Journal: Scientific Reports

Article Title: Tim-3 inhibits macrophage control of Listeria monocytogenes by inhibiting Nrf2

doi: 10.1038/srep42095

Figure Lengend Snippet: ( A , B ) Nrf2 mRNA ( A ) and protein ( B ) levels in RAW264.7 cells silenced for Nrf2 expression; ( C , D ) RAW264.7 cells treated with nonsense siRNA (NC) or Nrf2 siRNA were infected with CFSE-labeled L. monocytogenes for 2 h in the presence of 20 μg/ml of soluble sTim-3-Trx (sTim-3) or Trx control (Con), then the cells were analyzed for CFSE staining by flow cytometry; ( C ) shows a typical result, while ( D ) show the mean ± SD for triplicate wells. *p < 0.05. Similar results were obtained in three independent experiments. ( E ) RAW264.7 cells were incubated with medium or 20 μg/ml of sTim-3-Trx (sTim-3) or Trx control (Con) for 15 min, then were collected and analyzed for Nrf2 expression by Western blotting. ( F ) Normal RAW264.7 cells (NC) or RAW264.7 cells silenced for Tim-3 (Tim-3-si) were lysed and analyzed for Nrf2 expression by Western blotting. ( G ) Peritoneal macrophages isolated from WT or Tim-3-TG mice were analyzed for Nrf2 expression by Western blotting. ( H ) Peritoneal macrophages isolated from WT or Tim-3-TG mice were lysed, then Nrf2 protein was precipitated with specific antibody and examined for ubiquitination by Western blotting. ( I ) RAW264.7 cells were incubated with 20 μg/ml of sTim-3-Trx (sTim-3) or Trx control (Con) for 15 min, then were lysed and Nrf2 protein precipitated with specific antibody and examined for ubiquitination by Western blotting. ( J ) L monocytogenes - infected mice were injected intraperitoneally with 200 μg of sTim-3-Trx (sTim-3) or Trx control (Con) on the day of infection, then peritoneal macrophages were collected 3 days later and lysed, then Nrf2 protein was precipitated with specific antibody and examined for ubiquitination by Western blotting. ( K ) HEK-293-T cells transfected with a plasmid coding for Tim-3 or Tim-3 lacking the intracellular tail (Tim-3ΔIC) or the vector control were lysed and Nrf2 protein precipitated with specific antibody and examined for ubiquitination by Western blotting. In ( E – K ), the results shown are representative of those obtained in three independent experiments.

Article Snippet: For transient expression, the vector (pcDNA3.1) alone or plasmids coding for full length human Tim-3 (Tim3-pcDNA3.1) or a human Tim-3 construct lacking the tail region (hTim3-ΔIC-pcDNA3.1) were transfected into HEK-293-T cells (ATCC) for 48 h. For Nrf2 knockdown, the Nrf2 siRNA (Santa Cruz) were transfected into RAW264.7 cells for 48 h. For Tim-3 blockade studies, the cells were incubated for 12 h (for real-time PCR and FACS) or for 15 min (for Western blotting and co-immunoprecipitation studies) with 20 μg/ml of either “soluble Tim-3” (the extracellular domains of Tim-3 linked to His-tagged thioredoxin; sTim-3-Trx) or His-tagged Trx, both prepared as described previously , then were collected for analysis.

Techniques: Expressing, Infection, Labeling, Control, Staining, Flow Cytometry, Incubation, Western Blot, Isolation, Ubiquitin Proteomics, Injection, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: Tim-3 inhibits macrophage control of Listeria monocytogenes by inhibiting Nrf2

doi: 10.1038/srep42095

Figure Lengend Snippet: A549 cells expressing GFP-labeled Nrf2 were transfected with hTim3-pcDNA3.1 or vector control for 48 h, then were incubated with 20 μM menadione (VK) for 30 min and imaged and analyzed on a GE IN Cell Analyzer 2000. The activation of Nrf2 was expressed as the nucleus/cytosol GFP signal ratio. ( A ) Representative images showing VK-induced nuclear translocation of Nrf2. ( B ) Summarized data for the PCTACT in the presence of VK. The results, which are representative of those obtained in three independent assays, are the mean ± SD for triplicate wells; ***p < 0.001. ( C ) RAW264.7 cells were incubated with 20 μg/ml of sTim-3-Trx (sTim-3) or Trx control (Con) for 15 min, then nuclear and cytoplasmic extract were isolated to analyze for Nrf2 protein level by Western blotting.

Article Snippet: For transient expression, the vector (pcDNA3.1) alone or plasmids coding for full length human Tim-3 (Tim3-pcDNA3.1) or a human Tim-3 construct lacking the tail region (hTim3-ΔIC-pcDNA3.1) were transfected into HEK-293-T cells (ATCC) for 48 h. For Nrf2 knockdown, the Nrf2 siRNA (Santa Cruz) were transfected into RAW264.7 cells for 48 h. For Tim-3 blockade studies, the cells were incubated for 12 h (for real-time PCR and FACS) or for 15 min (for Western blotting and co-immunoprecipitation studies) with 20 μg/ml of either “soluble Tim-3” (the extracellular domains of Tim-3 linked to His-tagged thioredoxin; sTim-3-Trx) or His-tagged Trx, both prepared as described previously , then were collected for analysis.

