Journal: PLoS ONE

Article Title: Activation of ERAD Pathway by Human Hepatitis B Virus Modulates Viral and Subviral Particle Production

doi: 10.1371/journal.pone.0034169

Figure Lengend Snippet: HEK293T were transfected with pCiS, pCiM or pCiL, in the presence or absence of pCMVEDEM1. Controls (mock-transfected) cells were also included. Cell lysates were split in two and equal amounts of proteins were subjected to SDS-PAGE under reducing conditions, followed by Western blotting with anti-S (A) or anti-EDEM1 (B) Abs. Calnexin (Cnx) expression was used a total protein, gel loading control.

Article Snippet: The blots were incubated with mouse anti-preS1 (Santa Cruz, dilution 1/1000), rabbit anti-S (Europa Bioproducts, dilution 1/1000), goat anti-calnexin (Santa Cruz, dilution 1/1000), mouse anti-β actin (Abcam, dilution 1∶500), mouse anti-LC3 (NanoTools, dilution 1∶1000), or rabbit anti-EDEM1 (home-made, dilution 1/1000) antibodies (Abs) followed by anti-mouse (Santa Cruz, dilution, 1/10,000), anti-rabbit (Santa Cruz, dilution 1/10,000) or anti-goat (dilution 1/10,000) Abs conjugated to horseradish peroxidase.

Techniques: Transfection, SDS Page, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Activation of ERAD Pathway by Human Hepatitis B Virus Modulates Viral and Subviral Particle Production

doi: 10.1371/journal.pone.0034169

Figure Lengend Snippet: (A) HEK293T cells were transfected with pCiS in the presence or absence of pCMVEDEM1. Cell lysates were split in two and equal amounts of proteins were subjected to SDS-PAGE under reducing conditions, followed by Western blotting with anti-EDEM (upper panel) or anti-S (lower panel) Abs. Controls, mock-transfected cells were also included. Calnexin (Cnx) expression was used a total protein, gel loading control. (B) The relative band intensities were quantified and compared to control (considered 100%), using the “Quantity One” software from BioRad.

Article Snippet: The blots were incubated with mouse anti-preS1 (Santa Cruz, dilution 1/1000), rabbit anti-S (Europa Bioproducts, dilution 1/1000), goat anti-calnexin (Santa Cruz, dilution 1/1000), mouse anti-β actin (Abcam, dilution 1∶500), mouse anti-LC3 (NanoTools, dilution 1∶1000), or rabbit anti-EDEM1 (home-made, dilution 1/1000) antibodies (Abs) followed by anti-mouse (Santa Cruz, dilution, 1/10,000), anti-rabbit (Santa Cruz, dilution 1/10,000) or anti-goat (dilution 1/10,000) Abs conjugated to horseradish peroxidase.

Techniques: Transfection, SDS Page, Western Blot, Expressing, Software

Journal: PLoS ONE

Article Title: Activation of ERAD Pathway by Human Hepatitis B Virus Modulates Viral and Subviral Particle Production

doi: 10.1371/journal.pone.0034169

Figure Lengend Snippet: HEK293T were transfected with pCiS in the presence or absence of either EDEM1 siRNA (siEDEM1) or scrambled siRNA (siCtrl). Transfected cells were split in equal amounts and analysed for the efficiency of EDEM1 silencing by RT-real-time PCR (A) or the biosynthesis of the S, M and L proteins, by Western blotting using the anti-S Abs. When silencing of the over-expressed EDEM1 was investigated, the pCMVEDEM1 was added in the transfection mixture containing either pCiS or pCiS and EDEM1siRNA, as indicated (D, C). The expression level of EDEM1 (C) and S (D) was determined by Western blotting using the corresponding Abs. Calnexin (Cnx) level was used as total protein-gel loading control.

Article Snippet: The blots were incubated with mouse anti-preS1 (Santa Cruz, dilution 1/1000), rabbit anti-S (Europa Bioproducts, dilution 1/1000), goat anti-calnexin (Santa Cruz, dilution 1/1000), mouse anti-β actin (Abcam, dilution 1∶500), mouse anti-LC3 (NanoTools, dilution 1∶1000), or rabbit anti-EDEM1 (home-made, dilution 1/1000) antibodies (Abs) followed by anti-mouse (Santa Cruz, dilution, 1/10,000), anti-rabbit (Santa Cruz, dilution 1/10,000) or anti-goat (dilution 1/10,000) Abs conjugated to horseradish peroxidase.

Techniques: Transfection, Real-time Polymerase Chain Reaction, Western Blot, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Contrasting roles of SPARC-related granuloma in bacterial containment and in the induction of anti– Salmonella typhimurium immunity

doi: 10.1084/jem.20071734

Figure Lengend Snippet: SPARC −/− mice fail to develop an organized acute inflammatory reaction in response to S. typhimurium . SPARC +/+ (right) and SPARC −/− (left) mice were injected in the ear pinna with 10 7 CFU of attenuated S. typhimurium SL3261 AT. Immunohistochemical staining with anti-CD45 (top), anti-CD11b (middle), and anti– S. typhimurium (bottom) are shown on ear sections from mice killed 5 d after infection (brown stainings). Nuclei are stained in blue (hematoxylin). Images were merged by Photoshop to obtain whole ear sections. Original magnification of ear reconstructions was 5×, and of enlarged areas was 20×. Sections are representative of three independent experiments on four mice per group. Bar, 100 μm.

