human macrophage colony  (MedChemExpress)

Journal: Cell Death Discovery

Article Title: The chaperone GRP94 interacts with the proprotein convertase furin and regulates TGF-beta maturation in human primary M2 macrophages

doi: 10.1038/s41420-025-02866-2

Figure Lengend Snippet: PBMC of healthy volunteers differentiated in M2 macrophages and activated with IL-4 were treated with PU-WS13 (12.5 or 25 µM) during all the activation period. Western-blot analysis of pro-TGFβ expression in cell lysates ( A ) and active TGFβ secretion in supernatants ( B ) from M2 macrophages treated or not with PU-WS13 during 24 h (representative images, n = 10 for each experiment) ( C ) Furin enzymatic activity analysis in cell lysates of M2 macrophages treated or not 24 h with PU-WS13 25 µM ( n = 5). Fluorescence signals (expressed in relative fluorescence units, RFU) due to cleavage of the furin fluorogenic substrate, pERTKR-AMC, in cell lysates were recorded during 15 min ( D ) Western- blot analysis of MMP14 expression in M2 macrophages cell lysates (representative image, n = 10). (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Article Snippet: After adhesion, monocytes were differentiated in M2 macrophages with 100 ng/mL macrophage colony stimulating factor (M-CSF, 130-096-492, Miltenyi, Bergisch Gladbach, Germany) in RPMI medium with 10% heat inactivated FBS during 6 days, with differentiation medium renewal after 3 days.

Techniques: Activation Assay, Western Blot, Expressing, Activity Assay, Fluorescence

Journal: Cell Death Discovery

Article Title: The chaperone GRP94 interacts with the proprotein convertase furin and regulates TGF-beta maturation in human primary M2 macrophages

doi: 10.1038/s41420-025-02866-2

Figure Lengend Snippet: A Analysis by microscale thermophoresis of the specific binding between Nano-RED-labelled recombinant GRP94 (100 nM) and either recombinant furin (3.58 μM to 0.1 nM) or the negative control recombinant Hsp110 (2.33 μM to 0.07 nM). Changes in thermophoresis depending on ligand (furin or Hsp110) concentration from n = 3 experiments were plotted and expressed as ∆Fnorm (‰). B Staining and quantification of proximity ligation assays (Duolink™ PLA) of GRP94 and furin or the negative control Hsp110 in human PBMC-derived M2 macrophages ( n = 3 donors). Scale bars = 50 µm. The bar plot represents the mean number of spots for each cell analyzed +/− SEM. (**** p < 0.0001). C GRP94-furin co-immunoprecipitation in M2 macrophages. GRP94 was immunoprecipitated in M2 macrophages cell lysates using an anti-GRP94 antibody (IP:GRP94) or a non-relevant antibody (IgCT). Samples were analyzed by western-blot (representative images, n = 7).

Article Snippet: After adhesion, monocytes were differentiated in M2 macrophages with 100 ng/mL macrophage colony stimulating factor (M-CSF, 130-096-492, Miltenyi, Bergisch Gladbach, Germany) in RPMI medium with 10% heat inactivated FBS during 6 days, with differentiation medium renewal after 3 days.

Techniques: Microscale Thermophoresis, Binding Assay, Recombinant, Negative Control, Concentration Assay, Staining, Ligation, Derivative Assay, Immunoprecipitation, Western Blot

Journal: Cell Death Discovery

Article Title: The chaperone GRP94 interacts with the proprotein convertase furin and regulates TGF-beta maturation in human primary M2 macrophages

doi: 10.1038/s41420-025-02866-2

Figure Lengend Snippet: A Immunofluorescence staining of GRP94 (red channel) and LRRC33 (green channel) in human PBMC-derived M2 macrophages. Cells stained without primary antibodies were used as negative control. White arrows indicate colocalization areas. Scale bars = 20 µm. Representative images of n = 3 experiments. B Staining and quantification of proximity ligation assays (Duolink™ PLA) of GRP94 and LRRC33 or the negative control Hsp110 in human PBMC-derived M2 macrophages treated or not with PU-WS13 25 µM ( n = 6 donors). Scale bars = 20 µm. The bar plot represents the mean number of spots for each cell analyzed +/− SEM. (**** p < 0.0001).

Article Snippet: After adhesion, monocytes were differentiated in M2 macrophages with 100 ng/mL macrophage colony stimulating factor (M-CSF, 130-096-492, Miltenyi, Bergisch Gladbach, Germany) in RPMI medium with 10% heat inactivated FBS during 6 days, with differentiation medium renewal after 3 days.

