Journal: Stem Cell Reports

Article Title: Cytosine-5 RNA Methylation Regulates Neural Stem Cell Differentiation and Motility

doi: 10.1016/j.stemcr.2016.11.014

Figure Lengend Snippet: Expression of NSUN2 in the Human Developing Brain and NES Cells (A) DAPI-stained human embryo (6 weeks of gestation) marked for prosencephalon, mesencephalon, and rhombencephalon. Region in square is magnified in (B). Scale bar, 1 mm. (B) Prosencephalon labeled for NSUN2 and SOX1. Region in squares are magnified in (b′) and (b″). Arrows indicate NSUN2-positive cells. Scale bar, 100 μm. (C–F) Bright-field image (C) and immunofluorescence (D–F) of AF22 (upper panels) and Sai1 (lower panels) cells labeled for Nestin (D), SOX2 (E), and βIII-tubulin (F). Scale bar, 50 μm. (G and H) NES cells co-labeled for NSUN2 and Nestin (NES) (G) or SOX1 (H). (I) Differentiation protocol. (J–L) Differentiated AF22 and Sai1 cells (day 15) labeled for Nestin (NES; J), SOX2 (K), and βIII-tubulin (L). Scale bars: 50 μm. (M) Western blot for NSUN2, βIII-tubulin (TUBB3), GFAP, SOX2, and Nestin during differentiation (days). α-Tubulin served as loading control. Nuclei are counterstained with DAPI (A, B, D–F, J–L).

Article Snippet: The fast amplification protocol was performed with pre-designed probe sets (NSUN2: Hs00214829_m1; TUBB3/TUJ1: Hs00801390_s1) and TaqMan Fast Universal PCR Master Mix (2×) (Applied Biosystems).

Techniques: Expressing, Staining, Labeling, Immunofluorescence, Western Blot, Control

Journal: Stem Cell Reports

Article Title: Cytosine-5 RNA Methylation Regulates Neural Stem Cell Differentiation and Motility

doi: 10.1016/j.stemcr.2016.11.014

Figure Lengend Snippet: Reduction of Upper-Layer Neurons in the Nsun2 −/− Cortex (A) Mouse brain cortex at E18.5 showing the location of the indicated markers. Region in red rectangle is imaged in (B), (C), and (G). (B and C) E18.5 mouse cortex labeled for PAX6 (B) and TBR2 (C) in wild-type (left-hand panels) and Nsun2 −/− (right-hand panels) brains. The area between the dashed lines indicates marker-positive cells. (D) Average of TBR2-positive (TBR2 + ) cells in Nsun2 − / − (MBKW) and wild-type littermates during development (n = 9 mice; five sections per data point). (E and F) Increased thickness (E) and number (F) of TBR2 + cells was confirmed in an independent Nsun2 −/− knockout line (D014D11). PAX6-positive (PAX6 + ) cells were unaffected (E; left-hand panel). Data are mean ± SD (n = 3 sections from two mice per genotype). WT, wild-type. (G) Cortical section of wild-type (WT) and Nsun2 −/−D014D11 mouse brains at E18.5 labeled for markers for layer VI (TBR1), layer V (CTIP2), and layer IV (SATB2). (H) Quantification of (G) showing percentage of the indicated populations in the cortical plate. Data represent mean ± SD (n = 3 mice per genotype). Significance was assessed using Student's t test. ∗∗ p < 0.01. Scale bars, 50 μm. See also Figure S1 .

Article Snippet: The fast amplification protocol was performed with pre-designed probe sets (NSUN2: Hs00214829_m1; TUBB3/TUJ1: Hs00801390_s1) and TaqMan Fast Universal PCR Master Mix (2×) (Applied Biosystems).

