cd147 emmprin  (R&D Systems)

Journal: Frontiers in Immunology

Article Title: Serum EMMPRIN/CD147 promotes the lung pre-metastatic niche in a D2A1 mammary carcinoma mouse model

doi: 10.3389/fimmu.2025.1568578

Figure Lengend Snippet: EMMPRIN promotes primary tumor growth, and D2A1 cells do not metastasize into the lung in this model. (A) Female BALB/c mice were orthotopically injected with D2A1-WT cells, with D2A1-KD cells (2x10 5 cells each), or with no tumor cells (healthy). Alternatively, mice injected with D2A1-WT cells were i.p. treated with three injections of anti-EMMPRIN antibody (m161-pAb, 10 μg/ml/100μL, every 7 days), and healthy mice were i.p. injected with recombinant EMMPRIN (400 ng/ml/100μL, 6 times, every 4 days). After 28 days, mice were sacrificed, and their lungs were stained. (A) Representative images for the H&E staining (upper panel) or the immunohistochemical staining with the anti-mCherry antibody (lower panel). Bar size is 200 μm and 50 μm in the insets. The images demonstrate a denser and more congested lung structure in mice injected with the D2A1-WT cells or with the recombinant EMMPRIN, while mCherry remains unstained, indicating the lack of tumor cells in the lungs of all groups. (B) Tumor volumes or (C) tumor weights at the end of the experiment indicate that high levels of EMMPRIN promote tumor growth. (D) Wet lung weight was not changed, suggesting that metastatic mass was not added. Data are presented as mean ± SEM, and analyzed using one-way ANOVA followed by Bonferroni’s post hoc test, or the non-parametric two-tailed Mann-Whitney t test (n=14 for the D2A1-WT group, n=9 for the D2A1-KD group, n=9 for the healthy group, n=9 for the D2A1-WT + m161-pAb group, n=9 for the Healthy + rec. EMMRPIN group, in 2–3 biological repetitions).

Article Snippet: To simulate the effect of soluble EMMPRIN on the lung niche, healthy mice (Healthy) were intraperitoneally (i.p.) injected with 400 ng/ml/100μL of mouse recombinant EMMPRIN (R&D systems, Minneapolis, MN, USA) once every four days, starting on day 4, for a total of 6 injections.

Techniques: Injection, Recombinant, Staining, Immunohistochemical staining, Two Tailed Test, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Serum EMMPRIN/CD147 promotes the lung pre-metastatic niche in a D2A1 mammary carcinoma mouse model

doi: 10.3389/fimmu.2025.1568578

Figure Lengend Snippet: EMMPRIN serum levels correlate with tumor weight. Female BALB/c mice were orthotopically injected with D2A1-WT cells (n=13-14), with D2A1-KD cells (n=8-9), or with no tumor cells (healthy mice, n=9-10), or treated with the anti-EMMPRIN antibody (m161-pAb, n=9) or recombinant EMMPRIN protein (n=9) as described in the legend of Figure 1 . Mice were sacrificed at day 28 and EMMPRIN levels were determined by ELISA in the (A-C) serum, (D-F) lung lysates, and (G, H) tumor lysates. Serum, lung and tumor EMMPRIN levels were increased in the group injected with D2A1-WT cells relative to the group injected with D2A1-KD cells or to healthy mice, and these levels could be inhibited using the anti-EMMPRIN antibody or recapitulated by injecting recombinant EMMPRIN. Data are presented as mean ± SEM. Significance between three groups is analyzed using one-way ANOVA followed by Bonferroni’s post-hoc test, and two groups are analyzed using the non-parametric two-tailed Mann-Whitney t test. (I) Serum EMMPRIN levels were correlated with the primary tumor weight using the non-parametric Spearman correlation test. Blue and red dots indicate mice implanted with D2A1-WT or D2A1-KD cells, respectively.

Article Snippet: To simulate the effect of soluble EMMPRIN on the lung niche, healthy mice (Healthy) were intraperitoneally (i.p.) injected with 400 ng/ml/100μL of mouse recombinant EMMPRIN (R&D systems, Minneapolis, MN, USA) once every four days, starting on day 4, for a total of 6 injections.

