P , .01; triple asterisk, P , .001, compared with t 5 0. Osteopontin concentrations in culture supernatants after incubation of (C) MH-S cells and (D) primary alveolar macrophages with medium or growth- arrested S. pneumoniae (multiplicity of infection, 1:6 and 1:60 for MH-S cells; 1:20 and 1:200 for primary alveolar macrophages) for 4 h (MH-S cells) or 20 h (primary alveolar macrophages). Data are expressed as mean 6 SEM; n 5 3 per group. Asterisk, P ,.05, compared with medium. OPN, osteopontin.

Article Snippet: Effect of Osteopontin on S. pneumoniae Viability, Phagocytosis, and Phagolysosomal Fusion S. pneumoniae or Staphylococcus (S.) aureus (Newman strain) (1 3 106 bacteria/mL) was incubated in sterile normal saline in the presence of 0.8–800 ng/mL recombinant mouse osteopontin (rOPN;,1.0 endotoxin units per 1 lg as determined by the LAL assay; R&D Systems), 800 ng/mL boiled rOPN (30 min at 100 C), 800 ng/mL bovine serum albumin (BSA), or normal saline only at 37 C for 6 h. At indicated time points the number of bacteria was determined.

Techniques: Clinical Proteomics, Infection, Incubation

Journal: The Journal of infectious diseases

Article Title: Osteopontin impairs host defense during pneumococcal pneumonia.

doi: 10.1093/infdis/jir185

Figure Lengend Snippet: Figure 2. Prolonged survival and reduced bacterial growth in osteopontin knockout (KO) mice. A, Percentage survival of wild-type (WT) mice (filled symbols) and osteopontin KO mice (open symbols) after intranasal infection with 104 colony-forming units (CFU) of Streptococcus pneumoniae (n 5 14 mice per group). P value indicates the difference between groups. WT (gray) and osteopontin KO (white) mice were infected with 104 CFU of S. pneumoniae, and bacterial loads were determined 6, 24, and 48 h after infection in (B) lung, (C) blood, and (D) spleen. Data are expressed as box-and- whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile, and largest observation; n 5 8 mice per group; asterisk, P ,.05; double asterisk, P ,.01; triple asterisk, P ,.001, compared with WT mice. Note to panels C and D: none of the mice in either group displayed positive blood or spleen culture results 6 h after infection; at 24 h, S. pneumoniae could be cultured from samples of the blood of only 3 of 8 osteopontin KO mice, compared with 7 of 8 WT mice and from the spleen tissue of only 1 of 7 osteopontin KO mice, compared with 7 of 8 WT mice (P , .05 and P , .01, respectively). OPN, osteopontin.

Article Snippet: Effect of Osteopontin on S. pneumoniae Viability, Phagocytosis, and Phagolysosomal Fusion S. pneumoniae or Staphylococcus (S.) aureus (Newman strain) (1 3 106 bacteria/mL) was incubated in sterile normal saline in the presence of 0.8–800 ng/mL recombinant mouse osteopontin (rOPN;,1.0 endotoxin units per 1 lg as determined by the LAL assay; R&D Systems), 800 ng/mL boiled rOPN (30 min at 100 C), 800 ng/mL bovine serum albumin (BSA), or normal saline only at 37 C for 6 h. At indicated time points the number of bacteria was determined.

Techniques: Knock-Out, Infection, Whisker Assay, Cell Culture

Journal: The Journal of infectious diseases

Article Title: Osteopontin impairs host defense during pneumococcal pneumonia.

doi: 10.1093/infdis/jir185

Figure Lengend Snippet: Figure 3. Decreased lung histopathology in osteopontin knockout (KO) mice. Representative lung histology of wild-type (WT) (A, D, G) and osteopontin KO (B, E, H ) mice at 6 h (A–C ), 24 h (D–F ), and 48 h (G–I) after intranasal infection with 104 CFU of Streptococcus pneumoniae. The lung sections are representative for 8 mice per group per time point. Hematoxilin and eosin staining, original magnification, 310. Inflammation scores are expressed as mean 6 standard error of the mean (WT mice, black bars; osteopontin KO mice, white bars; n 5 8 mice per group). Double asterisk, P ,.01, compared with WT mice. OPN, osteopontin.

Article Snippet: Effect of Osteopontin on S. pneumoniae Viability, Phagocytosis, and Phagolysosomal Fusion S. pneumoniae or Staphylococcus (S.) aureus (Newman strain) (1 3 106 bacteria/mL) was incubated in sterile normal saline in the presence of 0.8–800 ng/mL recombinant mouse osteopontin (rOPN;,1.0 endotoxin units per 1 lg as determined by the LAL assay; R&D Systems), 800 ng/mL boiled rOPN (30 min at 100 C), 800 ng/mL bovine serum albumin (BSA), or normal saline only at 37 C for 6 h. At indicated time points the number of bacteria was determined.

