Journal: Cardiovascular research

Article Title: Nogo-A reduces ceramide de novo biosynthesis to protect from heart failure.

doi: 10.1093/cvr/cvac108

Figure Lengend Snippet: Figure 1 The loss of cardiomyocytes Nogo-A exacerbates pathological cardiac hypertrophy. Representative images of (A) whole heart and (B) H&E staining of longitudinal heart sections of Nogo-A/Bf/f and (Nogo-A/Bf/f)Mlc2v-Cre+ sham and 3-month TAC-operated mice. (C) Heart weight/tibial length (HW/TL) ratios of sham and 3-month TAC-operated mice. (D) Real-time (RT)-PCR for Nogo-A/B mRNA expression in CM isolated from sham and 7 days TAC-operated Nogo-A/Bf/f and (Nogo-A/Bf/f)Mlc2v-Cre+ mice. GAPDH was used as housekeeping gene (n = 7 mice/group). (E) Immunofluorescence staining for Nogo-A/B and Nogo-A of myocardial sections from sham and TAC-operated Nogo-A/Bf/f and (Nogo-A/Bf/f)Mlc2v-Cre+ mice. FITC-labelled wheat germ agglutinin (WGA) staining has been used to evidence cell membranes in myocardial sections. Nuclei are stained with DAPI. Data are expressed as mean ± SEM. ***P≤0.001; Statistical significance was determined by two-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: After permeabilization in 0.5% Triton X-100 in 5% BSA solution, frozen myocardial sections were incubated at 4°C o.n. with antibodies against Nogo-A/ B (1:100, #AF-6034; R&D Systems, Minneapolis, MN, USA) and Nogo-A (1:100, #MAB3098; R&D Systems) in 5% BSA in PBS solution, followed by secondary antibodies in PBS solution, Alexa Fluor-555 donkey antisheep (1:200, #A21436; Invitrogen, Waltham, MA, USA) and Alexa Fluor 568-conjugated donkey anti-mouse IgG (1:200, #A10037; Invitrogen), and with fluorescein isothiocyanate (FITC)-conjugated wheat germ agglutinin (40 μg/mL; #L4895; Sigma) in PBS for 1 h at room temperature.

Techniques: Staining, Quantitative RT-PCR, Expressing, Isolation, Immunofluorescence, Comparison

Journal: Cardiovascular research

Article Title: Nogo-A reduces ceramide de novo biosynthesis to protect from heart failure.

doi: 10.1093/cvr/cvac108

Figure Lengend Snippet: Figure 2 The absence of Nogo-A in CM worsen heart failure and decreases survival rate. Echocardiographic analysis of sham- and TAC-operated Nogo-A/Bf/f (n = 14) and (Nogo-A/Bf/f)Mlc2v-Cre+ (n = 12) mice at different time points. (A) Left-ventricle end-diastolic diameter (LVDd), (B) LV end- systolic diameter (LVDs), (C) fractional shortening (FS) measured at baseline and 2, 4, and 12 weeks after TAC. (D) Representative images of LV serial echo cardiography. (E) Survival curve of Nogo-A/Bf/f (n = 16) and (Nogo-A/Bf/f)Mlc2v-Cre+ (n = 18) post-TAC. Survival analysis was performed by the Kaplan– Meier method, and between-group differences in survival were evaluated by using the Gehan–Breslow–Wilcoxon test. Flow cytometry analysis of inflam matory cells in non-CM cell suspension from the hearts of sham-operated Nogo-A/Bf/f mice (n = 4), Nogo-A/Bf/f and (Nogo-A/Bf/f)Mlc2v-Cre+ at 7 days post-TAC (n = 8 mice/group). Total number of: (F) CD45+ haematopoietic cells; (G) CD45+, CD64Lo, Ly6CHi monocytes; (H) CD45+, Ly6CLo, Ly6G-, CD64Hi macrophages; (I) CD45+, CD64-, Ly6CMed, Ly6GHi neutrophils; (J ) CD4+ CD3+ T cells; (K) CD4- CD3+ T cells; and (L) CD19+ B cells. Data are expressed as mean ± SEM. *P≤0.05; **P≤0.01. In A–C, *** is statistic vs. time 0 of the same genotype, and $$$ is statistic between the genotypes. Statistical significance was determined by two-way ANOVA followed by Tukey’s multiple comparison test (A–C) and one-way ANOVA (F–L)