Techniques: Immunofluorescence, Staining, Derivative Assay, Negative Control, Ligation

Journal: Biomolecules

Article Title: Interleukin-38 Ameliorates Atherosclerosis by Inhibiting Macrophage M1-like Polarization and Apoptosis

doi: 10.3390/biom15121741

Figure Lengend Snippet: IL-38 affects macrophage cholesterol loading and the expression of inflammatory factors. ( A ) Relative mRNA expression of TREM2 in macrophages. ( B ) Representative Oil Red O staining of lipid droplets in macrophages. ( C ) Quantitative analysis of Oil Red O-positive area percentage. ( D ) Western blot of ABCA1, ABCG1 and SR-A in macrophages. ( E ) Quantitative analysis of protein expression levels. ( F ) Relative mRNA expression of ABCA1 , ABCG1 , SR-A , and CD36 in macrophages. ( G ) Relative mRNA expression of inflammatory cytokines and chemokines in macrophages. n = 6 per group. * p < 0.05, ** p < 0.01, ns, not significant. Error bars represent mean ± SEM.

Article Snippet: After lysis of erythrocytes using erythrocyte lysis buffer and subsequent washing with phosphate-buffered saline (PBS), the remaining cells were cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 20 ng/mL macrophage colony stimulating factor (M-CSF; MedChemExpress Co., Ltd., Monmouth Junction, NJ, USA) for 7 consecutive days to induce macrophage differentiation.

Techniques: Expressing, Staining, Western Blot

Journal: Biomolecules

Article Title: Interleukin-38 Ameliorates Atherosclerosis by Inhibiting Macrophage M1-like Polarization and Apoptosis

doi: 10.3390/biom15121741

Figure Lengend Snippet: IL-38 affects macrophage M1-like polarization in mouse spleen and bone marrow-derived macrophages. ( A ) Representative flow cytometry dot plots of CD45 + CD11b + F4/80 + total macrophages in mouse spleen. ( B ) Quantitative analysis of the proportion of CD45 + CD11b + F4/80 + macrophages in splenic immune cells. ( C ) Flow cytometry dot plots of CD45 + CD11b + F4/80 + CD86 + M1-like macrophages in mouse spleen. ( D ) Quantitative analysis of the proportion of splenic CD45 + CD11b + F4/80 + CD86 + M1-like macrophages. ( E ) Flow cytometry dot plots of CD45 + CD11b + F4/80 + CD206 + M2-like macrophages in mouse spleen. ( F ) Quantitative analysis of the proportion of splenic CD45 + CD11b + F4/80 + CD206 + M2-like macrophages. ( G ) Representative immunofluorescence images of CD68 + CD86 + M1-like macrophages in atherosclerotic plaques. ( H ) Quantitative assessment of CD68 + CD86 + M1-like macrophages per plaque section. ( I ) Representative immunofluorescence images of CD68 + CD206 + M2-like macrophages in atherosclerotic plaques. ( J ) Quantitative assessment of CD68 + CD206 + M2-like macrophages per plaque section. n = 6 per group. * p < 0.05, *** p < 0.001, ns, not significant. Error bars represent mean ± SEM.

Article Snippet: After lysis of erythrocytes using erythrocyte lysis buffer and subsequent washing with phosphate-buffered saline (PBS), the remaining cells were cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 20 ng/mL macrophage colony stimulating factor (M-CSF; MedChemExpress Co., Ltd., Monmouth Junction, NJ, USA) for 7 consecutive days to induce macrophage differentiation.

Techniques: Derivative Assay, Flow Cytometry, Immunofluorescence

Journal: Biomolecules

Article Title: Interleukin-38 Ameliorates Atherosclerosis by Inhibiting Macrophage M1-like Polarization and Apoptosis

doi: 10.3390/biom15121741

Figure Lengend Snippet: IL-38 affects M1-like macrophage polarization in ox-LDL-stimulated BMDMs. ( A ) Flow cytometry dot plots of CD45 + CD11b + F4/80 + CD86 + M1-like macrophages in BMDMs. ( B ) Representative flow cytometry dot plots of CD45 + CD11b + F4/80 + CD206 + M2-like macrophages in BMDMs. ( C ) Quantitative analysis of the proportion of CD45 + CD11b + F4/80 + CD86 + M1-like macrophages. ( D ) Quantitative analysis of the proportion of CD45 + CD11b + F4/80 + CD206 + M2-like macrophages. ( E ) Relative mRNA expression of M1-like macrophage markers. ( F ) Relative mRNA expression of M2-like macrophage markers. n = 6 per group. * p < 0.05, *** p < 0.001, **** p < 0.0001, ns, not significant. Error bars represent mean ± SEM.

Article Snippet: After lysis of erythrocytes using erythrocyte lysis buffer and subsequent washing with phosphate-buffered saline (PBS), the remaining cells were cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 20 ng/mL macrophage colony stimulating factor (M-CSF; MedChemExpress Co., Ltd., Monmouth Junction, NJ, USA) for 7 consecutive days to induce macrophage differentiation.

