Journal: Nature Communications

Article Title: Neuronal SphK1 acetylates COX2 and contributes to pathogenesis in a model of Alzheimer’s Disease

doi: 10.1038/s41467-018-03674-2

Figure Lengend Snippet: SphK1 is decreased in APP/PS1 mice neuron. a SphK1 ( n = 6–7 per group) and SphK2 mRNA ( n = 5–7 per group) and SphK activity ( n = 6–7 per group) in cortex of brain. b Detection of sphingosine ( n = 6–7 per group) and S1P ( n = 6–7 per group) in brain. c SphK1 and SphK2 mRNA ( n = 4 per group) and SphK activity ( n = 4–10 per group) in neurons, microglia, and astrocytes isolated from mouse brain. d Detection of sphingosine ( n = 4–8 per group) and S1P ( n = 4–8 per group) in neurons, microglia, and astrocytes isolated from mouse brain. e Detection of SphK activity, sphingosine and S1P in neurons isolated from WT, SphK1 −/− , SphK2 −/− and SphK1 tg mouse brain ( n = 4 per group). f Left, representative immunofluorescence images of cortex showing SphK1 (green) merged with neuron (NeuN, red). Scale bars, 20 μm. Right, quantification of neuronal SphK1 ( n = 5 per group). g Western blotting for SphK1 in neuron isolated from mouse brain ( n = 4 per group). All data analysis was performed on 9-month-old mice. a – g One-way analysis of variance, Tukey’s post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001. All error bars indicate s.e.m.

Article Snippet: PS1 and ApoE4-iPSCs were established from the patient’s skin fibroblasts (Coriell Institute) as previously described .

Techniques: Activity Assay, Isolation, Immunofluorescence, Western Blot

Journal: Nature Communications

Article Title: Neuronal SphK1 acetylates COX2 and contributes to pathogenesis in a model of Alzheimer’s Disease

doi: 10.1038/s41467-018-03674-2

Figure Lengend Snippet: Elevation of SphK1 reduces Aβ deposition and neuroinflammation in APP/PS1 mice. a Left, representative immunofluorescence images of thioflavin S (ThioS, Aβ plaques) in cortex and hippocampus of APP/PS1 and APP/PS1/SphK1 tg mice. Scale bars, 200 μm. Right, quantification of area occupied by Aβ plaques ( n = 7–8 per group). b Representative immunofluorescence images and quantification of 6E10 ( n = 5–6 per group; Scale bars, 80 μm). c Left, immunofluorescence images of microglia (Iba1) in cortex of WT, APP/PS1, APP/PS1/SphK1 tg or SphK1 tg mice brain. Low-magnification scale bars, 100 μm; High-magnification scale bars, 30 μm. Right, quantification of microglia ( n = 8–10 per group). d Left, immunofluorescence images of astrocyte (GFAP) in cortex of mice brain. Scale bars, 100 μm. Right, quantification of astrocytes ( n = 7–8 per group). e mRNA levels of inflammatory markers in cortex of mice brain ( n = 4–8 per group). Pro-inflammatory marker: TNF-α, IL-1β, IL-6 and iNOS , Immunoregulatory cytokine: IL-10 , Anti-inflammatory marker: IL-4, TGF-β and Arg1 . All data analysis was performed on 9-month-old mice. a , b Student’s t test. c – e One-way analysis of variance, Tukey’s post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001. All error bars indicate s.e.m.

Article Snippet: PS1 and ApoE4-iPSCs were established from the patient’s skin fibroblasts (Coriell Institute) as previously described .

Techniques: Immunofluorescence, Marker

Journal: Nature Communications

Article Title: Neuronal SphK1 acetylates COX2 and contributes to pathogenesis in a model of Alzheimer’s Disease

doi: 10.1038/s41467-018-03674-2

Figure Lengend Snippet: Neuronal SphK1 induces SPMs secretion by COX2 acetylation. a Protein levels of LxA4, RvE1, and RvD1 were detected by using ELISA in CM of neurons derived from WT, APP/PS1, APP/PS1/SphK1 tg, and SphK1 tg mice ( n = 6–8 per group). b mRNA levels of COX2 and LOX-15 in neurons derived from cortex of WT, APP/PS1, APP/PS1/SphK1 tg, and SphK1 tg mice ( n = 4–6 per group). c Quantification of neuronal COX2 ( n = 6 per group) and neuronal LOX-15 ( n = 4–6 per group). d Acetylation assay of COX2 protein in neurons derived from WT, APP/PS1, APP/PS1/SphK1 tg, and SphK1 tg mice. [ 14 C] aspirin-treated neuron was positive control. Sonicated neurons incubated in the presence of [ 14 C] acetyl-CoA for 2 h at 37 °C and then COX2 was purified and analyzed on scintillation counter ( n = 3–6 per group). e Representative chromatograms of blank, 15-R-LxA4 standard, and 15-R-LxA4 in WT samples (left panel). Molecular MS scanning from the peak at retention time 6.8 min (right upper panel) and MS/MS fragmentation pattern of 15-R-LxA4 from the peak at retention time 6.8 min (right lower panel). f Representative chromatograms (left panel) and quantification of 15-R-LxA4 in neurons derived from WT, APP/PS1, APP/PS1/SphK1 tg, and SphK1 tg mice with acetyl-coA treatment (24 h after 2.5 mM acetyl-CoA treatment) (right panel, n = 6 per group). All data analysis was done on 9-month-old mice. a − d , f One-way analysis of variance, Tukey’s post hoc test. * P < 0.05, *** P < 0.001. All error bars indicate s.e.m.