Article Snippet: After permeabilization in 0.5% Triton X-100 in 5% BSA solution, frozen myocardial sections were incubated at 4°C o.n. with antibodies against Nogo-A/ B (1:100, #AF-6034; R&D Systems, Minneapolis, MN, USA) and Nogo-A (1:100, #MAB3098; R&D Systems) in 5% BSA in PBS solution, followed by secondary antibodies in PBS solution, Alexa Fluor-555 donkey antisheep (1:200, #A21436; Invitrogen, Waltham, MA, USA) and Alexa Fluor 568-conjugated donkey anti-mouse IgG (1:200, #A10037; Invitrogen), and with fluorescein isothiocyanate (FITC)-conjugated wheat germ agglutinin (40 μg/mL; #L4895; Sigma) in PBS for 1 h at room temperature.

Techniques: Flow Cytometry, Suspension, Comparison

Journal: Cardiovascular research

Article Title: Nogo-A reduces ceramide de novo biosynthesis to protect from heart failure.

doi: 10.1093/cvr/cvac108

Figure Lengend Snippet: Figure 3 Nogo-A interacts with SPT and regulates ceramide de novo biosynthesis in cardiomyocytes. (A) Western blot (WB) analysis of transfected SPTLC1 and MYC-NOGO-A in HEK293T whole cell lysate (WCL) and after co-immunoprecipitation (co-IP) with anti-MYC antibody. (B) WB analysis of Nogo-A and SPTLC1 in NOGO-A/B-/- and WT myocardial microsomes, in WCL and after co-IP with anti-NOGO-A antibody. RT-PCR for (C) Tnnt2 and Nkx2.5, (D) Fsp1, and (E) Cd45 mRNA expression in CM vs. non-CM fractions. (F) RT-PCR for Cd31 mRNA expression in CM fraction vs. endo thelial cells (ECs). (G) WB analysis of NOGO-A in CM isolated from Nogo-A/Bf/f and (Nogo-A/Bf/f)Mlc2v-Cre+ mice sham, 7 days, 1 and 3 months post-TAC. EC lysate has been used as positive control for NOGO-B expression. (H) SPT activity in CM isolated from Nogo-A/Bf/f [sham n = 5; TAC (7d) n = 7; TAC (1m) n = 4; TAC (3m) n = 5] and (Nogo-A/Bf/f)Mlc2v-Cre+ [sham n = 5; TAC (7d) n = 10; TAC (1m) n = 4; TAC (3m) n = 5] mice. (I) Total ceramides, (J–L) specific ceramides, (M) C16:0-dihydro ceramide (dhC16:0-cer), (N) dihydrosphingosine (dhSph), and (O) sphingosine (Sph) mea sured in CM isolated from Nogo-A/Bf/f [sham n = 6; TAC (7d) n = 5, TAC (1m) n = 4, TAC (3m) n = 4] and (Nogo-A/Bf/f)Mlc2v-Cre+ [sham n = 4; TAC (7d) n = 6, TAC (1m) n = 4, TAC (3m) n = 4] mice. (P) WB analysis and (Q–S) relative quantification of ORMDLs, SPTLC1, and SPTLC2 expression, performed on CM isolated from sham and TAC(7d)-operated Nogo-A/Bf/f and (Nogo-A/Bf/f)Mlc2v-Cre+ mice (n ≥4/group). GAPDH was used as house keeping. (T ) Total diacylglycerols (DAGs) and (U) specific DAG measured in Nogo-A/Bf/f and (Nogo-A/Bf/f)Mlc2v-Cre+ (n = 4/group) CM at 7 days post-TAC. Data are expressed as mean ± SEM. *P≤0.05; **P≤0.01; ***P≤0.001. Statistical significance was determined by unpaired t test (C–F, T–U), and two-way ANOVA followed by Tukey’s multiple comparison test (H–O, Q–S).

Article Snippet: After permeabilization in 0.5% Triton X-100 in 5% BSA solution, frozen myocardial sections were incubated at 4°C o.n. with antibodies against Nogo-A/ B (1:100, #AF-6034; R&D Systems, Minneapolis, MN, USA) and Nogo-A (1:100, #MAB3098; R&D Systems) in 5% BSA in PBS solution, followed by secondary antibodies in PBS solution, Alexa Fluor-555 donkey antisheep (1:200, #A21436; Invitrogen, Waltham, MA, USA) and Alexa Fluor 568-conjugated donkey anti-mouse IgG (1:200, #A10037; Invitrogen), and with fluorescein isothiocyanate (FITC)-conjugated wheat germ agglutinin (40 μg/mL; #L4895; Sigma) in PBS for 1 h at room temperature.

