Journal: Cell Reports

Article Title: Two-Step Activation Mechanism of the ClpB Disaggregase for Sequential Substrate Threading by the Main ATPase Motor

doi: 10.1016/j.celrep.2019.05.075

Figure Lengend Snippet: ClpB Activation Triggers a Sequential Mode of ATP Hydrolysis (A) ClpB domain organization and monomer structure. The identity and position of mutated residues are indicated. (B) ATPase activities of ClpB wild-type (WT) and ClpB-K476C were determined in the absence and presence of 10 μM casein (± substrate). SDs are indicated; for some points, error bars are shorter than the height of the symbol and are not depicted. (C) MDH disaggregation activities of ClpB-WT and ClpB-K476C in the absence and presence of Hsp70. (D) ATPase activity of ClpB-WT and ClpB-K476C in absence and presence of casein (± substrate) as a function of ATP concentration. (E) v max of ATPase activities, derived Hill coefficient (h), and ATP concentrations at half-maximal ATPase activity ( K 0.5 ) for WT, pore 1 (Y251A), and pore 2 (Y653A) loop mutants of ClpB-WT and ClpB-K476C. (F and G) ATPase activities of ClpB-K476C/ClpB-K476C/E279A/E678A (F) and MDH disaggregation of ClpB-WT/ClpB-E279A/E678A (G) mixes were determined (red, blue). They are compared with curves calculated from a model (black to gray) that assumes that a mixed hexamer only displays ATPase or disaggregation activity if it contains the number of wild-type subunits indicated. Mixing ratios are indicated as number of E279A/E678A mutant subunits.

Article Snippet: ClpB-DWB-K476C bound to casein and ATPγS state KC-2A cryoEM map , This paper , EMDB: 4626.

Techniques: Activation Assay, Activity Assay, Concentration Assay, Derivative Assay, Mutagenesis

Journal: Cell Reports

Article Title: Two-Step Activation Mechanism of the ClpB Disaggregase for Sequential Substrate Threading by the Main ATPase Motor

doi: 10.1016/j.celrep.2019.05.075

Figure Lengend Snippet: Overview of Substrate-Bound ClpB-DWB-K476C (A) Left, top view, and middle and right, side views of the cryo-EM density map of the most populated conformation of casein-bound ClpB-DWB-K476C (KC-2). The six protomers form a closed ring with a helical arrangement of two stacked AAA tiers and a seam between subunits A and F. The flexible N-terminal domains, located above the AAA1 tier, are not visible at high contour level. M-domains are partly visible for protomers C–E. (B) Views of the cryo-EM maps of the three states of substrate-bound ClpB-DWB-K476C. Densities of protomers A and B are removed to show conformational changes in protomers AAA1E and AAA2F, highlighted by orange and red arrows, respectively. Orange and red hexagons show the position of moving AAA1E and AAA2F pore loops.

Article Snippet: ClpB-DWB-K476C bound to casein and ATPγS state KC-2A cryoEM map , This paper , EMDB: 4626.

Techniques: Cryo-EM Sample Prep

Journal: Cell Reports

Article Title: Two-Step Activation Mechanism of the ClpB Disaggregase for Sequential Substrate Threading by the Main ATPase Motor

doi: 10.1016/j.celrep.2019.05.075

Figure Lengend Snippet: Pore Loop Movements and Arginine Finger Contacts in the Three States of Substrate-Bound ClpB-DWB-K476C Suggest a Sequential Mechanism of ATP Hydrolysis and Substrate Threading (A) Pore loop interactions with the substrate in AAA1 (top panels) and AAA2 (bottom panels) rings. The pore loop AAA1E (orange) engages the substrate in KC-2, while the pore loop AAA2F (red) dissociates. AAA2F moves from the bottom to the top of the staircase of pore loops in KC-3. (B) Arginine finger engagements in the AAA1 and AAA2 ring. All protomers were aligned to the large lobe of AAA1 or AAA2 domain of protomer C to compare engagement of the arginine fingers with neighboring subunits. Arginine fingers of AAA1B–C and AAA2B–D are shown as gray ribbons and interact with the γ-phosphate of ATP bound at the active site of a neighboring subunit in all three states. Activity states of AAA2 protomers are indicated by green (active) and red (inactive) arrows. (C) Nucleotide densities for AAA2A, AAA2B, and AAA2F protomers and assigned nucleotide state.

Article Snippet: ClpB-DWB-K476C bound to casein and ATPγS state KC-2A cryoEM map , This paper , EMDB: 4626.

Techniques: Activity Assay

Journal: Cell Reports

Article Title: Two-Step Activation Mechanism of the ClpB Disaggregase for Sequential Substrate Threading by the Main ATPase Motor

doi: 10.1016/j.celrep.2019.05.075

Figure Lengend Snippet: Docked M-Domains Repress the Activity of ClpB-WT and Reduce the Range of AAA Domain Movements (A) Heterogeneity of M-domain conformations. Top and side views of the cryo-EM density maps for the two main M-domain conformations of the ClpB-DWB-K476C:casein complex and for the ClpB-DWB:casein complex. Detached M-domains are indicated by green arrows and docked M-domains by red arrows. (B) Docking states of M-domains: atomic models showing the predominant conformation of M-domains enclosing the AAA1 tier in ClpB-K476C (left) and ClpB-WT (right) states. In ClpB-WT, M-domains are docked in a horizontal conformation that is stabilized by head-to-tail interactions between motif 1 and motif 2 of neighboring M-domains. In ClpB-K476C, M-domains adopt a tilted conformation with motif1 contacting the AAA1 domain of the adjacent protomer. Head-to-tail interactions are broken, rendering M-domain motif 2 invisible in the cryo-EM maps. Here, full-length M-domains are shown, docked in the density of motif1, to emphasize the differences in M-domain docking states between ClpB-WT and ClpB-K476C.

Article Snippet: ClpB-DWB-K476C bound to casein and ATPγS state KC-2A cryoEM map , This paper , EMDB: 4626.

