Journal: Advanced Science

Article Title: Oncogenic Roles of Laminin Subunit Gamma‐2 in Intrahepatic Cholangiocarcinoma via Promoting EGFR Translation

doi: 10.1002/advs.202309010

Figure Lengend Snippet: High LAMC2 associated with EGFR signaling activation and LAMC2 promoted EGFR translation. A) GSEA analysis with significantly altered genes ( p < 0.01) between LAMC2 ‐high and LAMC2 ‐low iCCA patients. The top 20 enriched signatures were listed. B) The enrichment of LAMC2 ‐high patients in EGFR signaling activation and non‐activation groups subclassified by an EGF/EGFR signaling gene set. C) Western blot analysis in RBE and HUCCT1 cells transfected with control siRNA, si LAMC2 #1, or si LAMC2 #2 and treated with EGF. D) Western blot analysis in RBE and HUCCT1 cells transfected with control siRNA as well as si LAMC2 #1 and #2. E) Western blot analysis of RBE and HUCCT1 cells transfected with Ctrl vector or LAMC2 ‐HA, along with EGFR‐flag. F) iCCA tumor formation in AKT/YapS127A‐induced iCCA mouse model with or without silencing LAMC2 by shRNAs, upon with or without LAMC2 /EGFR L858R overexpression. Student's t ‐test was used. G) The flow chart of Boncat Assay with L‐AHA to detect the newly synthesized proteins. H) Boncat assay in RBE and HUCCT1 cells with LAMC2 overexpression. I) Boncat assay in RBE and HUCCT1 cells with LAMC2 silencing.

Article Snippet: Cells were also treated with 20 μg mL −1 CHX and 100 n m bafilomycin A1 (Cat# HY‐100558, MedChemExpress) for 12 h to measure EGFR degradation, as well as with 20 μg mL −1 CHX and 2 μ m CB‐5083 (Cat# HY‐12861, MedChemExpress) to examine the degradation of unglycosylated EGFR.

Techniques: Activation Assay, Western Blot, Transfection, Control, Plasmid Preparation, Over Expression, Synthesized

Journal: Advanced Science

Article Title: Oncogenic Roles of Laminin Subunit Gamma‐2 in Intrahepatic Cholangiocarcinoma via Promoting EGFR Translation

doi: 10.1002/advs.202309010

Figure Lengend Snippet: LAMC2 interacted with unglycosylated EGFR. A) Cells were co‐transfected with indicated vectors and IP was performed with anti‐HA beads. B) IP assay with anti‐ LAMC2 antibody in iCCA cells. C) N‐linked glycosylation sites of EGFR (up panel) and the flow chart of the N‐glycosylation process (bottom panel). The related N‐glycosylation inhibitors and N‐glycan removing enzyme were indicated in red color. D) IP assay with anti‐HA beads in cells co‐transfected with LAMC2 ‐HA and EGFR‐flag. Both cell lysates and IP products were treated with or without PNGase F for 1 h before analysis. E) Anti‐HA IP in cells co‐transfected with LAMC2 ‐HA and EGFR‐flag and treated with or without tunicamycin (0.5 µg mL −1 ) for 24 h. F) Anti‐HA IP in cells co‐transfected with LAMC2 ‐HA and EGFR‐flag and treated with or without NGI‐1 (10 µ m ) for 24 h. G) Anti‐ LAMC2 IP in RBE and HUCCT1 cells treated with tunicamycin (0.5 µg mL −1 ) or NGI‐1 (10 µ m ) for 24 h. H) Confocal microscopy images of endogenous LAMC2 , EGFR, and ER marker Calreticulin and their co‐localization in iCCA cells. I) Construction of EGFR 13Q ‐flag and anti‐HA IP assay in cells co‐transfected with LAMC2 ‐HA and EGFR 13Q ‐flag. J) Boncat assay in cells co‐transfected with LAMC2 ‐HA and EGFR 13Q ‐flag. SP, signal peptide; TM, transmembrane; N, Asparagine; Q, Glutamine.