Techniques: Histopathology, Knock-Out, Infection, Staining

Journal: The Journal of infectious diseases

Article Title: Osteopontin impairs host defense during pneumococcal pneumonia.

doi: 10.1093/infdis/jir185

Figure Lengend Snippet: Figure 4. Osteopontin stabilizes Streptococcus pneumoniae viability in vitro. A, S. pneumoniae in saline (106 colony-forming units [CFU]/mL) was incubated with increasing doses (0.8–800 ng/mL) of recombinant osteopontin (black symbols) or saline (white symbols), and the viability of S. pneumoniae was determined over 6 h at 37C. B, S. pneumoniae in saline (106 CFU/mL) was incubated with 800 ng/mL recombinant osteopontin (filled squares), 800 ng/mL boiled recombinant osteopontin (open squares), 800 ng/mL bovine serum albumin (triangles), or saline (circles), and the viability of S. pneumoniae was determined over 6 h at 37C. Dashed lines depict detection limits. C, Osteopontin binds to S. pneumoniae. Enzyme-linked immunosorbent assay plates were coated or not coated with 1 3 108 CFU/mL S. pneumoniae type 3 (ATCC 6303) or serotype 2 (D39); coating with anti-osteopontin IgG was used as positive control. Binding was assessed using biotin-labeled recombinant mouse osteopontin. Data are means 6 standard error (n 5 4–6). Double asterisk, P , .01 vs buffer. OPN, osteopontin.

Article Snippet: Effect of Osteopontin on S. pneumoniae Viability, Phagocytosis, and Phagolysosomal Fusion S. pneumoniae or Staphylococcus (S.) aureus (Newman strain) (1 3 106 bacteria/mL) was incubated in sterile normal saline in the presence of 0.8–800 ng/mL recombinant mouse osteopontin (rOPN;,1.0 endotoxin units per 1 lg as determined by the LAL assay; R&D Systems), 800 ng/mL boiled rOPN (30 min at 100 C), 800 ng/mL bovine serum albumin (BSA), or normal saline only at 37 C for 6 h. At indicated time points the number of bacteria was determined.

Techniques: In Vitro, Saline, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Positive Control, Binding Assay, Labeling

Journal: The Journal of infectious diseases

Article Title: Osteopontin impairs host defense during pneumococcal pneumonia.

doi: 10.1093/infdis/jir185

Figure Lengend Snippet: Figure 5. Similar bacterial growth during pneumococcal sepsis. Bacterial loads in (A) blood, (B) lung, (C) liver, and (D) spleen from wild-type (WT; gray) and osteopontin knockout (OPN KO; white) mice at 24 and 48 h after intravenous injection with 105 colony-forming units (CFU) of Streptococcus pneumoniae. Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile, and largest observation; n 5 8 mice per group. Dashed line depicts detection limit.

Article Snippet: Effect of Osteopontin on S. pneumoniae Viability, Phagocytosis, and Phagolysosomal Fusion S. pneumoniae or Staphylococcus (S.) aureus (Newman strain) (1 3 106 bacteria/mL) was incubated in sterile normal saline in the presence of 0.8–800 ng/mL recombinant mouse osteopontin (rOPN;,1.0 endotoxin units per 1 lg as determined by the LAL assay; R&D Systems), 800 ng/mL boiled rOPN (30 min at 100 C), 800 ng/mL bovine serum albumin (BSA), or normal saline only at 37 C for 6 h. At indicated time points the number of bacteria was determined.

Techniques: Knock-Out, Injection, Whisker Assay

Journal: Clinical and Translational Medicine

Article Title: Regulatory roles of osteopontin in human lung cancer cell epithelial‐to‐mesenchymal transitions and responses

doi: 10.1002/ctm2.486

Figure Lengend Snippet: Roles of PI3K/Akt and MAPK/Erk1/2 on OPN‐induced EMT. Total Akt and p‐Akt were detected after challenge with OPN at different concentrations (5, 50, or 500 ng/ml) (A), as well as for cells treated with 0.01, 0.1, or 1 μM of the PI3K inhibitor SHBM1009 (B). E‐cadherin (C) and vimentin (D) were measured 48 h after challenge with 500 ng/ml OPN and 0.01, 0.1, or 1 μM of the PI3K inhibitor SHBM1009. Total Erk1/2 and p‐Erk1/2 were detected after challenge with OPN at different concentrations (5, 50, or 500 ng/ml) (E), as well as for cells treated with 0.1, 1, or 10 μM of the Erk1/2 inhibitor PD98059 (F). E‐cadherin (G) and vimentin (H) were measured 48 h after challenge with 500 ng/ml OPN and 0.1, 1, or 10 μM of the PD98059. Data are represented as mean ± SEM. Differences between groups were assessed by the Student's t ‐test, after ANOVA analyses.* and ** stand for p‐ values less than .05 and .01 as compared with vehicle, and + and ++ stand for p ‐values less than .05 and .01, as compared with OPN challenge, respectively

Article Snippet: Human recombinant OPN (R&D, Shanghai, China), PI3K/Akt specific inhibitor SHBM1009 (Biovision, San Francisco, USA), Erk1/2 specific inhibitor PD98059 (Biovision, San Francisco, USA), mouse monoclonal anti‐vimentin antibody (Abcam, Hong Kong, China), and rabbit polyclonal to OPN (Abcam, Hong Kong, China) were used in this study.