Techniques: Labeling, Marker, Knock-Out

Journal: Stem Cell Reports

Article Title: Cytosine-5 RNA Methylation Regulates Neural Stem Cell Differentiation and Motility

doi: 10.1016/j.stemcr.2016.11.014

Figure Lengend Snippet: Loss of m 5 C in RNA of Nsun2 −/− Brains (A) Bisulfite conversion rate (cytosine to uracil) in tRNA, non-coding (ncRNA), and coding RNA (cRNA) in wild-type ( Nsun2 +/+ ) and knockout ( Nsun2 −/− ) brains at E18.5. Box plots show the median and interquartile range from minimum to maximum. (B) Level of methylation in tRNA, ncRNA and cRNA in wild-type ( Nsun2 +/+ ) and Nsun2 −/− brains (sites with >10% methylation in pooled wild-type samples). (C) Correlation of coverage in cRNAs from Nsun2 +/+ (WT) versus Nsun2 −/− (KO) samples. Red dots indicate pooled methylation differences >5% (WT-KO). (D) Number of m 5 C sites in tRNA (upper panel), ncRNA (middle panel), and cRNA (lower panel) with the indicated level of methylation in Nsun2 +/+ (WT; gray) and Nsun2 −/− (KO; red) brain samples. (E) Correlation of methylation level in WT and KO samples (sites >10 reads coverage and >15% methylation in WT [mouse 1] versus KO [mouse 4]). (F) Examples of NSUN2-targeted ncRNA (snoRNA DQ267102 ), cRNA ( Kcnmb3 ), and tRNAs (Gly GCC and Lys CTT ) in Nsun2 +/+ (upper panels) and Nsun2 −/− (lower panels) brains represented as heatmaps. Red, methylated cytosines; gray, unmethylated cytosines; x axis, cytosines; y axis, reads. Data are averaged/pooled from four mice per genotype (A–D, F).

Article Snippet: The fast amplification protocol was performed with pre-designed probe sets (NSUN2: Hs00214829_m1; TUBB3/TUJ1: Hs00801390_s1) and TaqMan Fast Universal PCR Master Mix (2×) (Applied Biosystems).

Techniques: Knock-Out, Methylation

Journal: Stem Cell Reports

Article Title: Cytosine-5 RNA Methylation Regulates Neural Stem Cell Differentiation and Motility

doi: 10.1016/j.stemcr.2016.11.014

Figure Lengend Snippet: tRNA-Derived Small Non-coding RNAs Accumulate in Nsun2 −/− Brains (A) Angiogenin-mediated tRNA cleavage in the absence of NSUN2-dependent methylation. (B) tRNA secondary structure highlighted for 5′-derived (blue) and 3′-derived (green) small ncRNAs (20–40 nucleotides). 5′ tRNA fragments start at position 1–10 and 3′ tRNA fragments start after position 30. VL, variable loop; red circle, methylated sites. (C) Enrichment (log 2 fold change [FC]) of 5′-derived tRNA fragments (blue) compared with 3′-derived fragments (green), other fragments (start at position 11–30; any length <70), and full-length tRNAs (70–100) at E13.5 and E18.5. Box plots: n = 4 mice per genotype. ∗∗∗∗ p < 0.0001, Mann-Whitney U test. (D) NES cells incubated with recombinant angiogenin (rANG) for the indicated time points. Arrows indicate angiogenin-positive. Scale bar, 25 μm (E) Percentage of cells with internalized rANG per image field. (F) Nucleolar localization (arrows) of NSUN2 in NES cells. Cells are counterstained with DAPI. Scale bar, 25 μm. (G) Fold change (FC) of mean NSUN2 RNA expression levels in the presence of rANG versus control-treated Sai1 cells at days 0, 4, and 8 after induction to differentiate (n = 3 experiments per time point).

Article Snippet: The fast amplification protocol was performed with pre-designed probe sets (NSUN2: Hs00214829_m1; TUBB3/TUJ1: Hs00801390_s1) and TaqMan Fast Universal PCR Master Mix (2×) (Applied Biosystems).