Article Snippet: Cells were also treated with 20 μg mL −1 CHX and 100 n m bafilomycin A1 (Cat# HY‐100558, MedChemExpress) for 12 h to measure EGFR degradation, as well as with 20 μg mL −1 CHX and 2 μ m CB‐5083 (Cat# HY‐12861, MedChemExpress) to examine the degradation of unglycosylated EGFR.

Techniques: Transfection, Glycoproteomics, Confocal Microscopy, Marker

Journal: Advanced Science

Article Title: Oncogenic Roles of Laminin Subunit Gamma‐2 in Intrahepatic Cholangiocarcinoma via Promoting EGFR Translation

doi: 10.1002/advs.202309010

Figure Lengend Snippet: LAMC2 N‐terminus interacted with the extracellular domain of EGFR, promoting EGFR translation. A) Mapping LAMC2 regions involved in EGFR binding via IP in cells co‐transfected with EGFR‐flag and LAMC2 ‐HA deletion mutants. B) Mapping LAMC2 N‐terminus regions involved in EGFR binding via IP in cells co‐transfected with EGFR‐flag and LAMC2 ‐HA deletion mutants. C) Mapping EGFR regions involved in LAMC2 binding via IP in cells co‐transfected with LAMC2 ‐HA and different EGFR‐flag vectors. D) Boncat assay in cells co‐transfected with LAMC2 ‐HA deletion mutants and EGFR‐flag. E) Boncat assay in cells co‐transfected with LAMC2 ‐HA deletion mutants and EGFR 13Q ‐flag. F) iCCA tumor formation in AKT/YapS127A‐induced iCCA mouse model with or without silencing LAMC2 by shRNAs, upon with or without C‐ LAMC2 /N‐ LAMC2 overexpression. Student's t ‐test was used.

Article Snippet: Cells were also treated with 20 μg mL −1 CHX and 100 n m bafilomycin A1 (Cat# HY‐100558, MedChemExpress) for 12 h to measure EGFR degradation, as well as with 20 μg mL −1 CHX and 2 μ m CB‐5083 (Cat# HY‐12861, MedChemExpress) to examine the degradation of unglycosylated EGFR.

Techniques: Binding Assay, Transfection, Over Expression

Journal: Advanced Science

Article Title: Oncogenic Roles of Laminin Subunit Gamma‐2 in Intrahepatic Cholangiocarcinoma via Promoting EGFR Translation

doi: 10.1002/advs.202309010

Figure Lengend Snippet: LAMC2 promoting EGFR translation was partially dependent on BiP. A) The flow chart of tandem IP followed by MS, and the top 4 candidates were listed based on the quantity of unique peptides. B) Anti‐HA IP in cells co‐transfected with LAMC2 ‐HA and BiP‐flag. C) Anti‐flag IP in cells co‐transfected with LAMC2 ‐HA and BiP‐flag. D) EGFR‐flag expression in cells co‐transfected with LAMC2 ‐HA and EGFR‐flag with or without silencing BiP. E) EGFR 13Q ‐flag expression in cells co‐transfected with LAMC2 ‐HA and EGFR 13Q ‐flag with or without silencing BiP. F) Boncat assay in cells co‐transfected with LAMC2 ‐HA and EGFR‐flag with or without silencing BiP. G) Boncat assay in cells co‐transfected with LAMC2 ‐HA and EGFR 13Q ‐flag with or without silencing BiP. H) Boncat assay in cells transfected with BiP‐flag and EGFR‐flag (left panel) or EGFR 13Q ‐flag (right panel). I) Boncat assay in RBE and HUCCT1 cells upon BiP silencing. J,K) Boncat assay in cells co‐transfected with BiP‐HA and EGFR‐flag (J) or EGFR 13Q ‐flag (K) with or without silencing LAMC2 .