Techniques:

Journal: Clinical and Translational Medicine

Article Title: Regulatory roles of osteopontin in human lung cancer cell epithelial‐to‐mesenchymal transitions and responses

doi: 10.1002/ctm2.486

Figure Lengend Snippet: OPN promotes cell migration and proliferation. Cell migration (A and B) was detected by transwell chambers after challenge with OPN with different concentrations of PI3K inhibitor SHBM1009 (0.01, 0.1, or 1 μM), and the dynamical movements (C) were detected by Cell‐IQ monitoring. Cell migration (D and E) was detected by transwell chambers after challenge with OPN with different concentrations of Erk1/2 inhibitor PD98059 (0.1, 1, or 10 μM), and the dynamical movements (F) were detected by Cell‐IQ monitoring. Dynamical cell proliferation was measured by CCK8 (G) and Cell‐IQ monitoring (I) in cells pretreated with OPN with different concentrations of SHBM1009. Dynamical cell proliferation was measured by CCK8 (H) and Cell‐IQ monitoring (J) in cells pretreated with OPN with different concentrations of PD98059. Data are represented as mean ± SEM. Differences between groups were assessed by the Student's t ‐test, after ANOVA analyses. * and ** stand for p‐ values less than .05 and .01, in comparison with untreated control cells, and + and ++ stand for p‐ values less than .05 and .01, as compared with OPN‐treated cells at 24 h, respectively

Article Snippet: Human recombinant OPN (R&D, Shanghai, China), PI3K/Akt specific inhibitor SHBM1009 (Biovision, San Francisco, USA), Erk1/2 specific inhibitor PD98059 (Biovision, San Francisco, USA), mouse monoclonal anti‐vimentin antibody (Abcam, Hong Kong, China), and rabbit polyclonal to OPN (Abcam, Hong Kong, China) were used in this study.

Techniques: Migration

Journal: Clinical and Translational Medicine

Article Title: Regulatory roles of osteopontin in human lung cancer cell epithelial‐to‐mesenchymal transitions and responses

doi: 10.1002/ctm2.486

Figure Lengend Snippet: OPN‐specific siRNA inhibits lung cancer xenograft growth in vivo. In vivo tumor growth in OPN‐specific siRNA mice was significantly inhibited compared with vehicle or nonspecific siRNA, whereas no significant difference in tumor volumes between vehicle or nonspecific siRNA (A and C) was found. Tumor growth curves were plotted during the experiment (B). Expression of E‐cadherin, vimentin, and OPN in nonspecific siRNA or OPN‐specific siRNA mice was detected by immunohistochemistry staining (D). Expressions of PI3K/Akt and Erk1/2 signaling pathway components were detected in the tumor from mice determined by Western blot. Data are represented as mean ± SEM. Differences between groups were assessed by the Student's t ‐test, after ANOVA analyses.** stands for p‐ values less than .01 as compared with vehicle, and ++ stands for p ‐values less than .01, as compared with nonspecific siRNA

Article Snippet: Human recombinant OPN (R&D, Shanghai, China), PI3K/Akt specific inhibitor SHBM1009 (Biovision, San Francisco, USA), Erk1/2 specific inhibitor PD98059 (Biovision, San Francisco, USA), mouse monoclonal anti‐vimentin antibody (Abcam, Hong Kong, China), and rabbit polyclonal to OPN (Abcam, Hong Kong, China) were used in this study.

Techniques: In Vivo, Expressing, Immunohistochemistry, Staining, Western Blot

Journal: Frontiers in Neural Circuits

Article Title: Intersectional Strategies for Targeting Amacrine and Ganglion Cell Types in the Mouse Retina

doi: 10.3389/fncir.2018.00066

Figure Lengend Snippet: The Pvalb-FlpE driver targeted multiple RGC types. (A) The Pvalb-FlpE driver was crossed with CMV-Cre and Ai65 mice. Immunostainings were performed by using antibody markers for 4 different RGC types: Osteopontin for alpha RGCs, CART for ooDSGCs, FOXP2 for F-RGCs, and melanopsin for ipRGCs. Scale bar, 20 μm. (B) Proportions of RGC types targeted in the Pvalb-FlpE driver. n = 6 retinas from 6 animals (6 litters). (C) RGC types extracted from CAGGCre-ER;Pvalb-FlpE;Ai65 retinas without tamoxifen administration. Flat-mount view (top) and side view (bottom) with ChAT (blue) labeling. Scale bar: 50 μm for the flat-mount view, 10 μm for the side view.