Techniques: Injection, Recombinant, Enzyme-linked Immunosorbent Assay, Two Tailed Test, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Serum EMMPRIN/CD147 promotes the lung pre-metastatic niche in a D2A1 mammary carcinoma mouse model

doi: 10.3389/fimmu.2025.1568578

Figure Lengend Snippet: EMMPRIN promotes the secretion of VEGF, MMP-9 and TGFβ in the lung PMN. Mice were injected with the different tumor cells and treatments as described in the legend of Figure 1 . The levels of (A-C) VEGF, (D-F) MMP-9, and (G-I) TGFβ, were evaluated in the lung lysates and normalized to the total protein (n=12–13 for the D2A1-WT group, n=8–9 for the D2A1-KD group, n=9 for the healthy group, n=9 for the D2A1-WT + m161-pAb group, n=9 for the Healthy + rec. EMMRPIN group). Data are presented as mean ± SEM. Three groups were analyzed using one-way ANOVA followed by Bonferroni’s post-hoc test, and two groups were compared using the non-parametric two-tailed Mann-Whitney t test. The three cytokines were increased in the group injected with D2A1-WT cells relative to the group injected with D2A1-KD cells or to healthy mice, and the involvement of EMMPRIN in their regulation was demonstrated by the use of the anti-EMMPRIN antibody or by injecting recombinant EMMPRIN.

Article Snippet: To simulate the effect of soluble EMMPRIN on the lung niche, healthy mice (Healthy) were intraperitoneally (i.p.) injected with 400 ng/ml/100μL of mouse recombinant EMMPRIN (R&D systems, Minneapolis, MN, USA) once every four days, starting on day 4, for a total of 6 injections.

Techniques: Injection, Two Tailed Test, MANN-WHITNEY, Recombinant

Journal: Frontiers in Immunology

Article Title: Serum EMMPRIN/CD147 promotes the lung pre-metastatic niche in a D2A1 mammary carcinoma mouse model

doi: 10.3389/fimmu.2025.1568578

Figure Lengend Snippet: EMMPRIN promotes angiogenesis in the lung PMN. Mice were injected with the different tumor cells and treatments as described in the legend of Figure 1 . (A) Representative images of the endothelial cell marker CD31 staining in the lungs, and (C) their quantitation (n=9 for the D2A1-WT group, n=8 for the D2A1-KD group, n=7 for the healthy group, n=7 for the D2A1-WT + m161-pAb group, n=9 for the Healthy + rec. EMMRPIN group). Bar size is 100 μm. (B) Representative images of bEND3 endothelial cell migration length after their incubation for 18 h with serum-starvation media containing 5µg of lung lysates from the different experimental groups. (D) Quantitation of the migration length (n=14 for the D2A1-WT group, n=10 for the D2A1-KD group, n=8 for the healthy group, n=9 for the D2A1-WT + m161-pAb group, n=7 for the Healthy + rec. EMMRPIN group). Bar size is 150 μm. Data are presented as mean ± SEM. Three groups were analyzed using one-way ANOVA followed by Bonferroni’s post-hoc test, and two groups were compared using the non-parametric two-tailed Mann-Whitney t test. The increased CD31 staining of endothelial cells in the mice implanted with the D2A1-WT cells and their longer migration distance suggests that they proliferate and migrate, two properties necessary for angiogenesis, more than the endothelial cells in healthy mice or mice implanted with the D2A1-KD cells. The importance of EMMPRIN to this process is exemplified by the results from the addition of h161-pAb or the injection of recombinant EMMPRIN.

Article Snippet: To simulate the effect of soluble EMMPRIN on the lung niche, healthy mice (Healthy) were intraperitoneally (i.p.) injected with 400 ng/ml/100μL of mouse recombinant EMMPRIN (R&D systems, Minneapolis, MN, USA) once every four days, starting on day 4, for a total of 6 injections.