Techniques: Activity Assay, Cryo-EM Sample Prep

Journal: Cell Reports

Article Title: Two-Step Activation Mechanism of the ClpB Disaggregase for Sequential Substrate Threading by the Main ATPase Motor

doi: 10.1016/j.celrep.2019.05.075

Figure Lengend Snippet: Overview of Substrate-Bound ClpB-DWB, Pore Loop Movements and Arginine Finger Contacts (A) Views of the cryo-EM maps of the three states of substrate-bound ClpB-DWB. Densities of protomers A and B were removed to show conformational changes in protomers AAA1E and AAA2F, highlighted by orange and red arrows, respectively. Orange/red hexagons show the position of moving pore loops. (B) Interactions of ClpB-WT pore loops of the AAA1 (upper panel) and AAA2 (lower panel) rings with the substrate. The pore loop AAA2F (red) dissociates from the substrate in WT-2A. The pore loop AAA1E (orange) binds substrate in WT-2B. (C) Activity states of ClpB-WT AAA1 (upper panels) AAA2 (lower panels) domains. All protomers were aligned to the large AAA1 (AAA2) domain of protomer C to compare engagement of the arginine fingers with neighboring subunits. Arginine fingers of AAA1A-C and AAA2B-D are shown as grey ribbons and interact with the γ-phosphate of ATP bound at the active site of a neighboring subunit in all three states. Activity states of AAA1/2 protomers are indicated by green (active) and red (inactive) arrows. (D) Comparison of ClpB-WT and ClpB-K476C pore loop positions of AAA1E and AAA2F for states WT-1, WT-2A to WT-2B (pale to bright colors) and for states KC-1, KC-2 to KC-3 (pale to bright colors).

Article Snippet: ClpB-DWB-K476C bound to casein and ATPγS state KC-2A cryoEM map , This paper , EMDB: 4626.

Techniques: Cryo-EM Sample Prep, Activity Assay

Journal: Cell Reports

Article Title: Two-Step Activation Mechanism of the ClpB Disaggregase for Sequential Substrate Threading by the Main ATPase Motor

doi: 10.1016/j.celrep.2019.05.075

Figure Lengend Snippet:

Article Snippet: ClpB-DWB-K476C bound to casein and ATPγS state KC-2A cryoEM map , This paper , EMDB: 4626.

Techniques: Recombinant, ATPase Assay, Plasmid Preparation, Gel Extraction, Software, Electron Microscopy

Journal: Nature Communications

Article Title: Exploiting loss of heterozygosity for allele-selective colorectal cancer chemotherapy

doi: 10.1038/s41467-020-15111-4

Figure Lengend Snippet: a Identification of enzymes with prevalent alternative alleles resulting in amino-acid substitution near functional sites. A series of filtering steps was applied to identify the subset of SNVs causing amino-acid substitutions near active sites in publicly available 3D protein structures. To enrich potential target proteins for LOH-based tumor targeting approaches, the candidate genes were further selected based on expression profile and the frequency of LOH in common cancer types. b Loss of heterozygosity at 8p22 can render CRCs deficient in NAT2 function. To confirm the prevalence of rs1799930 and the frequency of LOH at NAT2, genomic DNA from 74 CIN CRCs and patient-matched normal tissues was genotyped. Left, genotype distribution of normal tissues. Right, LOH events observed in the tumors of heterozygous individuals. c Structure of human NAT2 co-crystallized with the cofactor coenzyme A (light blue) (PDB: 2PFR). The side chains of the active site amino-acid residues involved in substrate transformation (Asp122, Cys68, and His107) and cofactor binding (Gly104, Thr103, Thr214, Tyr208, and Ser287) (dark blue) and the side chain of Arg197 (rs1799930, red) are shown. The figure was designed in PyMOL (v. 2.3.2). d Exploiting loss of a rapid NAT2 allele in tumor cells for anti-cancer therapy. Eligible patients are heterozygous for the slow (A, blue) and rapid (G, red) NAT2 alleles. During cancer progression, cancer cells may undergo loss of heterozygosity (LOH) and lose the rapid NAT2 allele (red). Treatment with a cytotoxic compound (triangle) that can only be processed by the rapid NAT2 enzymatic variant lost in cancer cells will result in selective tumor death.

Article Snippet: The expression of NAT2 , DYRK1A , AURKA , or CDK7 transcripts was detected by qPCR using TaqMan probes against NAT2 (Hs04194721_s1), DYRK1A (Hs00176369_m1), AURKA (Hs01582072_m1), or CDK7 (Hs00361486_m1) with ACTB (Hs01060665_g1) as an internal reference gene.

Techniques: Functional Assay, Expressing, Transformation Assay, Binding Assay, Variant Assay

Journal: Nature Communications

Article Title: Exploiting loss of heterozygosity for allele-selective colorectal cancer chemotherapy

doi: 10.1038/s41467-020-15111-4

Figure Lengend Snippet: a , b Validation of RKO ( a ) and DLD-1 ( b ) CRC cell models of rapid and slow NAT2. Cell lysates from clones and parental cells were subjected to PAGE and immunoblotting with an α-MYC-Tag antibody to detect recombinant NAT2 and α-tubulin detection as loading control. c , d Quantification of NAT2 catalytic activity in RKO and DLD-1 cell clones. The velocity at which the NAT2-specific substrates amonafide (yellow) and procainamide (blue) become acetylated by different enzymatic variants at 10 µM was measured by LC-MS/MS in RKO ( c ) and DLD-1 ( d ) clones. Mean of two independent experiments. e Workflow of the selection of the chemical compounds for screening. f , g The dose-response for APA is NAT2 dependent in both RKO ( f ) and DLD-1 ( g ) CRC cells. Rapid or slow acetylator NAT2 clones, parental cells and vector controls were treated with different concentrations of APA and cell viability was measured by MTT assay after 72 h. The mean and S.D. of three independent experiments is shown. Data were analyzed using two-way ANOVA. ** P = 0.0035 in ( f ) and 0.0074 in ( g ), **** P < 0.0001.

Article Snippet: The expression of NAT2 , DYRK1A , AURKA , or CDK7 transcripts was detected by qPCR using TaqMan probes against NAT2 (Hs04194721_s1), DYRK1A (Hs00176369_m1), AURKA (Hs01582072_m1), or CDK7 (Hs00361486_m1) with ACTB (Hs01060665_g1) as an internal reference gene.

Techniques: Biomarker Discovery, Clone Assay, Western Blot, Recombinant, Control, Activity Assay, Liquid Chromatography with Mass Spectroscopy, Selection, Plasmid Preparation, MTT Assay

