Journal: bioRxiv

Article Title: Modeling motor neuron resilience in ALS using stem cells

doi: 10.1101/399659

Figure Lengend Snippet: (A) Time line depicting in vitro protocols for resistant oculomotor neurons (OMN) and vulnerable spinal motor neuron (SC MN) generation. ESC column reports the four cell lines used in this study. Patterning was performed for 5 days after ESC expansion and EB formation. mRNA-seq coupled with FACS was performed on EB dissociation day (day 9 of the protocol). Survival assay was performed after five days from EB dissociation. (B-C) SC MNs generated from E14.1 ESCs express Hb9, Islet1 and NF200, OMNs generated from E14.1 ESCs over-expressing the transcription factor Phox2a under the Nestin enhancer co-express Islet1 and NF200 in absence of Hb9. Scale bar in c 60 μm. (D) Percentage of Hb9+/Islet1+ cells in SC MN (64.1±5.2%, mean±SEM, n=4) and OMN (6.3±1.3%, mean±SEM) cultures respectively. (For quantification, experiments were run in quadruplicates including two technical replicates per experiment, with at least 120 Islet1+ cells counted per condition). (E) Microphotographs showing Islet1+/Tuj1+ cells in OMN cultures, (F) OMNs also express the specific marker Phox2a as indicated by asterisks. Scale bars in e and g 100 μm. (G) Quantification of Islet1+ over Tuj1+ cells demonstrates that half neuronal population appears to be Islet1+ (47.5±5.9%, mean±SEM, experiments performed in quadruplicates with two technical replicates each, total number of Tuj1+ cells counted: 1325). Quantification of Phox2A+ over Islet1+ cells (experiments performed in quadruplicates with two technical replicates each, total number Islet1+ cells counted: 746) indicates that 62±5.2% (mean±SEM) of the Islet1+ population is also Phox2a+. All quantifications were performed 5 days after EB dissociation (mean ± SEM). (H) PCA of OMN and SC MN samples based on all genes expressed confirmed cell differential identities. mRNA-seq analysis of OMNs and SC MNs isolated by FACS was performed after 5 days of patterning (day 9 of the protocol). (I) 1,017 differentially expressed genes were found between the two different cell types (adjusted P<0.05). Heatmap shows the top 500 most significant differentially expressed genes by adjusted p-value. (J) Heatmap of expected progenitors and motor neuron transcripts but also OMN and SC MN specific transcripts obtained in the two generated motor neuron populations. (K) Using the top 100 DEGs obtained from our RNAseq analysis we could separate datasets originating from in vivo microarray studies on early postnatal (J) and adult (L) rodent OMNs and SC MNs. (M) Venn diagram showing gene sets enriched either in brain stem cultures or OMN-specific as revealed by PAGODA analysis, (N) gene sets preferentially found in spinal cord cultures or MN-specific.

Article Snippet: The following primary antibodies were used: NF200 (1:1000, #AB5539 Millipore), Tuj1 (1:1000, MRB-435P Covance), Hb9 (1:10, #81.5C10 DSHB), Islet1 (1:500, #ab20670 abcam; 1:100, #40.2D6 DSHB), Phox2A (1:1000, gift of Prof JF Brunet), GFP (1:1000, #ab13970 abcam), phospho-AKT (Ser473) (1:50, #3787 Cell Signaling), activated beta-catenin (1:1000, #05-665 Millipore), alpha-catenin (1:1000, #AB153721 AbCam), ESYT1 (1:100, #HPA016858), BrdU (1:10, #G3G4 DSHB).

Techniques: In Vitro, Clonogenic Cell Survival Assay, Generated, Expressing, Marker, Isolation, In Vivo, Microarray

