Journal:

Article Title: Biological Characterization of Lipopolysaccharide from Treponema pectinovorum

doi: 10.1128/IAI.70.1.211-217.2002

Figure Lengend Snippet: IL-6, IL-8, and MCP-1 secretion by HGFs (Gin-7) challenged with purified T. pectinovorum ATCC 33768 LPS at 0.1, 1, 10, and 100 μg per ml. Supernatants were collected at 1, 3, 6, 12, 24, and 48 h after LPS challenge. Each cytokine concentration was determined by sequencial ELISA. The cells were stimulated with 5 × 109 bacteria. The bars denote the mean level of cytokine detected during 12-and 24-h culture periods from triplicate determinations.

Article Snippet: Following incubation of the serum, the system was developed with biotinylated goat polyclonal antibody specific for each cytokine (IL-1β, TNF-α, and IL-6; R & D Systems), streptavidin-alkaline phosphatase (Zymed, San Francisco, Calif.), and p -nitrophenylphosphate (Sigma).

Techniques: Purification, Concentration Assay, Enzyme-linked Immunosorbent Assay, Bacteria

Journal:

Article Title: Biological Characterization of Lipopolysaccharide from Treponema pectinovorum

doi: 10.1128/IAI.70.1.211-217.2002

Figure Lengend Snippet: Cytokine (IL-1β, TNF-α, and IL-6) pattern in C3H/HeN Tac-MTV (LPS responder) mice after LPS treatment. Mice (n = 3) were injected i.p. with 5, 0.5, and 0.05 μg of T. pectinovorum ATCC 33768 LPS with GalN as indicated in Materials and Methods. Serum samples were taken at 3, 8, and 12 h after LPS administration. Each cytokine concentration was determined by ELISA. Results are the means and standard deviations for three mice at 12 h.

Article Snippet: Following incubation of the serum, the system was developed with biotinylated goat polyclonal antibody specific for each cytokine (IL-1β, TNF-α, and IL-6; R & D Systems), streptavidin-alkaline phosphatase (Zymed, San Francisco, Calif.), and p -nitrophenylphosphate (Sigma).

Techniques: Injection, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Biological Characterization of Lipopolysaccharide from Treponema pectinovorum

doi: 10.1128/IAI.70.1.211-217.2002

Figure Lengend Snippet: Proinflammatory-cytokine (IL-1β, TNF-α, and IL-6) patterns in C3H/HeN Tac-MTV (LPS responder) mice were analyzed after i.p. challenge infection with live (108 cells) and heat-killed (H-K) (108 cells) T. pectinovorum ATCC 33768 and live E. coli O111 (105 cells) with and without GalN as described in Materials and Methods. Serum samples were taken at 3, 8, and 12 h postinfection. Each cytokine concentration was determined by sequential ELISA. Results are the means and standard deviations for three mice at 12 h.

Article Snippet: Following incubation of the serum, the system was developed with biotinylated goat polyclonal antibody specific for each cytokine (IL-1β, TNF-α, and IL-6; R & D Systems), streptavidin-alkaline phosphatase (Zymed, San Francisco, Calif.), and p -nitrophenylphosphate (Sigma).

Techniques: Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Monocytes Augment Bacterial Species- and Strain-Dependent Induction of Tissue Factor Activity in Bacterium-Infected Human Vascular Endothelial Cells

doi: 10.1128/IAI.69.5.2797-2807.2001

Figure Lengend Snippet: Contribution of endogenous IL-1 or TNF-α to enhanced TFA of monocyte-EC cocultures. Monolayers of 2 × 105 EC/well were incubated for 24 h with culture medium alone (no bacteria) or with ∼4 × 107 cells of the indicated bacteria, washed, and incubated for about 15 min with culture medium with (+) or without (−) 1 μg of anti-human TNF-α MAb/ml and/or 30 ng of rIL-1ra/ml. Then, these cells were cocultured for 6 h with 2 × 105 monocytes in the absence (−) or presence (+) of anti-human TNF-α MAb and/or rIL-1ra. TFA was determined as described in Materials and Methods. Values are the means ± standard deviations for eight separate experiments with EC from different donors. P values were <0.03 (∗)and <0.02 (∗∗) versus the same stimulus in the absense of rIL-1ra and anti-human TNF-α MAb (open bars). All mean values for TFA determined in the presence of both blocking compounds were not significantly different (P > 0.05; n = 8) from values measured in the presence of rIL-1ra alone.

Article Snippet: Neutralizing MAb against human tumor necrosis factor alpha (TNF-α) was purchased from R&D Systems.

Techniques: Incubation, Bacteria, Blocking Assay

Journal: Pathogens

Article Title: Analysis of Porcine Pro- and Anti-Inflammatory Cytokine Induction by S. suis In Vivo and In Vitro

doi: 10.3390/pathogens9010040

Figure Lengend Snippet: Cytokine release by peripheral blood mononuclear cells (PBMC) is S. suis dose-dependent, and IL-6 and TNF-α secretion is limited by IL-10. ( A ) Isolated porcine PBMC were stimulated with strain 10 or the acapsular mutant strain 10cpsΔEF at a PBMC-to-bacteria ratio of 10:1 (10 5 cfu/well) or 1:1 (1 × 10 6 cfu/well) in the presence of antibiotics. Cytokines were measured in cell culture supernatants after 42 h of incubation ( n = 5). Median and individual pigs shown. ( B ) Cytokine levels in supernatants of PBMC stimulated with S. suis strains 10 ( cps2 ), 13-00283-02 ( cps7 ) or 16085/3b ( cps9 ) at the PBMC-to-bacteria ratios 10:1 and 1:1 in the presence of antibiotics. Cytokines were measured after 42 h of incubation ( n = 3). ( C ) TNF-α and IL-6 production in presence of neutralizing anti-IL-10 antibody or with an isotype control after the stimulation of porcine PBMC with S. suis strain 10 at the same PBMC-to-bacteria ratios in the presence of antibiotics. IL-10 production was determined for reference. Cytokines from supernatants were measured after 42 h of incubation ( n = 9–11). Graphs show pooled data from four independent experiments with seven to ten week old piglets. For statistical analyses, Mann Whitney test was performed (** p < 0.01).