Article Snippet: PS1 and ApoE4-iPSCs were established from the patient’s skin fibroblasts (Coriell Institute) as previously described .

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Acetylation Assay, Positive Control, Sonication, Incubation, Purification, Tandem Mass Spectroscopy

Journal: Nature Communications

Article Title: Neuronal SphK1 acetylates COX2 and contributes to pathogenesis in a model of Alzheimer’s Disease

doi: 10.1038/s41467-018-03674-2

Figure Lengend Snippet: Increased SphK1 improves microglial phagocytosis. a Colocalization of microglia (Iba1, red) with Aβ (Aβ42, green) and quantification. Scale bars, 10 μm; 3D reconstruction from confocal image stacks scale bars, 10 μm ( n = 7 per group). b Left, representative photomicrograph of live slice section incubated with fluorescent beads (green). Scale bar, 10 μm. White arrow point to phagocytotic microglia with fluorescent beads. Right, quantification of the number of microglial phagocytes normalized to the total number of microglia ( n = 4–6 per group). c Up, immunofluorescence images of thio S (Aβ plaques, green) encapsulated within Lamp1 + structures (phagolysosomes, blue) in microglia (Iba1, red) present in brains of APP/PS1 and APP/PS1/SphK1 tg mice. Low-magnification scale bars, 50 μm; High-magnification scale bars, 10 μm; 3D reconstruction from confocal image stacks scale bars, 10 μm. Down, quantitation of microglial volume occupied by Lamp1 + phagolysosomes, percent of microglia containing Aβ-loaded phagolysosomes and Aβ encapsulated in phagolysosomes ( n = 5 per group). d Morphology of microglia (Iba1, red) surrounding Aβ (ThioS, green) in cortex of APP/PS1 and APP/PS1/SphK1 tg mice. Up, high magnification (Scale bars, 10 μm) and Imaris-based three-dimensional images (Scale bars, 5 μm) of microglia surrounding Aβ. Down, Imaris-based automated quantification of microglial morphology ( n = 7–8 per group). e Morphometric analysis of Aβ plaques in APP/PS1 and APP/PS1/SphK1 tg mice ( n = 5–6 per group). Brain sections were labeled with thio S and plaques were counted and assigned to three mutually exclusive size categories based on maximum diameter: small <25 μm; medium 25–50 μm; or large >50 μm. All data analysis was performed on 9-month-old mice. a , c−e , Student’s t test. b One-way analysis of variance, Tukey’s post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001. All error bars indicate s.e.m.

Article Snippet: PS1 and ApoE4-iPSCs were established from the patient’s skin fibroblasts (Coriell Institute) as previously described .

Techniques: Incubation, Immunofluorescence, Quantitation Assay, Labeling

Journal: Cell Biology and Toxicology

Article Title: Chronic cadmium exposure promotes TRPM7-dependent acquisition of a myofibroblast-like phenotype in pancreatic stellate cells

doi: 10.1007/s10565-026-10169-0

Figure Lengend Snippet: Expression of pancreatic stellate cell markers in PS-1 cells chronically exposed to Cd. A Protein level expression of α-SMA studied by western-blot ( n = 6). B Protein level expression of vimentin studied by western-blot ( n = 7). C Protein level expression of desmin studied by western-blot ( n = 7). D Protein level expression of GFAP studied by western-blot ( n = 5). E Protein level expression of α-SMA studied by immunofluorescence ( n = 3). F Protein level expression of vimentin studied by immunofluorescence ( n = 3). G Protein level expression of desmin studied by immunofluorescence ( n = 3). H. Protein level expression of GFAP studied by immunofluorescence ( n = 3). Bar = 10 µm. * p < 0.05, unpaired Student t-tests

Article Snippet: PS-1 cells were seeded onto Labtek® chambers and allowed to adhere overnight.

