Journal: bioRxiv

Article Title: Inhibition of IL34 unveils tissue-selectivity and is sufficient to reduce microglial proliferation in chronic neurodegeneration

doi: 10.1101/2020.03.09.976118

Figure Lengend Snippet: Activation of the CSF1R signalling pathway by IL-34 and CSF-1 and inhibition of IL-34-mediated cell growth using IL-34 neutralizing antibodies. [A] N13 microglia cell line was stimulated with IL-34 (50 or 100 ng/mL), CSF-1 (50 or 100 ng/mL) or LPS (1 μg/mL) for 5 or 10 minutes. Cell lysates were subjected to Western blotting which showed increased phosphorylation of CSF1R and downstream ERK1/2 and AKT after IL-34 and CSF-1 stimulation. [B] IL-34 neutralizing antibodies used in this study (mouse monoclonal IgG2A (v1.1, Ma et al., 2012), rat monoclonal IgG2A (MAB5195, R&D Systems) and sheep polyclonal IgG (AF5195, R&D Systems)) prevented IL-34-dependent cell growth of M-NFS-60 in a similar nanomolar range. n=3, data shown represent mean ± SEM, two-way ANOVA followed by Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: In white clear bottom 96-well plates 10 μL IL-34 antibody (mouse monoclonal IgG2A (v1.1 manufactured by Genscript, Ma et al., 2012), rat monoclonal IgG2A (MAB5195, R&D Systems) and sheep polyclonal IgG (AF5195, R&D Systems) and 10 μL IL-34 stimulus (R&D systems, 5195-ML-CF) were incubated at 37°C for 30 minutes before 80 μL M-NFS-60 cells (10 3 cells/well) were added.

Techniques: Activation Assay, Inhibition, Western Blot, Phospho-proteomics, Comparison

Journal: bioRxiv

Article Title: Inhibition of IL34 unveils tissue-selectivity and is sufficient to reduce microglial proliferation in chronic neurodegeneration

doi: 10.1101/2020.03.09.976118

Figure Lengend Snippet: Intracerebral IL-34 antibody administration resulted in reduced microglia proliferation in ME7 prion mice. [A] Mice infected with prion disease received a single intracerebral injection of mouse- or human-specific anti-IL-34 (sheep polyclonal IgG) and brains were analysed one week later. [B] Histological analysis of BrdU/Iba1-positive microglial cells in the cortex showed a reduction after treatment with a mouse-, but not with a human-specific antibody. Scale bar 100 μm. Naïve n=6, ME7 n=12, ME7 + isotype n=10, ME7 + anti-mIL-34 n=8, ME7 + anti-hIL-34 n=7, data shown represent mean ± SEM, two-way ANOVA followed by Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: In white clear bottom 96-well plates 10 μL IL-34 antibody (mouse monoclonal IgG2A (v1.1 manufactured by Genscript, Ma et al., 2012), rat monoclonal IgG2A (MAB5195, R&D Systems) and sheep polyclonal IgG (AF5195, R&D Systems) and 10 μL IL-34 stimulus (R&D systems, 5195-ML-CF) were incubated at 37°C for 30 minutes before 80 μL M-NFS-60 cells (10 3 cells/well) were added.

Techniques: Infection, Injection, Comparison

Journal: Cancer Research Communications

Article Title: Pancreatic Ductal Adenocarcinoma Cells Regulate NLRP3 Activation to Generate a Tolerogenic Microenvironment

doi: 10.1158/2767-9764.CRC-23-0065

Figure Lengend Snippet: CSF-1R engagement induces IL1β and IL18 release. A, Correlation between CSF-1R and NLRP3 expression in PDAC ( n = 96) from TGCA dataset. B, Spontaneous CSF-1 and IL34 secretion in Panc-1 cells at 72 hours ( n = 3). C, IL1β and IL18 secretion in THP-1 cells following stimulation with PDAC-conditioned media obtained from Panc-1 cells in presence of IgG (control), α-CSF-1, α-IL34, α-CSF-1/IL34 ( n = 3). D, Phosphorylation levels of PyK2 (Tyrosine 402) in THP-1 stimulated in D and E ( n = 3). E, IL1β and IL18 secretion from THP-1 cells following stimulation with PDAC-conditioned media obtained from Panc-1 cells in presence of α-CSF-1R and an CSF-1R inhibitor (AZD; n = 3). F, IL1β secretion from freshly isolated PBMCs from healthy donors ( n = 4) following stimulation with PDAC-conditioned media obtained from Panc-1 cells in presence of α-CSF-1R. Cytokine levels were measured by specific ELISAs. Data expressed as mean ± SEM; ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Article Snippet: For blockade of CSF-1 and IL34 in the CM, neutralizing antibodies against human α-CSF-1 and α-IL34 (R&D Systems) were used at 10 μg/mL and incubated overnight at 4°C.

Techniques: Expressing, Control, Phospho-proteomics, Isolation