Article Snippet: Specimens were then incubated for 1 h with biotinylated primary antibodies or with rabbit anti– S. typhimurium antibody (ViroStat) or rabbit polyclonal antibody against mouse collagen type IV (AB756; Chemicon).

Techniques: Injection, Immunohistochemical staining, Staining, Infection

Journal: The Journal of Experimental Medicine

Article Title: Contrasting roles of SPARC-related granuloma in bacterial containment and in the induction of anti– Salmonella typhimurium immunity

doi: 10.1084/jem.20071734

Figure Lengend Snippet: Collagen IV staining reveals disorganized collagen deposition and more scattered infiltrate in SPARC −/− mice. SPARC +/+ (top row) and SPARC −/− (bottom row) mice were injected in the skin with 10 7 CFU of attenuated S. typhimurium SL3261 AT. Immunohistochemical staining with anti–collagen IV (brown) and counterstaining of nuclei with hematoxylin (blue) is shown on sections from mice killed 5 d after infection. Numbers show the original magnification of the sections. Boxes represent the magnified areas in the adjacent right images. Bars: (left) 50 μm; (middle) 25 μm; (right) 5 μm.

Article Snippet: Specimens were then incubated for 1 h with biotinylated primary antibodies or with rabbit anti– S. typhimurium antibody (ViroStat) or rabbit polyclonal antibody against mouse collagen type IV (AB756; Chemicon).

Techniques: Staining, Injection, Immunohistochemical staining, Infection

Journal: The Journal of Experimental Medicine

Article Title: Contrasting roles of SPARC-related granuloma in bacterial containment and in the induction of anti– Salmonella typhimurium immunity

doi: 10.1084/jem.20071734

Figure Lengend Snippet: Inflammatory cell infiltrate composition is very similar in SPARC +/+ and SPARC −/− mice. SPARC +/+ and SPARC −/− mice were injected intradermally in the back skin with 10 7 CFU of attenuated S. typhimurium SL3261 AT 48 and 72 h later, the sites characterized by an inflammatory reaction were excised, and cells were isolated by collagenase treatment. Cells were stained with fluorescent antibodies and analyzed by flow cytometry. (A) Y axes show percentage of marker-positive cells, x axes show the analyzed markers. (B) Y axes show total numbers of marker positive cells, x axes show the analyzed markers. Error bars represent the SD of three independent mice. One of three independent experiments is shown. SPARC −/− , black bars; SPARC +/+ , white bars.

Article Snippet: Specimens were then incubated for 1 h with biotinylated primary antibodies or with rabbit anti– S. typhimurium antibody (ViroStat) or rabbit polyclonal antibody against mouse collagen type IV (AB756; Chemicon).

Techniques: Injection, Isolation, Staining, Flow Cytometry, Marker

Journal: The Journal of Experimental Medicine

Article Title: Contrasting roles of SPARC-related granuloma in bacterial containment and in the induction of anti– Salmonella typhimurium immunity

doi: 10.1084/jem.20071734

Figure Lengend Snippet: DC migration from infected sites is inhibited by host-derived SPARC. (A) Endogenous DC migration is restored in SPARC −/− mice. 10 7 fluorescent latex (LX) microspheres/site were injected i.d. in mice in four different sites in the presence or absence of 10 7 CFU of S. typhimurium . Mice were killed at day 3 after treatment and the total number of IA/IE + cells carrying FITC-latex particles per DLNs was assessed after acquisition of the whole sample. The efficiency of latex + cell migration (the number of latex + cells in DLN of mice injected only with latex particles is considered as 100% of migration) to the DLNs is shown. The difference between efficiency of migration of latex + cells in the presence of bacteria (LX + BT) in SPARC +/+ (white bars) versus SPARC −/− (black bars) mice is highly significant (***, P < 0.0001). This is one experiment representative of three each with three mice per group. (B) BM-DCs from SPARC +/+ and SPARC −/− display the same kinetic of activation in vitro. BM-DCs were generated in GM-CSF–conditioned medium and activated with bacteria at a multiplicity of infection of 10:1 or with 1 μg of LPS. Up-regulation of costimulatory molecules was evaluated by flow cytometry at the indicated time points. One of two similar experiments is shown. (C) DC migration is inhibited by host-derived SPARC. BM-DCs from SPARC +/+ and SPARC −/− were loaded with green and red latex beads (LX), respectively. Mice were injected i.d. with BM-DCs from both strains in the presence or absence of bacteria (BT), and cell migration to the DLNs was assessed 2 d after injection. (left) The mean total efficiency of migration ± the SD of six different mice/group is shown. The difference in DC migration efficiency in the recipient SPARC +/+ mice in the presence of bacteria is not statistically significant (P > 0.05). (right) Representative dot plots showing total migrated cells from single DLNs in the different conditions. Recipient line shows the recipient background where BM-DCs from the two donor strains were injected in the presence or absence of bacteria (BT). (top) Dot plots show the recovery of Red LX laden SPARC −/− BM-DCs; (bottom) dot plots show the recovery of Green LX laden SPARC +/+ BM-DCs.

