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: For experiments, cells were seeded 24 h in growth conditions before being treated with GRP94 inhibitors, PU-WS13 or GRP94 inhibitor-1 (HY-112910, MedChemExpress), during 24 h in serum free DMEM.

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 Furin enzymatic activity analysis in cell lysates of MDA-MB-231 treated or not 24 h with GRP94 inhibitors, PU-WS13 12.5 or 25 µM or GRP94 inhibitor-1 (GRP94inh1) 2.5 or 5 µM ( n = 6). Fluorescence signal (expressed in relative fluorescence units, RFU) due to cleavage of the furin fluorogenic substrate, pERTKR-AMC, in cell lysates was recorded during 30 min. (**** p < 0.0001). Western- blot analysis of ( B ) active TGFβ secretion in supernatants (representative images, n = 7) and ( C ) pro-TGFβ expression in cell lysates (representative images, n = 5) from MDA-MB-231 treated or not 24 h with GRP94 inhibitors, PU-WS13 12.5 or 25 µM or GRP94 inhibitor-1 (GRP94inh1) 2.5 or 5 µM. (* p < 0.05; ** p < 0.01). D GRP94-furin co-immunoprecipitation in MDA-MB-231 cells. GRP94 was immunoprecipitated in MDA-MB-231 cell lysates using an anti-GRP94 antibody (IP:GRP94) or a non-relevant antibody (IgCT). Samples were analyzed by western-blot (representative images, n = 4). E Staining and quantification of proximity ligation assays (Duolink™ PLA) of GRP94 and furin or the negative control Hsp110 in MDA-MB-231 cells treated or not 24 h with GRP94 inhibitor-1 5 µM ( n = 3 experiments). Scale bars = 20 µm. The bar plot represents the mean number of spots for each cell analyzed +/− SEM. (**** p < 0.0001).

Article Snippet: For experiments, cells were seeded 24 h in growth conditions before being treated with GRP94 inhibitors, PU-WS13 or GRP94 inhibitor-1 (HY-112910, MedChemExpress), during 24 h in serum free DMEM.

Techniques: Activity Assay, Fluorescence, Western Blot, Expressing, Immunoprecipitation, Staining, Ligation, Negative Control

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: Impact of GRP94 inhibitors, PU-WS13 12.5 or 25 µM and GRP94 inhibitor-1 (GRP94inh1) 2.5 or 5 µM, on MDA-MB-231 cell migration was analyzed by wound healing assay. Wounds were realized in starved cells at confluency and treatments added in serum free medium. Using an Incucyte® S3 Live-Cell Analysis System, images of the wound areas were taken every hour during 24 h. Representative images at T = 0 h and T = 24 h of n = 6 experiments are shown in the upper panel with black bars highlighting the wound borders. Scale bars = 400 µm. Data are represented as relative wound densities over time in the lower panel ( n = 6) (**** p < 0.0001).

Article Snippet: For experiments, cells were seeded 24 h in growth conditions before being treated with GRP94 inhibitors, PU-WS13 or GRP94 inhibitor-1 (HY-112910, MedChemExpress), during 24 h in serum free DMEM.

Techniques: Migration, Wound Healing Assay, Cell Analysis

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: For experiments, cells were seeded 24 h in growth conditions before being treated with GRP94 inhibitors, PU-WS13 or GRP94 inhibitor-1 (HY-112910, MedChemExpress), during 24 h in serum free DMEM.

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

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: Macrophages were then activated with 20 ng/mL interleukine-4 (IL-4, 130-093-922, Miltenyi) in serum free Opti-MEM medium for 24 h. The GRP94 inhibitor PU-WS13 (HY-18680, MedChemExpress, Monmouth Junction, New Jersey, USA) was added 24 h before activation and during the whole activation period.

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 Furin enzymatic activity analysis in cell lysates of MDA-MB-231 treated or not 24 h with GRP94 inhibitors, PU-WS13 12.5 or 25 µM or GRP94 inhibitor-1 (GRP94inh1) 2.5 or 5 µM ( n = 6). Fluorescence signal (expressed in relative fluorescence units, RFU) due to cleavage of the furin fluorogenic substrate, pERTKR-AMC, in cell lysates was recorded during 30 min. (**** p < 0.0001). Western- blot analysis of ( B ) active TGFβ secretion in supernatants (representative images, n = 7) and ( C ) pro-TGFβ expression in cell lysates (representative images, n = 5) from MDA-MB-231 treated or not 24 h with GRP94 inhibitors, PU-WS13 12.5 or 25 µM or GRP94 inhibitor-1 (GRP94inh1) 2.5 or 5 µM. (* p < 0.05; ** p < 0.01). D GRP94-furin co-immunoprecipitation in MDA-MB-231 cells. GRP94 was immunoprecipitated in MDA-MB-231 cell lysates using an anti-GRP94 antibody (IP:GRP94) or a non-relevant antibody (IgCT). Samples were analyzed by western-blot (representative images, n = 4). E Staining and quantification of proximity ligation assays (Duolink™ PLA) of GRP94 and furin or the negative control Hsp110 in MDA-MB-231 cells treated or not 24 h with GRP94 inhibitor-1 5 µM ( n = 3 experiments). Scale bars = 20 µm. The bar plot represents the mean number of spots for each cell analyzed +/− SEM. (**** p < 0.0001).