Article Snippet: The primary antibodies used were as follows: rabbit anti-RFP (1:1000, Rockland 600-401-379), chicken anti-RFP Biotin conjugated (1:200, Rockland 600-906-379), guinea pig anti-RBPMS (1:500, PhosphoSolutions 1832-RBPMS), mouse anti-AP2 (3 μg/ml, DSHB 3B5), goat anti-Osteopontin (1:500, R&D 441-OP-050), rabbit anti-CART (1:1000, Phoenix pharmaceuticals H-003-62), goat anti-FOXP2 (1:500, Abcam ab1307), rabbit anti-melanopsin (1:1000, Advanced Targeting Systems AB N38), goat anti-choline acetyltransferase (1:500, Millipore AB144P), rabbit anti-GABA (1:2000, Sigma-Aldrich A2052), goat anti- GLYT1 (1:25, Santa Cruz Biotechnology sc-16703), rabbit anti-VIP (1:1000, ImmunoStar 20077), rat anti-SST (1:10, Millipore MAB354).

Techniques: Labeling

Journal: Frontiers in Neural Circuits

Article Title: Intersectional Strategies for Targeting Amacrine and Ganglion Cell Types in the Mouse Retina

doi: 10.3389/fncir.2018.00066

Figure Lengend Snippet: The Sst-FlpO driver targeted both RGCs and ACs. (A) (i) To identify targeted RGCs, the Sst-FlpO driver was crossed with Vglut2-Cre and Ai65 reporter mice. (ii) To identify targeted amacrine cells, the Sst-FlpO driver was crossed with Slc32a1-Cre and Ai65 mice. Scale bar, 50μm. (B) Staining for RGC markers (green). RGC constituents were probed with antibodies against Osteopontin, CART, FOXP2 and melanopsin in the Vglut2-Cre ; Sst-FlpO;Ai65 retinas. Scale bar, 20 μm. (C) Proportions of RGC types targeted in the Sst-FlpO driver. n = 7 retinas from 7 animals (6 litters). (D) Individual RGC types extracted from the Vglut2-Cre ; Sst-FlpO;Ai65 retinas. Flat-mount views (top) and side views (bottom) with ChAT (blue). Scale bar: 50 μm for the flat-mount view, 10 μm for the side view. (E–G) Amacrine cell types extracted from the UBC-CreER2;Sst-FlpO;Ai65 retinas, without tamoxifen administration. (E) Representative images of an SST-1 AC. (i) Flat-mount view of the soma and surrounding processes. (ii) Side view of the soma and surrounding “dendrite-like” and “axon-like” processes. (iii) Axon-like processes traveled across the IPL and ended at the INL border (iv) . An SST-1 AC was co-labeled with antibodies against GABA (v) and SST (vi) . (F) A starburst amacrine cell (SAC) in the GCL. Flat-mount view (top) and side view (bottom) with ChAT (blue). (G) SST-2 AC. Flat-mount view of the soma and surrounding processes. (ii) Side view of the soma and surrounding dendrite-like (white arrow) and axon-like processes (green arrow). (iii) Axon-like process end at the INL border. An SST-2 AC was co-labeled with antibodies against GABA (iv) and SST (v) . Scale bar for (E–G) : 50 μm for the flat-mount view, 10 μm for the side view.

Article Snippet: The primary antibodies used were as follows: rabbit anti-RFP (1:1000, Rockland 600-401-379), chicken anti-RFP Biotin conjugated (1:200, Rockland 600-906-379), guinea pig anti-RBPMS (1:500, PhosphoSolutions 1832-RBPMS), mouse anti-AP2 (3 μg/ml, DSHB 3B5), goat anti-Osteopontin (1:500, R&D 441-OP-050), rabbit anti-CART (1:1000, Phoenix pharmaceuticals H-003-62), goat anti-FOXP2 (1:500, Abcam ab1307), rabbit anti-melanopsin (1:1000, Advanced Targeting Systems AB N38), goat anti-choline acetyltransferase (1:500, Millipore AB144P), rabbit anti-GABA (1:2000, Sigma-Aldrich A2052), goat anti- GLYT1 (1:25, Santa Cruz Biotechnology sc-16703), rabbit anti-VIP (1:1000, ImmunoStar 20077), rat anti-SST (1:10, Millipore MAB354).

Techniques: Staining, Labeling

Journal: PLoS Biology

Article Title: A Toxoplasma gondii Pseudokinase Inhibits Host IRG Resistance Proteins

doi: 10.1371/journal.pbio.1001358

Figure Lengend Snippet: Peptides of ROP5 isoforms identified by MS after GST-Irga6 pull-down.