Article Snippet: Specimens were then incubated for 1 h with biotinylated primary antibodies or with rabbit anti– S. typhimurium antibody (ViroStat) or rabbit polyclonal antibody against mouse collagen type IV (AB756; Chemicon).

Techniques: Migration, Infection, Derivative Assay, Injection, Bacteria, Activation Assay, In Vitro, Generated, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Contrasting roles of SPARC-related granuloma in bacterial containment and in the induction of anti– Salmonella typhimurium immunity

doi: 10.1084/jem.20071734

Figure Lengend Snippet: Restored DC migration correlates with enhanced bacterial counts into DLN and increased T cell proliferation. (A) Bacterial load in SPARC −/− is higher than that in SPARC +/+ DLNs at days 2 and 3 after injection. DLNs of mice treated with bacteria were lysed with Na deoxycholate and plated on LB agar. Bacterial CFU were counted after overnight incubation at 37°C. The difference between bacterial counts at 48 and 72 h in DLN from SPARC +/+ versus SPARC −/− mice is highly significant (*, P < 0.01; **, P < 0.001). One of two similar experiments is shown. (B) OVA-expressing recombinant bacteria induce proliferation of OVA-specific CD4 T cells in SPARC −/− , but not in SPARC +/+ mice. SPARC WT and KO recipient mice were adoptively transferred with 3 × 10 6 CFSE labeled DO11.10 CD4 + T cells, and were injected i.d. 24 h later with recombinant S. typhimurium –expressing (SL-OVA) or not expressing (SL-pGEX) OVA. Proliferation of transferred T cells in DLNs was assessed 3 d after S. typhimurium injection. The number of CFSE + proliferating cells is shown ± the SD of 8 mice per group. The difference in T cell proliferation in SPARC +/+ versus SPARC −/− mice is highly significant (***, P < 0.001).

Article Snippet: Specimens were then incubated for 1 h with biotinylated primary antibodies or with rabbit anti– S. typhimurium antibody (ViroStat) or rabbit polyclonal antibody against mouse collagen type IV (AB756; Chemicon).

Techniques: Migration, Injection, Bacteria, Incubation, Expressing, Recombinant, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Contrasting roles of SPARC-related granuloma in bacterial containment and in the induction of anti– Salmonella typhimurium immunity

doi: 10.1084/jem.20071734

Figure Lengend Snippet: SPARC −/− mice are more resistant to low dose intradermally injected pathogenic S. typhimurium . Different amounts of bacteria, ranging from 10 3 to 10 7 CFUs, were injected intradermally and mouse survival was analyzed. Kaplan-Meyer survival curves are shown. Whereas at 10 4 to 10 7 CFU there was no difference in survival curves, at 10 3 CFU, the difference between SPARC +/+ and SPARC −/− mice was statistically significant (P < 0.01, Log-Rank). This is one representative of three different experiments, each performed with five mice per group.

Article Snippet: Specimens were then incubated for 1 h with biotinylated primary antibodies or with rabbit anti– S. typhimurium antibody (ViroStat) or rabbit polyclonal antibody against mouse collagen type IV (AB756; Chemicon).

Techniques: Injection, Bacteria

Journal: The Journal of Experimental Medicine

Article Title: Contrasting roles of SPARC-related granuloma in bacterial containment and in the induction of anti– Salmonella typhimurium immunity

doi: 10.1084/jem.20071734

Figure Lengend Snippet: SPARC −/− mice display reduced systemic bacterial dissemination. (A) Bacterial dissemination was evaluated in SPARC +/+ and SPARC −/− animals injected intradermally with 10 3 CFU of pathogenic S. typhimurium . DLNs, nonDLNs, and spleens were lysed and plated at different time points. Total bacterial count/organ is shown ± the SD of 12 different mice per group. The difference in bacterial recovery in SPARC +/+ and SPARC −/− animals is statistically significant (*, P < 0.01). (B) Intradermal immunization with attenuated S. typhimurium can protect SPARC −/− but not SPARC +/+ mice from subsequent challenge with pathogenic S. typhimurium . SPARC +/+ and SPARC −/− mice were vaccinated subcutaneously with 2 × 10 3 CFU of attenuated S. typhimurium or with PBS as a control. 2 wk after vaccination, all mice were challenged i.v. with 10 3 CFU of pathogenic S. typhimurium . Only SPARC-deficient mice vaccinated with bacteria survive upon challenge with virulent S. typhimurium . Kaplan-Meyer survival curves are shown. This is one representative of four different experiments each performed on five mice per group. The difference between the SPARC −/− -vaccinated group versus all the others is statistically significant (P < 0.01, Log-Rank).

Article Snippet: Specimens were then incubated for 1 h with biotinylated primary antibodies or with rabbit anti– S. typhimurium antibody (ViroStat) or rabbit polyclonal antibody against mouse collagen type IV (AB756; Chemicon).

Techniques: Injection, Control, Bacteria