Techniques: Derivative Assay, Methylation, MANN-WHITNEY, Incubation, Recombinant, RNA Expression, Control

Journal: Stem Cell Reports

Article Title: Cytosine-5 RNA Methylation Regulates Neural Stem Cell Differentiation and Motility

doi: 10.1016/j.stemcr.2016.11.014

Figure Lengend Snippet: NSUN2 and Angiogenin Affect Neural Stem Cell Differentiation (A) Transfection of siRNA ( NSUN2 and ctr), time course of NES cell differentiation, and sample collection. (B–D) NSUN2 (B) and TUBB3 (C, D) RNA expression at days 0, 4, and 8 after growth factor removal in the presence of an NSUN2 siRNA (B and C) or recombinant angiogenin (rANG) (D). Shown is ΔΔCt relative to GAPDH. Error bars denote SD (n = 3 transfections per time point). (E) Treatment with rANG, the siRNAs for NSUN2 , or a scrambled RNA as control (Ctrl), and time course of NES cell differentiation. (F) Flow cytometry for NCAM and CD24 in control cells and cells treated with rANG and siRNAs (si NSUN2 or siCtrl) at day 0 (left-hand panels) and day 8 (right-hand panels) of the differentiation protocol. (G) Quantification of cells shown in (F). n = 5 experiments per condition. Error bars denote SD. ∗∗∗ p < 0.001, Student's t test. See also Figure S2 .

Article Snippet: The fast amplification protocol was performed with pre-designed probe sets (NSUN2: Hs00214829_m1; TUBB3/TUJ1: Hs00801390_s1) and TaqMan Fast Universal PCR Master Mix (2×) (Applied Biosystems).

Techniques: Cell Differentiation, Transfection, RNA Expression, Recombinant, Control, Flow Cytometry

Journal: Stem Cell Reports

Article Title: Cytosine-5 RNA Methylation Regulates Neural Stem Cell Differentiation and Motility

doi: 10.1016/j.stemcr.2016.11.014

Figure Lengend Snippet: Reduced Migration of Neuroepithelial Stem Cells in the Absence of NSUN2 (A) Western blot for NSUN2 after transfecting Sai1 or AF22 cells with NSUN2 or scrambled (Ctr) siRNAi constructs using two different amounts of transfection reagents (in μL). (B) Boyden chamber assay to measure migration in the presence or absence of FGF2. A total of 10,000 cells were plated. (C) Quantification of migrated cells at the indicated time points shown as mean ± SD (n = 3 wells per time point and condition). (D) Example image of migrated cells in the presence of FGF2 after 6 hr. Nuclei are stained with DAPI. Ctr, cells transfected with scrambled RNAi.

Article Snippet: The fast amplification protocol was performed with pre-designed probe sets (NSUN2: Hs00214829_m1; TUBB3/TUJ1: Hs00801390_s1) and TaqMan Fast Universal PCR Master Mix (2×) (Applied Biosystems).

Techniques: Migration, Western Blot, Construct, Transfection, Boyden Chamber Assay, Staining

Journal: Stem Cell Reports

Article Title: Cytosine-5 RNA Methylation Regulates Neural Stem Cell Differentiation and Motility

doi: 10.1016/j.stemcr.2016.11.014

Figure Lengend Snippet: Summary of NSUN2 Functions in NES and Progenitor Cells Shown are effects on tRNA methylation (blue dots), cell differentiation, and migration, as well as thickness of cortical layers in the presence (NSUN2-positive) and absence (NSUN2-negative) of NSUN2. Dotted lines show the reduction of migration and differentiation. Orange dot represents the chemoattractant.

Article Snippet: The fast amplification protocol was performed with pre-designed probe sets (NSUN2: Hs00214829_m1; TUBB3/TUJ1: Hs00801390_s1) and TaqMan Fast Universal PCR Master Mix (2×) (Applied Biosystems).

