Journal: bioRxiv

Article Title: FTY720 requires vitamin B 12 -TCN2-CD320 signaling in astrocytes to reduce disease in an animal model of multiple sclerosis

doi: 10.1101/2022.01.10.475450

Figure Lengend Snippet: A , Disease course of EAE-induced WT fos , S1P 1 -AsCKO fos , and FTY720-treated WT fos (WT fos +FTY720) mice (n = 50, 43, and 32 animals, respectively). Day 0 was designated as the day of clinical sign onset (mean ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001 by two-way ANOVA with Bonferroni’s multiple comparisons test). The x-axis represents “days post onset,” which is different from “days after immunization.” B , Fluorescence-activated nuclei sorting (FANS) to isolate NeuN-GFP + singlet nuclei from EAE spinal cords at 5-days post onset that is equivalent to 5-days off doxycycline. C , Nuclear RNA-seq. Heatmap is shown as 2-fold up- and down-regulated genes in S1P 1 -AsCKO fos and WT fos +FTY720 vs. WT fos . Top 4 significantly enriched gene ontologies identified by ClueGO of Cytoscape are shown. Expression levels of Cd320 (count per million, CPM) are plotted. D , Expression of Cd320 mRNA in EAE SCs determined by qPCR (mean ± SEM, * p < 0.05, ** p < 0.01 and *** p < 0.001 by t test for top panel, and one-way ANOVA with Tukey’s multiple comparisons test for bottom panel). E , Cd320 mRNA expression in human MS normalized to control brains (mean ± SEM, n = 5 and 2 brains, respectively. * p < 0.01 by one sample t test). F-I , Immunohistochemistry (IHC) for CD320 and GFAP in human control brain ( F and G ) and MS brain disease plaques ( H ). Scale bar, 100 μm ( F and H ) and 5 μm ( G ). CD320 immunolabeling is significantly reduced in human MS plaques as compared to control brain ( I ). Each symbol represents an assessed region of interest in the sections. * p < 0.001 by Mann-Whitney U test. J-K , Cd320 mRNA expression in primary astrocytes stimulated with 10 μg/mL poly(I:C) in the presence of S1P ( J ) or S1P 1 specific agonist, RP001 ( K ). Data are from three independent experiments of three technical replicates (mean ± SEM, ** p < 0.01 and *** p < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test). L , EAE disease course of CD320-WT and CD320-KO mice (n = 6 and 5 animals, respectively). Data are from three independent experiments (mean ± SEM, *, statistical significance was analyzed by two-way ANOVA; interaction, p = 0.90; time, p <0.0001; genotype, p < 0.001).

Article Snippet: Cells were washed with DMEM and stimulated with FTY720 and sphingosine in the presence or absence of serum for 1 h. Cells were detached and sequentially stained with anti-CD320 antibody (1:100 dilution: Proteintech, cat # 10343-1-AP) as the primary antibody and Alexa Fluor 488-conjugated Goat Anti-Rabbit IgG (1:1000 dilution: ThermoFishre Scientific, cat #A-11008) as a secondary antibody.

Techniques: Fluorescence, RNA Sequencing, Expressing, Control, Immunohistochemistry, Immunolabeling, MANN-WHITNEY

Journal: bioRxiv

Article Title: FTY720 requires vitamin B 12 -TCN2-CD320 signaling in astrocytes to reduce disease in an animal model of multiple sclerosis

doi: 10.1101/2022.01.10.475450

Figure Lengend Snippet: A , FTY720 treatment on EAE-induced CD320-WT (n = 8) and CD320-KO mice (n = 14). Data are combined from two independent experiments (mean ± SEM; *, statistical significance was analyzed by two-way ANOVA between day 9 to 20; interaction, p = 0.16; time, p <0.01; genotype, p < 0.01). B, Recovery rate with FTY720 treatment (*, p < 0.05 by one-way ANOVA with Bonferroni’s multiple comparisons test). C, IFN-β mRNA expression in EAE SCs (*, p < 0.05 by one-way ANOVA with Bonferroni’s multiple comparisons test; N.S ., non-significant). D, Therapeutic treatment of EAE-induced B 12 def mice with vehicle, FTY720, and FTY720+B 12 (n = 5, 5, 6 animals, respectively). Data are representative of 2 independent experiments (mean ± SEM; * p < 0.05 vs. Vehicle by two-way ANOVA, interaction, p = 0.27, time, p = 0.74; diet, p < 0.0001 with Bonferroni’s multiple comparisons test). E, Recovery rate with FTY720 treatment (*, p < 0.05 by one-way ANOVA with Bonferroni’s multiple comparisons test). F, IFN-β mRNA expression in EAE SCs (*, p < 0.05 by one-way ANOVA with Bonferroni’s multiple comparisons test).

Article Snippet: Cells were washed with DMEM and stimulated with FTY720 and sphingosine in the presence or absence of serum for 1 h. Cells were detached and sequentially stained with anti-CD320 antibody (1:100 dilution: Proteintech, cat # 10343-1-AP) as the primary antibody and Alexa Fluor 488-conjugated Goat Anti-Rabbit IgG (1:1000 dilution: ThermoFishre Scientific, cat #A-11008) as a secondary antibody.