Article Snippet: Macrophages were then activated with 20 ng/mL interleukine-4 (IL-4, 130-093-922, Miltenyi) in serum free Opti-MEM medium for 24 h. The GRP94 inhibitor PU-WS13 (HY-18680, MedChemExpress, Monmouth Junction, New Jersey, USA) was added 24 h before activation and during the whole activation period.

Techniques: Activity Assay, Fluorescence, Western Blot, Expressing, Immunoprecipitation, Staining, Ligation, Negative Control

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: Impact of GRP94 inhibitors, PU-WS13 12.5 or 25 µM and GRP94 inhibitor-1 (GRP94inh1) 2.5 or 5 µM, on MDA-MB-231 cell migration was analyzed by wound healing assay. Wounds were realized in starved cells at confluency and treatments added in serum free medium. Using an Incucyte® S3 Live-Cell Analysis System, images of the wound areas were taken every hour during 24 h. Representative images at T = 0 h and T = 24 h of n = 6 experiments are shown in the upper panel with black bars highlighting the wound borders. Scale bars = 400 µm. Data are represented as relative wound densities over time in the lower panel ( n = 6) (**** p < 0.0001).

Article Snippet: Macrophages were then activated with 20 ng/mL interleukine-4 (IL-4, 130-093-922, Miltenyi) in serum free Opti-MEM medium for 24 h. The GRP94 inhibitor PU-WS13 (HY-18680, MedChemExpress, Monmouth Junction, New Jersey, USA) was added 24 h before activation and during the whole activation period.

Techniques: Migration, Wound Healing Assay, Cell Analysis

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: Macrophages were then activated with 20 ng/mL interleukine-4 (IL-4, 130-093-922, Miltenyi) in serum free Opti-MEM medium for 24 h. The GRP94 inhibitor PU-WS13 (HY-18680, MedChemExpress, Monmouth Junction, New Jersey, USA) was added 24 h before activation and during the whole activation period.

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

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: For experiments, cells were seeded 24 h in growth conditions before being treated with GRP94 inhibitors, PU-WS13 or GRP94 inhibitor-1 (HY-112910, MedChemExpress), during 24 h in serum free DMEM.

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 Furin enzymatic activity analysis in cell lysates of MDA-MB-231 treated or not 24 h with GRP94 inhibitors, PU-WS13 12.5 or 25 µM or GRP94 inhibitor-1 (GRP94inh1) 2.5 or 5 µM ( n = 6). Fluorescence signal (expressed in relative fluorescence units, RFU) due to cleavage of the furin fluorogenic substrate, pERTKR-AMC, in cell lysates was recorded during 30 min. (**** p < 0.0001). Western- blot analysis of ( B ) active TGFβ secretion in supernatants (representative images, n = 7) and ( C ) pro-TGFβ expression in cell lysates (representative images, n = 5) from MDA-MB-231 treated or not 24 h with GRP94 inhibitors, PU-WS13 12.5 or 25 µM or GRP94 inhibitor-1 (GRP94inh1) 2.5 or 5 µM. (* p < 0.05; ** p < 0.01). D GRP94-furin co-immunoprecipitation in MDA-MB-231 cells. GRP94 was immunoprecipitated in MDA-MB-231 cell lysates using an anti-GRP94 antibody (IP:GRP94) or a non-relevant antibody (IgCT). Samples were analyzed by western-blot (representative images, n = 4). E Staining and quantification of proximity ligation assays (Duolink™ PLA) of GRP94 and furin or the negative control Hsp110 in MDA-MB-231 cells treated or not 24 h with GRP94 inhibitor-1 5 µM ( n = 3 experiments). Scale bars = 20 µm. The bar plot represents the mean number of spots for each cell analyzed +/− SEM. (**** p < 0.0001).

Article Snippet: For experiments, cells were seeded 24 h in growth conditions before being treated with GRP94 inhibitors, PU-WS13 or GRP94 inhibitor-1 (HY-112910, MedChemExpress), during 24 h in serum free DMEM.

Techniques: Activity Assay, Fluorescence, Western Blot, Expressing, Immunoprecipitation, Staining, Ligation, Negative Control

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: Impact of GRP94 inhibitors, PU-WS13 12.5 or 25 µM and GRP94 inhibitor-1 (GRP94inh1) 2.5 or 5 µM, on MDA-MB-231 cell migration was analyzed by wound healing assay. Wounds were realized in starved cells at confluency and treatments added in serum free medium. Using an Incucyte® S3 Live-Cell Analysis System, images of the wound areas were taken every hour during 24 h. Representative images at T = 0 h and T = 24 h of n = 6 experiments are shown in the upper panel with black bars highlighting the wound borders. Scale bars = 400 µm. Data are represented as relative wound densities over time in the lower panel ( n = 6) (**** p < 0.0001).

Article Snippet: For experiments, cells were seeded 24 h in growth conditions before being treated with GRP94 inhibitors, PU-WS13 or GRP94 inhibitor-1 (HY-112910, MedChemExpress), during 24 h in serum free DMEM.

Techniques: Migration, Wound Healing Assay, Cell Analysis

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: For experiments, cells were seeded 24 h in growth conditions before being treated with GRP94 inhibitors, PU-WS13 or GRP94 inhibitor-1 (HY-112910, MedChemExpress), during 24 h in serum free DMEM.

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