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