Techniques: Western Blot, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation, Positive Control, Activity Assay, Quantitative Proteomics, Comparison

Journal: Cardiovascular research

Article Title: Nogo-A reduces ceramide de novo biosynthesis to protect from heart failure.

doi: 10.1093/cvr/cvac108

Figure Lengend Snippet: Figure 4 Cardiomyocyte Nogo-A preserves beneficial autophagy by refraining ceramide de novo biosynthesis. (A) WB analysis of LC3B-II and NOGO-A/ B and (B) BECLIN-1 expression in CM from Nogo-A/Bf/f and (Nogo-A/Bf/f)Mlc2v-Cre+ mice. GAPDH was used as housekeeping. (C) Quantification of LC3B-II expression in Nogo-A/Bf/f [sham n = 7; TAC (7d) n = 11] and (Nogo-A/Bf/f)Mlc2v-Cre+ [sham n = 7; TAC (7d) n = 12] CM. (D) Quantification of BECLIN-1 expression in Nogo-A/Bf/f [sham n = 6; TAC (7d) n = 10] and (Nogo-A/Bf/f)Mlc2v-Cre+ [sham n = 6; TAC (7d) n = 8] CM. (E) WB analysis of LC3B-II and BECLIN-1 expression in CM from Nogo-A/Bf/f and (Nogo-A/Bf/f)Mlc2v-Cre+ mice at 7 days post-TAC, with or without CQ. GAPDH was used as housekeeping. Quantification of (F) LC3B-II and (G) BECLIN-1 expression in Nogo-A/Bf/f and (Nogo-A/Bf/f)Mlc2v-Cre+ CM (n = 5). Quantification of (H) mitochondrial ROS in Nogo-A/Bf/f [sham n = 16; TAC (7d) n = 91] and (Nogo-A/Bf/f)Mlc2v-Cre+ [sham n = 109; TAC (7d) n = 59] CM and (I) cel lular ROS in Nogo-A/Bf/f [sham n = 11; TAC (7d) n = 153] and (Nogo-A/Bf/f)Mlc2v-Cre+ [sham n = 77; TAC (7d) n = 86] CM. Data are expressed as mean ± SEM. *P≤0.05; **P≤0.01; ***P≤0.001. Statistical significance was determined by two-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: After permeabilization in 0.5% Triton X-100 in 5% BSA solution, frozen myocardial sections were incubated at 4°C o.n. with antibodies against Nogo-A/ B (1:100, #AF-6034; R&D Systems, Minneapolis, MN, USA) and Nogo-A (1:100, #MAB3098; R&D Systems) in 5% BSA in PBS solution, followed by secondary antibodies in PBS solution, Alexa Fluor-555 donkey antisheep (1:200, #A21436; Invitrogen, Waltham, MA, USA) and Alexa Fluor 568-conjugated donkey anti-mouse IgG (1:200, #A10037; Invitrogen), and with fluorescein isothiocyanate (FITC)-conjugated wheat germ agglutinin (40 μg/mL; #L4895; Sigma) in PBS for 1 h at room temperature.

Techniques: Expressing, Comparison

Journal: Cardiovascular research

Article Title: Nogo-A reduces ceramide de novo biosynthesis to protect from heart failure.