Techniques: Flow Cytometry, Expressing

Journal: Biomolecules

Article Title: Interleukin-38 Ameliorates Atherosclerosis by Inhibiting Macrophage M1-like Polarization and Apoptosis

doi: 10.3390/biom15121741

Figure Lengend Snippet: Analysis of gene regulation of IL-38 on macrophages. ( A ) Volcano plot showing the number and expression trends of DEGs in BMDMs treated with IL-38. ( B ) Heatmap of DEGs in BMDMs treated with IL-38. ( C ) KEGG enrichment analysis of DEGs, listing the top 20 significantly enriched signaling pathways ( D ) GSEA plots showing the significantly enriched gene sets in foam cells compared with non-foam cells. Data for foam/non-foam cell signature genes were retrieved from the public GEO database (accession ID: GSE116239 ). ( E ) GSEA plots showing the significantly enriched gene sets in macrophages co-stimulated with ox-LDL and IL-38.

Article Snippet: After lysis of erythrocytes using erythrocyte lysis buffer and subsequent washing with phosphate-buffered saline (PBS), the remaining cells were cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 20 ng/mL macrophage colony stimulating factor (M-CSF; MedChemExpress Co., Ltd., Monmouth Junction, NJ, USA) for 7 consecutive days to induce macrophage differentiation.

Techniques: Expressing, Protein-Protein interactions

Journal: Biomolecules

Article Title: Interleukin-38 Ameliorates Atherosclerosis by Inhibiting Macrophage M1-like Polarization and Apoptosis

doi: 10.3390/biom15121741

Figure Lengend Snippet: IL-38 inhibits NF-κB pathway activity in mouse bone marrow-derived macrophages (BMDMs). Experimental groups: (1) NC (negetive control): BMDMs without any stimulation; (2) ox-LDL group: BMDMs stimulated with 50 μg/mL ox-LDL for 48 h; (3) ox-LDL+IL-38 group: BMDMs stimulated with 50 μg/mL ox-LDL plus 50 ng/mL IL-38 for 48 h; (4) ox-LDL+IL-38+NF-κB activator 1 group: BMDMs pre-stimulated with 30 ng/mL NF-κB activator 1 for 6 h, followed by co-stimulation with 50 μg/mL ox-LDL and 50 ng/mL IL-38 for 48 h. ( A ) Immunofluorescence staining of p65 in BMDMs. ( B ) Quantitative analysis of p65 nuclear translocation. ( C ) Western blot of p-p65 and total p65 in BMDMs. ( D ) Quantitative analysis of p-p65/p65 protein expression ratio. n = 6 per group. ** p < 0.01, *** p < 0.001,**** p < 0.0001, ns, not significant. Error bars represent mean ± SEM.

Article Snippet: After lysis of erythrocytes using erythrocyte lysis buffer and subsequent washing with phosphate-buffered saline (PBS), the remaining cells were cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 20 ng/mL macrophage colony stimulating factor (M-CSF; MedChemExpress Co., Ltd., Monmouth Junction, NJ, USA) for 7 consecutive days to induce macrophage differentiation.

Techniques: Activity Assay, Derivative Assay, Control, Immunofluorescence, Staining, Translocation Assay, Western Blot, Expressing

Journal: Biomolecules

Article Title: Interleukin-38 Ameliorates Atherosclerosis by Inhibiting Macrophage M1-like Polarization and Apoptosis

doi: 10.3390/biom15121741

Figure Lengend Snippet: IL-38 affects apoptosis of mouse macrophages in atherosclerotic lesions and in vitro models. ( A ) CD68/TUNEL double immunofluorescence co-localization staining of mouse aortic roots. ( B ) Quantitative analysis of apoptotic macrophages. ( C ) Western blot of Bcl2 and Bax in mouse macrophages. ( D ) Quantitative analysis of the Bcl2/Bax protein expression ratio. ( E ) Flow cytometry dot plots of macrophage apoptosis. ( F ) Quantitative analysis of total macrophage apoptosis rate and the proportion of cells in each apoptotic stage. n = 6 per group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Error bars represent mean ± SEM.

Article Snippet: After lysis of erythrocytes using erythrocyte lysis buffer and subsequent washing with phosphate-buffered saline (PBS), the remaining cells were cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 20 ng/mL macrophage colony stimulating factor (M-CSF; MedChemExpress Co., Ltd., Monmouth Junction, NJ, USA) for 7 consecutive days to induce macrophage differentiation.

Techniques: In Vitro, TUNEL Assay, Immunofluorescence, Staining, Western Blot, Expressing, Flow Cytometry

Journal: Biomolecules

Article Title: Interleukin-38 Ameliorates Atherosclerosis by Inhibiting Macrophage M1-like Polarization and Apoptosis

doi: 10.3390/biom15121741

Figure Lengend Snippet: IL-38 ameliorates the process of atherosclerosis by inhibiting the polarization of macrophages to M1-like phenotype and apoptosis of macrophages. It also inhibits NF-κB pathway activity and alleviates inflammation during atherosclerosis.

Article Snippet: After lysis of erythrocytes using erythrocyte lysis buffer and subsequent washing with phosphate-buffered saline (PBS), the remaining cells were cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 20 ng/mL macrophage colony stimulating factor (M-CSF; MedChemExpress Co., Ltd., Monmouth Junction, NJ, USA) for 7 consecutive days to induce macrophage differentiation.

Techniques: Activity Assay