Journal: Circulation Research

Article Title: Hemoglobin Scavenger Receptor CD163 Mediates Interleukin-10 Release and Heme Oxygenase-1 Synthesis

doi: 10.1161/01.res.0000109414.78907.f9

Figure Lengend Snippet: Figure 3. Induction of HO-1 by Hb:Hp via a CD163 receptor IL-10 autocrine mechanism. A, Representative HO-1 and HO-2 immunoblots are shown from in vitro differentiated day 7 macrophages stimulated with Hb, Hp, Hb:Hp, or Hb:Hp plus blocking anti–IL-10, IgG control, or RM3/1 antibody. Induction of the HO-1 isomer was established relative to con- stitutive expression of the HO-2 isomer by probing blots with polyclonal antibody against HO-1, followed by stripping and reprobing with anti–HO-2. B, Lysates in A were subjected to quantification of intracellular HO-1 levels by HO-1–spe- cific ELISA and normalized with respect to total cell protein. Values shown repre- sent meanSEM. Results are represen- tative of 3 similar experiments.

Article Snippet: Neutralizing anti- human IL-10 polyclonal antibody and control goat IgG antibody were purchased from R&D Systems (Abingdon, UK).

Techniques: Western Blot, In Vitro, Blocking Assay, Control, Expressing, Stripping Membranes, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Negative Immunomodulatory Effects of Type 2 Porcine Reproductive and Respiratory Syndrome Virus-Induced Interleukin-1 Receptor Antagonist on Porcine Innate and Adaptive Immune Functions

doi: 10.3389/fimmu.2019.00579

Figure Lengend Snippet: PRRSV-induced IL-1Ra was not involved in suppression of IFN-γ-producing T lymphocytes in both (A) polyclonal and (B) recalled CSFV responses. The supernatants obtained from type 2 PRRSV or mock (MARC-145 cell lysate) were pretreated with anti-IL-1Ra Ab for 2 h prior to addition into the culture. PBL or PBMC were cultured with PHA, CSFV or controls for 48 h, in the presence of the pretreated supernatants. ± indicates presence/absence of indicated treatment within the culture. Data represents mean ± SD from 5 pigs. Statistical significance was analyzed using ANOVA followed by Tukey's test. * indicates significant difference at p < 0.05.

Article Snippet: In some experimental conditions, cells were pre-treated with final concentration of 10 ng/mL polyclonal goat anti-porcine IL-1Ra antibody (R&D system, clone AF780) or polyclonal goat IgG isotype control antibody (R&D system) at 2 h post-inoculation to neutralize PRRSV-induced IL-1Ra which was then cultured for another 22 h. To determine the effect of PRRSV-induced IL-1Ra on MoDC phagocytic activity, the antibody pre-treated MoDC (2 × 10 6 cell) were further incubated with inactivated E. coli -FITC (ThermoFisher Scientific) in complete RPMI at a MoDC: E. coli ratio of 1:50 for 10 min at 37°C.

Techniques: Cell Culture

Journal: bioRxiv

Article Title: Tumor- and Nerve-Derived Axon Guidance Molecule Promotes Pancreatic Ductal Adenocarcinoma Progression and Metastasis through Macrophage Reprogramming

doi: 10.1101/2023.10.24.563862

Figure Lengend Snippet: SEMA3D reprograms macrophages indirectly though ARF6 signaling and lactate production in PDA tumor cells a. Western blot analysis of the expression of ARF6 and active ARF6-GTP protein in CTRL-AP and SEMA3D-AP treated KPC tumor cells. b. qRT-PCR analysis of the expression of LDHB , LDHC , and LDHD genes in CTRL-AP or SEMA3D-AP treated KPC cells which were first pre-treated with anti- PLXND1 neutralizing antibody or isotype IgG control antibody. c. Lactate concentration in supernatant from KPC and KPCS cells treated with CTRL-AP or SEMA3D-AP, respectively. d,e. qRT-PCR analysis of gene expression markers for M1 (d) and M2 (e) polarization, respectively, in BMDM cells polarized with M1 or M2 polarizing cytokines and subsequently co-cultured with CTRL-AP or SEMA3D-AP treated KPC cells in the double-chamber.

Article Snippet: Neutralizing antibody against goat anti-PlexinD1 or goat IgG isotype control (Novus) was used during cell culture.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Control, Concentration Assay, Gene Expression, Cell Culture

Journal: bioRxiv

Article Title: Tumor- and Nerve-Derived Axon Guidance Molecule Promotes Pancreatic Ductal Adenocarcinoma Progression and Metastasis through Macrophage Reprogramming

doi: 10.1101/2023.10.24.563862

Figure Lengend Snippet: Nerve-derived SEMA3D impacts macrophage polarization in vitro a. ELISA analysis of secreted SEMA3D was performed on media cultured with and without DRG cells. b. Western blot analysis of the expression of ARF6 and active ARF6-GTP proteins in KPC cells treated with control and DRG-conditioning media, respectively. c. Western blot analysis of ARF6 and active ARF6-GTP proteins in KPC cells treated with CTRL-AP, SEMA3D-AP or the DRG- conditioning media supplemented with anti-PLXND1 neutralizing antibody or isotype IgG control. d. qRT-PCR analysis of gene expression of GLUT-1 , HK-2 , and LDHA in KPC cells treated with CTRL-AP, SEMA3D-AP or the DRG-conditioning media supplemented with anti-PLXND1 neutralizing antibody or isotype IgG control. e. qRT-PCR analysis of gene expression markers for M1 and M2 polarization in M1- and M2- polarized macrophages, as indicated, after co-culture with KPC or KPCS cells treated with control or DRG-conditioning media.

Article Snippet: Neutralizing antibody against goat anti-PlexinD1 or goat IgG isotype control (Novus) was used during cell culture.

Techniques: Derivative Assay, In Vitro, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot, Expressing, Control, Quantitative RT-PCR, Gene Expression, Co-Culture Assay