Article Snippet: For functional studies, recombinant porcine TNF-α (10 ng/mL) or neutralizing anti-TNF-α antibody (4 μg/mL; both R&D Systems Inc., Minneapolis, MN, USA) was added.

Techniques: Isolation, Mutagenesis, Bacteria, Cell Culture, Incubation, Control, MANN-WHITNEY

Journal: Pathogens

Article Title: Analysis of Porcine Pro- and Anti-Inflammatory Cytokine Induction by S. suis In Vivo and In Vitro

doi: 10.3390/pathogens9010040

Figure Lengend Snippet: Monocytes are the main producers of TNF-α in response to encapsulated S. suis. ( A ) Levels of TNF-α, IL-6 and IL-10 secreted by PBMC and CD14-positive monocytes after stimulation with the wt S. suis strain 10 ( n = 4–7) or the acapsular mutant strain 10cpsΔEF for 42 h in the presence of antibiotics. PBMC were cultivated at 1 × 10 6 cells/well (200 µL) and monocytes were cultivated at 5 × 10 4 cells/well. Bacteria were always used at 1 × 10 6 cfu/well, which is equivalent to a PBMC-to-bacteria ratio of 1:1. Graphs show pooled data of four independent experiments with seven to 10 week old piglets. Depending on the resulting cell yield of the monocyte separation, fractions were stimulated with S. suis strain 10 and/or 10cpsΔEF. ( B ) Density gradient-purified granulocytes (1 × 10 6 cells/well) from different donors, but pigs of the same herd ( n = 11) were stimulated under the same conditions as the PBMC and the monocytes. The cytokines were quantified from cell culture supernatant by ELISA. Graphs show data from two independent experiments with seven to 10 week old piglets. * p < 0.05; ** p < 0.01; *** p < 0.001 according to Mann Whitney test of control (medium) versus S. suis -stimulated samples.

Article Snippet: For functional studies, recombinant porcine TNF-α (10 ng/mL) or neutralizing anti-TNF-α antibody (4 μg/mL; both R&D Systems Inc., Minneapolis, MN, USA) was added.

Techniques: Mutagenesis, Bacteria, Purification, Cell Culture, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Control

Journal: Pathogens

Article Title: Analysis of Porcine Pro- and Anti-Inflammatory Cytokine Induction by S. suis In Vivo and In Vitro

doi: 10.3390/pathogens9010040

Figure Lengend Snippet: TNF-α does not contribute to bacterial killing in vitro, not even in the absence of S. suis -specific antibodies. ( A ) Kinetics of TNF-α induction and bacterial survival after infection of whole blood with 2 × 10 6 cfu/mL S. suis strain 10 ( n = 3). ( B ) Blood samples of piglets with low specific IgG ( n = 2–4) or blood cells reconstituted with colostrum-deprived serum (CDS, see Methods) (no specific IgG; n = 3–5) were infected with the viable S. suis strain 10 alone or additionally treated with porcine recombinant TNF-α (rTNF-α, 10 ng/mL) or neutralizing anti-TNF-α antibody (4 µg/mL). Bacterial survival was determined after 2 h. ( C ) Relative units per mL (RU/mL) of S. suis strain 10-specific IgG in sera of different groups of piglets. RU were calculated relative to a reference serum. Blood and sera were categorized based on the amount of S. suis strain 10-specific IgG: IgG-high > 60/mL RU ( n = 3); IgG-low < 60 RU/mL ( n = 4); No IgG = 0 RU/mL, using CDS with no detectable S. suis strain 10-specific IgG antibodies for the reconstitution of porcine blood cells. Graphs show data of four independent experiments with six to eight week old piglets.

Article Snippet: For functional studies, recombinant porcine TNF-α (10 ng/mL) or neutralizing anti-TNF-α antibody (4 μg/mL; both R&D Systems Inc., Minneapolis, MN, USA) was added.

Techniques: In Vitro, Infection, Recombinant

Journal: mSystems

Article Title: Association of intratumoral microbiome diversity with hepatocellular carcinoma prognosis

doi: 10.1128/msystems.00765-24

Figure Lengend Snippet: Bacterial LPS and LTA are present inside HCC tissues. (a) Representative images of multiplex immunofluorescence (multiplex IF, 20×) staining with an Opal kit. The bacteria were stained with antibodies against LPS and LTA, while CD45 and CD68 were used to identify immune cells. The area circled by the green curve indicates the IRR. Scale bars, 50 µm. (b) The boxplot of bacterial LPS and LTA intensity. Nine ROIs were selected.

Article Snippet: Staining was conducted manually using primary antibodies against the following markers: CD45 (anti-CD45, eBioscience #14-0459-82), CD68 (anti-CD68, Invitrogen #MA5-12407), LPS (anti-LPS, abcam #ab35654), and LTA (anti-LTA, Santa Cruz #sc57752).

