Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Recapitulating Cholangiopathy-Associated Necroptotic Cell Death In Vitro Using Human Cholangiocyte Organoids

doi: 10.1016/j.jcmgh.2021.10.009

Figure Lengend Snippet: Necroptotic mediators are associated with hepatic pathogenesis. ( A ) Representative microscopic images of immunohistochemical staining with cytokeratin-19 (cholangiocyte marker) and RIPK3 ( black arrows ), as well as pMLKL ( yellow arrows ), on consecutive sections of biopsy specimens from liver transplantation, including donation after brain death (DBD) (n = 3) and donation after cardiac death (DCD) (n = 3) donor livers and explant livers from recipients undergoing liver retransplantation as a result of hepatic artery thrombosis (HAT), ischemic-type biliary lesions (ITBL) (n = 1), and primary graft nonfunction (PNF) (n = 1). Detailed images are shown in the right panel (magnification, 800×). ( B ) Sections from patients with end-stage liver diseases, including NASH (n = 3), ALD (n = 3), and PSC (n = 3) are shown. ( C ) Sections from nonischemic liver tissue obtained from a living donor (LDLT) are shown (n = 1). All detailed images are shown in the right panel (magnification, 800×). ( D ) Expression heatmap of selected genes associated with hepatocyte (Hep), cholangiocyte (Cho), pro-apoptosis, prosurvival, and necroptosis machinery in primary cholangiocytes derived from the common bile duct (CBD; n = 4) and primary human hepatocytes (PHHs; n = 2). KRT19, cytokeratin-19.

Article Snippet: p–NF-κB2 p100 , Human , IF , Rabbit , Polyclonal , Cell Signaling (4810S) , 1:50.

Techniques: Immunohistochemical staining, Staining, Marker, Transplantation Assay, Expressing, Derivative Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Recapitulating Cholangiopathy-Associated Necroptotic Cell Death In Vitro Using Human Cholangiocyte Organoids

doi: 10.1016/j.jcmgh.2021.10.009

Figure Lengend Snippet: Transcriptional profiles of inflammatory genes and NF-κB signaling in necroptotic hICOs. hICOs were stimulated as indicated for 6 hours. ( A ) Gene expression levels of inflammatory genes were measured by quantitative polymerase chain reaction (n = 5). Fold change relative to TNF-α–treated hICOs was presented. ( B ) Representative confocal images of p-IKKα/β, p–NF-κB2 p100, and p65–NF-κBactive staining in treated hICOs are shown. ( C ) Quantification of positive cells in fluorescent images was performed using ImageJ (at least 15 high-power fields in each condition). Data are means ± SD. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, ( A ) 1-way analysis of variance test followed by the Tukey post hoc test and ( C ) the Kruskal–Wallis test followed by the Dunn post hoc test. DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: p–NF-κB2 p100 , Human , IF , Rabbit , Polyclonal , Cell Signaling (4810S) , 1:50.

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Recapitulating Cholangiopathy-Associated Necroptotic Cell Death In Vitro Using Human Cholangiocyte Organoids

doi: 10.1016/j.jcmgh.2021.10.009

Figure Lengend Snippet: List of Antibodies Used in This Study

Article Snippet: p–NF-κB2 p100 , Human , IF , Rabbit , Polyclonal , Cell Signaling (4810S) , 1:50.

Techniques:

Journal: Communications Chemistry

Article Title: Supercritical fluid chromatography-mass spectrometry enables simultaneous measurement of all phosphoinositide regioisomers

doi: 10.1038/s42004-022-00676-6

Figure Lengend Snippet: a – c MEFs were treated with 1 μM VPS34-IN, 200 nM Apilimod, or vehicle (DMSO) for 90 min, then PIPs were extracted and analyzed using SFC-MS/MS system. a The content of individual PIPs in MEFs (1 × 10 6 cells) treated with 1 μM VPS34-IN, 200 nM Apilimod or vehicle (DMSO) ( n = 3). b , c MRM chromatogram [987.6 → 605.6 ( b ) and 1117.6 → 627.6 ( c )] of extracts from MEFs (1 × 10 6 cells) treated with VPS34-IN, Apilimod or vehicle (DMSO). d – g MEFs were treated with 10 μM PAO, 1 μM A23187, or vehicle (DMSO) for 30 min, and then PIPs were extracted and analyzed using SFC-MS/MS system (see “Methods”). d , e The content of individual PIPs in MEFs (1 × 10 6 cells) treated with 10 μM PAO, 1 μM A23187 or vehicle (DMSO) ( n = 4). f , g Overlay of MRM chromatogram [987.6 → 605.6 ( f ) and 1117.6 → 627.6 ( g )] of extracts from MEFs (1 × 10 6 cells) treated with PAO, A23187, or vehicle (DMSO). h Contents of individual PIPs in MEFs (1 × 10 6 cells) transfected with the indicated siRNA ( n = 3). PIPs were extracted at 48 h after transfection. i , j Intracellular 38:4-PI(3,4,5)P 3 content ( i ) and species ( j ) in MEFs. MEF cells cultured in complete medium were serum-starved for 12 h (starved) and then treated with 10% FBS for 10 min (stimulated). PIPs were extracted from MEFs at each step and analyzed using SFC-MS/MS system. Data were collected using a QTRAP4500 mass spectrometer. Values are mean ± s.e.m. Data were analyzed by one-way ANOVA with Dunnett’s test. * P < 0.05, ** P < 0.01, and *** P < 0.001 vs vehicle (DMSO). Data are from one set of experiments.

Article Snippet: The membranes were blocked with 5% (w/v) BSA in TTBS buffer [10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% (w/v) Tween 20] and incubated with an antibody to GAPDH (6C5; Calbiochem), monoclonal mouse anti-LPIAT1 antibody (clone: FT10) , VPS34 (sc-365404; Santa Cruz), Pikfyve (sc-100408; Santa Cruz), PI4Kα (12411-1-AP; Proteintech), and PI4Kβ (611816; BD laboratory).

Techniques: Tandem Mass Spectroscopy, Transfection, Cell Culture, Mass Spectrometry

Journal: Journal of Neuroinflammation

Article Title: Circular RNA METTL9 contributes to neuroinflammation following traumatic brain injury by complexing with astrocytic SND1

doi: 10.1186/s12974-023-02716-x

Figure Lengend Snippet: CircMETTL9 interacts with SND1 protein in astrocytes. A Experimental procedure for protein pull-down by circMETTL9. First, circMETTL9 probe is biotinylated and added to lysate from LPS-stimulated astrocytes. Magnetic beads with bound streptavidin are then added to capture biotinylated probe and associated bound proteins. Finally, proteins are obtained by washing the magnetic beads. B KEGG pathway analysis of the top twenty enriched proteins bound to circMETTL9 according to mass spectrometry. C Overlap of proteins in catRAPID with Interaction Propensity > 70 and proteins with -10lgP > 70 by mass spectrometry (potential circMETTL9-interacting proteins). D Silver staining showing the presence of SND1. E Detection of SND1 by western blot after pull-down from astrocyte lysate by circMETTL9 probe but not negative probe. F Ion peak pattern showing the SND1 peptide, it is the secondary map of SND1 protein to a specific peptide. G Co-localization of SND1 (green) with circMETTL9 (red) in astrocyte cytoplasm by combined IF-FISH. Scale bar = 50 µm. H Representative western blots of SND1 expression in astrocytes after knockdown of circMETTL9. * P < 0.05, ** P < 0.01, and *** P < 0.001 by one-way ANOVA

Article Snippet: Membranes were blocked with 5% non-fat skim milk for 3 h, incubated sequentially in primary antibody against SND1 (1:5000; Proteintech, 60265-1-Ig) or β-actin (1:5,000; Proteintech, 66009-1-Ig) at 4 °C overnight and then in secondary antibodies for 1 h, and treated with enhanced chemiluminescence reagent (Proteintech, PK10001).