Journal: Nature Communications

Article Title: Exploiting loss of heterozygosity for allele-selective colorectal cancer chemotherapy

doi: 10.1038/s41467-020-15111-4

Figure Lengend Snippet: a NAT2 mediates APA acetylation. The velocity of NAPA formation was determined by LC-MS/MS in slow (green), rapid (yellow) NAT2 clones, vector control (blue), and parental RKO cells (black) after incubation with APA at indicated concentrations. b APA is selectively metabolized by NAT2. Enzyme kinetics of NAPA formation by human recombinant NAT1 (black) and NAT2 (red) proteins. c , d siRNA-mediated gene silencing of protein kinases with differential inhibition by APA and NAPA expressed in RKO CRC cells. Relative growth of RKO ( c ) and DLD-1 ( d ) cells transfected with a pool of siRNA targeting DYRK1A , AURKA , CDK7 , non-target RNA control and mock transfection as percentage of the growth of untransfected cells. Mean and S.D. (error bars) of three independent experiments is shown. Data were analyzed by one-way ANOVA. * P = 0.0435, ** P = 0.0013, *** P = 0.0004. e APA (2 µM) reduces the phospho-Thr288 Aurora kinase A (pAURKA) staining in NAT2-deficient cells but not in rapid NAT2 clones. Mitotic spindles were visualized by α-tubulin staining (green) and pAURKA levels (red). Representative images of three independent repeats are shown for each cell type. Scale bar, 10 µm. f Quantification of pAURKA signal in parental DLD-1 and slow and rapid NAT2 clones after APA treatment (2 µM). The pAURKA levels are shown as a fold of the DMSO control group. Mean and S.D. (error bars) of three independent experiments. Data were analyzed using two-way ANOVA. n.s., P = 0.0619 and **** P < 0.0001. g APA (2 µM), similarly to the AURKA selective inhibitor S1451 (10 µM) reduces the total protein level of AURKA in contrast to nocodazole (100 ng mL −1 ). DLD-1 cells in different treatments were subjected to immunoblot analysis with different antibodies for detection of total AURKA, PCNA (loading control), histone H3 pSer10, and total histone H3. Representative immunoblot from at least two independent experiments is shown.

Article Snippet: The expression of NAT2 , DYRK1A , AURKA , or CDK7 transcripts was detected by qPCR using TaqMan probes against NAT2 (Hs04194721_s1), DYRK1A (Hs00176369_m1), AURKA (Hs01582072_m1), or CDK7 (Hs00361486_m1) with ACTB (Hs01060665_g1) as an internal reference gene.

Techniques: Liquid Chromatography with Mass Spectroscopy, Clone Assay, Plasmid Preparation, Control, Incubation, Recombinant, Inhibition, Transfection, Staining, Western Blot

Journal: Nature Communications

Article Title: Exploiting loss of heterozygosity for allele-selective colorectal cancer chemotherapy

doi: 10.1038/s41467-020-15111-4

Figure Lengend Snippet: a , b Treatment with liposomal APA selectively inhibits growth of slow NAT2 tumors in vivo. Athymic nude mice carrying subcutaneous slow (left flank) and rapid (right flank) NAT2 RKO cell clones received intravenous injections of liposomal APA on days 0 and 3. Results are shown for one representative experiment conducted in 9 mice for the treatment group and 10 mice for the control group. Data were analyzed using one-way ANOVA, n.s., P = 0.9013 and * P = 0.0220. c Primary CRC tumors show sensitivity toward APA treatment. The survival index (SI) of primary tumor cells and organoids from 12 different CRC patients was determined when exposed to increasing concentrations of APA (log of concentration in μM). Samples encoding rapid (blue), intermediate (orange), and slow (gray) acetylator phenotypes are shown with their respective NAT2 haplotype. d A conceptual way of exploiting NAT2 loss of heterozygosity. Anti-cancer drugs benefit from reduced toxicity and improved tumor uptake when encapsulated in stealth liposomes. First, liposomal APA is injected intravenously in a rapid/slow NAT2 patient whose tumor has lost the rapid allele . The stealth liposomes extravasate through the leaky tumor vessels and accumulate in tumor tissues due to the enhanced permeability and retention (EPR) effect . The lipid bilayer of the liposomes fuses with cell membranes and deliver APA to the colorectal tumor cells and adjacent normal epithelial cells. The adjacent normal epithelial cells express rapid NAT2 and are able to process APA into its nontoxic product NAPA. In contrast, cancer cells expressing only slow NAT2 are not capable of detoxifying APA and die. Liposomal drug formulations also accumulate in the liver, where APA is cleared by rapid NAT2 . Every element of ( d ) was created in Adobe Illustrator 2019.

Article Snippet: The expression of NAT2 , DYRK1A , AURKA , or CDK7 transcripts was detected by qPCR using TaqMan probes against NAT2 (Hs04194721_s1), DYRK1A (Hs00176369_m1), AURKA (Hs01582072_m1), or CDK7 (Hs00361486_m1) with ACTB (Hs01060665_g1) as an internal reference gene.

Techniques: In Vivo, Clone Assay, Control, Concentration Assay, Liposomes, Injection, Permeability, Expressing

Journal: Cell reports

Article Title: Both enantiomers of β-aminoisobutyric acid BAIBA regulate Fgf23 via MRGPRD receptor by activating distinct signaling pathways in osteocytes

doi: 10.1016/j.celrep.2024.114397

Figure Lengend Snippet:

Article Snippet: Taqman assay probe for Mepe , Thermo Fisher Scientific , Mm02525159_s1.

Techniques: Recombinant, Modification, Protease Inhibitor, Saline, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Membrane, cAMP Assay, TaqMan Assay, Software

Journal: iScience

Article Title: Nrf2 activation reprograms macrophage intermediary metabolism and suppresses the type I interferon response

doi: 10.1016/j.isci.2022.103827

Figure Lengend Snippet: Nrf2 is an endogenous suppressor of IFN-β in inflammatory macrophages (A) IFN-β, IL-6, and TNF protein levels in LPS-stimulated Nrf2-KO and Keap1-KD compared to WT cells (n = 3 biological replicates). (B) Ifnb and Ifit2 mRNA levels in LPS-stimulated Nrf2-KO and Keap1-KD compared to WT cells (n = 5-6 biological replicates). (A-B) Data are mean ± SEM p value determined by unpaired t test, corrected for multiple comparisons by Holm-Sidak test. (C) IFN-β in Poly(I:C) stimulated Nrf2-KO and Keap1-KD compared to WT cells (n = 3 biological replicates). Data are mean ± SEM p value determined by two-tailed unpaired t test. (D) Ifnb and Ifit2 mRNA levels in WT, Nrf2-KO, and Keap1-KD cells that had been pre-treated for 1 h with the Nrf2 activator TBE-31 (30 nM) and stimulated with LPS (100 ng/mL) for a further 4 h (n = 3 biological replicates). (E) Ifnb and Ifit2 mRNA levels in WT, Nrf2-KO, and Keap1-KD cells that had been pre-treated for 24 h with the Nrf2 activator TBE-31 (20 nM) and stimulated with LPS (100 ng/mL) for a further 4 h (n = 2-3 biological replicates). (D-E) Data are mean ± SEM p value determined by one-way ANOVA, corrected for multiple comparisons by Tukey statistical test. (F) Nqo1 mRNA levels in WT, Nrf2-KO, and Keap1-KD cells that had been pre-treated for 24 h with the Nrf2 activator TBE-31 (20 nM) (n = 2-3 biological replicates). Data are mean ± SEM p value determined by a two-tailed unpaired t test. p <0.05∗; p <0.01∗∗; p <0.001∗∗∗.

Article Snippet: IFNB1 (human) , Applied Biosystems , Hs01077958_s1.