Article Snippet: Immunoreagents used in this study were: 3E2 mouse monoclonal antibody against ROP5 isoforms , anti-ROP18 rat antiserum (El Hajj and Dubremetz, unpublished), affinity-purified rabbit sera 87558 against (pT108)Irga6 and 87555 against (pT102)Irga6 , 165/3 rabbit antiserum , and 10E7 mouse monoclonal antibody against Irga6 , 141/1 rabbit antiserum against Irgb6 , 201.1 rat antiserum against T. gondii GRA7 (unpublished), and rabbit anti-calnexin antiserum (Calbiochem).

Techniques:

Journal: PLoS Biology

Article Title: A Toxoplasma gondii Pseudokinase Inhibits Host IRG Resistance Proteins

doi: 10.1371/journal.pbio.1001358

Figure Lengend Snippet: Glutathione Sepharose 4B beads loaded at 50 µg protein/100 µl 1∶1 bead suspension with bacterially expressed GST-IRG fusion proteins as bait were incubated at 4°C o/n with whole postnuclear lysates from RH-YFP strain T. gondii . Beads loaded with GST served as negative control. Bound proteins were separated by SDS-PAGE and monoclonal antibody 3E2 was used for detection of ROP5 in subsequent Western blot analysis. (A) In vitro pull-down of ROP5 with bacterially expressed and purified GST-Irga6. A single lysate, equivalent to 50×10 6 organisms per track, was used for GST-Irga6 and GST alone control beads (B) pull-down of ROP5 by Irga6 was markedly enhanced in the presence of 1 mM GDP (left hand blot). Lysates with and without GDP, equivalent to 25×10 6 organisms per track, were prepared from a single batch of T. gondii ; the right hand blot shows equal ROP5 signals from the supernatants of the pull-downs with GST alone with or without nucleotide. T. gondii calnexin provided the loading controls. (C) GST-Irgb6 and GST-Irgb10 also pulled down ROP5, though more weakly than GST-Irga6. One lysate, equivalent to 50×10 6 organisms per track, was used. All four tracks were run on a single gel; the vertical line indicates excision of irrelevant tracks.

Article Snippet: Immunoreagents used in this study were: 3E2 mouse monoclonal antibody against ROP5 isoforms , anti-ROP18 rat antiserum (El Hajj and Dubremetz, unpublished), affinity-purified rabbit sera 87558 against (pT108)Irga6 and 87555 against (pT102)Irga6 , 165/3 rabbit antiserum , and 10E7 mouse monoclonal antibody against Irga6 , 141/1 rabbit antiserum against Irgb6 , 201.1 rat antiserum against T. gondii GRA7 (unpublished), and rabbit anti-calnexin antiserum (Calbiochem).

Techniques: Suspension, Incubation, Negative Control, SDS Page, Western Blot, In Vitro, Purification, Control

Journal: PLoS Biology

Article Title: A Toxoplasma gondii Pseudokinase Inhibits Host IRG Resistance Proteins

doi: 10.1371/journal.pbio.1001358

Figure Lengend Snippet: Western blot of detergent lysates from IFNγ–induced L929 cells either uninfected (no) or infected with RHΔ rop18 , RHΔ rop5 , or wt RH–YFP T . gondii strains. Efficient phosphorylation of Irga6 is detectable with (A) anti-phosphothreonine 102 (anti–pT102) and (B) anti–phosphothreonine 108 (anti–pT108) antibodies only after RH–YFP–infection (black arrowheads). (C) Signals for ROP18 (upper panel) and ROP5 (lower panel) are shown in the three infected cell lysates. GRA7 (middle panel) in all infected cell lysates indicates essentially equivalent levels of infection. Calnexin provided loading controls.

Article Snippet: Immunoreagents used in this study were: 3E2 mouse monoclonal antibody against ROP5 isoforms , anti-ROP18 rat antiserum (El Hajj and Dubremetz, unpublished), affinity-purified rabbit sera 87558 against (pT108)Irga6 and 87555 against (pT102)Irga6 , 165/3 rabbit antiserum , and 10E7 mouse monoclonal antibody against Irga6 , 141/1 rabbit antiserum against Irgb6 , 201.1 rat antiserum against T. gondii GRA7 (unpublished), and rabbit anti-calnexin antiserum (Calbiochem).

Techniques: Western Blot, Infection, Phospho-proteomics

Journal: PLoS Biology

Article Title: A Toxoplasma gondii Pseudokinase Inhibits Host IRG Resistance Proteins