Article Snippet: Cells were also treated with 20 μg mL −1 CHX and 100 n m bafilomycin A1 (Cat# HY‐100558, MedChemExpress) for 12 h to measure EGFR degradation, as well as with 20 μg mL −1 CHX and 2 μ m CB‐5083 (Cat# HY‐12861, MedChemExpress) to examine the degradation of unglycosylated EGFR.

Techniques: Transfection, Expressing

Journal: Advanced Science

Article Title: Oncogenic Roles of Laminin Subunit Gamma‐2 in Intrahepatic Cholangiocarcinoma via Promoting EGFR Translation

doi: 10.1002/advs.202309010

Figure Lengend Snippet: LAMC2 , EGFR, and BiP interacted with each other, contributing to EGFR translation. A) Anti‐flag IP in cells co‐transfected with BiP‐flag and EGFR‐HA with or without tunicamycin treatment. B) Anti‐HA IP in cells co‐transfected with BiP‐HA and EGFR 13Q ‐flag. C) Schematic diagram of BiP functional domain and a group of BiP truncations. D) Mapping BiP regions involved in LAMC2 binding via IP in cells co‐transfected with LAMC2 ‐HA and different BiP‐flag vectors. E) Mapping BiP regions involved in EGFR binding via IPs in cells co‐transfected EGFR‐HA and different BiP‐flag vectors with tunicamycin treatment. F) Anti‐HA IP in cells co‐transfected with BiP‐flag and different LAMC2 vectors. G) An illustrated interacting model of LAMC2 , BiP, and nascent EGFR in promoting EGFR translation in ER. H) Boncat assay in cells co‐transfected with BiP‐flag/EGFR‐flag and an intact LAMC2 , or N‐ LAMC2 . I) Boncat assay in cells co‐transfected with LAMC2 ‐HA /EGFR‐flag and an intact BiP, or BiP ΔSBD and BiP Δ501‐650 . SP, signal peptide; NBD, nucleotide‐binding domain; SBD, substrate binding domain.

Article Snippet: Cells were also treated with 20 μg mL −1 CHX and 100 n m bafilomycin A1 (Cat# HY‐100558, MedChemExpress) for 12 h to measure EGFR degradation, as well as with 20 μg mL −1 CHX and 2 μ m CB‐5083 (Cat# HY‐12861, MedChemExpress) to examine the degradation of unglycosylated EGFR.

Techniques: Transfection, Functional Assay, Binding Assay

Journal: Advanced Science

Article Title: Oncogenic Roles of Laminin Subunit Gamma‐2 in Intrahepatic Cholangiocarcinoma via Promoting EGFR Translation

doi: 10.1002/advs.202309010

Figure Lengend Snippet: LAMC2 ‐high iCCA tumors had poor prognosis, but were sensitive to EGFR TKIs treatment. A) Kaplan–Meier survival analysis of iCCA patients from Cohort 4 based on LAMC2 IHC staining score and LAMC2 copy number. B) Kaplan–Meier survival analysis of iCCA patients from Cohort 5 based on LAMC2 protein level (the median cut‐off). C) EGFR IHC staining and the spearman correlation of EGFR and LAMC2 in Cohort 4. Student's t ‐test was used for group comparison. D,E) Cell viability of iCCA cells with different LAMC2 levels under treatment of EGFR TKI Gefitinib (C) or EGFR neutralizing antibody Cetuximab (D). Two‐way ANOVA analysis was used. F) Representative images and quantitative analysis of orthoptic iCCA tumor formation in AKT/YapS127A‐induced iCCA mouse model with or without LAMC2 , upon with or without Gefitinib treatment. Student's t ‐test was used. NS, not significant. G) The schematic model summarized LAMC2 as a key oncogenic event in iCCA.

Article Snippet: Cells were also treated with 20 μg mL −1 CHX and 100 n m bafilomycin A1 (Cat# HY‐100558, MedChemExpress) for 12 h to measure EGFR degradation, as well as with 20 μg mL −1 CHX and 2 μ m CB‐5083 (Cat# HY‐12861, MedChemExpress) to examine the degradation of unglycosylated EGFR.

Techniques: Immunohistochemistry, 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: 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