Journal: bioRxiv

Article Title: The active secretion of a subunit of IL-12 by tissue cells is regulated by Valosin-Containing Protein and intracellular calcium redistribution

doi: 10.64898/2026.01.28.702376

Figure Lengend Snippet: The mechanisms of p35 release identified here operate in primary murine tissues. (A) Luciferase assay of supernatants from p40KO muscle fibroblasts transduced with p35NL and treated with titration of ionomycin for 24 hours. (B) Viability of p35NL primary p40KO fibroblasts treated with ionomycin for 24 hours. (C) Luciferase assay of supernatants from p40KO muscle fibroblasts transduced with NLO and treated with titration of ionomycin for 24 hours. (D) Viability of NLO primary p40KO fibroblasts treated with ionomycin for 24 hours. (E) Luciferase assay of supernatants from p40KO muscle fibroblasts transduced with p35NL and treated with titration of CB-5083 for 24 hours. (F) Viability of p35NL primary p40KO fibroblasts treated with CB-5083 for 24 hours. (G) Luciferase assay of supernatants from p40KO muscle fibroblasts transduced with NLO and treated with titration of CB-5083 for 24 hours. (H) Viability of NLO primary p40KO fibroblasts treated with CB-5083 for 24 hours. (I) Live cell imaging of p40KO muscle fibroblast transduced with p35-Scarlet and imaged at 100X magnification. (J) A theoretical model for independent p35 release initiated by pathogen-induced VCP inhibition. We propose a model of p35 release from stromal cells which is initiated by pathogen-induced VCP inhibition. Inhibition of VCP promotes gene expression that increases intracellular calcium flux, potentially through the upregulation of an ER-associated channel like IP 3 R. From this point, our data suggests two possible modes of release. In the first, intracellular calcium activates a calcium-responsive cargo adaptor and promotes the selective passage of p35 through the conventional secretory pathway. In a second mode of release, we propose that the vacuole-like structures induced by VCP inhibition may be critical in allowing a Golgi-independent pathway to release of p35. In this mechanism, an imbalance of intracellular calcium promoted by VCP inhibition leads to the dilation of the ER into vacuoles containing p35. ER dilation promotes the release of p35 through an unknown, perhaps novel pathway in manner that does not rely on cell death. Data is represented as raw Relative Luminescence Units. Significant elevations in luminescence were determined through one-way ANOVA of the mean in comparison to vehicle controls (DMSO). Significant changes in cell viability were determined through an ordinary one-way ANOVA of the mean in comparison to vehicle controls (DMSO). *, p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns = not significant

Article Snippet: Cells were exposed to titrations of ionomycin (Tocris), CB-5083 (Tocris), or vehicle for 24 hours.

Techniques: Luciferase, Transduction, Titration, Live Cell Imaging, Inhibition, Gene Expression, Comparison

Journal: bioRxiv

Article Title: The active secretion of a subunit of IL-12 by tissue cells is regulated by Valosin-Containing Protein and intracellular calcium redistribution

doi: 10.64898/2026.01.28.702376

Figure Lengend Snippet: Viable cells that do not express p40 can be triggered to release the p35 subunit: Ionomycin, a calcium ionophore, was validated as a pharmacological trigger of p35 release from viable cells. p35NL and NLO L cells were treated with concentrations of ionomycin ranging from 50-0.78µM. (A) Luciferase assay of supernatants from p35NL cells treated with ionomycin for 24 hours (B) Viability of p35NL cells after 24-hour ionomycin treatment. (C) Luciferase assay of supernatants from NLO cells treated with ionomycin for 24 hours (D) Viability of NLO cells after 24-hour ionomycin treatment (n=3). CB-5083, an inhibitor of the multi-functional ATPase VCP (p97), was validated as a pharmacological trigger of p35 release from viable cells. p35NL and NLO L cells were treated with concentrations of CB-5083 ranging from 50-0.39µM. (E) Luciferase assay of supernatants from p35NL cells treated with CB-5083 for 24 hours (F) Viability of p35NL cells after 24-hour CB-5083 treatment. (G) Luciferase assay of supernatants from NLO cells treated with CB-5083 for 24 hours (H) Viability of NLO cells after 24-hour CB-5083 treatment (n=3). (I) Luciferase assay showing the kinetics of p35NL release in response to ionomycin. (J) Luciferase assay showing the kinetics of NLO release in response to ionomycin. (K) Luciferase assay showing the kinetics of p35NL release in response to CB-5083 (L) Luciferase assay showing the kinetics of NLO release in response to CB-5083 (n=3). RLUs are normalized internally and represented as a heat map (M) p35NL cells were pre-treated with media, actinomycin D, or cycloheximide for 60 minutes before the addition of vehicle or 6.25µM ionomycin. Supernatants were collected after 24 hours and used for luciferase assay. (N) p35NL cells were pre-treated with media, actinomycin D, or cycloheximide for 60 minutes before the addition of vehicle or 1.56µM CB-5083. Supernatants were collected after 24 hours and used for luciferase assay. Data (A-H, M, N) is represented as raw Relative Luminescence Units. Significant elevations in luminescence were determined through one-way ANOVA of the mean in comparison to vehicle controls (DMSO). Significant changes in cell viability were determined through an ordinary one-way ANOVA of the mean in comparison to vehicle controls (DMSO). *, p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns = not significant

Article Snippet: Cells were exposed to titrations of ionomycin (Tocris), CB-5083 (Tocris), or vehicle for 24 hours.