Techniques: Expressing, Labeling, Transfection, Plasmid Preparation, Control, Incubation, Activation Assay, Translocation Assay, Isolation, Western Blot

Journal: Scientific Reports

Article Title: Tim-3 inhibits macrophage control of Listeria monocytogenes by inhibiting Nrf2

doi: 10.1038/srep42095

Figure Lengend Snippet: ( A ) Peritoneal macrophages isolated from WT or Tim-3-TG mice were examined for CD36 expression by flow cytometry. ( B , C ) RAW264.7 cells (NC) or Nrf2-knockdown (Nrf2-si) RAW264.7 cells were incubated with 20 μg/ml of soluble sTim-3-Trx (sTim-3) or Trx control (Con) for 12 h (for PCR) or for 24 h (for FACS), then were collected and analyzed for CD36 mRNA expression by real-time PCR ( B ) and for CD36 protein expression ( C ) by flow cytometry. ( D , E ) RAW 264.7 cells were incubated with 20 μg/ml of sTim-3-Trx (sTim-3) or Trx control (Con) and either DMSO (vehicle) or the deubiquitination inhibitor PR619 (20 μM) for 12 h (for PCR) or for 24 h (for FACS), then were collected and analyzed for CD36 mRNA expression by real-time PCR ( D ) and for CD36 protein expression ( E ) by flow cytometry. ( F , G ) RAW264.7 cells infected with CFSE-labeled L. monocytogenes were incubated with PBS or the CD36 inhibitor SSO (20 μM) for 12 h in the presence of 20 μg/ml of sTim-3-Trx (sTim-3) or Trx control (Con), then were collected and analyzed for CFSE staining in macrophages by flow cytometry. ( G ) Representative data showing CFSE staining of the macrophages in ( F ). In ( B , D , F ), the results are expressed as the mean ± SD of triplicate wells. *p < 0.05; **p < 0.01; ***p < 0.001. The results shown are representative of those obtained in three independent experiments.

Article Snippet: For transient expression, the vector (pcDNA3.1) alone or plasmids coding for full length human Tim-3 (Tim3-pcDNA3.1) or a human Tim-3 construct lacking the tail region (hTim3-ΔIC-pcDNA3.1) were transfected into HEK-293-T cells (ATCC) for 48 h. For Nrf2 knockdown, the Nrf2 siRNA (Santa Cruz) were transfected into RAW264.7 cells for 48 h. For Tim-3 blockade studies, the cells were incubated for 12 h (for real-time PCR and FACS) or for 15 min (for Western blotting and co-immunoprecipitation studies) with 20 μg/ml of either “soluble Tim-3” (the extracellular domains of Tim-3 linked to His-tagged thioredoxin; sTim-3-Trx) or His-tagged Trx, both prepared as described previously , then were collected for analysis.

Techniques: Isolation, Expressing, Flow Cytometry, Knockdown, Incubation, Control, Real-time Polymerase Chain Reaction, Infection, Labeling, Staining

Journal: Scientific Reports

Article Title: Tim-3 inhibits macrophage control of Listeria monocytogenes by inhibiting Nrf2

doi: 10.1038/srep42095

Figure Lengend Snippet: ( A ) Peritoneal macrophages isolated from WT and Tim-3-TG mice were analyzed for HO-1 mRNA levels by real-time-PCR. ( B ) Control or RAW264.7 cells silenced for Nrf2 as in were examined for HO-1 mRNA levels by real-time PCR. ( C ) RAW264.7 cells or Nrf2 knockdown RAW264.7 cells were incubated with 20 μg/ml of sTim-3-Trx (sTim-3) or Trx control (Con) for 12 h, then were collected and HO-1 mRNA levels examined by real-time PCR. ( D ) RAW264.7 cells were incubated with 20 μg/ml of sTim-3-Trx (sTim-3) or Trx control (Con) in the presence of PR619 (20 μM) or vehicle (DMSO) for 12 h, then were collected and HO-1 mRNA levels examined by real-time PCR. ( E ) RAW264.7 cells were incubated with 20 μg/ml of sTim-3-Trx (sTim-3) or Trx control (Con) in the presence or absence of an HO-1 inhibitor (Snpp-IX, 5 μM) or vehicle (DMSO) for 12 h, then were infected with L. monocytogenes (1 × 10 6 CFU) for 12 h and IL-1β mRNA levels examined by real-time PCR. ( F , G ) Tim-3 blockade promotes macrophages killing of L. monocytogenes . Raw264.7 cells were infected with L. monocytogenes ( F ) or CFSE-labeled L. monocytogenes ( G ) for 2 h, then washed, and incubated with Ampicillin to kill extracellularly located bacteria in the presence of sTim-3 or control protein for 24 h. Macrophages were either homogenized for CFU counting ( F ) or were imaged using confocal laser scanning microscope to examine the living bacteria (CFSE-bright spot) ( G ). The data are expressed as the mean ± SD for triplicate wells; **p < 0.01; ***p < 0.001, and are representative of the results obtained in three independent experiments.

Article Snippet: For transient expression, the vector (pcDNA3.1) alone or plasmids coding for full length human Tim-3 (Tim3-pcDNA3.1) or a human Tim-3 construct lacking the tail region (hTim3-ΔIC-pcDNA3.1) were transfected into HEK-293-T cells (ATCC) for 48 h. For Nrf2 knockdown, the Nrf2 siRNA (Santa Cruz) were transfected into RAW264.7 cells for 48 h. For Tim-3 blockade studies, the cells were incubated for 12 h (for real-time PCR and FACS) or for 15 min (for Western blotting and co-immunoprecipitation studies) with 20 μg/ml of either “soluble Tim-3” (the extracellular domains of Tim-3 linked to His-tagged thioredoxin; sTim-3-Trx) or His-tagged Trx, both prepared as described previously , then were collected for analysis.

Techniques: Isolation, Real-time Polymerase Chain Reaction, Control, Knockdown, Incubation, Infection, Labeling, Bacteria, Laser-Scanning Microscopy