Journal: bioRxiv

Article Title: Modeling motor neuron resilience in ALS using stem cells

doi: 10.1101/399659

Figure Lengend Snippet: (A) Oculomotor neurons (OMNs) and spinal motor neurons (SC MNs) express similar mRNA levels of glutamate ionotropic receptor AMPA, NMDA and kainate type subunits. The heatmap shows log2 RPKM values of these subunits and no separate clustering of the two cell types is observed. (B-C) Immunohistochemistry performed on generated OMNs and SC MNs at D1 in vitro in control conditions; similar levels of glutamate ionotropic receptor kainate type subunit 5 (Grik5) are found in both cell types. Scale bar in d 60 μm. (D-E) Microphotographs presenting SC MN and OMN response to kainic acid induced toxicity (20 μM) for a week. Scale bars in f = 100 μm. (F) Curves represent percentages of MN survival over time in OMN and SC MN cultures. OMNs were visualized as NF200+Islet1+Hb9- clls, while SC MNs as NF200+Islet1+Hb9+ cells. OMNs show increased survival to KA toxicity at D7 (mean ± SEM, 2way ANOVA and Tukey’s multiple comparison test, F(9, 56)=2.333, *P=0.0261, SC MNs n=4; OMNs n=5) when compared to SC MNs (experiments were performed at least in quadruplicates, with technical replicates and with at least 130 motor neurons counted per condition in each experiment). Analysis of the length of neuronal processes in both oculomotor and spinal motor neuron cultures exposed to kainic acid for seven days. showed that oculomotor neurons were unaffected by kainic acid while spinal motor neurons displayed a shortening of neurites (G). (H-K) Sholl analysis was performed on OMN at D7 survival assay in control and KA20 conditions to further assess individual MN arborization complexity during toxicity. Scale bar = 100 μm. (I) Sholl mask was applied to individual OMNs after specifying the radius from the center of the soma of the neuron and created concentric circles every 25 μm of increasing radius. (J) Comparison of average number of neurite intersections of OMN in control and KA20 toxicity conditions with radial step size of 25 μm. OMNs did not show reduction in arborization (multiple t test, n = 10 per condition). (K) Schematic depicting identification of neurite segments by Sholl analysis. Color code is assigned depending on arbor localization from the soma in an inside-out manner following the given radius. Multiple intersections within the same segment display the same colour.

Article Snippet: The following primary antibodies were used: NF200 (1:1000, #AB5539 Millipore), Tuj1 (1:1000, MRB-435P Covance), Hb9 (1:10, #81.5C10 DSHB), Islet1 (1:500, #ab20670 abcam; 1:100, #40.2D6 DSHB), Phox2A (1:1000, gift of Prof JF Brunet), GFP (1:1000, #ab13970 abcam), phospho-AKT (Ser473) (1:50, #3787 Cell Signaling), activated beta-catenin (1:1000, #05-665 Millipore), alpha-catenin (1:1000, #AB153721 AbCam), ESYT1 (1:100, #HPA016858), BrdU (1:10, #G3G4 DSHB).

Techniques: Immunohistochemistry, Generated, In Vitro, Clonogenic Cell Survival Assay

Journal:

Article Title: Derivation of midbrain dopamine neurons from human embryonic stem cells

doi: 10.1073/pnas.0404700101

Figure Lengend Snippet: Phenotypic characterization of midbrain dopaminergic differentiation. (A and B) RT-PCR analyses of undifferentiated hES cells, passage 0 cells (day 28, Rosette), passage 1 cells during cell expansion (day 35, P1 exp.), passage 2 cells during cell expansion (day 42, P2 exp.), and passage 2 cells during cell differentiation (day 50, P2 diff.). Shown are ES-cell, neural, and neuronal markers (A) and markers of midbrain DA neuron development (B). ALDH1A1, aldehyde dehydrogenase 1; Nurr1, nuclear orphan receptor 1. (C) Pax2-positive cells (red) at passage 1 did not coexpress En1 (green). (D and E) Cells at passage 2 expressed Ki67 (green) and nestin (red) (D) and coexpressed Pax2 (red) and En1 (green) (E). (F) Pax6 (red) and Lmx (green) expression in passage 2 expansion cells. (G–N) Differentiated cells at passage 2: TH/Tuj1-positive (red, TH; green, Tuj1) neurons with radially oriented processes emanating from a rosette (G); Pax2- or Pax5-positive (red) immature precursors next to TH-positive (green) neurons (H and I); En1-p (green) cells coexpressing TH (red) (J); TH-positive (red) cells coexpressing AADC (green) (K) or VMAT2 (L); TH-positive neurons expressing the synaptic markers SV2 (green) (M) or synapsin (red) (N). Similar results were obtained with three independent hES lines. C–E show H9-derived cells; G–I and M show H1-derived cells; and F, J–L, and N show HES-3-derived cells. (Scale bars, 50 μm.)

