grp94 inhibitor pu-ws13  (Millipore)

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: Biomedicines

Article Title: Defective Proinsulin Handling Modulates the MHC I Bound Peptidome and Activates the Inflammasome in β-Cells

doi: 10.3390/biomedicines10040814

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 and ( C , D ) statistical comparison for RT1.A expression INS-1E cells in tested conditions versus relevant controls. N = 3 for C and = 6 for D. MFI = mean fluorescence intensity. Drugs/cytokines dose and exposure: GRP94i = 20 µM, IL-1β = 15 pg/mL, IFNγ = 10 ng/mL for 24 h. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.

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. ® , Burlington, MI, USA) or exposed to recombinant human interleukin-1β (IL-1β, Sino Biological Inc. ® , Beijing, China) at 15 pg/mL or 10 ng/mL recombinant rat IFNγ (R&D ® , Minneapolis, MN, USA) for 24 h prior to harvesting cells for flow cytometry and collection of pellets for immunopeptidomics.

Techniques: Flow Cytometry, Comparison, Expressing, Fluorescence

Journal: Biomedicines

Article Title: Defective Proinsulin Handling Modulates the MHC I Bound Peptidome and Activates the Inflammasome in β-Cells

doi: 10.3390/biomedicines10040814

Figure Lengend Snippet: ( A ) Summary table of RT1.A-bound 8–15 amino acids length peptides from INS-1E cells under tested conditions. ( B ) Venn diagrams showing peptides overlap between the individual replicates per condition. n = 2 for all groups except for 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. ® , Burlington, MI, USA) or exposed to recombinant human interleukin-1β (IL-1β, Sino Biological Inc. ® , Beijing, China) at 15 pg/mL or 10 ng/mL recombinant rat IFNγ (R&D ® , Minneapolis, MN, USA) for 24 h prior to harvesting cells for flow cytometry and collection of pellets for immunopeptidomics.

Techniques:

Journal: Biomedicines

Article Title: Defective Proinsulin Handling Modulates the MHC I Bound Peptidome and Activates the Inflammasome in β-Cells

doi: 10.3390/biomedicines10040814

Figure Lengend Snippet: Venn diagrams showing number of MHC I-eluted peptides distinct or overlapping between ( A ) parental INS-1E, control KO & GRP94 KO, ( B ) parental INS-1E, control KO & IL-1β exposure (15 pg/mL for 24 h), ( C ) parental INS-1E, control KO & IFNγ exposure (10 ng/mL for 24 h), 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. ® , Burlington, MI, USA) or exposed to recombinant human interleukin-1β (IL-1β, Sino Biological Inc. ® , Beijing, China) at 15 pg/mL or 10 ng/mL recombinant rat IFNγ (R&D ® , Minneapolis, MN, USA) for 24 h prior to harvesting cells for flow cytometry and collection of pellets for immunopeptidomics.

Techniques: Control

Journal: Biomedicines

Article Title: Defective Proinsulin Handling Modulates the MHC I Bound Peptidome and Activates the Inflammasome in β-Cells

doi: 10.3390/biomedicines10040814

Figure Lengend Snippet: Length distribution for 8–15 amino acid-length peptides between parental INS-1E, control and confirmed GRP94 KO INS-1E, 24 h 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. ® , Burlington, MI, USA) or exposed to recombinant human interleukin-1β (IL-1β, Sino Biological Inc. ® , Beijing, China) at 15 pg/mL or 10 ng/mL recombinant rat IFNγ (R&D ® , Minneapolis, MN, USA) for 24 h prior to harvesting cells for flow cytometry and collection of pellets for immunopeptidomics.

Techniques: Control

Journal: Biomedicines

Article Title: Defective Proinsulin Handling Modulates the MHC I Bound Peptidome and Activates the Inflammasome in β-Cells

doi: 10.3390/biomedicines10040814

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 h), ( D ) GRP94 KO INS-1E clone, ( E ) IL-1β exposure (15 pg/mL for 24 h), and ( F ) IFNγ exposure (10 ng/mL for 24 h). 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. ® , Burlington, MI, USA) or exposed to recombinant human interleukin-1β (IL-1β, Sino Biological Inc. ® , Beijing, China) at 15 pg/mL or 10 ng/mL recombinant rat IFNγ (R&D ® , Minneapolis, MN, USA) for 24 h prior to harvesting cells for flow cytometry and collection of pellets for immunopeptidomics.