Techniques: Two Tailed Test

Journal: iScience

Article Title: Nrf2 activation reprograms macrophage intermediary metabolism and suppresses the type I interferon response

doi: 10.1016/j.isci.2022.103827

Figure Lengend Snippet:

Article Snippet: IFNB1 (human) , Applied Biosystems , Hs01077958_s1.

Techniques: Recombinant, Control, Negative Control, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Data-independent acquisition, Quantitative Proteomics, Software, Gene Expression

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: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Flrt2 mRNA detection by fluorescence in situ hybridization (FISH) in P8 wild-type mouse retina in the optic nerve head (ONH) area, where the central artery and vein enter the retina, and in the superficial vascular plexus (SVP) (upper panel); and in P8 wild-type cerebral cortex (upper cortical layers and pial vasculature) (lower panel). Blood vessels were detected by immunostaining with podocalyxin (Podxl) (general vessel marker). (b) Coronal section of the cerebral cortex from P6 wild-type mouse stained for FLRT2 and NeuN as neuronal marker (neuronal layers I-VI annotated). Blood vessels were visualized with isolectin-B4 (IB4) staining. Arrows show FLRT2 positive blood vessels (right). (c) Flat-mounted P7-P8 retinas from control and Flrt2 iΔEC littermate mice injected with 4-hydroxytamoxifen (Tmx) from P1 to P3 and stained with IB4. (d - f) Quantification of radial vascular length ratio (d), total retinal vessel length (e), and total number of branch points (f) per retina. (g) Representative images of P7-P8 control and Flrt2 iΔEC flat-mounted retinas stained for IB4. Veins (V) and arteries (A) are indicated. (h) Quantification of capillary network density between veins and arteries. (i) Glut1 staining of the vasculature in control and Flrt2 iΔEC brain cortices from P7-P8 mice after Tmx administration from P1 to P3. ( j - l ) Quantification of vessel density (j), vessel length (k) and number of branch points (l). Scale bars: 20 μm (a), 50 μm (b), 500 μm (c), 200 μm (g), 100 μm (i). n = 7-12 (d), 6-7 (e, f), 8 (h), 7 (j-k), 5-6 (l) animals per genotype. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired t-test.

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Fluorescence, In Situ Hybridization, Immunostaining, Marker, Staining, Control, Injection

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Schematic representation of the Flrt2 endothelial specific knockout strategy in Cdh5(PAC)-CreERT2:Flrt2 lox/lox ( Flrt2 iΔEC ) mice. (b) Experimental design of Flrt2 gene deletion by Cre recombination. 4-hydroxytamoxifen (Tmx) was applied from postnatal day 1 (P1) to 3 and tissue collected at P5 and P7 or 8. Cartoon created with Biorender.com. (c, e) Flat-mounted retina (c) and neocortical brains slice (e) from P7 Cdh5-CreERT2:Rosa26tdTomato mice injected from P1 to P3 with Tmx. Cells undergoing Cre-mediated recombination expressed the fluorescent protein tdTomato, which was used to assess specificity and efficiency in blood vessels, co-stained with IB4 (c) or Podxl (e). (d, f) Quantification of the percentage of tdTomato-positive signal per IB4-positive (d) and Podxl-positive signal (f). (g) Immunoblot showing FLRT2 protein levels in primary lung ECs isolated from P8 control and Flrt2 iΔEC mice. FLRT2-specific antibody detects the full-size protein, the cleaved extracellular domain, and glycosylated forms. Pan-cadherin was used as loading control. (h) Flrt2 and Flrt3 mRNA levels from primary mouse brain ECs (pmBECs) isolated from P5 control and Flrt2 iΔEC mice. (i) pmBECs isolated from P7 control and Flrt2 iΔEC mice stained for FLRT2 and cell nuclei (DAPI). (j) FLRT2 fluorescence intensity quantification in control and Flrt2 iΔEC pmBECs. (k) Body weight measured from control and Flrt2 iΔEC male and female mice at P7-8. (l) Example of whole fixed brains from control and Flrt2 iΔEC P7-8 mice. (m, n) Quantifications of brain length (m) and width (n) of control and Flrt2 iΔEC P7-8 mice. Scale bar: 200 μm (c), 100 μm (e), 10 μm (i), 1 mm (l). n = 8 mice (d), 3 mice (f), 4-10 animals per genotype (h), 14-16 pictures per animal from 2 mice per genotype, one litter (j), 6-13 mice per genotype and sex (k), 9-10 mice per genotype (m, n). Horizontal bar shows the median value (d, f). Data are shown as mean ± SEM. **P < 0.01, ***P > 0.001, ns= not significant, unpaired t-test (h ( Flrt3 ), j, k, m, n), Mann-Whitney test (h, Flrt2 ).

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Knock-Out, Injection, Staining, Western Blot, Isolation, Control, Fluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Flat mounted retinas stained for Collagen IV (Col IV) and IB4 to visualize blood vessel regression. Col IV+ IB4-empty sleeves are marked with yellow arrows. (b) Quantification of vessel regression as Col IV+ IB4-empty sleeves per imaging field. (c) Representative images of retinas from control and Flrt2 iΔEC P7-P8 littermates stained with IB4. Arteries and veins are highlighted in pink and light blue, respectively. (d) Quantification of the total number of veins and arteries per retina in mutant and control mice at P7-8. (e) Negative Flrt2 (grey) expression in a cortical artery (A) expressing the Gkn3 marker (red) and positive Flrt2 signal in a vein (V) expressing Scl38a5 (blue). Podxl immunostaining is used as a general marker of the vasculature. (f) Representative images of recombinant tip cells in the retinal vascular front of Cdh5-CreERT2:Rosa26tdTomato (control) and Flrt2 iΔEC :Rosa26tdTomato P7-P8 mice. Upon Cre-mediated recombination ECs express tdTomato reporter protein (depicted as red). (g) Quantification of the number of filopodia per tip cell. (h) Retinal vascular fronts from control and Flrt2 iΔEC P7-P8 mice with proliferative cells labelled with EdU, EC nuclei stained with ERG, and blood vessels with IB4. (i) Quantification of % EdU + ERG + cells in total ERG + cells per image. Scale bars: 100 μm (a), 500 μm (c), 25 μm (e, f, h). n = 6-7 animals per genotype (b, d), 19-21 cells per genotype (g), 7-9 animals per genotype (i). Data are shown as mean ± SEM. ***P > 0.001, ns = not significant, two-way ANOVA (d), unpaired t-test (b, g i).