Techniques: Multiplex Assay, Immunofluorescence, Staining, Bacteria

Journal: Antimicrobial Agents and Chemotherapy

Article Title: A short antimicrobial peptides family demonstrates efficacy to infection via a multimodal mechanism of action

doi: 10.1128/aac.01343-25

Figure Lengend Snippet: In vivo sepsis efficacy evaluation of SAMPs. ( A ) Bacterial quantification in the MDR- E. coli -infected liver, spleen, lung, kidney, and PLF after treatment with PBS, FA, Amcill-s, or KR (5 mg kg -1 ). ( B ) Body weight changes of mice during treatment. Relative organ index (organ mass/weight of mice before execution) of ( C ) liver, ( D ) spleen, ( E ) lung, and ( F ) kidney after treatment. The ( G ) TNF-α and ( H ) IL-6 levels in mouse serum after treatment. Data are presented as mean ± SD ( n = 6); * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.005. ( I ) Immunofluorescence (TNF-α and IL-6) images of liver, spleen, lung, and kidney after treatment, Scale bar: 100 μm. ( J ) Gram-stained images and ( K ) H&E-stained images of liver, spleen, lung, and kidney after treatment, Scale bar: 200 μm.

Article Snippet: 10× PBS solution was procured from BioSharp (China); yeast extract and tryptone was obtained from Thermo Fisher (USA); Agar powder, sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid disodium salt (EDTA-2Na), Triton X-100, ampicillin sodium, kanamycin sulfate, crystal violet, PI solution, 10× DNA loading buffer, 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI), calcein-AM/PI Dead/Live Cell Dual Staining Kit and Annexin V-FITC Apoptosis Detection Kit were obtained from Solarbio (China); 3,3'′-dipropylthiadicarbocyanine Iodide (DiSC 3 -5) was purchased from AAT BioQuest (USA); HEPES buffer was purchased from Yuanye (China); methanol, 25% aqueous glutaraldehyde solution, absolute ethanol, sodium chloride (NaCl), and potassium chloride (KCl) were obtained from Sinopharm Chemical Reagent Co., Ltd. (China); TFE was obtained from Sigma Aldrich (USA); 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and dimethyl sulfoxide (DMSO) were obtained from J&K Chemical Technology (China); Micro-MDA Assay Reagent Kit was purchased from KeyGEN Biotech (China); Cell Counting Kit-8 (CCK8) was purchased from GlpBio (USA); Bacterial Genomic DNA Extraction Kit was purchased from TIANGEN (China); Bacteria RNA Extraction Kit, HiScript III RT SuperMix for qPCR(+gDNA wiper) and Taq Pro Universal SYBR qPCR Master Mix were obtained from Vazyme (China); 2 kb DNA ladder (100-2000 bp) was obtained from Baiaolaibo (China); Mouse TNF-α ELISA kit and Mouse IL-6 ELISA kit were obtained from Shanghai Enzyme-linked Biotechnology Co., Ltd. (China); Anti-IL-6 antibody (ab233706) and goat Anti-rabbit IgG H&L (ab150078, Alexa Fluor 555) were obtained from Abcam (UK); Anti-TNF-α (D2D4) XP rabbit monoclonal antibody was obtained from Cell Signaling Technology (USA); All the mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (China) and kept in a specific pathogen-free environment.

Techniques: In Vivo, Infection, Immunofluorescence, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Nonproteolytic ubiquitination regulates chromatin occupancy by the NCoR/SMRT/HDAC3 corepressor complex in MCF-7 breast cancer cells.

doi: 10.1073/pnas.2502805122

Figure Lengend Snippet: Fig. 5. (A) Western blotting with anti-TRAF6 Ab to visualize the nuclear translocation of TRAF6 in MCF-7 cells treated with IL-1β for 5 min. WBs for HDAC2 and β-tubulin were run on the cytoplasmic and nuclear extracts as a control. (B) TRAF6 interaction with NCoR in IL-1β-treated MCF7 cells by immunoprecipitation with anti-NCoR Ab. (C) Graphic representation of the NCoR protein showing the putative TRAF6 interaction motif, and sequence alignment of similar motifs found in human RIP2, IRAK2, and NCoR across species. (D) Interaction between TRAF6 and the identified interaction motif in HEK293T cells transfected with Myc-TRAF6 and wild type (Left) or PxExxDAxAxxA mutant (Right) GAL4-NCoR516-811 plasmids. Immunoprecipitation was performed either with anti-Myc, anti-GAL4, or control IgG, and blotted with anti-GAL4, anti-TRAF6, or anti-Myc Ab. (E) ChIP experiment showing rapid and transient recruitment of TRAF6 and Ubc13 to the BMP7 promoter in response to IL-1β in MCF7 pretreated with E2 for 1 h. (F) Validation of siRNA efficiency in MCF7 cells. Cells are transfected with siRNA (siCtl, siTRAF6, or siUbc13) for 48 h and relative expression measured by RTqPCR, with normalization to b-actin. (G) BMP7 derepression by IL-1β, as measured by RT-PCR, requires both TRAF6 and Ubc13. MCF7 cells transfected with either control, TRAF6, or Ubc13 siRNA, were treated with E2 and/or IL-1β for 6 h. (H) Overexpression of dominant-negative TRAF6 or mutated TAB2 abrogates derepression by IL-1β in U2OS-ERα cells transiently transfected with BMP7promoter reporter and treated with E2 and vehicle (PBS) or IL-1β for 36 h. Luciferase activity was assayed and normalized to b-gal activity. (I) MEKK1’s ubiquitin-interacting motif (UIM) interacts with K63-linked, but not K48-linked, polyubiquitin chains. GST and GST-MEKK1-UIM proteins were expressed in bacteria, purified, and used for GST pulldown with recombinant K63- (Left) or K48-linked (Right) polyubiquitin chains, followed by western blotting with anti-ubiquitin or anti-GST Ab. (J) Ubc13 is required for accumulation of K63-linked polyubiquitin chains and for the recruitment of MEKK1 to the BMP7 promoter in MCF7 cells transfected with control or Ubc13 siRNA and pretreated with E2 for 1 h prior to treatment with IL-1β. ChIP was performed with anti-K63-ubiquitin or anti-MEKK1 Ab or protein A beads alone as control. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 vs. vehicle control, Student’s t test.