Techniques: Injection, Marker, Staining, Quantitation Assay, Migration, Incubation, Two Tailed Test, MANN-WHITNEY, Recombinant

Journal: Frontiers in Immunology

Article Title: Serum EMMPRIN/CD147 promotes the lung pre-metastatic niche in a D2A1 mammary carcinoma mouse model

doi: 10.3389/fimmu.2025.1568578

Figure Lengend Snippet: EMMPRIN promotes neutrophil infiltration and fibroblast activation in the lung PMN. Mice were injected with the different tumor cells and treatments as described in the legend of Figure 1 . (A) Representative images of the neutrophil marker Ly6G and (B) of the fibroblasts activation marker αSMA staining in the lungs. Bar size is 150 μM. (C-E) Quantification of neutrophil infiltration, and (F-H) of fibroblast activation in the different experimental groups (n=9–10 for the D2A1-WT group, n=8–9 for the D2A1-KD group, n=8–10 for the healthy group, n=6 for the D2A1-WT + m161-pAb group, n=6–8 for the Healthy + rec. EMMRPIN group). Data are presented as mean ± SEM. Three groups were analyzed using one-way ANOVA followed by Bonferroni’s post-hoc test, and two groups were compared using the non-parametric two-tailed Mann-Whitney t test. Infiltrated neutrophils and fibroblasts were activated more in the lungs of mice implanted with the D2A1-WT cells than healthy mice or mice implanted with D2A1-KD cells. EMMPRIN’s involvement in these processes is also demonstrated by the results of the administration of the anti-EMMPRIN antibody and the injection of recombinant EMMPRIN.

Article Snippet: To simulate the effect of soluble EMMPRIN on the lung niche, healthy mice (Healthy) were intraperitoneally (i.p.) injected with 400 ng/ml/100μL of mouse recombinant EMMPRIN (R&D systems, Minneapolis, MN, USA) once every four days, starting on day 4, for a total of 6 injections.

Techniques: Activation Assay, Injection, Marker, Staining, Two Tailed Test, MANN-WHITNEY, Recombinant

Journal: Frontiers in Immunology

Article Title: Serum EMMPRIN/CD147 promotes the lung pre-metastatic niche in a D2A1 mammary carcinoma mouse model

doi: 10.3389/fimmu.2025.1568578

Figure Lengend Snippet: EMMPRIN regulates the expression of interstitial collagens. Mice were injected with the different tumor cells and treatments as described in the legend of Figure 1 . (A) Representative images of lung sections from the different experimental groups that were stained with Masson’s Trichrome. Blue fibers indicate presence of collagen. Bar size is 150 μm. (B-D) Quantification of the collagen fibers stained by Masson’s Trichrome (n=9 for the D2A1-WT group, n=8 for the D2A1-KD group, n=9 for the healthy group, n=9 for the D2A1-WT + m161-pAb group, n=7 for the Healthy + rec. EMMRPIN group). Total RNA was extracted from the lungs, reverse transcribed and amplified for the determination of mRNA expression of (E-G) collagen 1A, (H-J) collagen 3A (K-M) LOX (n=11–12 for the D2A1-WT group, n=8–9 for the D2A1-KD group, n=9–10 for the healthy group, n=8–9 for the D2A1-WT + m161-pAb group, n=8–9 for the Healthy + rec. EMMRPIN group). Data are presented as mean ± SEM. Three groups were analyzed using one-way ANOVA followed by Bonferroni’s post-hoc test, and two groups were compared using the non-parametric two-tailed Mann-Whitney t test. The ECM in mice implanted with the D2A1-WT cells was denser with more collagen fibers, and the expression of interstitial collagens Col1A and Col3A, as well as the crosslinking enzyme LOX were higher than the healthy mice or mice implanted with D2A1-KD cells. Again, EMMPRIN’s involvement was demonstrated by the results of administration of the m161-pAb and the injection of recombinant EMMPRIN.

Article Snippet: To simulate the effect of soluble EMMPRIN on the lung niche, healthy mice (Healthy) were intraperitoneally (i.p.) injected with 400 ng/ml/100μL of mouse recombinant EMMPRIN (R&D systems, Minneapolis, MN, USA) once every four days, starting on day 4, for a total of 6 injections.

