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: 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: 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: 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: bioRxiv

Article Title: Defective proinsulin handling modulates the MHC I bound peptidome and activates the inflammasome in β-cells

doi: 10.1101/2021.12.20.472059

Figure Lengend Snippet: Flow cytometry comparison for RT1.A expression in INS-1E cells under tested conditions. ( A & B ) INS-1E cells compared with relevant controls for RT1.A expression under tested conditions and ( C & D ) Mean fluorescence intensity statistical comparison for RT1.A expression in INS-1E cells in tested conditions versus relevant controls. N = 3 for C and = 6 for D . Data is presented as mean ± SD. MFI = mean fluorescence intensity. Drugs/cytokines dose and exposure: GRP94i = 20µM, IL-1β = 15pg/ml, IFNγ = 10ng/ml for 24 hours.

Article Snippet: To induce GRP94 functional loss or mimic inflammatory conditions, INS-1E cells were treated with either 20 µM GRP94 inhibitor (GRP94i) PU-WS13 (EMD Millipore Corp. ® ) or exposed to 15 pg/ml recombinant human interleukin-1β (IL-1β, Sino Biological Inc. ® ) or 10 ng/ml recombinant rat IFNγ (R&D ® ) for 24 hours prior to harvesting cells for flow cytometry or immunopeptidomics.

Techniques: Flow Cytometry, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Defective proinsulin handling modulates the MHC I bound peptidome and activates the inflammasome in β-cells

doi: 10.1101/2021.12.20.472059

Figure Lengend Snippet: Venn diagrams showing number of source proteins for RT1.A eluted peptides distinct or overlapping between (A) Parental INS-1E, control KO & GRP94 KO and (B) Parental INS-1E, control KO & IL-1β exposure (15 pg/ml for 24 hours) (C) Parental INS-1E, control KO & IFNγ exposure (10 ng/ml for 24 hours) and (D) RT1.A contributing proteins exclusive for IL-1β, IFNγ exposed and GRP94 KO INS-1E groups. N = 2 for all except GRP94 KO where N = 4. * = IL-1β group proteins excluding parental INS-1E and control KO clone ** = IFNγ group proteins excluding parental INS-1E and control KO clone *** = GRP94 KO group proteins excluding parental INS-1E and control KO clone

Article Snippet: To induce GRP94 functional loss or mimic inflammatory conditions, INS-1E cells were treated with either 20 µM GRP94 inhibitor (GRP94i) PU-WS13 (EMD Millipore Corp. ® ) or exposed to 15 pg/ml recombinant human interleukin-1β (IL-1β, Sino Biological Inc. ® ) or 10 ng/ml recombinant rat IFNγ (R&D ® ) for 24 hours prior to harvesting cells for flow cytometry or immunopeptidomics.

Techniques:

Journal: bioRxiv

Article Title: Defective proinsulin handling modulates the MHC I bound peptidome and activates the inflammasome in β-cells

doi: 10.1101/2021.12.20.472059

Figure Lengend Snippet: Length distribution for 8 – 15 amino acid-length peptides between parental INS-1E, control and confirmed GRP94 KO INS-1E, 24 hour treatment with GRP94 inhibitor (20 µM), or INS-1E cells exposed to cytokines IL-1β (15 pg/ml) or IFNγ (10 ng/ml). N = 2 for all except GRP94 KO where N = 4.

Article Snippet: To induce GRP94 functional loss or mimic inflammatory conditions, INS-1E cells were treated with either 20 µM GRP94 inhibitor (GRP94i) PU-WS13 (EMD Millipore Corp. ® ) or exposed to 15 pg/ml recombinant human interleukin-1β (IL-1β, Sino Biological Inc. ® ) or 10 ng/ml recombinant rat IFNγ (R&D ® ) for 24 hours prior to harvesting cells for flow cytometry or immunopeptidomics.

Techniques:

Journal: bioRxiv

Article Title: Defective proinsulin handling modulates the MHC I bound peptidome and activates the inflammasome in β-cells

doi: 10.1101/2021.12.20.472059

Figure Lengend Snippet: Venn diagrams showing number of MHC I-eluted peptides distinct or overlapping between (A) Parental INS-1E, control KO & GRP94 KO and (B) Parental INS-1E, control KO & IL-1β exposure (15 pg/ml for 24 hours) (C) Parental INS-1E, control KO & IFNγ exposure (10 ng/ml for 24 hours) and (D) Peptides exclusive for IL-1β, IFNγ exposed and GRP94 KO INS-1E groups. N = 2 for all except GRP94 KO where N = 4 * = IL-1β peptides excluding parental INS-1E and control KO clone ** = IFNγ peptides excluding parental INS-1E and control KO clone *** = GRP94 KO peptides excluding parental INS-1E and control KO clone

Article Snippet: To induce GRP94 functional loss or mimic inflammatory conditions, INS-1E cells were treated with either 20 µM GRP94 inhibitor (GRP94i) PU-WS13 (EMD Millipore Corp. ® ) or exposed to 15 pg/ml recombinant human interleukin-1β (IL-1β, Sino Biological Inc. ® ) or 10 ng/ml recombinant rat IFNγ (R&D ® ) for 24 hours prior to harvesting cells for flow cytometry or immunopeptidomics.