Article Snippet: Commercial antibodies used for western blotting, immunoprecipitation, and ChIP experiments include HDAC3 rabbit polyclonal antibody #ab7030 (Abcam); anti- HDAC3(H- 99) rabbit polyclonal antibody #sc- 11417 (Santa Cruz Biotechnology); anti- ubiquitin mouse monoclonal antibody (P4D1 clone, Cell Signaling Technology); anti- β- tubulin mouse monoclonal antibody #T0198 (Sigma- Aldrich); anti- HDAC2 rabbit polyclonal antibody #ab16032 (Abcam); anti- TAB2 mouse monoclonal antibody sc- 398188, goat polyclonal antibody sc- 11850, and rabbit polyclonal sc- 20756 (Santa Cruz Biotechnology); anti- K63 ubiquitin rabbit monoclonal antibody #05- 1308 (Millipore); anti- TRAF6 rabbit polyclonal antibody sc- 7221 (Santa Cruz Biotechnology); anti- Ubc13 rabbit polyclonal antibody #10243- 1- AP (ProteinTech); anti- Ubc13 (131- 148) rabbit polyclonal antibody #PA1- 41188 (Thermo Scientific); anti- FLAG mouse monoclonal antibody, clone M2, #F3165, and anti- Flag- HRP #A8592 (Sigma- Aldrich); antiMYC(9E10) mouse monoclonal antibody #sc- 40 (Santa Cruz Biotechnology); and anti- GST(56C1) mouse monoclonal antibody #sc- 80998 (Santa Cruz Biotechnology).

Techniques: Western Blot, Translocation Assay, Control, Immunoprecipitation, Sequencing, Transfection, Mutagenesis, Biomarker Discovery, Expressing, Reverse Transcription Polymerase Chain Reaction, Over Expression, Dominant Negative Mutation, Luciferase, Activity Assay, Ubiquitin Proteomics, Bacteria, Purification, Recombinant

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Nonproteolytic ubiquitination regulates chromatin occupancy by the NCoR/SMRT/HDAC3 corepressor complex in MCF-7 breast cancer cells.

doi: 10.1073/pnas.2502805122

Figure Lengend Snippet: Fig. 6. Graphical abstract representation of TRAF6 nuclear translocation and recruitment to the TRAF-interaction domain (TID) on NCoR/SMRT in response to IL-1β (red arrows). Within the NCoR/SMRT complex, GPS2 maintains TRAF6 activity under negative regulation (black solid line), while HDAC3 and TAB2 were identified as targets of TRAF6-mediated ubiquitination (green arrows). Dotted black arrows represent previously reported interactions between components of the NCoR/SMRT corepressor complex and associated TFs. RD1, RD2, and RD3 represent repressor domains; DAD is the deacetylase activation domain, while interaction with recruiting TFs occurs through the C’-terminal nuclear receptor–interacting domains I and II.

Article Snippet: Commercial antibodies used for western blotting, immunoprecipitation, and ChIP experiments include HDAC3 rabbit polyclonal antibody #ab7030 (Abcam); anti- HDAC3(H- 99) rabbit polyclonal antibody #sc- 11417 (Santa Cruz Biotechnology); anti- ubiquitin mouse monoclonal antibody (P4D1 clone, Cell Signaling Technology); anti- β- tubulin mouse monoclonal antibody #T0198 (Sigma- Aldrich); anti- HDAC2 rabbit polyclonal antibody #ab16032 (Abcam); anti- TAB2 mouse monoclonal antibody sc- 398188, goat polyclonal antibody sc- 11850, and rabbit polyclonal sc- 20756 (Santa Cruz Biotechnology); anti- K63 ubiquitin rabbit monoclonal antibody #05- 1308 (Millipore); anti- TRAF6 rabbit polyclonal antibody sc- 7221 (Santa Cruz Biotechnology); anti- Ubc13 rabbit polyclonal antibody #10243- 1- AP (ProteinTech); anti- Ubc13 (131- 148) rabbit polyclonal antibody #PA1- 41188 (Thermo Scientific); anti- FLAG mouse monoclonal antibody, clone M2, #F3165, and anti- Flag- HRP #A8592 (Sigma- Aldrich); antiMYC(9E10) mouse monoclonal antibody #sc- 40 (Santa Cruz Biotechnology); and anti- GST(56C1) mouse monoclonal antibody #sc- 80998 (Santa Cruz Biotechnology).