Techniques: Expressing, Injection, Staining, Reverse Transcription, Amplification, Two Tailed Test, MANN-WHITNEY, Recombinant

Journal: Frontiers in Immunology

Article Title: Serum EMMPRIN/CD147 promotes the lung pre-metastatic niche in a D2A1 mammary carcinoma mouse model

doi: 10.3389/fimmu.2025.1568578

Figure Lengend Snippet: EMMPRIN induces the STAT3 and ERK1/2 signaling pathways. The activation of different signaling pathways was assessed in lung lysates obtained from healthy mice or healthy mice injected with recombinant EMMPRIN from previous experiments, by determining the phosphorylation of key proteins in these pathways. The phosphorylation of (A) STAT3 and (B) ERK1/2 were evaluated using Intracellular DuoSet ELISA kits (n=7). Additionally, representative images of western blot analyses of two repetitions of the phosphorylation of (C) Akt 1/2/3, (D) ERK1/2, and (E) IκBα, and their quantification (n=5). Recombinant EMMPRIN increased the phosphorylation of STAT3 and ERK1/2, but not of Akt or IκBα, suggesting that the PI3K and the NF-κB pathways do not mediate EMMPRIN’s effect on the lung PMN.

Article Snippet: To simulate the effect of soluble EMMPRIN on the lung niche, healthy mice (Healthy) were intraperitoneally (i.p.) injected with 400 ng/ml/100μL of mouse recombinant EMMPRIN (R&D systems, Minneapolis, MN, USA) once every four days, starting on day 4, for a total of 6 injections.

Techniques: Protein-Protein interactions, Activation Assay, Injection, Recombinant, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: (A) NF-kB activity in Hek-p65-luc cells transfected with human SOD1 WT , SOD1 G93A , or empty plasmid (mock) for 48h and treated with 0.5nM PPIA during the last 24h. Data are mean±SEM of n=3-4 independent experiments. Two-Way Anova followed by Tukey’s multiple comparisons test. (B) NF-kB activity in Hek-p65-luc cells transfected with siRNA control (siCTR) or against EMMPRIN (siEMN) for 72h, including 48h transfection with human SOD1 WT or SOD1 G93A and 24h treatment with 0.5nM PPIA. Data are mean±SEM of n=5 independent experiments. Two-Way Anova followed by Bonferroni’s multiple comparisons test. (C) NF-kB activity in Hek-p65-luc cells transfected with human SOD1 WT or SOD1 G93A plasmids for 48h and treated with a combination of 0.5nM PPIA and 0.5nM of control (CTR Ab) or anti-EMMPRIN (EMN Ab) antibody for the last 24h. Data are mean±SEM of n=3-4 independent experiments. Two-Way Anova followed by Tukey’s multiple comparisons test. For all experiments: All data were obtained by luciferase assay. Data are expressed as fold of Mock untreated cells. Relative luminescence units (RLU) were normalized on total proteins (TP, μg); *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Control, Luciferase

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: (A-D) Representative western blot of (A) lysates from 72h transfected Hek cells expressing human SOD1 WT , SOD1 G93A , or empty plasmid (mock) and relative quantification of PPIA (B) , low-glycosylated (37kDa) (C) and high-glycosylated (50kDa) (D) forms of EMMPRIN (EMN). Data are mean±SEM of n=3 independent experiments. One-Way Anova followed by uncorrected Fisher’s LSD test. (E-G) Representative western blot of (E) media from 72h transfected Hek cells expressing human SOD1 WT , SOD1 G93A , or empty plasmid (mock) and relative quantification of extracellular PPIA (ePPIA) (F) and soluble EMMPRIN (sEMN) (G) . Data are mean±SEM of n=3-4 independent experiments. One-Way Anova followed by uncorrected Fisher’s LSD test. (H) Luciferase assay for NF-kB activity in Hek-p65-luc cells transfected with human SOD1 WT or SOD1 G93A plasmids for 72h and treated with 0.5nM of control (CTR Ab) or anti-EMMPRIN (EMN Ab) antibody for the last 24h. Data are mean±SEM of n=3 independent experiments expressed as fold of Mock untreated cells. Two-Way Anova followed by Tukey’s multiple comparisons test. For all experiments: Target protein intensity was normalized on total transferred proteins (TTP). Relative luminescence units (RLU) were normalized on total proteins (TP, μg). *, p<0.05; **, p<0.01, ***, p<0.001.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation, Quantitative Proteomics, Luciferase, Activity Assay, Control