Techniques: Methylation, Cell Differentiation, Migration

Journal: Human Gene Therapy

Article Title: Long-Term Sustained Effect of Liver-Targeted Adeno-Associated Virus Gene Therapy for Mitochondrial Neurogastrointestinal Encephalomyopathy

doi: 10.1089/hum.2017.133

Figure Lengend Snippet: Vector genomes and thymidine phosphorylase (TP) activity in the liver. Quantitative polymerase chain reaction–assessed AAV genome copies in the liver from treated KO mice using a human TYMP cDNA probe (a), and TP activity in the liver of wt, untreated, and treated KO mice 8 and 21 months (88 weeks) after treatment (b). The number of animals in consecutive groups of panel (a) are, from left to right, n = 6, 5, 6, 4, 5, and 5. The number of animals in consecutive groups of panel (b) are, from left to right, n = 6, 3, 4, 11, 6, 5, 6, 4, 5, and 5. Horizontal lines represent medians. Doses are indicated on the x-axis (vg/kg). Asterisks indicate statistical differences between groups (*p < 0.05, Mann-Whitney U-test). To facilitate matching the results of each mouse between different figures, the same symbol identifies a particular mouse in Figs. 1, ​,3,3, and ​and44.

Article Snippet: Detection and quantification of vector genome copies per cell was performed by quantitative PCR (qPCR) in the ABI PRISM 7900 sequence detection system (Applied Biosystems, Foster City, CA). hcTYMP DNA was quantified using the pre-designed TaqMan MGB gene expression assay Hs00157317_m1 (Applied Biosystems), and was referred to the single copy nuclear gene Ang1 using the predesigned TaqMan MGB gene expression assay Mm00833184_s1 (Applied Biosystems).

Techniques: Plasmid Preparation, Activity Assay, Real-time Polymerase Chain Reaction, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Reduced Bone Mass and Muscle Strength in Male 5α-Reductase Type 1 Inactivated Mice

doi: 10.1371/journal.pone.0021402

Figure Lengend Snippet: Real-time PCR showing the relative mRNA levels of Srd5a1 (type 1), Srd5a2 (type 2), and Srd5a3 (type 3) in epididymis (Ep), prostate (Pr), liver (Li), bone (Bo) and heart (He). Values are given as % of levels in epididymis and are means ± SEM (males n = 5, females n = 5).

Article Snippet: We used pre-designed RT-PCR assays from Applied Biosystems for the analysis of Srd5a1 (Mm00614213_ml), Srd5a2 (Mm01237407_ml), Srd5a3 (Mm00491099_m1), prolactin receptor (Mm00599957_m1), LH receptor (Mm00442931_m1) and GAPDH (internal control, 4352339E) mRNA levels (Applied Biosystems).

Techniques: Real-time Polymerase Chain Reaction

Journal: Nature

Article Title: Suppression of proteolipid protein rescues Pelizaeus-Merzbacher disease

doi: 10.1038/s41586-020-2494-3

Figure Lengend Snippet: a , Schematic of CRISPR Plp1 targeting in jimpy . Black arrows indicate predicted sgRNA cutting sites. Hashed boxes show the CR- impy 80 base-pair complex deletion (see ). b , Kaplan-Meier plot comparing lifespans between genotypes. n=25, 23, 18 for wild-type, CR- impy , and jimpy mice, respectively. p-values calculated using log-rank test. For source data, see Supplementary Data 1 . c , Immunohistochemical images of whole-brain sagittal sections showing MBP + myelin (green) and DAPI + nuclei (blue). Scale bars, 2mm. d-e , Quantification of MyRF + and Sox10 + cells at ( d ) 3 weeks and ( e ) 6 months. n=3–6 mice. For representative source images, see – . f-g , Electron micrograph images showing myelination at ( f ) 3 weeks and ( g ) 18 months. Higher magnification of red boxed area shown in subsequent row. Scale bar, 0.5μm; except first row, 5μm. h , Quantification of myelinated axons at 3 weeks (n=3–4 mice) and 18 months (n=2 mice). p-values calculated with unpaired, two-sided t-tests. i , Optic nerve conduction velocities at 3 weeks (n=5–6 mice) and 6 months (n=4 mice). j , Polynomial trendline illustrating conduction velocity vs . brain myelination in CR- impy relative to min-max scaling of values from jimpy and wild-type. Data from 3-week time point of ( h ) and ( i ), with same n. k-l , ( k ) Accelerating rotarod or ( l ) open field performance. n=25, 20, 12 (3 weeks) for wild-type, CR- impy , jimpy mice, respectively. n= 25, 23 (2 months); n=25, 21 (6 months); n=4, 5 (18 months) mice for wild-type, CR- impy , respectively. Biological replicates (individual mice) indicated by open circles. Graph bars indicate mean ± standard deviation. p-values calculated using one-way ANOVA with Tukey correction at 3 weeks or two-way, an unpaired two-sided t-test at later time points, except where indicated. p-values stated for p<0.1, otherwise not significant (n.s).