Article Snippet: Rabbit polyclonal antibodies included TH and vesicular monoamine transporter 2 (VMAT2, Pel-Freez Biologicals); nestin 130 (R. McKay, National Institutes of Health, Bethesda); glial fibrillary acidic protein and DA (Chemicon); GABA and serotonin (Sigma); Pax2, Pax6, and β-tubulin III (Covance); Pax5 (clone A2; M. Busslinger, Institute of Molecular Pathology, Vienna); aromatic l -amino acid decarboxylase (AADC, Protos Biotech, New York); synaptic vesicle 2 (SV2, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City); and synapsin (Calbiochem).

Techniques: Reverse Transcription Polymerase Chain Reaction, Cell Differentiation, Expressing, Derivative Assay

Journal: Frontiers in Pharmacology

Article Title: A frequent CYP2D6 variant promotes skipping of exon 3 and reduces CYP2D6 protein expression in human liver samples

doi: 10.3389/fphar.2023.1186540

Figure Lengend Snippet: Frequency of CYP2D6 diplotype in liver samples.

Article Snippet: CYP2D6 protein was detected with a rabbit anti-CYP2D6 antibody (1:10, Novus NBP2-67020) followed by an HRP-conjugated anti-rabbit secondary antibody (Biotechne, San Jose, CA, United States).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: A frequent CYP2D6 variant promotes skipping of exon 3 and reduces CYP2D6 protein expression in human liver samples

doi: 10.3389/fphar.2023.1186540

Figure Lengend Snippet: Associations between CYP2D6 genotype and levels of CYP2D6∆E3, E6-containing CYP2D6, and total CYP2D6 mRNA in liver samples using multiple linear regression. Only significant variables were included in the regression models after screening, as described in the method section. The p -value for each variable is shown, and the coefficient is in a log10 scale.

Article Snippet: CYP2D6 protein was detected with a rabbit anti-CYP2D6 antibody (1:10, Novus NBP2-67020) followed by an HRP-conjugated anti-rabbit secondary antibody (Biotechne, San Jose, CA, United States).

Techniques: Real-time Polymerase Chain Reaction

Journal: Frontiers in Pharmacology

Article Title: A frequent CYP2D6 variant promotes skipping of exon 3 and reduces CYP2D6 protein expression in human liver samples

doi: 10.3389/fphar.2023.1186540

Figure Lengend Snippet: The frequent synonymous SNP rs1058164 G is associated with higher levels of the CYP2D6 splice isoform missing exon 3 (CYP2D6∆E3). (A) CYP2D6∆E3 (log) levels in liver samples grouped by rs1058164 genotypes, adjusted by rs16947 and rs3892097 genotypes. The differences between groups were analyzed with multiple linear regression. The box and horizontal lines show the 25th and 75th percentiles surrounding the mean, and the whiskers show values up to 1.5*IQR. (B) The expression levels of CYP2D6∆E3 in HEK293 T cells after transfection with CYP2D6 genomic DNA expression plasmids containing rs1085164 G or C alleles. Data are combined results from different clones (4 for each genotype) and two independent experiments with triplicates. The horizontal bar indicates the average (mean).

Article Snippet: CYP2D6 protein was detected with a rabbit anti-CYP2D6 antibody (1:10, Novus NBP2-67020) followed by an HRP-conjugated anti-rabbit secondary antibody (Biotechne, San Jose, CA, United States).

Techniques: Expressing, Transfection, Clone Assay

Journal: Frontiers in Pharmacology

Article Title: A frequent CYP2D6 variant promotes skipping of exon 3 and reduces CYP2D6 protein expression in human liver samples

doi: 10.3389/fphar.2023.1186540

Figure Lengend Snippet: Testing mRNA and protein stabilities of full-length CYP2D6 and its splice isoforms. (A) Time course of the relative expression levels of full-length CYP2D6, CYP2D6∆E3, and CYP2D6∆E6 in HepG2 cells after actinomycin D treatment. (B) Western blot image of CYP2D6 protein isoforms in HEK293 T cells transfected with plasmid DNAs expressing full-length (HEK2D6F), CYP2D6∆E3 (HEK2D6S), a mix of the two (mix-2D6F/2D6S), or a mix of the two and treated with cycloheximide (Cyclo-mix-2D6F/2D6S).