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: (A) Representative western blot and relative quantification of the high-glycosylated (50kDa) (B) and the low-glycosylated (37kDa) (C) forms of EMMPRIN (EMN) in the ventral horns of the lumbar spinal cord of Ntg (black dots) and SOD1 G93A (red dots) mice. Two-Way Anova: HG-EMN (interaction, p=0.084; age, p<0.0001; genotype, p<0.0001) followed by T-test for genotype comparison: HG-EMN (PS, p=0.4986; ON, **, p=0.0064; SY, ***, p=0.0005; ES, **, p=0.0052); LG-EMN (interaction, p=0.6868; age, p=0.825; genotype, p=0.831). One-Way Anova for linear trend: HG-EMN, p<0.0001; LG-EMN, p=0.0636. Target protein intensity was normalized on total transferred proteins (TTP). Data are mean±SEM of n=5 mice/stage. (D) Representative image of EMMPRIN (EMN, gray) expression in neuronal cells (NeuN, green) in the ventral horn of the lumbar spinal cord of NTg and SOD1 G93A mice at the onset of the disease. Large neurons, i.e motoneurons, are labeled by anti-EMMPRIN antibody. Experiments have been performed in n=3 mice/group. (E) Representative image of EMMPRIN (EMN, gray) expression in astrocytes (GFAP, green) in the ventral horn of the lumbar spinal cord of NTg and SOD1 G93A mice at an advanced symptomatic stage. (F) Representative image of EMMPRIN (EMN, gray) expression in microglia (Iba1, red) in the ventral horn of the lumbar spinal cord of NTg and SOD1 G93A mice at an advanced symptomatic stage. Of note, E and F are the same image but with split channels and performed appropriate merge. (G) Relative quantification of the percentage of astrocytes (GFAP) or microglia (Iba1) expressing EMMPRIN. Two-Way Anova (interaction, p=0.0003; age, p<0.0001; cell type, p<0.0001) followed by Bonferroni multiple comparison test for cell type comparison (***PS, p=0.0001; ON, SY, ES ****, p<0.0001). One-Way Anova for linear trend: GFAP, p<0.0001; Iba1, p<0.0001. Data are mean±SEM of n=4-5 mice/stage. For all experiment: PS, presymptomatic; ON, onset; SY, symptomatic; ES, end-stage. For D-F scale bar = 100μm.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Western Blot, Quantitative Proteomics, Comparison, Expressing, Labeling

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: (A) Representative image of primary cultures of NTg and SOD1 G93A astrocytes. Most of the cells present in the preparation are astrocytes (GFAP), and only a very small percentage of microglia (Iba1) cells is detected. Scale bar = 100μm. (B) Representative western blot and (C) relative quantification of EMMPRIN (EMN) in NTg astrocytes treated 24h with 0.5nM recombinant PPIA. Data are mean±SEM of n=3 independent experiments (dots) expressed as fold of NTg cells. Target protein intensity was normalized on total transferred proteins (TTP). *, p<0.05 by unpaired T-Test. (D) Relative quantification of factors released by NTg astrocytes after 24h of treatment with 0.5nM recombinant PPIA (N:P) compared to untreated NTg astrocytes (N:U). Red (upregulated), black (unchanged), blue (downregulated). Pooled media of n=5 preparations in duplicate. Data are expressed as fold of NTg untreated cells (N:U). *, p<0.05 versus untreated NTg astrocytes by unpaired T-Test. Relative fold change, p-value, difference and q-value are listed in Supplementary Table 1. (E) Pie chart of upregulated, unchanged or downregulated proteins in NTg astrocytes treated with PPIA compared to untreated NTg astrocytes. (F) Significant leading pathways related to the 43 upregulated proteins found in NTg astrocytes treated with PPIA. Proteins associated with the pathways are listed in Supplementary Table 2.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Western Blot, Quantitative Proteomics, Recombinant