Article Snippet: Real-Time PCR was then performed on an Applied Biosystems 7300 Real-time PCR system with 10ng cDNA per sample in quadruplicate using Taqman gene expression master mix (4369016, ThermoFisher) and the following pre-designed Taqman gene expression assays (4351370, ThermoFisher): Plp1 (Mm01297210_m1), Mbp (Mm01266402_m1) and Actb (Mm00607939_s1) (endogenous control).

Techniques: CRISPR, Immunohistochemical staining, Standard Deviation

Journal: Nature

Article Title: Suppression of proteolipid protein rescues Pelizaeus-Merzbacher disease

doi: 10.1038/s41586-020-2494-3

Figure Lengend Snippet: a , Annotated Sanger sequencing traces of wild-type, jimpy , and CR- impy mice showing the complex, frameshift in Plp1 exon 3 from dual cutting of CRISPR/spCas9 sgRNAs in CR- impy mice as well as the jimpy point mutation in intron 4. sgRNA 3 and 7 sequences outlined by black boxes with the predicted double strand break site shown a black arrow. b , Table showing the top predicted on- and off-target sites for sgRNAs 3 and 7. CRISPR-induced indels were detected by whole genome sequencing of the CR- impy founder and three independent CR- impy F2 generation males, and consisted of an on-target 80bp complex deletion (CR- impy deletion) in exon 3 of Plp1 (green), an off-target 1 bp insertion in chromosome 6 (red), and an off-target 1 bp insertion in chromosome 11 (yellow). c-e , Integrative Genomics Viewer browser images showing aligned reads for the CR- impy founder, the jimpy control, and three CR- impy F2 males along with the detected indels at the on-target locus at exon 3 of Plp1 on chromosome X ( c ), and off-targets on chromosome 6 ( d ) and chromosome 11 ( e ) depicted by the dashed green, red, and yellow boxes, respectively. sgRNA 3 or sgRNA 7 targeted sequences are depicted by black bars.

Article Snippet: Real-Time PCR was then performed on an Applied Biosystems 7300 Real-time PCR system with 10ng cDNA per sample in quadruplicate using Taqman gene expression master mix (4369016, ThermoFisher) and the following pre-designed Taqman gene expression assays (4351370, ThermoFisher): Plp1 (Mm01297210_m1), Mbp (Mm01266402_m1) and Actb (Mm00607939_s1) (endogenous control).

Techniques: Sequencing, CRISPR, Mutagenesis

Journal: Nature

Article Title: Suppression of proteolipid protein rescues Pelizaeus-Merzbacher disease

doi: 10.1038/s41586-020-2494-3

Figure Lengend Snippet: a , RT-qPCR data showing the levels of Plp1 transcript at 6 months (n=3 mice). b , Western blot data demonstrating the levels of MBP protein at 3 weeks (n=3 mice). c , RT-qPCR data showing the levels of Mbp transcript at 6 months (n=3 mice). d , Western blot data demonstrating the levels of MBP protein at 6 months (n=3 mice). Individual data points represent the mean value of 4 technical replicates for each biological replicate ( a, c ) or independent biological replicates ( b, d ). Biological replicates (individual mice) indicated by open circles. Graph bars indicate mean ± standard deviation. p-values calculated using one-way ANOVA with Tukey correction at 3 weeks or two-way, an unpaired two-sided t-test at later time points. p-values stated for p<0.1, otherwise not significant (n.s). See for full western blot images for all samples.