Techniques: Magnetic Beads, Mass Spectrometry, Silver Staining, Western Blot, Expressing, Knockdown

Journal: Journal of Neuroinflammation

Article Title: Circular RNA METTL9 contributes to neuroinflammation following traumatic brain injury by complexing with astrocytic SND1

doi: 10.1186/s12974-023-02716-x

Figure Lengend Snippet: SND1 mediates the LPS-induced inflammatory response in astrocytes. A Treatment of astrocytes with 500 ng/mL LPS for 3, 12, and 24 h elevated SND1 expression as measured by qPCR. ** P < 0.01 by one-way ANOVA. B Two of three siRNAs targeting SND1 successfully knocked down SND1 in astrocytes after transfection. * P < 0.05 and ** P < 0.01 by one-way ANOVA. C – G Astrocyte expression levels of CCL2, CXCL1, CCL3, CXCL3, and CXCL10 were elevated by LPS, while 2 different siRNAs reversed these effects. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 by one-way ANOVA

Article Snippet: Membranes were blocked with 5% non-fat skim milk for 3 h, incubated sequentially in primary antibody against SND1 (1:5000; Proteintech, 60265-1-Ig) or β-actin (1:5,000; Proteintech, 66009-1-Ig) at 4 °C overnight and then in secondary antibodies for 1 h, and treated with enhanced chemiluminescence reagent (Proteintech, PK10001).

Techniques: Expressing, Transfection

Journal: Journal of Neuroinflammation

Article Title: Circular RNA METTL9 contributes to neuroinflammation following traumatic brain injury by complexing with astrocytic SND1

doi: 10.1186/s12974-023-02716-x

Figure Lengend Snippet: SND1 is regulated by circMETTL9. A – E Astrocytes transfected with circMETTL9 overexpression plasmid demonstrated stronger LPS-induced inflammatory responses than astrocytes co-transfected with circMETTL9 overexpression plasmid and an SND1-targeted siRNA as evidenced by expression levels of CCL2, CXCL1, CCL3, CXCL3, and CXCL10. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 by one-way ANOVA

Article Snippet: Membranes were blocked with 5% non-fat skim milk for 3 h, incubated sequentially in primary antibody against SND1 (1:5000; Proteintech, 60265-1-Ig) or β-actin (1:5,000; Proteintech, 66009-1-Ig) at 4 °C overnight and then in secondary antibodies for 1 h, and treated with enhanced chemiluminescence reagent (Proteintech, PK10001).

Techniques: Transfection, Over Expression, Plasmid Preparation, Expressing

Journal: Journal of Neuroinflammation

Article Title: Circular RNA METTL9 contributes to neuroinflammation following traumatic brain injury by complexing with astrocytic SND1

doi: 10.1186/s12974-023-02716-x

Figure Lengend Snippet: Knockdown of circMETTL9 mitigates cortical neuroinflammation in TBI model rats by downregulating SND1. A Representative western blots of SND1 expression in the cerebral cortex of rats 7 days post-TBI. * P < 0.05 and ** P < 0.01 by one-way ANOVA. B – F Expression levels of CCL2, CXCL1, CCL3, CXCL3, and CXCL10 in cortex 7 days post-TBI as measured by qPCR. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 by one-way ANOVA

Article Snippet: Membranes were blocked with 5% non-fat skim milk for 3 h, incubated sequentially in primary antibody against SND1 (1:5000; Proteintech, 60265-1-Ig) or β-actin (1:5,000; Proteintech, 66009-1-Ig) at 4 °C overnight and then in secondary antibodies for 1 h, and treated with enhanced chemiluminescence reagent (Proteintech, PK10001).

Techniques: Knockdown, Western Blot, Expressing