Techniques: Translocation Assay, Activity Assay, Ubiquitin Proteomics, Histone Deacetylase Assay, Activation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Renal collecting duct epithelial cells react to pyelonephritis-associated Escherichia coli by activating distinct TLR4-dependent and -independent inflammatory pathways.

doi: 10.4049/jimmunol.177.7.4773

Figure Lengend Snippet: FIGURE 7. Differential activation of TRAF2, TRAF6, and MAPKs by the UPEC AL511 strain in Lpsn and Lpsd MCD cells. Western blot analyses of the time-dependent expression of TRIF-, TRAF6-, and -actin-labeled, phosphorylated (p-) and total p38-la- beled, and ERK1/2-labeled bands (A) and TRAF2, -actin, and phosphorylated (p-) and total ASK1 and JNK (B) in cultured Lpsn and Lpsd MCDs incubated with AL511 (5 105 bacteria per well) for 3 h. C, Bars are mean ratio values (arbitrary units) of densitometric analyses of phosphorylated (p-) over total p38-, ERK1/ 2-, ASK1-, and JNK-labeled bands and TRAF2 or TRAF6 over -actin-labeled bands. Values are means SEM from three or four separate cultures of MCDs dissected from the kidneys of 2–4 mice in each group tested. , p 0.05 vs time 0 values.

Article Snippet: Abs against IkB- , p38, TRAF2, MAPK-activated protein kinase 2 (MAPKAPK-2), c-Jun (Cell Signaling Technology), TRIF (Imgenex), JNK, TRAF6, apoptosis signal regulatory kinase 1 (ASK1), ERK1/2 (Santa Cruz Biotechnology), and -actin (Sigma-Aldrich) were used to detect the corresponding Ags.

Techniques: Activation Assay, Western Blot, Expressing, Labeling, Cell Culture, Incubation, Bacteria

Journal: Nucleic acids research

Article Title: Metabolically-targeted dCas9 expression in bacteria.

doi: 10.1093/nar/gkac1248

Figure Lengend Snippet: Figure 2. Repression of GusA activity in E. coli with dCas9. (A) Schematic of the arabinose-regulated pBAD-dCas9. Large right-facing arrows represent genes and small right-facing arrows represent promoters. CmR, chloramphenicol resistance gene; pTet, promoter from the tetracycline resistance gene; pBAD, arabinose-inducible promoter. (B) Schematic of pGreg-dCas9 with the E. coli gus operon (not to scale) depicted on top with dashed line indicating the gusA regulatory region cloned upstream of dCas9. O1 and O2, GusR operator binding sites; pGusA, promoter from the gusA regulatory region. (C) Expression of dCas9 from E. coli harbouring pBAD-dCas9 under the indicated conditions assessed by western blot with an anti-Cas9 antibody. (D) Expression of dCas9 under the indicated conditions with E. coli harbouring pBad-dCas9 or pGreg-dCas9 assessed by western blots with an anti-Cas9 antibody. The positive control is 5 ng of purified Streptococcus pyogenes Cas9. (E) Impact of tiling sgRNAs along the length of the gusA gene on GusA activity. Plot of GusA activity (left) in control strains expressing dCas9 without an sgRNA and in the absence or presence of 1 mM pNPG. Plot of GusA activity (right) in dCas9-sgRNA strains for 74 individual sgRNAs targeted to the upstream or coding region of GusA on either the template (orange circles) or non-template (purple diamonds) strand. sgRNAs 349, 373, 451 and 980 used in later experiments are indicated. (F) Schematic of sgRNA multi-array to express sgRNA349, sgRNA451, sgRNA373 and sgRNA980. Right-facing coloured arrows represent different promoters for each sgRNA. H, sgRNA handle sequence; T, sgRNA terminator sequence. Note that promoter, handle and terminator sequences are different for each sgRNA. (G) Knockdown of E. coli GusA activity with pGreg-dCas9 and four different sgRNAs, or an array consisting of the same four sgRNAs. (H) Western blot of dCas9 and GusA expression in pGreg-dCas9 strains without (NG) or with the indicated sgRNAs. Uncropped images for gel images in panels (C), (D) and (H) are in Supplementary Figures S3, S4 and S5, respectively. For panels (E) and (G) data points are mean values from three biological replicates with whiskers indicating the mean plus or minus the standard deviation. *P < 0.05 calculated by Welch’s t-test.

Article Snippet: Anti-Cas9 rabbit polyclonal primary antibody (Rockland, 600-401-GK0S) was used for dCas9 blots, anti- -glucuronidase N-terminal rabbit polyclonal primary antibody (Sigma-Aldrich, G5420) was used for GusA blots, and anti-HA-tag rabbit monoclonal primary antibody (Invitrogen, MA5-27915) was used for GusR blots.

Techniques: Activity Assay, Clone Assay, Binding Assay, Expressing, Western Blot, Positive Control, Purification, Control, Sequencing, Knockdown, Standard Deviation

Journal: Nucleic acids research

Article Title: Metabolically-targeted dCas9 expression in bacteria.

