Journal: PLoS ONE

Article Title: Tissue Kallikrein Mediates Pro-Inflammatory Pathways and Activation of Protease-Activated Receptor-4 in Proximal Tubular Epithelial Cells

doi: 10.1371/journal.pone.0088894

Figure Lengend Snippet: Cells were incubated with 0.1 or 0.5/ml AGE-BSA for 6 h, and gene expression was determined by real-time PCR analysis. AGE induced KLK1 mRNA expression in PTEC (A). Cells were transfected with KLK1-specific siRNA, and the endogenous protein level was determined by Western blot analysis (B). Transfected cells were incubated with 0.5 mg/ml AGE for 48 h, and protein expression of cytokine was detected in culture medium by ELISA. AGE -induced IL-8 (C) and ICAM-1 (D) protein expression was inhibited by KLK1 gene silencing. ***p<0.001 compared with control, δ P<0.05 compared with mock transfection and # p<0.05; ## p<0.01 compared with mock transfection incubated with AGE.

Article Snippet: PTEC were transfected with KLK1 or negative control of Silence®Select Pre-designed siRNA (Applied Biosystems) using Lipofectamine™ 2000 according to manufacturer’s instructions.

Techniques: Incubation, Expressing, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Human Mutation

Article Title: Weaver Syndrome‐Associated EZH2 Protein Variants Show Impaired Histone Methyltransferase Function In Vitro

doi: 10.1002/humu.22946

Figure Lengend Snippet: Weaver syndrome mutants are impaired in their histone methyltransferase activity in vitro . Histone methyltransferase reactions were performed using 2 μg purified core histones and 0.67 μM 3 H‐S‐adenosyl‐methionine ( 3 H‐SAM). Each reaction was incubated with 250 ng of either wild‐type (WT) or a mutant HMTase complex (or no enzyme controls). Histone methyltransferase activity was measured based on the incorporation of 3 H‐labeled methyl groups, represented in scintillation counts per minute. Counts were normalized by subtracting background counts (i.e., no enzyme) from the total counts. A : Incorporation of tritiated methyl groups from 3 H‐SAM onto core histones is shown for each complex: EZH2 WT • , p.(Phe672Ile) × , p.(Pro132Ser) ★ , p.(Tyr153del) △, p.(His694Tyr) ▽, p.(Glu745Lys) ▴, p.(Ala682Thr) ▾, p.(Arg684Cys) ▪, p.(Tyr133Cys) □, and p.(Asp185His) ◇. Error bars represent standard deviation (SD) within the groups “EZH2 WT” and “EZH2 mutants.” Unpaired t‐test showed statistically significant difference between the two groups (P value < 0.0001). B : Incorporation of tritiated methyl groups from 3 H‐SAM onto core histones is shown for the positive control EZH2 WT, the negative control EZH2 (p.Phe672Ile), and the mutant complex with activity closest to WT, namely, EZH2 (p.Pro132Ser). Error bars represent SD of four independent replicates for the controls, and three independent replicates for the mutant EZH2 (p.Pro132Ser). One‐way ANOVA showed statistically significant difference between all groups (overall P value < 0.0001; P values between WT and p.(Phe672Ile), between p.(Phe672Ile) and p.(Pro132Ser), and between WT and p.(Pro132Ser) were all <0.05).

Article Snippet: To test our hypothesis, we designed recombinant human EZH2 proteins, had them preassembled into PRC2 complexes (BPS Bioscience, San Diego CA), and tested their activity in vitro using a well‐accepted in vitro assay [Ernst et al., ; Yap et al., ; Score et al., ].

Techniques: Activity Assay, In Vitro, Purification, Incubation, Mutagenesis, Labeling, Standard Deviation, Positive Control, Negative Control

Journal: European Journal of Translational Myology

Article Title: A New Semi-Automatic Approach to Find Suitable Virtual Electrodes in Arrays Using an Interpolation Strategy

doi: 10.4081/ejtm.2016.6029

Figure Lengend Snippet: Overview of the closed-loop system. A controller K adjusts the global intensity u based on the error e between the angular output of one DoF y and the reference angle r. The interpolation function f i (u, p ,d) assigns an individual intensity q i to the elements in the plant G, which consists of the FES system, the array, the patient, and a motion measurement system. The motion in the remaining DoF y' can be observed by the therapist/patient who can adjust the position p and diameter d of the VE.

Article Snippet: The controller design and implementation were performed in MATLAB/Simulink.

Techniques:

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