Article Snippet: Real-Time PCR was then performed on an Applied Biosystems 7300 Real-time PCR system with 10ng cDNA per sample in quadruplicate using Taqman gene expression master mix (4369016, ThermoFisher) and the following pre-designed Taqman gene expression assays (4351370, ThermoFisher): Plp1 (Mm01297210_m1), Mbp (Mm01266402_m1) and Actb (Mm00607939_s1) (endogenous control).

Techniques: Quantitative RT-PCR, Western Blot, Standard Deviation

Journal: Nature

Article Title: Suppression of proteolipid protein rescues Pelizaeus-Merzbacher disease

doi: 10.1038/s41586-020-2494-3

Figure Lengend Snippet: a-b , Immunohistochemical images of brain sagittal sections showing ASO + staining and DAPI + nuclei (blue) of ( a ) wtASO Plp1.a , wtASO Plp1.b , and wild-type uninjected or ( b ) jpASO Plp1.a , jpASO Plp1.b , and uninjected jimpy mice, 3 weeks post-ASO injection (30μg dose at birth). Scale bar, 50μm.

Article Snippet: Real-Time PCR was then performed on an Applied Biosystems 7300 Real-time PCR system with 10ng cDNA per sample in quadruplicate using Taqman gene expression master mix (4369016, ThermoFisher) and the following pre-designed Taqman gene expression assays (4351370, ThermoFisher): Plp1 (Mm01297210_m1), Mbp (Mm01266402_m1) and Actb (Mm00607939_s1) (endogenous control).

Techniques: Immunohistochemical staining, Staining, Injection

Journal: Nature

Article Title: Suppression of proteolipid protein rescues Pelizaeus-Merzbacher disease

doi: 10.1038/s41586-020-2494-3

Figure Lengend Snippet: a-b , Immunostaining of HDAC2 + (red) and ( a ) NG2 + OPCs (green; arrows) in the spinal cord or ( b ) CC1 + oligodendrocytes (green; arrows) in the corpus callosum from 8-week-old wild-type mice injected with PBS control or Hdac2 -targeting ASO, 2 weeks post-injection. Scale bar, 20 μm. c , Depiction of Plp1 pre-mRNA demonstrating the approximate binding locations of two independent ASOs in intron 5 and the 3’ UTR. d , Schematic of the design for ASO experiments in this figure. e , RT-qPCR data showing wild-type spinal cord Plp1 transcript levels, 3 weeks after injection with the indicated ASO doses (10μg, 30μg, and 60μg) or PBS controls at postnatal day 1 (n=2–6 mice). f , RT-qPCR data showing the levels of Plp1 transcript and g , western blot data showing the levels of PLP protein, 3 weeks post-ASO injection (30μg dose) at birth in wild-type (n=3 mice). Individual data points represent the mean value of 4 technical replicates for each biological replicate ( e, f ) or independent biological replicates ( g ). Biological replicates (individual mice) indicated by open circles. Graph bars indicate mean ± standard deviation. p-values calculated using one-way ANOVA with Dunnett’s correction. p-values stated for p<0.1, otherwise not significant (n.s). See for full western blot source images.

Article Snippet: Real-Time PCR was then performed on an Applied Biosystems 7300 Real-time PCR system with 10ng cDNA per sample in quadruplicate using Taqman gene expression master mix (4369016, ThermoFisher) and the following pre-designed Taqman gene expression assays (4351370, ThermoFisher): Plp1 (Mm01297210_m1), Mbp (Mm01266402_m1) and Actb (Mm00607939_s1) (endogenous control).