Techniques: Expressing

Journal: bioRxiv

Article Title: FTY720 requires vitamin B 12 -TCN2-CD320 signaling in astrocytes to reduce disease in an animal model of multiple sclerosis

doi: 10.1101/2022.01.10.475450

Figure Lengend Snippet: A-C, Specific binding curves between CD320 vs. TCN2 and B 12 ( A ), FTY720 and FTY720P ( B ), sphingosine (Sph) and S1P ( C ). D-F, Specific binding curves between TCN2 vs. B 12 ( D ), FTY720 and FTY720P ( E ), sphingosine (Sph) and S1P ( F ). Compensated Interferometric Reader (CIR) signals are plotted against concentrations of binding partners and fitted by nonlinear regression using the Michaelis-Menten equation (mean ± SEM, n = 6-7). G, Computational modeling of FTY720 binding site on TCN2 (PDB ID, 4ZRP). Cartoon representation of TCN2 (light grey) and LDLR domains of CD320 (dark grey) ( left ) and electrostatic surface of these complex ( right ). H, Magnified view of a potential FTY720 binding site in TCN2. Interactions are represented by dashed lines with distances between atoms (Å). I, A representative FCM histogram of CD320 expression on HASTR/ci35 cells stimulated with or without FTY720 (1 μM) in the presence or absence of FBS ( left ). Normalized CD320 expression levels (mean ± SEM, n = 6-7, pooled from 2 independent experiments). *, p < 0.05 by Kruskal-Wallis test with Dunn’s multiple comparisons test; N.S., non-significant.

Article Snippet: Cells were washed with DMEM and stimulated with FTY720 and sphingosine in the presence or absence of serum for 1 h. Cells were detached and sequentially stained with anti-CD320 antibody (1:100 dilution: Proteintech, cat # 10343-1-AP) as the primary antibody and Alexa Fluor 488-conjugated Goat Anti-Rabbit IgG (1:1000 dilution: ThermoFishre Scientific, cat #A-11008) as a secondary antibody.

Techniques: Binding Assay, Expressing

Journal: bioRxiv

Article Title: FTY720 requires vitamin B 12 -TCN2-CD320 signaling in astrocytes to reduce disease in an animal model of multiple sclerosis

doi: 10.1101/2022.01.10.475450

Figure Lengend Snippet: Pink arrows indicate newly identified pathways that contribute to the direct CNS effects of FTY720 beyond functional antagonism of astrocyte S1P 1 . ; S1P 1 inhibition in peripheral lymphocytes is the originally proposed CNS MOA. ; FTY720 is complexed with B 12 -TCN2. ; This FTY720 complex induces CD320 internalization and, along with B 12 , is taken up via astrocyte CD320. ; B 12 is essential for astrocyte IFN-I sensitivity and microglial IFN-β production. ; Astrocyte SK1/2 phosphorylates FTY720 to form FTY720P, which is identified in the accompanying Resource paper . ; The resulting FTY720P may be transported via SPNS2 and functionally antagonizes S1P 1 in an autocrine/paracrine manner. ; FTY720P functionally antagonizes astrocyte S1P 1 . ; Astrocytic S1P 1 inhibition increases CD320 expression, enabling increased engagement of, and astrocyte internalization, of the B 12 -TCN2-FTY720 complex.

Article Snippet: Cells were washed with DMEM and stimulated with FTY720 and sphingosine in the presence or absence of serum for 1 h. Cells were detached and sequentially stained with anti-CD320 antibody (1:100 dilution: Proteintech, cat # 10343-1-AP) as the primary antibody and Alexa Fluor 488-conjugated Goat Anti-Rabbit IgG (1:1000 dilution: ThermoFishre Scientific, cat #A-11008) as a secondary antibody.

Techniques: Functional Assay, Inhibition, Expressing

Journal: BMC Cancer

Article Title: Identification of a Novel CD8 + T cell exhaustion-related gene signature for predicting survival in hepatocellular carcinoma

doi: 10.1186/s12885-023-11648-x

Figure Lengend Snippet: Expression of STAM, ANXA5 and MAD2L2 in HCC cell lines. A The expressions of STAM, ANXA5 and MAD2L2 in normal hepatocytes and hepatoma cell lines were detected using Western blotting. Knocking down STAM, ANXA5 and MAD2L2 inhibited the proliferation and migration of HCC cells. B The knockdown efficiency of STAM, ANXA5 and MAD2L2 was detected using Western Blotting. C Plate cloning experiment showed that the number of cloned cell clusters formed by hepatocellular carcinoma cells after knockdown of STAM, ANXA5 and MAD2L2 was significantly reduced; D The results of CCK8 experiment showed that inhibiting the expression of STAM, ANXA5 and MAD2L2 decreased the proliferative ability of HCC cells; E Scratch assay showed that knockdown of STAM, ANXA5 and MAD2L2 inhibited the migration of hepatocellular carcinoma cells; F Transwell experiment showed that the migration ability of hepatocellular carcinoma cells was weakened after inhibiting the expression of STAM, ANXA5 and MAD2L2 .* P < 0.05, ** P < 0.01, *** P < 0.001, there was no significant difference in ns

Article Snippet: Anti- MAD2L2 (BM5428) and anti- STAM (A00864-1) antibodies were purchased from BosterBio (Pleasanton, CA, USA), while Anti- TBL1XR1 (MBS850368), Anti- ANXA5 (MBS474163), Anti- FKBP1A (MBS9404059) and Anti- PPM1G (MBS626576) antibodies were purchased from MyBioSource (San Diego, CA, USA).

Techniques: Expressing, Western Blot, Migration, Knockdown, Cloning, Clone Assay, Wound Healing Assay