doi: 10.1093/nar/gkac1248

Figure Lengend Snippet: Figure 5. dCas9 expression with pGreg-GusR-dCas9.7 is limited to GUS-positive bacteria. (A) Strategy to examine dCas9 expression in GUS-positive or GUS-negative bacteria by conjugation of pGreg-GusR-dCas9. CmR, chloramphenicol resistance. The dap- donor strain has a knockout of the dapA gene that is used for counter selection during conjugation. (B) Western blots of dCas9 expression in exconjugants with pGreg-GusR-dCas9.7 in the indicated strains grown in liquid media supplemented with 0.2% D-glucose (Glu), 0.2% L-arabinose (Ara) or 1 mM MetGluc (MG). The Cas9+ lane is 5 ng of purified Cas9. (C) As for panel (B) but with exconjugants harbouring pBAD-dCas9. (D) GusA activity assays with the indicated strains harbouring either pBAD-dCas9 (pBAD) or pGreg-GusR-dCas9.7 (pGreg) without (NG) or with sgRNA349. Open and filled circles represent data for pBAD, and open and filled triangles represent data for pGreg. (E) SN-38G processing by E. coli OBEAV1 cell extracts harbouring pGreg-GusR-dCas9 with (orange circles) or without (black circles) sgRNA349. SN-38G hydrolysis in the E. coli gusA strain is plotted for comparison. (F) Plot of SN-38G hydrolysis for the indicated cell extracts calculated using the data shown in panel (D). For panels (D), (E) and (F), data points are the mean of three biological replicates with the whiskers indicating the mean plus or minus the standard deviation. N.S. not significant, *P < 0.05, **P < 0.005 calculated by Welch’s t-test. Uncropped gel images in panels (B) and (C) are in Supplementary Figures S11 and S12, respectively, and uncropped gel images of the native microbiome species (to ensure no cross-reactivity occurred with the anti-Cas9 antibody) are in Supplementary Figure S13.

Article Snippet: Anti-Cas9 rabbit polyclonal primary antibody (Rockland, 600-401-GK0S) was used for dCas9 blots, anti- -glucuronidase N-terminal rabbit polyclonal primary antibody (Sigma-Aldrich, G5420) was used for GusA blots, and anti-HA-tag rabbit monoclonal primary antibody (Invitrogen, MA5-27915) was used for GusR blots.

Techniques: Expressing, Bacteria, Conjugation Assay, Knock-Out, Selection, Western Blot, Purification, Activity Assay, Comparison, Standard Deviation

Journal: Journal of Biological Chemistry

Article Title: Reduced Display of Tumor Necrosis Factor Receptor I at the Host Cell Surface Supports Infection with Chlamydia trachomatis

doi: 10.1074/jbc.m708422200

Figure Lengend Snippet: FIGURE 1. Reduced surface exposure of TNFR1 on cells infected with C. trachomatis. A, reduction of TNFR1 on infected cells. Hep-2 cells were infected with C. trachomatis (acute infection) or UV-inactivated C. trachomatis as indicated. 24 h post-infection TNFR1 was stained with a monoclonal antibody labeled with phycoerythrin and analyzed by FACS under non-permeabilizing conditions.B, time course of TNFR1 reduction. HEp-2 cells were mock-infected or infected with C. trachomatis for 2, 4, 10, 15, 24, or 30 h or infected with UV-inactivated C. trachomatis for 10 or 24 h and stained for TNFR1 under non-permeabilizing conditions. The mean fluorescence values for infected cells were calculated relative to mock-infected HEp-2 cells (100%). Error bars represent the mean S.D. of three independent experiments (**, p 0.001). C, cell-type independent reduction of TNFR1 by infection. HUVEC (left side), the monocytic cell line U937 (middle), and Jurkat T-cells (right side) were mock-infected or infected with C. trachomatis for 24 h, and surface-exposed TNFR1 was measured by FACS analysis. D, quantification of the experiments shown in C. The mean fluorescence of uninfected cells was set as 100%. Error bars represent the mean S.D. of three independent experiments (**, p 0.001). E, effect of infection on the surface exposure of other receptors. Surface exposure of TNFR2 on HEp-2 cells (bottom left), TRAIL-R2 on HUVEC (top left), FAS-R (CD95) on Jurkat T-cells (top right), and IL-8-R on HEp-2 cells (bottom right) was measured in infected and control cells asdescribedabove.Theblacklinesdemonstratetheisotypecontrolofmock-infectedcells,thedottedlinedemonstratestheisotypecontrolofinfectedcells,the light gray-filled area demonstrates the level of the receptor on the surface of mock-infected cells, and the dark gray-filled area shows the level of the receptors on the surface of infected cells in all FACS histograms of the figure.

Article Snippet: The following antisera or antibodies were used: rabbit polyclonal anti-human TNFR1 and rabbit polyclonal antihuman ERK1/2 (Santa Cruz Biotechnology); mouse monoclonal anti-human -actin or mouse monoclonal anti-human -tubulin (Sigma Aldrich); mouse monoclonal anti-human phospho-p44/42 ERK1/2 (Thr-202/Tyr-204); rabbit polyclonal anti-human phospho-MEK1/2 (Ser-217/221) or rabbit polyclonal anti-human MEK1/2 (Cell Signaling).

Techniques: Infection, Staining, Labeling, Fluorescence, Control

Journal: Journal of Biological Chemistry

Article Title: Reduced Display of Tumor Necrosis Factor Receptor I at the Host Cell Surface Supports Infection with Chlamydia trachomatis

doi: 10.1074/jbc.m708422200

Figure Lengend Snippet: FIGURE 2. Expression of TNFR1 and TNFR2 is increased in infected cells. A, increased mRNA levels of TNFR1 and TNFR2 in infected cells. HEp-2 cells were either mock-infected or infected with C. trachomatis, and the relative (rel.) amount of TNFR1 or TNFR2 mRNA was analyzed by quantitative real- time RT-PCR. The mean mRNA level of TNFR1 increased 3-fold; that of TNFR2 5-fold upon infection. Error bars show the S.D. of three independent experi- ments. B–D, level of TNFR1 is elevated in infected cells. FACS analysis of intra- cellular TNFR1 (B) and subsequent quantification (C) revealed an up-regula- tion of TNFR1 24 h post-infection. In B, the black line represents the isotype control from mock-infected cells, the dotted line represents the isotype con- trol from infected cells, the light gray-filled graph indicates TNFR1 in mock- infected cells, and the dark gray-filled graph shows TNFR1 from infected cells. Error bars in C represent the S.D. of three independent experiments (*, p 0.05). D, consistently, immunoblot analysis showed a strong increase in the amount of TNFR1 protein, and an additional band appeared above the main TNFR1 band (indicated with an arrow) in infected cells compared with mock- infected cells and cells infected with UV-inactivated bacteria.