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Staining, Imaging, Control, Mutagenesis, Expressing, Marker, Immunostaining, Recombinant

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Representative images of main arteries and veins (identified by their morphology) from control and Flrt2 iΔEC P7-P8 retinas stained with IB4. Red dots indicate branch points from the corresponding mother vessel. (b) Quantification of the number of branch points in arteries (left) and veins (right) per vessel length. (c) Expanded P7 retina stained for FLRT2 and collagen IV (Col IV) showing FLRT2 expression in retinal vessels. Note the expression of FLRT2 at the EC membrane in the vein but its absence in the artery. (d) Representative images of retinal vascular front from control and Flrt2 iΔEC mice stained with IB4. Red dots indicate cellular protrusions identified as angiogenic sprouts. (e) Quantification of number of sprouts per 100 μm of retinal vascular front. (f) Vascular fronts from control and Flrt2 iΔEC P7-P8 retinas showing blood vessels labelled with IB4 and EC nuclei stained for ERG. (g) Quantification of the number of tip cells per stalk cells at the vascular front. (h) Glut1 staining visualizing vessel sprouts in P7-P8 control and Flrt2 iΔEC cerebral cortices. ( i ) Quantification of the number of sprouts per vessel density in the cerebral cortex. Scale bars: 50 μm (a, f), 90 μm (c), 40 μm (d), 100 μm (h). n = 17-20 (b), 18-22 (e), 8-10 (g), 5-6 (i) animals per genotype. Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001, ns = not significant, unpaired t-test.

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Control, Staining, Expressing, Membrane

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) P5 flat-mounted retinas from control and Flrt2 iΔEC littermates injected with 4-hydroxytamoxifen (Tmx) from P1 to P3, stained with IB4 to visualize blood vessels. (b , c) Quantification of the vascular radial growth ratio (b), and the capillary density (c) as the area covered by IB4 staining in P5 control and mutant retinas. (d) Glut1 staining of control and Flrt2 iΔEC cortices at P4-P5 after Tmx administration from P1 to P3. (e - g) Quantification of vessel density (e), vessel length (f) and number of branch points (g) in mouse cerebral cortices. (h) Representative images of arteries and veins from control and Flrt2 iΔEC retinas stained with IB4 at P5. Red dots indicate branch points from the mother vessel. (i) Quantification of the number of branch points in main arteries (left) and veins (right) per vessel length. (j) P5 retinal vascular fronts stained with IB4. Red dots indicate cellular protrusions identified as angiogenic sprouts. (k) Quantification of the number of sprouts per 100 μm of vascular front. ( l) Control and Flrt2 iΔEC cerebral cortices at P5 stained for Glut1 to visualize vessel sprouts. ( m ) Quantification of the number of sprouts per vessel density at P5 in Flrt2 iΔEC and control littermates. Scale bars: 500 μm (a), 100 μm (d), 50 μm (h, j, l). n = 10-13 (b, i), 7-9 (c), 10-11 (e), 6 -10 (f, g), 10-12 (k), 6-10 (m) animals per genotype. Data are shown as mean ± SEM. **P < 0.01, ns = not significant, unpaired t-test.

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Control, Injection, Staining, Mutagenesis

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a, b) Flat mounted retinas (a) and cortical brain slices (c) stained for apoptosis marker cleaved caspase-3 (cleaved casp-3) and blood vessels (IB4 or Podxl). (b, d) Quantification of cleaved caspase-3+ ECs in control and Flrt2 iΔEC P5 retinas (b) and P4-P5 brain neocortices (d). (e, g) Flat mounted retinas (e) and cortical brain slices (g) stained for cell cycle arrest marker p21 and blood vessels (IB4 or Podxl). (f, h) Quantification of p21+ ECs in control and Flrt2 iΔEC P5 retinas (f) and P4-P5 brain neocortices (h). Scale bars: 50 μm. n = 4-5 animals per genotype (b, f), 6-7 animals per genotype (d), 5-6 animals per genotype (h). Data are shown as mean ± SEM. *P < 0.05, ns = not significant, unpaired t-test (b, d, h), Mann-Whitney test (f).

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Staining, Marker, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Proximity ligation assay (PLA) in HUVEC cultures. White puncta indicate FLRT2 and VE-cadherin being in close proximity (< 40 nm). Cells were stained with phalloidin and DAPI to visualize actin filaments and nuclei, respectively. (b) PLA signal quantification as puncta per cell number in the field (identified by DAPI nuclei). (c) Immunoprecipitation (IP) of VE-cadherin and immunodetection of FLRT2 and VE-cadherin from total mouse brain lysates. TL, total lysate. (d) Representative immunoblot showing FLRT2 protein reduced expression in HUVECs treated with Flrt2 siRNA compared to control treated cells. β-actin was used as loading control. (e) Quantification of FLRT2 protein levels in control and Flrt2 siRNA treated HUVECs. (f) Antibody feeding assay in HUVEC transfected with control and Flrt2 -specific siRNA treated with chloroquine. Cells were immunostained for internalized VE-cadherin, total VE-cadherin and cell nuclei (DAPI). Intensity of internalized VE-cadherin is shown in arbitrary units (AU, upper panels). (g) Quantification of the fluorescence intensity of internalized VE-cadherin per cell. (h) Quantification of VE-cadherin intensity per cell-junction length. (i) Representative immunoblot showing VE-cadherin, its cleaved form, and α-tubulin as loading control in HUVEC transfected with control and Flrt2 -specific siRNAs. (j) Quantification of VE-cadherin/loading control and cleaved VE-cadherin/loading control ratios. ( k) Primary mouse brain ECs (pmBEC) from control and Flrt2 iΔEC littermates stained for Calpain-2 and DAPI. (l) Quantification of the fluorescence intensity of Calpain-2 staining per cell. (m) Neocortical blood vessels stained for Calpain-2 and IB4 from control and Flrt2 iΔEC littermates. (n) Quantification of Calpain-2 fluorescence intensity in the blood vessels. Scale bars: 40 μm (a, upper panels), 15 μm (a, lower panels), 10 μm (f, k), 5 μm (m). n = 20-23 pictures per condition from three different experiments (b), 6 independent experiments (e), 84-115 cells from 3 independent experiments (g), 18-22 pictures per condition from 3 independent experiments (h), 8-11 independent experiments (j), 71-72 cells per condition, from 1 control and 1 Flrt2 iΔE littermate (l), 4-5 animals per genotype (n). Data are shown as mean ±. *P < 0.05, **P < 0.01, SEM. ***P < 0.001, ns = not significant, unpaired t-test (b, e, h, j, n), Mann-Whitney test (g, l).