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: Relative quantification of extracellular PPIA (ePPIA) (A) , EMMPRIN (EMN) (B) and the phosphorylated form pf p65/NF-kB (p-p65) (C) in NTg and SOD1 G93A astrocytes. Data are mean±SEM of n=3 independent experiments (dots) expressed as fold of NTg cells. Target protein intensity was normalized on total transferred proteins (TTP). *, p<0.05, **, p<0.01 by unpaired T-Test. (D) Relative quantification of factors released in 24h conditioned medium from SOD1 G93A astrocytes (G:U) compared to NTg astrocytes (N:U). Red (upregulated), black (unchanged), blue (downregulated). Pooled media of n=5 preparations in duplicate. Data are expressed as fold of Ntg untreated cells (N:U). *, p<0.05 versus untreated NTg astrocytes by unpaired T-Test. Relative fold change, p-value, difference and q-value are listed in Supplementary Table 3. (E) Pie chart of upregulated, unchanged or downregulated proteins in SOD1 G93A astrocytes compared to untreated NTg astrocytes. (F) Venn diagram showing 42 commonly secreted proteins between NTg astrocytes treated with PPIA (N:P) and SOD1 G93A astrocytes untreated (G:U). List of common proteins is present in Supplementary Table 4. (G) Significant leading pathways related to the 62 upregulated proteins found in SOD1 G93A astrocytes. Pathways found also in NTg astrocytes treated with PPIA are labelled with a star. Proteins associated with the pathways are listed in Supplementary Table 5.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Quantitative Proteomics

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: Relative quantification of extracellular PPIA (ePPIA) (A) , EMMPRIN (EMN) (B) and the phosphorylated form pf p65/NF-kB (p-p65) (C) in SOD1 G93A astrocytes treated 24h with 0.5nM anti-EMMPRIN (EMN Ab) or isotype control (CTR Ab) antibodies. Data are mean±SEM of n=3-4 independent experiments (dots) expressed as fold of NTg cells. Target protein intensity was normalized on total transferred proteins (TTP). *, p<0.05, **, p<0.01, ***, p<0.001 by unpaired T-Test. (D) Relative quantification of the 42 commonly secreted factors between untreated SOD1 G93A and NTg astrocytes treated with PPIA (List in Supplementary Table 4) released from SOD1 G93A astrocytes after 24h treatment with 0.5nM anti-EMMPRIN (G:E) or isotype control (G:C) antibodies. Red (upregulated), black (unchanged), blue (downregulated). Pooled media of n=4 preparations in duplicate. Data are expressed as fold of Ntg untreated cells (N:U). *, p<0.05; ** versus SOD1 G93A astrocytes treated with control antibody by unpaired T-Test. Relative fold change, p-value, difference and q-value are listed in Supplementary Table 6. (E) Pie chart of upregulated, unchanged or downregulated proteins in SOD1 G93A astrocytes treated with anti-EMMPRIN compared to control antibody treated SOD1 G93A astrocytes. (F) Significant leading pathways related to the 30 downregulated proteins found in SOD1 G93A astrocytes after anti-EMMPRIN treatment. Proteins associated with the pathways are listed in Supplementary Table 7.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Quantitative Proteomics, Control

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: (A) NF-kB activity in Hek-p65-luc cells transfected with human TDP-43 WT , TDP-43 A315T , or empty plasmid (mock) for 48h and treated with 0.5nM PPIA during the last 24h. Data are mean±SEM of n=6-8 independent experiments. Two-Way Anova followed by Tukey’s multiple comparisons test. (B) NF-kB activity in Hek-p65-luc cells transfected with siRNA control (siCTR) or against EMMPRIN (siEMN) for 72h, including 48h transfection with human TDP-43 WT or TDP-43 A315T and 24h treatment with 0.5nM PPIA. Data are mean±SEM of n=3 independent experiments. Two-Way Anova followed by Bonferroni’s multiple comparisons test. (C) NF-kB activity in Hek-p65-luc cells transfected with human TDP-43 WT or TDP-43 A315T plasmids for 48h and treated with a combination of 0.5nM PPIA and 0.5nM of control (CTR Ab) or 0.5nM anti-EMMPRIN (EMN Ab) antibody for the last 24h. Data are mean±SEM of n=3-6 independent experiments. Two-Way Anova followed by Tukey’s multiple comparisons test. For all experiments: All data were obtained by luciferase assay. Data are expressed as fold of Mock untreated cells. Relative luminescence units (RLU) were normalized on total proteins (TP, μg); *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Control, Luciferase