Techniques: Binding Assay, Control

Journal: Biomedicines

Article Title: Defective Proinsulin Handling Modulates the MHC I Bound Peptidome and Activates the Inflammasome in β-Cells

doi: 10.3390/biomedicines10040814

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 h), ( D ) GRP94 KO INS-1E clone, ( E ) IL-1β exposure (15 pg/mL for 24 h), and ( F ) IFNγ exposure (10 ng/mL for 24 h). 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. ® , Burlington, MI, USA) or exposed to recombinant human interleukin-1β (IL-1β, Sino Biological Inc. ® , Beijing, China) at 15 pg/mL or 10 ng/mL recombinant rat IFNγ (R&D ® , Minneapolis, MN, USA) for 24 h prior to harvesting cells for flow cytometry and collection of pellets for immunopeptidomics.

Techniques: Binding Assay, Control

Journal: Biomedicines

Article Title: Defective Proinsulin Handling Modulates the MHC I Bound Peptidome and Activates the Inflammasome in β-Cells

doi: 10.3390/biomedicines10040814

Figure Lengend Snippet: RT1.A eluted proinsulin peptides from INS-1E cells derived from (pre)-proinsulin overlapping or unique for GRP94 KO/KD or cytokines exposure. ‘✓’refers to the presence of peptide in the group whereas empty column indicates absence of the peptides in the specific group. n = 2 for all groups 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. ® , Burlington, MI, USA) or exposed to recombinant human interleukin-1β (IL-1β, Sino Biological Inc. ® , Beijing, China) at 15 pg/mL or 10 ng/mL recombinant rat IFNγ (R&D ® , Minneapolis, MN, USA) for 24 h prior to harvesting cells for flow cytometry and collection of pellets for immunopeptidomics.

Techniques: Derivative Assay, Control

Journal: Biomedicines

Article Title: Defective Proinsulin Handling Modulates the MHC I Bound Peptidome and Activates the Inflammasome in β-Cells

doi: 10.3390/biomedicines10040814

Figure Lengend Snippet: Western blot analysis for the expression of NLRP1, IκBα, and IL-1β in GRP94 KO INS-1E and control KO cells. ( A ) SDS-PAGE for expression of NLRP1 (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 NLRP1, IκBα, and pro-IL-1β/IL-1β, quantified and normalized to tubulin in tested conditions. n = 4–6 for different proteins of interest. ( B – E ) were generated using the ratio of quantified protein of interest to tubulin while ( F ) was generated based on quantified IL-1β only from the supernatant from equal cell numbers cultured and volume of culturing media. * p ≤ 0.05, *** = p ≤ 0.01.

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. ® , Burlington, MI, USA) or exposed to recombinant human interleukin-1β (IL-1β, Sino Biological Inc. ® , Beijing, China) at 15 pg/mL or 10 ng/mL recombinant rat IFNγ (R&D ® , Minneapolis, MN, USA) for 24 h prior to harvesting cells for flow cytometry and collection of pellets for immunopeptidomics.

Techniques: Western Blot, Expressing, Control, SDS Page, Generated, Cell Culture

Journal: Biomedicines

Article Title: Defective Proinsulin Handling Modulates the MHC I Bound Peptidome and Activates the Inflammasome in β-Cells

doi: 10.3390/biomedicines10040814

Figure Lengend Snippet: Proposed mechanism of autoimmunity induction in T1D. A defect in the ability of GRP94 to process proinsulin leads to an accumulation of mishandled proinsulin in β-cells. This leads to ER stress, activation of inflammatory pathways, assembly of int-proteasome, and secretion of GRP94-PI complexes. Simultaneously, the increased MHC-I expression on β-cells make them more visible to immune cells. The secreted GRP94-PI complexes are taken up by APCs and cross-presented to CD8 + T-cells to trigger immune attack against β-cells.

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. ® , Burlington, MI, USA) or exposed to recombinant human interleukin-1β (IL-1β, Sino Biological Inc. ® , Beijing, China) at 15 pg/mL or 10 ng/mL recombinant rat IFNγ (R&D ® , Minneapolis, MN, USA) for 24 h prior to harvesting cells for flow cytometry and collection of pellets for immunopeptidomics.

Techniques: Activation Assay, Expressing