Article Snippet: The following antisera or antibodies were used: rabbit polyclonal anti-human TNFR1 and rabbit polyclonal antihuman ERK1/2 (Santa Cruz Biotechnology); mouse monoclonal anti-human -actin or mouse monoclonal anti-human -tubulin (Sigma Aldrich); mouse monoclonal anti-human phospho-p44/42 ERK1/2 (Thr-202/Tyr-204); rabbit polyclonal anti-human phospho-MEK1/2 (Ser-217/221) or rabbit polyclonal anti-human MEK1/2 (Cell Signaling).

Techniques: Expressing, Infection, Quantitative RT-PCR, Control, Western Blot, Bacteria

Journal: Journal of Biological Chemistry

Article Title: Reduced Display of Tumor Necrosis Factor Receptor I at the Host Cell Surface Supports Infection with Chlamydia trachomatis

doi: 10.1074/jbc.m708422200

Figure Lengend Snippet: FIGURE 3. Reduction of surface TNFR1 on infected HEp-2 cells is controlled by the MEK-ERK pathway. A, activation of the MEK-ERK pathway correlates with the modification of TNFR1. HEp-2 cells were either mock-infected or infected with C. trachomatis for the indicated time points and subsequently subjected to immunoblot analysis for phosphorylated (p) ERK1/2p42/44, ERK1/2p42/44, phosphorylated MEK1/2, and MEK1/2. The increase in ERK1/2p42/44 and MEK1/2 phosphorylation correlated with an increase in the overall amount of TNFR1 protein and the appearance of an additional band, the TNFR1 band indicated with arrows. B, infected cells contain high amounts of phosphorylated ERK1/2p42/44 (red). Cells were stained with an antibody against phosphorylated ERK1/2p42/44 24 h post-infection and analyzed by confocal laser scanning microscopy. Cells containing chlamydial inclusions (marked with an arrow) also have phosphorylated ERK1/2p42/44. C–F, HEp-2 cells were infected for 24 h and treated with U0126 as indicated. Inhibition of ERK activation decreased infec- tion-induced TNFR1 levels and modification (C). Quantification of the band intensity from the Western blot, presented in C. Band intensity was calculated using the AIDA Image Analyzer Version 4.03 software (D). U0126 reversedtheinfection-inducedreductionofsurfaceTNFR1.TNFR1wasanalyzedinMe2SO-treatedcontrolcells, cells treated with 10 M U0126, and 25 M U0126 by FACS. The black line shows the isotype control from mock-infected cells, the dotted line shows the isotype control from infected cells, the light gray-filled graph shows the level of the receptor on the surface of mock-infected cells, and the dark gray-filled area shows the amount of the receptors on the surface of infected cells (E). Quantification of the experiment is shown in F. Error bars represent S.D. of three independent experiments.

Article Snippet: The following antisera or antibodies were used: rabbit polyclonal anti-human TNFR1 and rabbit polyclonal antihuman ERK1/2 (Santa Cruz Biotechnology); mouse monoclonal anti-human -actin or mouse monoclonal anti-human -tubulin (Sigma Aldrich); mouse monoclonal anti-human phospho-p44/42 ERK1/2 (Thr-202/Tyr-204); rabbit polyclonal anti-human phospho-MEK1/2 (Ser-217/221) or rabbit polyclonal anti-human MEK1/2 (Cell Signaling).

Techniques: Infection, Activation Assay, Modification, Western Blot, Phospho-proteomics, Staining, Confocal Laser Scanning Microscopy, Inhibition, Software, Control

Journal: Journal of Biological Chemistry

Article Title: Reduced Display of Tumor Necrosis Factor Receptor I at the Host Cell Surface Supports Infection with Chlamydia trachomatis

doi: 10.1074/jbc.m708422200

Figure Lengend Snippet: FIGURE 4. TNFR1 colocalizes with the inclusion membrane and the Golgi apparatus. A, HEp-2 cells were infected with C. trachomatis or mock-infected for 24 h. Cells were fixed and stained to visualize Golgi-58K (green) and TNFR1 (red). Chlamydia were visualized using the DNA dye DRAQ5 (blue, see arrowheads). Colocalization of Golgi with TNFR1 appears yellow in the overlay; that of TNFR1 with chlamydial inclusions appears purple. B, HEp-2 cells were infected with C. trachomatis or mock-infected for 24 h and treated with 2.5 M brefeldin A at the time of infection. Cells were fixed and stained to visualize Golgi-58K (red) and TNFR1 (green). Chlamydia were visualized using the DNA dye DRAQ5 (blue, see arrowheads). C, Golgi disruption causes down-regulation of surface-exposed TNFR1. Brefeldin A causes the reduction of TNFR1 from the surface in uninfected cells similar to the reduction in infected cells. HEp-2 cells were infected with C. trachomatis (Ctr) or mock-infected. Mock-infected cells were additionally treated with 2.5 M brefeldin A (BreA) as indicated. The distribution of TNFR1 on the surface was measured by FACS analysis. The mean fluorescence values for infected cells were calculated relative to mock-infected HEp-2 cells (100%). Error bars representthemeanS.D.ofthreeindependentexperiments(**,p0.001).D,releaseofsTNFR1byC. trachomatisinfectedcells.HEp-2cellswereinfectedwith C. trachomatisormock-infectedfor6,16,24,and48handtreatedwith25MU0126asindicated.ThesupernatantwastestedforthepresenceofsolubleTNFR1 by ELISA. Error bars represent the S.D of two different measurements (p 0.05). The graph is representative of three different biological replicates. E, release of sTNFR1 by C. trachomatis is inhibited by the metalloprotease inhibitor TAPI-1. HEp-2 cells were infected with C. trachomatis (Ctr) or mock-infected and treated with 1 or 10 M TAPI-1 for 24 h as indicated, and the presence of sTNFR1 was tested in the supernatant. Error bars represent the S.D. of three independent experiments. F, reduction of surface-exposed TNFR1 by TACE inhibition. Cells either left untreated (-) or infected with C. trachomatis (Ctr) or treated with 10 M TAPI-1 or infected and treated with both were analyzed by FACS for surface-exposed TNFR1. Shown are the results from one representative experiment of three.