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Proximity Ligation Assay, Staining, Immunoprecipitation, Immunodetection, Western Blot, Expressing, Control, Feeding Assay, Transfection, Fluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Immunoprecipitation of VE-cadherin and immunodetection of FLRT2 and VE-cadherin from HUVEC cultures. (b) PLA in primary mouse brain EC (pmBECs) cultures. White puncta indicate FLRT2 and VE-cadherin being in close proximity (< 40 nm). Cells were stained with Pecam1 and DAPI to visualize EC junctions and nuclei, respectively. Note the major presence of PLA puncta along the Pecam1+ junctions in the cells isolated form control mice, indicating that FLRT2 and VE-cadherin interact at the cell surface. (c) PLA signal quantification as puncta per cell. (d) HUVEC treated with control and Flrt2 -specific siRNAs immunostained for VE-cadherin and DAPI. (e) Quantification of VE-cadherin intensity per cellular junction length. (f) mRNA expression of Flrt2 and Cdh5 in HUVEC transfected with control and Flrt2 -specific siRNAs measured by qPCR. (g) HUVEC cultures transfected with control and Flrt2 -specific siRNAs stained for Calpain-1 and DAPI. (h) Quantification of the fluorescence intensity of Calpain-1 staining per cell. Scale bars: 10 μm (b), 15 μm (d), 20 μm (g). n = 71-72 cells per condition from 2 animals per genotype (c), 27-30 images per condition from 3 independent experiments (e), 7 independent experiments (f), 33-46 cells, 1 representative experiment from 3 independents experiments (h). Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001, Mann-Whitney test (c, h), unpaired t-test (e, f).

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Immunoprecipitation, Immunodetection, Staining, Isolation, Control, Expressing, Transfection, Fluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Proximity ligation assay (PLA) in EC cultures. White puncta indicate Numb and FLRT2 and Numb and VE-cadherin being in close proximity (< 40 nm). Cells were stained with phalloidin and DAPI to visualize actin filaments and nuclei, respectively. (b) Expansion microscopy on HUVEC cultures stained for FLRT2 and Numb showing expression of both proteins in cell-cell contacts and along filopodia extensions. (c) mRNA expression of Flrt2 and Numb in ECs transfected with control and Flrt2 -specific siRNAs measured by RT-qPCR. (d) Representative immunoblot showing FLRT2 and Numb protein levels in ECs transfected with control and Flrt2 -specific siRNAs. (e) FLRT2 and Numb protein levels quantification in ECs transfected with control and Flrt2 -specific siRNAs. (f) mRNA expression quantification of Numb from primary mouse brain ECs (pmBECs) isolated from control and Flrt2 iΔEC littermates. (g) Scheme showing the quantification of Golgi orientation in migrating cells after scratch assay and in retinal vascular fronts. Cell was classified as polarized if the angle formed between the scratch or vascular front and Golgi located within a 120°. (h) Scratch assay on HUVECs stained for VE-cadherin, Golgi apparatus (GM130) and cell nuclei (DAPI). Yellow stars in lower panels indicate cells polarized towards the wound area. The 3 first cell rows were considered for quantification. (i) Quantification of the percentage of cells per image polarized towards the wound. (j) Representative images of retinal vascular front from control and Flrt2 iΔEC littermates stained for blood vessels, EC nuclei and Golgi apparatus with IB4, ERG and GM130, respectively. White arrows indicate cellular orientation identified with GM130 position relative to ERG staining. (k) Quantification of the percentage of cells polarized towards the vascular front. The 3 first cell rows were considered for quantification. Scale bars: 20 μm (a, b, h, j). n = 4 independent experiments (c), 7 independent experiments (e), 5-10 animals per genotype (f), 27-28 images from 3 independent experiments (i), 4 animals per genotype (k). Data are shown as mean ± SEM. *P > 0.05, ***P < 0.001, unpaired t-test (c, e, f), Mann-Whitney test (i, k).

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Proximity Ligation Assay, Staining, Microscopy, Expressing, Transfection, Control, Quantitative RT-PCR, Western Blot, Isolation, Wound Healing Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a and c) Vascular front of P5 wild-type retinas (a) and remodeling capillaries in cerebral cortex (c) from control and Flrt2 iΔEC littermates stained for VE-cadherin antibody. Blood vessels visualized with IB4. (b and d) Quantification of VE-cadherin activity in retina (b) and cerebral cortex (d) blood vessels. 15 x 15 μm regions of interest (ROIs) were blindly classified to a VE-cadherin activity category: low (smooth pattern), medium (irregular pattern), high (rough pattern). Example images of the three VE-cadherin activity categories in retina (b) and cortex (d) vessels are shown in correlation with the graph. Scale bars: 20 μm (a), 15 μm (c). n = 53-66 images per genotype from three litters b), 78-80 images per genotype from two litters (d). Data are shown as mean ± SEM. *P > 0.05, **P < 0.01, ns = not significant, two-way ANOVA (b and d).

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Control, Staining, Activity Assay

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Expansion microscopy 3D-visualization of a large vessel stained for VE-cadherin, FLRT2 and Numb in the cerebral cortex. Higher magnification (lower panels) showing the colocalization of the three proteins at EC junction. (b and c) Expansion microscopy 3D-visualization of a large vessel stained for VE-cadherin and FLRT2 (b) and VE-cadherin and Numb (c) in the retina. (d) Expansion microscopy 3D-visualization of cerebral cortex capillaries stained for VE-cadherin and Glut1 in control (left) and Flrt2 iΔEC (right) mice. Higher magnifications (1-5) showing x-y planes (first row) and y-z planes (second row) exposing VE-cadherin pattern in a control capillary (1-2), a control tip cells (3), and a Flrt2 iΔEC capillary (4-5). Scale bars: 4 μm (a upper panel), 1 μm (a lower pannels), 15 μm (b, c), 30 μm (d).

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Microscopy, Staining, Control

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Representative immunoblot of the cytosolic and nuclear fractions obtained from bEnd.3 cells treated with control and Flrt2 siRNA showing protein levels of β-catenin and FoxO1, and α-tubulin and Lamin A/C as cytosolic and nuclear controls, respectively. (b and c) β-catenin (b) and FoxO1 (c) protein levels relative to the loading controls. (d) Representative immunoblot showing FLRT2 and Claudin-5, and β-actin as loading control in bEnd.3 cells treated with control and Flrt2 siRNA. (e) Quantification of FLRT2 and Claudin-5 protein levels relative to the loading control. ( f) Representative immunoblot showing Claudin 5, and α-tubulin as loading control in total brain lysates from control and Flrt2 iΔEC littermates. (g) Quantification of Claudin-5 protein levels relative to the loading control. (h) Neocortical blood vessels stained for Claudin-5 and VE-cadherin. Note the colocalization of both protein in the control vessel, compared to the split Claudin-5 signal in Flrt2 iΔEC blood vessel. (i) Quantification of the ratio of split Claudin-5 junction length to the total junctional length. (j) Representative fluorescent whole-brain images of control and Flrt2 iΔEC littermates injected with AlexaFluor TM 555-conjugated cadaverine at P7-P8. (k) Quantification of cadaverine whole-brain intensity in P7-P 8control and Flrt2 iΔEC littermates. (l) Representative images of Collagen IV (Col IV) immunostaining and cadaverine signal in control and Flrt2 iΔEC cortices showing cadaverine leakage in Flrt2 iΔEC mice. (m) TEM representative images of brain capillaries showing an EC junction in control and Flrt2 iΔEC mice. Note the abnormal junctions often associated with the presence of vacuoles (red arrows) in the Flrt2 iΔEC vessels. (n) Incidence (in percentage) of abnormal junctions (left) and junctions with vacuoles (right) in TEM images of control and Flrt2 iΔEC brain capillaries. Scale bars: 10 μm (h), 1 mm (j), 100 μm (l), 200 nm (m). n = 3 independent experiments (b, c), 5-6 independent experiments (e), 8 animals per genotype (g), 5-7 animals per genotype (i), 16-18 animals per genotype (k), 3 animals per genotype (n). Data are shown as mean ± SEM. *p < 0.05, **P < 0.01, ***P > 0.001, unpaired t-test (b, c, e, i, k), Mann-Whitney test (g), 2-way ANOVA (n).

