Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: TRPV6-mediated Store-operated Ca 2+ Entry Participates in Pancreatic Acinar Cell Injury During Acute Pancreatitis

doi: 10.1016/j.jcmgh.2025.101620

Figure Lengend Snippet: TRPV6 and STIM1 interact in PACs. ( A ) Representative IHC staining images and quantification of STIM1 staining performed on mouse pancreas tissue (n = 4 sections from 3 mice per group). ( B ) Representative IHC staining images and quantification of STIM1 performed on human pancreas tissue (n = 3 sections from 3 patients per group). ( C and D ) Representative images ( C ) and quantification ( D ) of TRPV6 and STIM1 colocalization in PACs obtained by structured illumination microscopy (n = 7–8 regions of interest [ROIs] from 3 independent experiments per group). ( E and F ) Blots ( E ) and quantification ( F ) of Co-IP experiments performed by immunoprecipitating TRPV6 or STIM1 in freshly isolated PAC lysates (n = 3 blots from 3 independent experiments per group). ( G and H ) Representative whole-cell TRPV6 current traces ( G ) and quantification ( H ) of current density at −74 mV under different holding potentials in TRPV6- and STIM1-transfected HEK293T cells (n = 8–10 cells from 4 independent experiments per group). Data are the mean ± SEM; n.s., non-significance, ∗ P < .05, ∗∗ P < .01.

Article Snippet: Then, slides were incubated for 1 hour at room temperature using a primary antibody of STIM1 dilution of 1:100 (NB110-55300, Novus), and then for another hour with biotinylated secondary antibodies (1:1000).

Techniques: Immunohistochemistry, Staining, Microscopy, Co-Immunoprecipitation Assay, Isolation, Transfection

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: TRPV6-mediated Store-operated Ca 2+ Entry Participates in Pancreatic Acinar Cell Injury During Acute Pancreatitis

doi: 10.1016/j.jcmgh.2025.101620

Figure Lengend Snippet: Pharmacological TRPV6 inhibition ameliorates CER-induced AP. ( A ) Schematic of the in vivo experiments in AP and control mice. ( B ) Line chart displaying the effect of SORC27 on von Frey filament (VFF) test withdrawal thresholds at 0.5, 1.5, 135 2.5, and 3.5 hours following the last PBS injection (n = 6 mice per group). ( C ) Serum amylase, pancreatic MPO, and trypsin activity (n = 6 mice per group). ( D and E ) Representative images ( D ) and histological scores ( E ) of pancreas tissue sections (n = 6 mice per group). ( F ) Line chart displaying the effect of SORC27 on VFF test withdrawal thresholds at 0.5, 1.5, 2.5, and 3.5 hours following the last CER injection (n = 6 mice per group). ( G ) Serum amylase, pancreatic MPO, and trypsin activity (n = 6 samples per group). ( H ) Representative H&E-stained images and histological scores of pancreas tissue sections (n = 6 sections from 6 mice per group). ( I and J ) Representative images of pancreas ( I ) and lung ( J ) tissue section staining for F4/80 + macrophages ( left ) and quantitative results ( right ) (n = 6 sections from 6 mice per group). ( K ) Representative images of lung injury from CER-AP (CER), low (SOR-L) and high (SOR-H) -dose-SORC27 treated mice AP modeling. ( L ) Western blot (WB) images and quantification, and gene expression ( M ) of pancreatic STIM1 after SORC27 treatment (n = 4–6 mice per group). Data are the mean ± SEM; n.s., non-significance, ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001.

Article Snippet: Then, slides were incubated for 1 hour at room temperature using a primary antibody of STIM1 dilution of 1:100 (NB110-55300, Novus), and then for another hour with biotinylated secondary antibodies (1:1000).

Techniques: Inhibition, In Vivo, Control, Injection, Activity Assay, Staining, Western Blot, Gene Expression

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: TRPV6-mediated Store-operated Ca 2+ Entry Participates in Pancreatic Acinar Cell Injury During Acute Pancreatitis