Article Snippet: CYP2D6 protein was detected with a rabbit anti-CYP2D6 antibody (1:10, Novus NBP2-67020) followed by an HRP-conjugated anti-rabbit secondary antibody (Biotechne, San Jose, CA, United States).

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation

Journal: Frontiers in Pharmacology

Article Title: A frequent CYP2D6 variant promotes skipping of exon 3 and reduces CYP2D6 protein expression in human liver samples

doi: 10.3389/fphar.2023.1186540

Figure Lengend Snippet: Association between CYP2D6 genotype and full-length CYP2D6 protein level in liver samples using simple or multiple linear regression.

Article Snippet: CYP2D6 protein was detected with a rabbit anti-CYP2D6 antibody (1:10, Novus NBP2-67020) followed by an HRP-conjugated anti-rabbit secondary antibody (Biotechne, San Jose, CA, United States).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: A frequent CYP2D6 variant promotes skipping of exon 3 and reduces CYP2D6 protein expression in human liver samples

doi: 10.3389/fphar.2023.1186540

Figure Lengend Snippet: Comparison of full-length CYP2D6 protein expression in liver samples between different genotypes. (A) Full-length CYP2D6 protein levels in liver samples (adjusted by rs5758550 and rs16947 genotypes) grouped by rs1058164 genotype. (B) Full-length CYP2D6 protein expression in CYP2D6*1 samples (carrying rs1058164 G) and CYP2D6*2 samples (carrying rs16947, rs1058164 C, and rs5758550). Only samples with a single copy of a functional CYP2D6 gene were analyzed. In both plots, the box and horizontal lines show the 25th and 75th percentiles surrounding the mean, and the whiskers show values up to 1.5*IQR.

Article Snippet: CYP2D6 protein was detected with a rabbit anti-CYP2D6 antibody (1:10, Novus NBP2-67020) followed by an HRP-conjugated anti-rabbit secondary antibody (Biotechne, San Jose, CA, United States).

Techniques: Comparison, Expressing, Functional Assay

Journal: Frontiers in Pharmacology

Article Title: A frequent CYP2D6 variant promotes skipping of exon 3 and reduces CYP2D6 protein expression in human liver samples

doi: 10.3389/fphar.2023.1186540

Figure Lengend Snippet: Relationship between CYP2D6 CNV and full-length CYP2D6 protein expression in liver samples. Samples with duplicated null alleles (*3, *4, *6) and *41 CNV >4 were excluded.

Article Snippet: CYP2D6 protein was detected with a rabbit anti-CYP2D6 antibody (1:10, Novus NBP2-67020) followed by an HRP-conjugated anti-rabbit secondary antibody (Biotechne, San Jose, CA, United States).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: A frequent CYP2D6 variant promotes skipping of exon 3 and reduces CYP2D6 protein expression in human liver samples

doi: 10.3389/fphar.2023.1186540

Figure Lengend Snippet: Association between full-length CYP2D6 protein level and predicted expression scores (ES) based on the three models using simple linear regression.

Article Snippet: CYP2D6 protein was detected with a rabbit anti-CYP2D6 antibody (1:10, Novus NBP2-67020) followed by an HRP-conjugated anti-rabbit secondary antibody (Biotechne, San Jose, CA, United States).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: A frequent CYP2D6 variant promotes skipping of exon 3 and reduces CYP2D6 protein expression in human liver samples

doi: 10.3389/fphar.2023.1186540

Figure Lengend Snippet: Relationship between full-length CYP2D6 protein expression and expression score derived from genotype. (A) CYP2D6 protein level as predicted by ES using Model 1. (B) ES-based prediction of CYP2D6 protein via model 2. (C) Model 3 ES-based prediction of CYP2D6 protein. In all three plots, open circles represent samples that did not move ES values compared to model 1, while closed squares are samples which moved to new ES values based on the particular model.

Article Snippet: CYP2D6 protein was detected with a rabbit anti-CYP2D6 antibody (1:10, Novus NBP2-67020) followed by an HRP-conjugated anti-rabbit secondary antibody (Biotechne, San Jose, CA, United States).

Techniques: Expressing, Derivative Assay