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Western Blot, Control, Staining, Injection, Immunostaining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) mRNA fold change levels of Flrt2 and tight junction protein claudin5 ( Cldn5 ) in bEnd.3 cells treated with control and Flrt2 siRNA. (b) mRNA expression quantification of Cldn5 in control and Flrt2 iΔEC total brains at P7-P8. (c, e) Representative immunoblots from total brain lysates of control and Flrt2 iΔEC mice showing tight junctions proteins ZO-1 (c) and JAM-A (e) levels. (d, f) Quantification of protein ZO-1 (d) and JAM-A (f) levels in total brain lysates from control and Flrt2 iΔEC mice. (g, h) Neocortical blood vessels stained for ZO-1 (g) and JAM-A (h) showing no differences in tight junction proteins distribution between control and Flrt2 mutant mice. (i) Representative fluorescent whole-brain images of control and Flrt2 iΔEC littermates injected with AlexaFluor555nm-conjugated ovalbumin (45 kDa) at P7-8. (j) Quantification of ovalbumin whole-brain intensity in control and Flrt2 iΔEC mice at P7-8. (k) Representative fluorescent whole-brain images of control and Flrt2 iΔEC littermates injected with AlexaFluor555nm-conjugated cadaverine at P5. (l) Quantification of cadaverine whole-brain intensity in control and Flrt2 iΔEC mice at P5. Scale bars: 10 μm (g, h), 1 mm (i, k). n = 5 independent experiments (a), 6 animals per genotype (b), 5 animals per genotype (d), 4 animals per genotype (f, j), 7-8 animals per genotype (l). Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ns = not significant, unpaired t-test.

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Control, Expressing, Western Blot, Staining, Mutagenesis, Injection

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a, c, e,) Cerebral cortices of control and vascular Flrt2 mutant mice stained with the pericyte marker PDGFRβ (a), astrocytic end-feet marker Aquaporin-4 (Aqp4) (c) and extracellular matrix marker Collagen IV (Col IV) (e) and blood vessel markers Glut1 (a) or Podxl (c, e). (b, d, f) Quantification of PDGFRβ (b), Aqp4 (d) or Col IV (f) coverage of cortical vasculature. Scale bars: 50 μm (a), 10 μm (c, e). n = 7 animals per genotype (b), 6 animals per genotype (d, f). Data are shown as mean ± SEM. ns = not significant, unpaired t-test.

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Control, Mutagenesis, Staining, Marker

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: Representative image of an endothelial-specific Flrt2 mutant cerebral cortex section showing a leakage area where the punch was performed and further processed for transmission electron microscopy (TEM) analysis. After assessing the quality of the tissue, imaging was processed focusing in capillary EC tight junctions. Cartoon created with Biorender.com. Scale bars: 1mm, 250 μm, 2500 nm, 250 nm

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Mutagenesis, Transmission Assay, Electron Microscopy, Imaging

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Schematic timeline representation of the vascular events derived from FLRT2 deletion in ECs. (b) Schematic representation of the molecular mechanisms regulated by FLRT2 in postnatal CNS vasculature. In control conditions, FLRT2 forms a complex with VE-cadherin and the endocytic adaptor Numb which allows the dynamic turnover of VE-cadherin necessary for angiogenic sprouting. By contrast, in FLRT2-deficient cells VE-cadherin cytoplasmic tail is cleaved by Calpains and subsequently fated to lysosomal degradation, while a compensatory biosynthesis accumulates VE-cadherin at the cell membrane impairing vascular sprouts. In addition, FLRT2 deletion facilitates the nuclear translocation of β-catenin, repressing Claudin-5 expression and triggering increased size-selective BBB permeability.

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Derivative Assay, Control, Membrane, Translocation Assay, Expressing, Permeability

Journal: eLife

Article Title: Induction of osteogenesis by bone-targeted Notch activation

doi: 10.7554/eLife.60183

Figure Lengend Snippet: ( A ) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified Dll4-Asp 8x recombinant proteins. From left to right, protein molecular mass marker (Marker), extract of untransfected HEK293 cells (Cells (-)) OR pcDNA3.1-Dll4-Asp 8x -transfected cells (Cells (+)), supernatant after culture of untransfected (Supernatant (-)) OR pcDNA3.1-Dll4-Asp 8 -transfected cells (Supernatant (+)), flow-through after loading of Supernatant (+) on His-tag protein purification column (Flow-through), wash buffer (Wash), elute (Elute), elute after dialysis (Elute (AD)), and Opti-MEM culture medium (Medium) were loaded. ( B, C ) RT-qPCR analysis of DLL4 , HEY1 , HEY2 , HES1 , EFNB2 , and NRARP transcripts in confluent human umbilical vascular endothelial cells (HUVECs) stimulated with soluble ( B ) or in sub-confluent HUVECs stimulated with immobilized Dll4-Asp 8x or BSA with poly-L-lysine coating ( C ). Data represent mean ± s.e.m. (n = 4 for ( B ) and n = 6 for ( C )) (p-values determined by multiple unpaired t-test with Welch’s correction). Figure 1—figure supplement 1—source data 1. Source data for . Figure 1—figure supplement 1—source data 2. Images of uncropped blots and gels with the relevant bands labeled for , , and . Figure 1—figure supplement 1—source data 3. ZIP file containing the full raw unedited blots and gels for , , and .

Article Snippet: The following FAM-conjugated TaqMan gene expression probes (ThermoFisher) were used in combination with SsoAdvanced Universal Probes Supermix (BIO-RAD): DLL4 (Hs00184092_m1), HEY1 (Hs00232618_m1), HEY2 (Hs00232622_m1), HES1 (Hs00172878_m1), EFNB2 (Hs00187950_m1), NRARP (Hs01104102_s1).