Techniques: Immunostaining, Injection, Binding Assay, Quantitative RT-PCR, Western Blot, Standard Deviation

Journal: Nature

Article Title: Suppression of proteolipid protein rescues Pelizaeus-Merzbacher disease

doi: 10.1038/s41586-020-2494-3

Figure Lengend Snippet: a-b , RT-qPCR data showing the level of (a) Plp1 transcript levels or (b) expression levels of off-target transcripts (up to 3 base mismatches) in the spinal cord for Plp1 -tageting ASOs, including Xylt1 (off-target for ASO Plp1 .a), Scfd1 , or Tpk1 (off-targets for ASO Plp1 .b), 2 weeks post-injection of Plp1 -targeting ASOs (30μg, 100μg, and 300μg doses) or PBS control in 8 week old adult wild-type (wt) mice (n=3 mice). c-d , RT-qPCR data showing Plp1 transcript levels or tolerability by expression levels of Gfap, Aif1 , and Cd68 transcripts in the cerebral cortex and spinal cord, 8 weeks post-injection with the indicated ASOs (300μg dose) or PBS control in 8 week old wild-type mice (n=3 mice). e-h , Immunohistochemistry images with hematoxylin counterstain showing Iba1 + or GFAP + astrocytes in ( e ) Cortical layers I-IV (Iba1), ( f ) cortical layers I-III (GFAP), ( g ) spinal cord dorsal horn grey/white matter intersection (Iba1), and ( h ) spinal cord (GFAP), 8 weeks post-injection with the indicated ASOs (300μg dose) or PBS control in 8 week old wild-type mice. Scale bar, 500μm. Biological replicates (individual mice) indicated by open circles, representing the mean value of 3 technical replicates. Graph bars indicate mean ± standard deviation. p-values calculated using one-way ANOVA with Dunnett’s correction for multiple comparisons. p-values stated for p<0.1, otherwise not significant (n.s).

Article Snippet: Real-Time PCR was then performed on an Applied Biosystems 7300 Real-time PCR system with 10ng cDNA per sample in quadruplicate using Taqman gene expression master mix (4369016, ThermoFisher) and the following pre-designed Taqman gene expression assays (4351370, ThermoFisher): Plp1 (Mm01297210_m1), Mbp (Mm01266402_m1) and Actb (Mm00607939_s1) (endogenous control).

Techniques: Quantitative RT-PCR, Expressing, Injection, Immunohistochemistry, Standard Deviation

Journal: Nature

Article Title: Suppression of proteolipid protein rescues Pelizaeus-Merzbacher disease

doi: 10.1038/s41586-020-2494-3

Figure Lengend Snippet: a , Schematic of ASO experimental design used in Figures 3- . b , Kaplan-Meier plot depicting the lifespan of contemporaneous wtASOctr, uninjected jimpy , jpASOctr, jpASO Plp1 .a, and jpASO Plp1 .b (n=12, 14, 5, 5 mice, respectively). p-values calculated using the log-rank test. See for source metadata. c-d , Immunohistochemical images of ( c ) 3-week and ( d ) 8-month whole-brain sagittal sections showing MBP + myelin (green) and DAPI + nuclei (blue) staining. Scale bar, 2mm. See – for higher magnification. e-f , Quantification of ( e ) MyRF + oligodendrocytes and ( f ) Sox10 + glial lineage cells at 3 weeks of age (n=3 mice). For representative source images, see – . g-h , ( g ) Electron micrograph images and ( h ) quantification of myelinated axons at 3 weeks of age (n=3–5 mice). Scale bar, 0.5μm. Biological replicates (individual mice) indicated by open circles. Graph bars indicate mean ± standard deviation. p-values calculated using one-way ANOVA with Dunnett’s correction, except where indicated. p-values stated for p<0.1, otherwise not significant (n.s).