Techniques: Luciferase, Functional Assay, Comparison

Journal: bioRxiv

Article Title: The active secretion of a subunit of IL-12 by tissue cells is regulated by Valosin-Containing Protein and intracellular calcium redistribution

doi: 10.64898/2026.01.28.702376

Figure Lengend Snippet: (A) Percentage of UPR+ 7-AAD-L cells (GFP+ for XBP-1 binding to Unfolded Protein Response Element) after treatment with vehicle (DMSO), 3.12μM ionomycin, or 1.56μM CB-5083 then analyzed with flow cytometry. (B) Luciferase assay of supernatants from p35NL cells pre-treated with PERK inhibitor AMG PERK 44 for 2 hours then stimulated with 3.12 μM ionomycin for 24 hours. (C) Luciferase assay of supernatants from p35NL cells pre-treated with PERK inhibitor AMG PERK 44 for 2 hours then stimulated with 1.56μM CB-5083 for 24 hours. (D) Luciferase assay of supernatants from p35NL cells pre-treated with IRE1 inhibitor MKC8866 for 2 hours then stimulated with 3.12 μM ionomycin for 24 hours. (E) Luciferase assay of supernatants from p35NL cells pre-treated with IRE1 inhibitor MKC8866 for 2 hours then stimulated with 1.56μM CB-5083 for 24 hours. (F) Luciferase assay of supernatants from p35NL cells pre-treated with ATF6 inhibitor Ceapin-A7 for 2 hours then stimulated with 3.12 μM ionomycin for 24 hours. (G) Luciferase assay of supernatants from p35NL cells pre-treated with ATF6 inhibitor Ceapin-A7 for 2 hours then stimulated with 1.56μM CB-5083 for 24 hours. (n=3). Data is represented as raw Relative Luminescence Units (RLUs). Significant changes in luminescence were determined through ordinary one-way ANOVAs of cells stimulated with ionomycin only or CB-5083 only (control). *, p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns = not significant

Article Snippet: Cells were exposed to titrations of ionomycin (Tocris), CB-5083 (Tocris), or vehicle for 24 hours.

Techniques: Binding Assay, Flow Cytometry, Luciferase, Control

Journal: bioRxiv

Article Title: The active secretion of a subunit of IL-12 by tissue cells is regulated by Valosin-Containing Protein and intracellular calcium redistribution

doi: 10.64898/2026.01.28.702376

Figure Lengend Snippet: VCP inhibition leads to aggregation of p35-Scarlet near Golgi markers and the formation of unique vacuolated structures. Subcellular localization of p35 under CB-5083-induced release conditions. (A) Representative live cell imaging of p35-Scarlet/Golgi-eGFP/ER-Halotag L cells treated with either vehicle (DMSO) or 1.56μM CB-5083 (CB) for 24 hours. 10 μm scale bars (B) Representative live cell imaging of p35-Scarlet/Golgi-eGFP/BiP-BFP L cell treated with treated with either vehicle or 1.56μM CB-5083 for 8 hours (n=20). 10 μm scale bars (C) Quantification of colocalization between p35-Scarlet and Golgi-eGFP using Pearson’s coefficient (n=20). (D) Quantification of colocalization between p35-Scarlet and BiP-BFP using Pearson’s coefficient (n=20). (E) Quantification of colocalization between BiP-BFP and Golgi-eGFP using Pearson’s coefficient (n=20). (F) Transmission electron microscopy of L cells treated with vehicle, 3.12μM ionomycin, or 1.56μM CB-5083 for 24 hours. (G) Ultrastructure of vacuoles from CB-5083-treated L cells after 24 hours. (H) Representative live cell images from p35-Scarlet/VCP1-eGFP L cells treated with either vehicle, 3.12μM ionomycin, or 1.56μM CB-5083 for 12 hours (n=1). Significant changes in colocalization were determined using an unpaired t-test of the mean in comparison to vehicle controls (DMSO). Each image analyzed was a 100X frame containing 5-10 cells. *, p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns = not significant

Article Snippet: Cells were exposed to titrations of ionomycin (Tocris), CB-5083 (Tocris), or vehicle for 24 hours.

Techniques: Inhibition, Live Cell Imaging, Transmission Assay, Electron Microscopy, Comparison