Techniques: Polyacrylamide Gel Electrophoresis, SDS Page, Purification, Recombinant, Marker, Transfection, Protein Purification, Quantitative RT-PCR, Labeling

Journal: eLife

Article Title: Induction of osteogenesis by bone-targeted Notch activation

doi: 10.7554/eLife.60183

Figure Lengend Snippet: ( A ) Diagram showing the amino acid sequence of mouse and rat Dll4 extracellular domain (ECD). Amino acid replacements shown in red were introduced into the murine sequence for the generation of Dll4 (E12) with increased Notch-binding affinity. ( B ) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified Dll4 (E12) recombinant protein. From left to right, protein molecular mass marker (Marker), supernatant after culture of untransfected (Supernatant (-)) OR pcDNA3.1-Dll4 (E12) -transfected HEK293 cells (Supernatant (+)), flow-through after loading of Supernatant (+) on His-tag protein purification column (Flow-through), wash buffer (Wash), elute (Elute), elute after dialysis (Elute (AD)), and Opti-MEM culture medium (Medium) were loaded. ( C, D ) RT-qPCR analysis of DLL4 , HEY1 , HEY2 , HES1 , EFNB2 , and NRARP transcripts in confluent human umbilical vascular endothelial cells (HUVECs) stimulated with soluble ( C ) or in sub-confluent HUVECs stimulated with immobilized Dll4 (E12) , Dll4-Asp 8x , and BSA by poly-L-lysine coating ( D ). Data represent mean ± s.e.m. (n = 3 for ( C ) and n = 4 for ( D )) (p-values determined by ordinary one-way ANOVA with Tukey’s multiple comparison test). ( E ) Western blot analysis of Dll4 (E12) protein expression in liver lysate of pLIVE-Dll4 (E12) and control-injected mice. GAPDH is used as loading control. Figure 1—figure supplement 4—source data 1. Source data for .

Article Snippet: The following FAM-conjugated TaqMan gene expression probes (ThermoFisher) were used in combination with SsoAdvanced Universal Probes Supermix (BIO-RAD): DLL4 (Hs00184092_m1), HEY1 (Hs00232618_m1), HEY2 (Hs00232622_m1), HES1 (Hs00172878_m1), EFNB2 (Hs00187950_m1), NRARP (Hs01104102_s1).

Techniques: Sequencing, Binding Assay, Polyacrylamide Gel Electrophoresis, SDS Page, Purification, Recombinant, Marker, Transfection, Protein Purification, Quantitative RT-PCR, Comparison, Western Blot, Expressing, Control, Injection

Journal: eLife

Article Title: Induction of osteogenesis by bone-targeted Notch activation

doi: 10.7554/eLife.60183

Figure Lengend Snippet: ( A ) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified Jag1-Asp 8x recombinant protein. From left to right, protein molecular mass marker (Marker), extract of untransfected HEK293 cells (Cells (-)) OR pcDNA3.1-Jag1-Asp 8x -transfected cells (Cells (+)), supernatant after culture of untransfected (Supernatant (-)) OR pcDNA3.1-Jag1-Asp 8x -transfected cells (Supernatant (+)), flow-through after loading of Supernatant (+) on His-tag protein purification column (Flow-through), wash buffer (Wash), elution of Jag1-Asp 8x (Elute), and Opti-MEM culture medium (Medium) were loaded. ( A, B ) SDS-PAGE analysis of purified Jag1-Asp 8x ( A ) and Jag1 (JV1) ( B ) recombinant proteins. From left to right, protein molecular mass marker (Marker), extract of untransfected (Cells (-)) or transfected HEK293 cells (Cells (+)), supernatant after culture of untransfected (Supernatant (-)) or transfected cells (Supernatant (+)), flow-through after loading of Supernatant (+) on His-tag protein purification column (Flow-through), wash buffer (Wash), elution of recombinant Jag1 (Elute) and Opti-MEM culture medium (Medium) were loaded. ( C ) Western blot analysis of eluted Jag1-Asp 8x and Jag1 (JV1) recombinant proteins used for in vitro stimulation experiments ( D, E ). Proteins detected at the expected molecular weight with anti-Jag1 and anti-His1 antibodies. ( D, E ) RT-qPCR analysis of human ( HS) DLL4 , HEY1 , HEY2 , HES1 , EFNB2 , and NRARP transcripts in confluent human umbilical vascular endothelial cells (HUVECs) after stimulation with soluble ( D ) or immobilized (poly-L-lysine coating) Jag1-Asp 8x , Jag1 (JV1) , and BSA ( E ). Data represent mean ± s.e.m. (n = 3) (p-values determined by ordinary one-way ANOVA with Tukey’s multiple comparison test). Figure 5—figure supplement 1—source data 1. Source data for .

Article Snippet: The following FAM-conjugated TaqMan gene expression probes (ThermoFisher) were used in combination with SsoAdvanced Universal Probes Supermix (BIO-RAD): DLL4 (Hs00184092_m1), HEY1 (Hs00232618_m1), HEY2 (Hs00232622_m1), HES1 (Hs00172878_m1), EFNB2 (Hs00187950_m1), NRARP (Hs01104102_s1).

Techniques: Polyacrylamide Gel Electrophoresis, SDS Page, Purification, Recombinant, Marker, Transfection, Protein Purification, Western Blot, In Vitro, Molecular Weight, Quantitative RT-PCR, Comparison

Journal: eLife

Article Title: Induction of osteogenesis by bone-targeted Notch activation

doi: 10.7554/eLife.60183

Figure Lengend Snippet:

Article Snippet: The following FAM-conjugated TaqMan gene expression probes (ThermoFisher) were used in combination with SsoAdvanced Universal Probes Supermix (BIO-RAD): DLL4 (Hs00184092_m1), HEY1 (Hs00232618_m1), HEY2 (Hs00232622_m1), HES1 (Hs00172878_m1), EFNB2 (Hs00187950_m1), NRARP (Hs01104102_s1).

Techniques: Recombinant, Variant Assay, Plasmid Preparation, Sequencing, Control, Gene Expression, Bicinchoninic Acid Protein Assay, cDNA Synthesis, Amplification, Magnetic Beads, Protease Inhibitor, Western Blot, Software, Staining

Journal: iScience

Article Title: Dietary fish oil enriched in very-long-chain polyunsaturated fatty acid reduces cardiometabolic risk factors and improves retinal function

doi: 10.1016/j.isci.2023.108411

Figure Lengend Snippet:

Article Snippet: Acot1 (Mm01622471_s1) , Life Technologies , Cat# 4331182.

Techniques: Recombinant, Sample Prep, Software, Diagnostic Assay, Chromatography, Spectrophotometry, Nuclear Magnetic Resonance, Spectroscopy, Mass Spectrometry