doi: 10.1371/journal.pbio.1001358

Figure Lengend Snippet: C57BL/6 mouse embryonic fibroblasts were induced with 3 U ml −1 IFNγ for 24 h and infected for 2 h with wt RH strain T. gondii , or RHΔ rop5 or RHΔ rop18 . Irga6 and Irgb6 positive vacuoles were identified microscopically on slides double stained with mouse mAb 10E7 (anti-Irga6) and 141/1 rabbit antiserum (anti-Irgb6) and appropriate fluorescent tagged species-specific secondary antibodies. (A, D) Left hand panels: fluorescent images of vacuoles from RH, RHΔ rop5 and RHΔ rop18 with Irga6 (A, green) and Irgb6 (D, red) fluorescence intensities indicated (white arrows). Right hand panels: phase contrast images. Nuclei stained with DAPI. The fluorescence intensities on vacuoles indicated with *** were too close to the background to be measured. (B, E) The percentage of vacuoles loaded with Irga6 (B) and Irgb6 (E) was determined by visual inspection of coded slides in two separate experiments (I and II, black and grey bars); (C, F) the fluorescence signal intensity of Irga6 (C) and Irgb6 (F) at individual vacuoles was estimated from experiment II (grey bars) of on coded slides using Volocity automatic software (see ). For technical reasons the automatic threshold for vacuole detection was set at 16,000 pixel units (horizontal line), which loses many vacuoles visibly but weakly loaded with Irga6 (see ). Bars across the intensity dot-plots indicate the arithmetic mean intensities of above-threshold vacuoles. N values above and below the threshold indicate the number of vacuoles recorded in these two categories. In , ** and *** indicate numbers of vacuoles loaded with Irga6 and Irgb6, respectively, that are significantly greater than RH at <0.01 and <0.001 levels, respectively . In , *** indicates differences significant at p <0.001 in loading intensity of Irga6 and Irgb6 between the indicated data sets by the Mann-Whitney U-test. NS, not significant.

Article Snippet: Immunoreagents used in this study were: 3E2 mouse monoclonal antibody against ROP5 isoforms , anti-ROP18 rat antiserum (El Hajj and Dubremetz, unpublished), affinity-purified rabbit sera 87558 against (pT108)Irga6 and 87555 against (pT102)Irga6 , 165/3 rabbit antiserum , and 10E7 mouse monoclonal antibody against Irga6 , 141/1 rabbit antiserum against Irgb6 , 201.1 rat antiserum against T. gondii GRA7 (unpublished), and rabbit anti-calnexin antiserum (Calbiochem).

Techniques: Infection, Staining, Fluorescence, Software, MANN-WHITNEY

Journal: PLoS Biology

Article Title: A Toxoplasma gondii Pseudokinase Inhibits Host IRG Resistance Proteins

doi: 10.1371/journal.pbio.1001358

Figure Lengend Snippet: C57BL/6 strain mouse embryonic fibroblasts were induced for 24 h with 10 U ml −1 IFNγ and infected with RH, RH-YFP, RHΔ rop5 , or RHΔ rop18 at an MOI of 0.3. After 24 h, 3 H-uracil was added and the assay harvested after a further 24 h culture. Growth inhibition was calculated as in relative to parallel cultures of similarly infected cells not induced with IFNγ. Growth of the RHΔ rop5 and RHΔ rop18 parasites was massively inhibited, while RH and RH-YFP were completely insensitive to growth inhibition. Error bars represent the standard deviations of 3 H-uracil incorporation from triplicate cultures.

Article Snippet: Immunoreagents used in this study were: 3E2 mouse monoclonal antibody against ROP5 isoforms , anti-ROP18 rat antiserum (El Hajj and Dubremetz, unpublished), affinity-purified rabbit sera 87558 against (pT108)Irga6 and 87555 against (pT102)Irga6 , 165/3 rabbit antiserum , and 10E7 mouse monoclonal antibody against Irga6 , 141/1 rabbit antiserum against Irgb6 , 201.1 rat antiserum against T. gondii GRA7 (unpublished), and rabbit anti-calnexin antiserum (Calbiochem).

Techniques: Infection, Inhibition

Journal: PLoS Biology

Article Title: A Toxoplasma gondii Pseudokinase Inhibits Host IRG Resistance Proteins