Article Snippet: Real-Time PCR was then performed on an Applied Biosystems 7300 Real-time PCR system with 10ng cDNA per sample in quadruplicate using Taqman gene expression master mix (4369016, ThermoFisher) and the following pre-designed Taqman gene expression assays (4351370, ThermoFisher): Plp1 (Mm01297210_m1), Mbp (Mm01266402_m1) and Actb (Mm00607939_s1) (endogenous control).

Techniques: Immunohistochemical staining, Staining, Standard Deviation

Journal: Nature

Article Title: Suppression of proteolipid protein rescues Pelizaeus-Merzbacher disease

doi: 10.1038/s41586-020-2494-3

Figure Lengend Snippet: a-b , Electron micrograph images showing myelination of wtASOctr or jpASO Plp1.b at ( a ) 2 months and ( b ) 8 months. For a, scale bar, 0.5μm. For b, lower panel is a higher magnification of red boxed area in the upper panel. Upper panel scale bar, 5μm and lower panel scale bar, 0.5μm.

Article Snippet: Real-Time PCR was then performed on an Applied Biosystems 7300 Real-time PCR system with 10ng cDNA per sample in quadruplicate using Taqman gene expression master mix (4369016, ThermoFisher) and the following pre-designed Taqman gene expression assays (4351370, ThermoFisher): Plp1 (Mm01297210_m1), Mbp (Mm01266402_m1) and Actb (Mm00607939_s1) (endogenous control).

Techniques:

Journal: Nature

Article Title: Suppression of proteolipid protein rescues Pelizaeus-Merzbacher disease

doi: 10.1038/s41586-020-2494-3

Figure Lengend Snippet: a-b , Performance for ( a ) open field testing ( b ) accelerating rotarod (n=5–8 mice). See for raw data values. p-values calculated using one-way ANOVA with Dunnett’s correction. c , Optic nerve conduction velocity at 3 weeks of age (n = 3, 8, and 4 wtASOctr, jpASOctr, and jpASO Plp1 .b mice, respectively). p-values calculated using one-sided, unpaired t-test. d , Polynomial trendline illustrating conduction velocity vs. brain myelination in jpASO Plp1 .b relative to min-max scaling of values from jpASOctr and wtASOctr. Source data from ( c and ) with same n. e , Trace of a jimpy seizure during hypercapnic challenge (respiratory flow rate on y-axis). f , Minute ventilation per body weight (MV/g) in mL/g/min in 5% CO 2 (hypercapnia) and 10.5% O 2 (hypoxia), representing all repeated measurements from n=9, 6, 7 wtASOctr, jpASOctr, and jpASO Plp1 .b mice, respectively. Violin plots indicate median (center white lines) ± quartiles (border white lines). p-values calculated using Brown and Forsythe’s test. g-j , Baseline MV/g (mL/g/min) in ( g ) normal air, ( h ) 15–30 minutes after transitioning from normal air to 5% CO 2 , ( i ) 0–3 minutes and ( j ) 8–9 minutes after transitioning from normal air to 10.5% O 2 . Post-mortality hypoxia data were not included. For g-i, n=9, 6, 7 and j, n=9, 3, 7 wtASOctr, jpASOctr, and jpASO Plp1 .b mice, respectively. k , Kaplan-Meier plot depicting survival during hypoxia. n=12, 8, 9 wtASOctr, jpASOctr, and jpASO Plp1 .b mice, respectively. p-values calculated using log-rank test. Biological replicates (individual mice) indicated by open circles. Graph bars indicate mean ± standard deviation, except where indicated. p-values stated for p<0.1, otherwise not significant (n.s).

Article Snippet: Real-Time PCR was then performed on an Applied Biosystems 7300 Real-time PCR system with 10ng cDNA per sample in quadruplicate using Taqman gene expression master mix (4369016, ThermoFisher) and the following pre-designed Taqman gene expression assays (4351370, ThermoFisher): Plp1 (Mm01297210_m1), Mbp (Mm01266402_m1) and Actb (Mm00607939_s1) (endogenous control).

Techniques: Standard Deviation