Techniques: Expressing, Western Blot, Immunofluorescence

Journal: Cell Biology and Toxicology

Article Title: Chronic cadmium exposure promotes TRPM7-dependent acquisition of a myofibroblast-like phenotype in pancreatic stellate cells

doi: 10.1007/s10565-026-10169-0

Figure Lengend Snippet: Expression of matrix extracellular proteins and inflammatory cytokines in PS-1 cells chronically exposed to Cd. A Protein level expression of type I collagen studied by western-blot ( n = 8). B Protein level expression of elastin studied by western-blot ( n = 11). C Protein level expression of fibronectin studied by western-blot ( n = 8). D Protein level expression of type I collagen studied by immunofluorescence ( n = 3). E Protein level expression of elastin studied by immunofluorescence ( n = 3). F Protein level expression of fibronectin studied by immunofluorescence ( n = 3). G Expression of secreted IL-8 ( n = 3). H Expression of secreted IL-10 by ELISA assays ( n = 3). Bar = 100 µm. ** p < 0.01; *** p < 0.001, Mann–Whitney (B, H) or unpaired Student t-tests (A, C, G)

Article Snippet: PS-1 cells were seeded onto Labtek® chambers and allowed to adhere overnight.

Techniques: Expressing, Western Blot, Immunofluorescence, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Cell Biology and Toxicology

Article Title: Chronic cadmium exposure promotes TRPM7-dependent acquisition of a myofibroblast-like phenotype in pancreatic stellate cells

doi: 10.1007/s10565-026-10169-0

Figure Lengend Snippet: Effect of Cd exposure on PS-1 cell migration. A MMP-2 secretion in control and Cd exposed PS-1 ( n = 4). B uPA secretion in control and Cd exposed PS-1 ( n = 4). C Cell migration of control and Cd exposed PS-1 cells assessed by wound healing assay. Photographs were taken at magnification × 50 right after the wound (T0) and 24 h after (T24) ( n = 4) D Cell migration of control and Cd exposed PS-1 cells evaluated in Boyden chambers ( n = 6). Bar = 10 µm. *** p < 0.001, Mann–Whitney tests

Article Snippet: PS-1 cells were seeded onto Labtek® chambers and allowed to adhere overnight.

Techniques: Migration, Control, Wound Healing Assay, MANN-WHITNEY

Journal: Cell Biology and Toxicology

Article Title: Chronic cadmium exposure promotes TRPM7-dependent acquisition of a myofibroblast-like phenotype in pancreatic stellate cells

doi: 10.1007/s10565-026-10169-0

Figure Lengend Snippet: Role of TRPM7 in PS-1 cell exposed to cadmium. A Protein level expression of TRPM7 studied by western-blot ( n = 5). B Averaged current–voltage relationship of MIC currents recorded between −100 and + 100 mV (left) and current-densities measured at + 100 mV in control and Cd exposed PS-1 cells (right; n = 5). C Effect of TRPM7 silencing on TRPM7 mRNA expression ( n = 6). D Averaged current–voltage relationship of MIC currents recorded between −100 and + 100 mV (left) and current-densities measured at + 100 mV in control and Cd exposed PS-1 cells transfected with siTRPM7 or scrambled siRNA (siCTL) (right; n = 4 for control cells; n = 3 for Cd exposed cells). E Representative photographs of migrating cells through Boyden chambers exposed to the different experimental conditions. F Effect of Cd exposure on control ( n = 3) and TRPM7 silenced ( n = 3) cell migration. G Effect of Cd exposure on control ( n = 5) and NS8593 treated ( n = 5) cell migration. Bar = 10 µm. * p < 0.05; ** p < 0.01; *** p < 0.001, Mann–Whitney (A) or unpaired Student t-tests (B), or 2-ways ANOVA followed by Šídák's multiple comparisons test (C, D, F, G)

Article Snippet: PS-1 cells were seeded onto Labtek® chambers and allowed to adhere overnight.

Techniques: Expressing, Western Blot, Control, Transfection, Migration, MANN-WHITNEY

Journal: Cell Biology and Toxicology

Article Title: Chronic cadmium exposure promotes TRPM7-dependent acquisition of a myofibroblast-like phenotype in pancreatic stellate cells

doi: 10.1007/s10565-026-10169-0

Figure Lengend Snippet: Effect of Cd exposure on the interaction between pancreatic stellate cells and cancer cells. A Schematic representation of the co-culture protocol (top panel) and representative photographs of migrating MIA PaCa-2 through Boyden chambers (low panel). B Measurement of MIA PaCa-2 migration stimulated by PS-1 exposed to Cd ( n = 5). C Schematic representation of the co-culture protocol (top panel) and representative photographs of migrating MIA PaCa-2 through Boyden chambers (low panel). D Effect of NS8593 on MIA PaCa-2 cell migration stimulated by PS-1 cells ( n = 3). E Schematic representation of the co-culture protocol (top panel) and representative photographs of migrating MIA PaCa-2 through Boyden chambers (low panel). F Effect of TRPM7 silencing on MIA PaCa-2 cell migration stimulated by PS-1 cells ( n = 6). Bar = 10 µm. * p < 0.05; *** p < 0.001, ANOVA followed by Tukey's multiple comparisons test (B) or 2-ways ANOVA followed by Šídák's multiple comparisons test (D and F)

Article Snippet: PS-1 cells were seeded onto Labtek® chambers and allowed to adhere overnight.

Techniques: Co-Culture Assay, Migration