doi: 10.1371/journal.pbio.1001358

Figure Lengend Snippet: (A) IFNγ-stimulated L929 cells were left uninfected (no) or infected for 2 h with either of the two deletion strains, RHΔ rop18 , RHΔ rop5 , or the RHΔ rop5 strain transgenic for ROP5 isoforms RHΔ rop5 +A III /A III , or RHΔ rop5 +A III /B III (see for further details) or RH-YFP. Lysates were resolved on SDS-PAGE and stained on Western blots with anti-pT102 (upper panel) and anti-pT108 (middle panel) antibodies. Expression of ROP5 isoform A III in RHΔ rop5 +A III /A III resulted in detectably increased phosphorylation of Irga6 compared with RHΔ rop5- infected cells. In cells infected with RHΔ rop5 +A III /B III , phosphorylation of Irga6 was restored almost to wt levels. Arrows on the RHΔ rop5 tracks indicate what is probably residual kinase activity of ROP18 on Irga6 in the absence of ROP5 since these signals are absent from cells infected with RHΔ rop18 (and unpublished data). Signals of ROP18 serve as infection controls for the ROP18-expressing strains (lower panel). Calnexin serves as a loading control. (B, C, D, E) MEFs were induced with 3 U ml −1 IFNγ for 24 h and infected with wt RH, RHΔ rop5 , RHΔ rop5 +A III /A III , or RHΔ rop5 +A III /B III for 2 h in two independent experiments (I and II, black and grey bars) and positive vacuoles were identified with mAb 10E7 (anti-Irga6) (B) or 141/1 rabbit antiserum (anti-Irgb6) (D). Fluorescent signal intensities shown for Irga6 (C) and Irgb6 (E) for individual vacuoles were determined for visually identified positive vacuoles from experiment II as described in . Presentation of the data is as described in the legend to . As already seen in , ROP5 deletion and restoration had relatively modest effects on the proportion of vacuoles loaded with Irga6 (5B), while the effects on loading of Irgb6 (5D) were striking. Again as seen in , loss of ROP5 caused a highly significant increase in the intensity of Irga6 loading relative to which RHΔ rop5 +A III /A III -infection had little effect (5C). However, following infection with RHΔ rop5 +A III /B III the intensity of Irga6 loading was reduced to the levels of wild-type RH. As already seen in , loading intensities of the few wild-type RH vacuoles loaded with Irgb6 were the same as for the RHΔ rop5 vacuoles, as well as for the RHΔ rop5 +A III /A III and RHΔ rop5 +A III /B III vacuoles. Significances shown in are all relative to the number of Irga6 or Irgb6 loaded vacuoles of the RH strain. Statistical analysis of numerical data for (B) and (D) is given in . The significances of differences between the datasets shown in were estimated by the Mann-Whitney U-test.

Article Snippet: Immunoreagents used in this study were: 3E2 mouse monoclonal antibody against ROP5 isoforms , anti-ROP18 rat antiserum (El Hajj and Dubremetz, unpublished), affinity-purified rabbit sera 87558 against (pT108)Irga6 and 87555 against (pT102)Irga6 , 165/3 rabbit antiserum , and 10E7 mouse monoclonal antibody against Irga6 , 141/1 rabbit antiserum against Irgb6 , 201.1 rat antiserum against T. gondii GRA7 (unpublished), and rabbit anti-calnexin antiserum (Calbiochem).

Techniques: Infection, Transgenic Assay, SDS Page, Staining, Western Blot, Expressing, Phospho-proteomics, Activity Assay, Control, MANN-WHITNEY

Journal: PLoS Biology

Article Title: A Toxoplasma gondii Pseudokinase Inhibits Host IRG Resistance Proteins

doi: 10.1371/journal.pbio.1001358

Figure Lengend Snippet: (A) HSQC spectra of [ 13 C]-Met labeled Irga6 in the absence (black) or presence (blue) of Mg 2+ and GDP. Peaks that shift are indicated with arrows. Peaks that have been assigned by mutagenesis are indicated. (B) HSQC spectra of [ 13 C]-Met labeled Irga6 in the absence (black) or presence (red) of ROP5B I , with no Mg 2+ or GDP. Peaks that exhibit chemical shift changes upon addition of ROP5 are starred. (C) HSQC spectra of [ 13 C]-Met labeled Irga6 in the absence (blue) or presence (purple) of ROP5B I , with Mg 2+ and GDP. Peaks that are sensitive to only GDP are indicated with a black arrowhead, those sensitive to ROP5 only are starred, and those sensitive to both are indicated with an open arrow. (D) A surface representation of the GDP-bound Irga6 structure. Surface exposed methionines are indicated in red, and buried methionines are shown as red spheres. The bound Mg 2+ is shown as a green sphere and the bound GDP as sticks.

Article Snippet: Immunoreagents used in this study were: 3E2 mouse monoclonal antibody against ROP5 isoforms , anti-ROP18 rat antiserum (El Hajj and Dubremetz, unpublished), affinity-purified rabbit sera 87558 against (pT108)Irga6 and 87555 against (pT102)Irga6 , 165/3 rabbit antiserum , and 10E7 mouse monoclonal antibody against Irga6 , 141/1 rabbit antiserum against Irgb6 , 201.1 rat antiserum against T. gondii GRA7 (unpublished), and rabbit anti-calnexin antiserum (Calbiochem).

Techniques: Labeling, Mutagenesis

Journal: PLoS Biology

Article Title: A Toxoplasma gondii Pseudokinase Inhibits Host IRG Resistance Proteins