Article Snippet: The following antisera or antibodies were used: rabbit polyclonal anti-human TNFR1 and rabbit polyclonal antihuman ERK1/2 (Santa Cruz Biotechnology); mouse monoclonal anti-human -actin or mouse monoclonal anti-human -tubulin (Sigma Aldrich); mouse monoclonal anti-human phospho-p44/42 ERK1/2 (Thr-202/Tyr-204); rabbit polyclonal anti-human phospho-MEK1/2 (Ser-217/221) or rabbit polyclonal anti-human MEK1/2 (Cell Signaling).

Techniques: Membrane, Infection, Staining, Disruption, Fluorescence, Enzyme-linked Immunosorbent Assay, Inhibition

Journal: Journal of Biological Chemistry

Article Title: Reduced Display of Tumor Necrosis Factor Receptor I at the Host Cell Surface Supports Infection with Chlamydia trachomatis

doi: 10.1074/jbc.m708422200

Figure Lengend Snippet: FIGURE5.Delayed signal transduction through TNFR1. A, delayed trans- location of TNF-induced NF-B (p65) in infected cells. HEp-2 cells were infected with C. trachomatis (bottom panel) or mock (top panel)-infected for 24 h. TNF was added, and cells were fixed at different time points and stained for NF-B (p65). Nuclear translocation of NF-B (p65) (red staining) is delayed in infected cells (green staining) compared with non-infected cells. B, time course of NF-B (p65) translocation induced by 50 g/ml TNF in infected and non-infected cells. C, TNF-induced IL-8 secretion is not affected in infected cells. HEp-2 cells were infected with C. trachomatis or mock-infected and 24 h post-infection treated with 40 ng/ml TNF or 25 M U0126. The release of IL-8 was measured by ELISA 8 or 24 h after the addition of TNF and/or U0126. Error bars represent the S.D. of three independent experiments.

Article Snippet: The following antisera or antibodies were used: rabbit polyclonal anti-human TNFR1 and rabbit polyclonal antihuman ERK1/2 (Santa Cruz Biotechnology); mouse monoclonal anti-human -actin or mouse monoclonal anti-human -tubulin (Sigma Aldrich); mouse monoclonal anti-human phospho-p44/42 ERK1/2 (Thr-202/Tyr-204); rabbit polyclonal anti-human phospho-MEK1/2 (Ser-217/221) or rabbit polyclonal anti-human MEK1/2 (Cell Signaling).

Techniques: Transduction, Infection, Staining, Translocation Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biological Chemistry

Article Title: Reduced Display of Tumor Necrosis Factor Receptor I at the Host Cell Surface Supports Infection with Chlamydia trachomatis

doi: 10.1074/jbc.m708422200

Figure Lengend Snippet: FIGURE6.DepletionofTNFR1orincreasingsTNFR1protectsC. trachoma- tis from bactericidal TNF effects. A, MAPK inhibition reduces and TNFR1 knockdown increases C. trachomatis infectivity. Chlamydia were grown in HEp-2 cells transfected with a siRNA against Luciferase, TNFR1 or TNFR2, or treated with U0126 (25 M) or TNF (25 ng/ml) as described under “Experi- mental Procedures.” The bacteria were then transferred to fresh, non-treated Hep-2cells,andtheinfectivitywasanalyzedbyimmunofluorescencestaining ofchlamydialinclusions.ShownareimmunofluorescencepicturesofHoechst stained HEp-2 cells (blue) and chlamydial inclusions (red). B, quantification of the experiments shown in A. The number of inclusions was determined by automated microscopy and expressed as a percentage of siLuciferase-trans- fected, -infected cells. Shown is the result of one representative of five inde- pendent experiments.

Article Snippet: The following antisera or antibodies were used: rabbit polyclonal anti-human TNFR1 and rabbit polyclonal antihuman ERK1/2 (Santa Cruz Biotechnology); mouse monoclonal anti-human -actin or mouse monoclonal anti-human -tubulin (Sigma Aldrich); mouse monoclonal anti-human phospho-p44/42 ERK1/2 (Thr-202/Tyr-204); rabbit polyclonal anti-human phospho-MEK1/2 (Ser-217/221) or rabbit polyclonal anti-human MEK1/2 (Cell Signaling).

Techniques: Inhibition, Knockdown, Infection, Transfection, Luciferase, Bacteria, Staining, Microscopy