Journal: bioRxiv

Article Title: Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS

doi: 10.1101/2025.02.23.639749

Figure Lengend Snippet: Representative dot blot (A) and relative quantification ( B) of extracellular PPIA in the CSF of Ntg and TDP-43 A315T mice. One-Way Anova followed by Tukey’s multiple comparison test. *, p=0.0248. One-Way Anova for linear trend in TDP-43 A315T mice: p=0.1466. Representative western blot ( C ) and relative quantification of the high-glycosylated (50kDa) (D) and the low-glycosylated (37kDa) (E) forms of EMMPRIN (EMN) in the lumbar spinal cord of Ntg and TDP-43 A315T mice. One-Way Anova followed by Tukey’s multiple comparison test. **, p=0.0020; ***, p=0.0001. One-Way Anova for linear trend in TDP-43 A315T mice: HG-EMN, p=0.0051; LG-EMN, p=0.0054. For A-E, target protein intensity was normalized on total transferred proteins (TTP). Data are mean±SEM of n=3-4 mice/stage (6 months, onset; 10 months, early symptomatic; 13 months, late-symptomatic). (F) Representative image of EMMPRIN (EMN, gray) expression in neuronal cells (NeuN, green) in the ventral horn of the lumbar spinal cord of NTg and TDP-43 A315T mice at the onset of the disease (6 months). Large neurons, i.e motoneurons, are labeled by anti-EMMPRIN antibody. (G) Representative image of EMMPRIN (EMN, green) expression in astrocytes (GFAP, red) or microglia (Iba1, gray) in the ventral horn of the lumbar spinal cord of TDP-43 A315T mice at the early symptomatic stage (10 months). Please note that due to antigen retrieval we observed some Iba1 leaking signal in neurons (yellow arrow heads). For F, G: experiments have been performed in n=3 mice/group. Scale bar = 100μm.

Article Snippet: Antibodies used for immunoblot, (western/dot blot) (IB), immunofluorescence (IF) are as follows: rat monoclonal anti-EMMPRIN antibody (1:1000 for IB; 1:500 for IF; Bio-Rad, #MCA2283), rabbit polyclonal anti-EMMPRIN antibody (1:1000 for IB; ProteinTech, #11989-1-AP), rabbit polyclonal anti-PPIA antibody (1:5000 for IB; ProteinTech, #10720-1-AP), rabbit polyclonal anti-NF-kB p65 subunit antibody (1:1000 for IB; Santa Cruz Biotechnology, #sc-8008), rabbit polyclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3033), mouse monoclonal anti-phospo-NF-kB p65 (Ser536) antibody (1:1000 for IB; Cell Signaling Technology, #3036), rabbit polyclonal anti-Iba-1 antibody (1:500 for IF; Wako, #019-19741), mouse monoclonal anti-GFAP antibody (1:500 for IF; Cell Signaling Technology, #3670), mouse monoclonal anti-PPIA antibody (1:500 for IF; Invitrogen, #39-1100), rabbit monoclonal anti-NeuN antibody (1:500 for IF; Cell Signaling Technology, #12943), goat polyclonal anti-choline acetyltransferase antibody (1:500 for IF; Millipore, #AB114P), goat anti-mouse, anti-rabbit or anti-rat peroxidase-conjugated secondary antibodies (1:5000 for IB; Jackson Immunoresearch Lab), goat Alexa Fluor 647 or 555 or 488 anti-mouse or anti-rabbit or anti-rat or anti-goat fluorophore-conjugated secondary antibodies (1:500 for IF; Invitrogen).

Techniques: Dot Blot, Quantitative Proteomics, Comparison, Western Blot, Expressing, Labeling