doi: 10.1371/journal.pbio.1001358

Figure Lengend Snippet: (A) In vitro pull-down of ROP5 with bacterially expressed and purified wt or mutant GST-Irga6 (at 25 µg protein/100 µl 1∶1 bead suspension) from RH-YFP strain T. gondii -lysates. GST was used as a negative control. Values reflect the mean ROP5 signal intensity and standard deviations of 2–3 pull-down experiments based on quantitated Western blots using 3E2 antibody (anti-ROP5). The signal given by GST was subtracted and the value obtained for each mutant normalized against wt Irga6. The values were correlated with a color in a spectrum ranging from green (100% of wt intensity) to red (0% of wt intensity). (B) Monomeric structural model of Irga6 based on Irga6-M173A in the GDP-bound form (PDB 1TQ6, ). The G-domain with the residues analyzed in (A) is depicted as spheres colored according to the values given in . Residues not assayed for inhibition of ROP5 binding are shown in blue. The rest of the protein is shown in ribbon structure (N-terminal helical domain in light blue, C-terminal helical domain in cyan). (C) Irga6-M173A-GDP in the same orientations as , with the residues contributing to the activation interface shown in red. (D) ROP5B I inhibits oligomerization of Irga6 in vitro. GTP-dependent oligomerization of 20 µM wt Irga6 was monitored by dynamic light scattering (DLS) in the presence or absence of equal or 4-fold molar excess amounts of ROP5B I pseudokinase domain protein. Values shown give the mean hydrodynamic radius of molecular complexes in solution. Monomeric Irga6 in the presence of GDP has a hydrodynamic radius of 3.4 nm. Oligomerization of wt Irga6 is almost completely inhibited by addition of a 4-fold molar excess of ROP5B I .

Article Snippet: Immunoreagents used in this study were: 3E2 mouse monoclonal antibody against ROP5 isoforms , anti-ROP18 rat antiserum (El Hajj and Dubremetz, unpublished), affinity-purified rabbit sera 87558 against (pT108)Irga6 and 87555 against (pT102)Irga6 , 165/3 rabbit antiserum , and 10E7 mouse monoclonal antibody against Irga6 , 141/1 rabbit antiserum against Irgb6 , 201.1 rat antiserum against T. gondii GRA7 (unpublished), and rabbit anti-calnexin antiserum (Calbiochem).

Techniques: In Vitro, Purification, Mutagenesis, Suspension, Negative Control, Western Blot, Inhibition, Binding Assay, Activation Assay

Journal: PLoS Biology

Article Title: A Toxoplasma gondii Pseudokinase Inhibits Host IRG Resistance Proteins

doi: 10.1371/journal.pbio.1001358

Figure Lengend Snippet: Bacterially expressed and purified wt GST-Irga6 (A) or the GST-Irga6-D164A mutant (B) that is unable to bind ROP5 (see ) and GST-ROP18-Ty were incubated in the absence or presence of purified pseudokinase domain protein ROP5B I in an in vitro kinase reaction supplemented with 1 mM ATP (left panels). Each kinase reaction was analyzed by Western blot for phosphorylation of Irga6 using anti-pT108 antibody. (A) Enhanced ROP18-mediated phosphorylation of wt Irga6 can be detected after addition of ROP5B I . (B) With Irga6-D164A mutant the presence of ROP5B I does not result in enhanced phosphorylation. Addition of BSA instead of ROP5B I served as a control for an effect of total protein in the reaction system (right panels). In the absence of ROP18 there was no detectable phosphorylation of either wt Irga6 or Irga6D164A (right panels). Vertical lines indicate excision of irrelevant tracks from the images of the two gels.

Article Snippet: Immunoreagents used in this study were: 3E2 mouse monoclonal antibody against ROP5 isoforms , anti-ROP18 rat antiserum (El Hajj and Dubremetz, unpublished), affinity-purified rabbit sera 87558 against (pT108)Irga6 and 87555 against (pT102)Irga6 , 165/3 rabbit antiserum , and 10E7 mouse monoclonal antibody against Irga6 , 141/1 rabbit antiserum against Irgb6 , 201.1 rat antiserum against T. gondii GRA7 (unpublished), and rabbit anti-calnexin antiserum (Calbiochem).

Techniques: Purification, Mutagenesis, Incubation, In Vitro, Western Blot, Phospho-proteomics, Control

Journal: PLoS Biology

Article Title: A Toxoplasma gondii Pseudokinase Inhibits Host IRG Resistance Proteins

doi: 10.1371/journal.pbio.1001358

Figure Lengend Snippet: ROP5 slows the rate of IRG oligomerization, holding it in a GDP-bound monomeric conformation that facilitates phosphorylation by ROP18. According to this model, ROP18 is unable to phosphorylate the GTP-bound activated IRG protein and thus unable to prevent oligomerization at the PVM. It is important to note that all steps are in equilibrium except for phosphorylation (starred). It is also important to note that both ROP5 and ROP18 associate with the PVM through their RAH domains, which, for clarity, we have not included in the figure.

Article Snippet: Immunoreagents used in this study were: 3E2 mouse monoclonal antibody against ROP5 isoforms , anti-ROP18 rat antiserum (El Hajj and Dubremetz, unpublished), affinity-purified rabbit sera 87558 against (pT108)Irga6 and 87555 against (pT102)Irga6 , 165/3 rabbit antiserum , and 10E7 mouse monoclonal antibody against Irga6 , 141/1 rabbit antiserum against Irgb6 , 201.1 rat antiserum against T. gondii GRA7 (unpublished), and rabbit anti-calnexin antiserum (Calbiochem).

Techniques: Phospho-proteomics