doi: 10.1093/cvr/cvac108

Figure Lengend Snippet: Figure 5 Inhibition of the ceramide de novo biosynthesis with myriocin restores CM autophagy and prevents HF in absence of Nogo-A. (A) WB analysis and quantification of (B) LC3B-II and (C) BECLIN-1 expression in CM from Nogo-A/Bf/f [sham n = 6, TAC (7d) n = 12, TAC (7d + My) n = 8] and (Nogo-A/ Bf/f)Mlc2v-Cre+ [sham n = 6, TAC (7d) n = 8, TAC (7d + My) n = 8] mice. GAPDH was used as housekeeping. Echocardiographic analysis of vehicle- and My-treated TAC-operated Nogo-A/Bf/f (vehicle n = 13; My n = 15) and (Nogo-A/Bf/f)Mlc2v-Cre+ (vehicle n = 12; My n = 12) mice. (D) LVDd, (E) LVDs, (F) FS, and (G) HW/TL ratios of vehicle- and My-treated Nogo-A/Bf/f (vehicle n = 8; My n = 12) and (Nogo-A/Bf/f)Mlc2v-Cre+ mice (vehicle n = 12, My n = 7) measured at 1 month post-TAC. (H) Representative images of LV serial echocardiography. (I) RT-PCR for Ppara, Glut4, Cpt1B, and Cd36 in CM isolated from Nogo-A/Bf/f [sham n = 4, TAC (7d) n = 7; TAC (7d)+My n = 7] and (Nogo-A/Bf/f)Mlc2v-Cre+ [sham n = 4, TAC (7d) n = 4; TAC (7d)+My n = 7] mice. Data are expressed as mean ± SEM. *P≤0.05; **P≤0.01; ***P≤0.001. Statistical significance was determined by two-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: After permeabilization in 0.5% Triton X-100 in 5% BSA solution, frozen myocardial sections were incubated at 4°C o.n. with antibodies against Nogo-A/ B (1:100, #AF-6034; R&D Systems, Minneapolis, MN, USA) and Nogo-A (1:100, #MAB3098; R&D Systems) in 5% BSA in PBS solution, followed by secondary antibodies in PBS solution, Alexa Fluor-555 donkey antisheep (1:200, #A21436; Invitrogen, Waltham, MA, USA) and Alexa Fluor 568-conjugated donkey anti-mouse IgG (1:200, #A10037; Invitrogen), and with fluorescein isothiocyanate (FITC)-conjugated wheat germ agglutinin (40 μg/mL; #L4895; Sigma) in PBS for 1 h at room temperature.

Techniques: Inhibition, Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Comparison

Journal: The Journal of Neuroscience

Article Title: Limiting Neuronal Nogo Receptor 1 Signaling during Experimental Autoimmune Encephalomyelitis Preserves Axonal Transport and Abrogates Inflammatory Demyelination

doi: 10.1523/JNEUROSCI.1760-18.2019

Figure Lengend Snippet: Primary antibodies used in study

Article Snippet: Mouse anti-NgR 1:100 MAB1208 R&D Systems Human post-mortem MS brain immunofluorescence Rabbit anti-proteolipid (PLP) 1:500 Ab28486 Abcam Human post-mortem MS brain immunofluorescence Rabbit anti-pCRMP2-T555 1:40,000 ( Petratos et al., 2012 ; Mokhtar et al., 2018 ) Western blot Mouse anti-CRMP2 1:40,000 11096 IBL Western blot Rabbit anti-KIF5c 1:4000 LS- {"type":"entrez-nucleotide","attrs":{"text":"C81914","term_id":"33650884","term_text":"C81914"}} C81914 LSBio Western blot Mouse anti-β-actin 1:50,000 MAB1501 Millipore Western blot Mouse anti-Kinesin light chain-1 (KLC-1) 1:2000 MAB1617 Millipore Western blot Mouse anti-α-tubulin 1:10,000 05-829 Millipore Western blot Biotinylated anti-GFP 1:200 Ab6658 Abcam Oligodendrocyte apoptosis assay Mouse anti-CC1 1:50 {"type":"entrez-nucleotide","attrs":{"text":"M11217","term_id":"333925"}} M11217 Calbiochem Oligodendrocyte apoptosis assay Rabbit anti-cleaved caspase-3 1:400 9661 Cell Signaling Technology Oligodendrocyte apoptosis assay Open in a separate window Primary antibodies used in study table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Antibody Dilution Catalogue Supplier Application AlexaFluor 488 conjugated goat anti-rabbit IgG (H + L) 1:500 A-11008 Life Technologies Retina whole-mount, Optic nerve immunofluorescence AlexaFluor 546 conjugated goat anti-mouse IgG (H + L) 1:500 A-11030 Life Technologies Retina whole-mount, Optic nerve immunofluorescence, Oligodendrocyte apoptosis assay AlexaFluor 647 conjugated goat anti-rat IgG (H + L) 1:500 A-21247 Life Technologies Retina whole-mount, Optic nerve immunofluorescence, Oligodendrocyte apoptosis assay HRP-conjugated goat anti-mouse IgG (H + L) 1:20,000 AB308P Millipore Western blot HRP-conjugated goat anti-rabbit IgG (H + L) 1:20,000 AB307P Millipore Western blot Streptavidin, AlexaFluor 488 conjugated 1:500 {"type":"entrez-protein","attrs":{"text":"S11223","term_id":"112468","term_text":"pir||S11223"}} S11223 Life Technologies Oligodendrocyte apoptosis assay Open in a separate window Secondary antibodies used in study

Techniques: Immunofluorescence, Western Blot, Apoptosis Assay