Techniques:

Journal: bioRxiv

Article Title: Defective proinsulin handling modulates the MHC I bound peptidome and activates the inflammasome in β-cells

doi: 10.1101/2021.12.20.472059

Figure Lengend Snippet: Nonamers binding motif for INS-1E cells under tested conditions. (A) parental INS-1E (B) control GRP94 KO clone (C) INS-1E treated with GRP94 inhibitor (20 µM for 24 hours) (D) GRP94 KO INS-1E clone (E) IL-1β exposure (15 pg/ml for 24 hours) and (F) IFNγ exposure (10 ng/ml for 24 hours). N = 2 for all except GRP94 KO where N = 4.

Article Snippet: To induce GRP94 functional loss or mimic inflammatory conditions, INS-1E cells were treated with either 20 µM GRP94 inhibitor (GRP94i) PU-WS13 (EMD Millipore Corp. ® ) or exposed to 15 pg/ml recombinant human interleukin-1β (IL-1β, Sino Biological Inc. ® ) or 10 ng/ml recombinant rat IFNγ (R&D ® ) for 24 hours prior to harvesting cells for flow cytometry or immunopeptidomics.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Defective proinsulin handling modulates the MHC I bound peptidome and activates the inflammasome in β-cells

doi: 10.1101/2021.12.20.472059

Figure Lengend Snippet: Decamers binding motif for INS-1E under tested conditions. (A) parental INS-1E (B) control GRP94 KO clone (C) INS-1E treated with GRP94 inhibitor (20 µM for 24 hours) (D) GRP94 KO INS-1E clone (E) IL-1β exposure (15 pg/ml for 24 hours) and (F) IFNγ exposure (10 ng/ml for 24 hours). N = 2 for all except GRP94 KO where N = 4.

Article Snippet: To induce GRP94 functional loss or mimic inflammatory conditions, INS-1E cells were treated with either 20 µM GRP94 inhibitor (GRP94i) PU-WS13 (EMD Millipore Corp. ® ) or exposed to 15 pg/ml recombinant human interleukin-1β (IL-1β, Sino Biological Inc. ® ) or 10 ng/ml recombinant rat IFNγ (R&D ® ) for 24 hours prior to harvesting cells for flow cytometry or immunopeptidomics.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Defective proinsulin handling modulates the MHC I bound peptidome and activates the inflammasome in β-cells

doi: 10.1101/2021.12.20.472059

Figure Lengend Snippet: Gibbscluster analysis for RT1.A-bound peptides in INS-1E cells under tested conditions. (A) parental INS-1E (B) control GRP94 KO clone (C) INS-1E treated with GRP94 inhibitor (20 µM for 24 hours) (D) GRP94 KO INS-1E clone (E) IL-1β exposure (15 pg/ml for 24 hours) and (F) IFNγ exposure (10 ng/ml for 24 hours). N = 2 for all except GRP94 KO where N = 4.

Article Snippet: To induce GRP94 functional loss or mimic inflammatory conditions, INS-1E cells were treated with either 20 µM GRP94 inhibitor (GRP94i) PU-WS13 (EMD Millipore Corp. ® ) or exposed to 15 pg/ml recombinant human interleukin-1β (IL-1β, Sino Biological Inc. ® ) or 10 ng/ml recombinant rat IFNγ (R&D ® ) for 24 hours prior to harvesting cells for flow cytometry or immunopeptidomics.

Techniques:

Journal: bioRxiv

Article Title: Defective proinsulin handling modulates the MHC I bound peptidome and activates the inflammasome in β-cells

doi: 10.1101/2021.12.20.472059

Figure Lengend Snippet: Western blot analysis for the expression of NALP1, IκBα and IL-1β in GRP94 KO INS-1E and control KO cells. (A) SDS-PAGE for expression of NALP1 (fully assembled version (165 kDa) and without LRR (70 kDa)), IκBα and IL-1β in tested conditions. ( B-F) Statistical analysis (unpaired t-test) for the expression of NALP1, IκBα and pro-IL-1β/IL-1β quantified and normalized to tubulin in tested conditions. N = 4 – 6 for different proteins of interest.

Article Snippet: To induce GRP94 functional loss or mimic inflammatory conditions, INS-1E cells were treated with either 20 µM GRP94 inhibitor (GRP94i) PU-WS13 (EMD Millipore Corp. ® ) or exposed to 15 pg/ml recombinant human interleukin-1β (IL-1β, Sino Biological Inc. ® ) or 10 ng/ml recombinant rat IFNγ (R&D ® ) for 24 hours prior to harvesting cells for flow cytometry or immunopeptidomics.

Techniques: Western Blot, Expressing, SDS Page