doi: 10.1016/j.jcmgh.2025.101620

Figure Lengend Snippet: Pancreatic Trpv6 knockdown alleviates CER-induced AP. ( A ) Schematic of the in vivo experiments in si Trpv6 and siNC mice. ( B ) Pancreatic Trpv6 gene expression (n = 5–6 mice per group). ( C ) Line chart displaying the effect of SORC27 on VFF test withdrawal thresholds at 0.5, 1.5, 2.5, and 3.5 hours following the last PBS injection (n = 6–7 mice per group). ( D ) Serum amylase, pancreatic MPO, and trypsin activity (n = 4–6 mice per group). ( E and F ) Representative images ( E ) and histological scores ( F ) of pancreas tissue sections (n = 5–6 mice per group). ( G ) Line chart indicating VFF withdrawal thresholds observed at 0.5, 1.5, 2.5, and 3.5 hours following the last CER injection (n = 8, 12 mice per group). ( H ) Serum amylase, pancreatic MPO, and trypsin activity (n = 8–12 samples per group). ( I and J ) Representative H&E-stained images ( I ) and histological scores ( J ) of pancreas tissue sections (n = 77–88 sections from at least 8 mice per group). ( K and L ) Representative images and quantitative results of pancreas ( K ) and lung tissue ( L ) F4/80 + staining for macrophages (n = 3 sections from 3 mice per group). ( M ) Representative images of lung injury. ( N ) Immunoblotting images and quantification of STIM1 levels in pancreas tissue (n = 4 samples per group). ( O ) Representative IHC staining images and quantitative results of STIM1 levels in pancreas tissue (n = 3 sections from 3 mice per group). Data are the mean ± SEM; n.s., non-significance, ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001.

Article Snippet: Then, slides were incubated for 1 hour at room temperature using a primary antibody of STIM1 dilution of 1:100 (NB110-55300, Novus), and then for another hour with biotinylated secondary antibodies (1:1000).

Techniques: Knockdown, In Vivo, Gene Expression, Injection, Activity Assay, Staining, Western Blot, Immunohistochemistry

Journal: Journal of Lipid Research

Article Title: Crosstalk between ORMDL3, serine palmitoyltransferase, and 5-lipoxygenase in the sphingolipid and eicosanoid metabolic pathways

doi: 10.1016/j.jlr.2021.100121

Figure Lengend Snippet: Identification of the ORMDL3/5-LO/SPTLC1/SPTLC2 complex. A: ORMDL3-MYC immunoprecipitated from lysates of BMMCL expressing ORMDL3-MYC or empty vectors (control) and activated or not with thapsigargin. The immunoprecipitates were size-fractionated by SDS-PAGE and stained by Coomassie blue. ←5-LO indicates position of 5-LO as identified by mass spectrometry. B: 5-LO interacts with ORMDL3, SPTLC1, and SPTLC2. Twin-Strep-tag (TS) affinity purification from lysates of HEK293FT cells stably transduced with FLAG-ORMDL3 alone or in combination with TS-5-LO and activated for 0, 3, 15, or 25 min with thapsigargin. The proteins were analyzed by immunoblotting using the corresponding antibodies. C: Colocalization of endogenous 5-LO with ORMDLs (left panel) or STIM1 (right panel) in nonactivated (0 min) or activated HMC-1.1 cells for 10 or 30 min with ionomycin. Scale bars represent 5 μm. D: Physical interaction of 5-LO with ORMDL3. FLAP, ORMDL3, LTC4S, and 5-LO were tagged with GST and expressed in bacteria. GST-tagged proteins and GST alone were purified on glutathione-coated beads in the presence of in vitro translate expressing 35 S-5-LO. SDS-PAGE followed by autoradiography was used to detect 35 S-5-LO. The gel was stained with Coomassie brilliant blue (CB staining) to determine the loading levels of GST and GST-tagged proteins. E: SDS-PAGE of lysates from HEK293 cells stably transduced with empty vector (control), murine 5-LO, or murine ORMDL3 developed with the indicated protein-specific antibodies. F and G: LC-ESI-MS/MS analysis of sphingolipids in resting HEK293 cells stably transduced with murine 5-LO (n = 13), murine ORMDL3 (n = 7), or empty vector (control, n = 12). F: Total sphingosines, the sum of C18:1 and C18:0 is calculated. G: The sum of total ceramide fatty acid chain molecular species (including 2-hydroxy ceramide molecular species), derived from d18:1 sphingosine, was calculated. Data in B and D are representative of three independent experiments. Quantitative data in F and G are mean ± SEM, calculated from n, which show numbers of biological replicates of independently transduced cells. P values were determined by one-way ANOVA with Bonferoni post hoc test.

Article Snippet: Antibodies against HA tag (NB600-363) and stromal interaction molecule 1 (STIM1) (NB110-60547S) were obtained from Novus Biologicals.

Techniques: Immunoprecipitation, Expressing, Control, SDS Page, Staining, Mass Spectrometry, Strep-tag, Affinity Purification, Stable Transfection, Transduction, Western Blot, Bacteria, Purification, In Vitro, Autoradiography, Plasmid Preparation, Tandem Mass Spectroscopy, Derivative Assay