Journal: Pulmonary Circulation

Article Title: High gestational leucine level dampens WDPCP/MAPK signaling to impair the EMT and migration of cardiac microvascular endothelial cells in congenital heart defects

doi: 10.1002/pul2.70013

Figure Lengend Snippet: Neonatal CHDs are associated with elevated plasma leucine levels and dysregulation of the WDPCP/EMCN axis. (a) The analysis of leucine levels in plasma samples of healthy controls and neonatal CHD patients ( n = 10 in each category). (b) Western blot analysis of the protein levels of WDPCP, and (c) EMCN in plasma samples of healthy controls and neonatal CHD patients ( n = 3 in each category). (d) Detection of phospho‐p38 and (e) phospho‐ERK levels by ELISA in plasma samples of healthy controls and neonatal CHD patients ( n = 10 in each category) were quantified ELISA. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Phospho‐p38 MAPK and phospho‐ERK levels were measured using the Human/Mouse/Rat Phospho‐ERK1/2, JNK, p38 MAPK Cell‐Based ELISA kit (#CBEL‐ERK‐SK, RayBiotech) and the ERK1/ERK2 (Phospho) Multispecies InstantOneTM ELISA Kit (#85‐86013‐11, ThermoFisher Scientific.

Techniques: Clinical Proteomics, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Pulmonary Circulation

Article Title: High gestational leucine level dampens WDPCP/MAPK signaling to impair the EMT and migration of cardiac microvascular endothelial cells in congenital heart defects

doi: 10.1002/pul2.70013

Figure Lengend Snippet: High‐leucine diet in pregnant female mice triggers CHD‐like features in the neonatal mice. Pregnant female mice ( n = 5 in each group) were fed the standard diet or a high‐leucine diet. (a) The plasma leucine levels were determined in the neonatal mice (2 weeks old) from the normal diet (control) and the high‐leucine diet (model) groups. (b) 320‐slice spiral CT analysis of epicardial adipose tissue volume (EATV) in neonatal mice from the control and model groups. (c) Analysis of WDPCP and EMCN protein expression in neonatal plasma samples of the control and model groups. (d) Detection of phospho‐p38, and (e) phospho‐ERK levels in plasma samples of neonatal mice from the the control and model groups. (f) Plasma VEGF‐A and ANG2 levels were analyzed by ELISA in the control and model groups. (g) Hematoxylin & Eosin staining in the cardiac tissues of neonatal mice from the control and model groups. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Phospho‐p38 MAPK and phospho‐ERK levels were measured using the Human/Mouse/Rat Phospho‐ERK1/2, JNK, p38 MAPK Cell‐Based ELISA kit (#CBEL‐ERK‐SK, RayBiotech) and the ERK1/ERK2 (Phospho) Multispecies InstantOneTM ELISA Kit (#85‐86013‐11, ThermoFisher Scientific.

Techniques: Clinical Proteomics, Control, Expressing, Enzyme-linked Immunosorbent Assay, Staining

Journal: Pulmonary Circulation

Article Title: High gestational leucine level dampens WDPCP/MAPK signaling to impair the EMT and migration of cardiac microvascular endothelial cells in congenital heart defects

doi: 10.1002/pul2.70013

Figure Lengend Snippet: WDPCP overexpression and MAPK activation ameliorate high‐leucine‐induced CHDs. Pregnant female mice ( n = 5 in each group) were fed the standard diet or a high‐leucine diet. Neonatal mice from the high‐leucine diet group was administered an AAV‐carrying WDPCP expression construct or a MAPK signaling pathway activator (C16‐PAF) for 2 weeks. (a) Western blot analysis of WDPCP and EMCN protein levels in plasma samples of the neonatal mice in each group. (b) 320‐slice spiral CT analysis of epicardial adipose tissue volume (EATV) in the neonatal mice of each group. (c) Phospho‐p38 and (d). phospho‐ERK levels were examined in plasma samples of the neonatal mice by ELISA. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. AAV, adeno‐associated virus.

Article Snippet: Phospho‐p38 MAPK and phospho‐ERK levels were measured using the Human/Mouse/Rat Phospho‐ERK1/2, JNK, p38 MAPK Cell‐Based ELISA kit (#CBEL‐ERK‐SK, RayBiotech) and the ERK1/ERK2 (Phospho) Multispecies InstantOneTM ELISA Kit (#85‐86013‐11, ThermoFisher Scientific.

Techniques: Over Expression, Activation Assay, Expressing, Construct, Western Blot, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Virus

Journal: Pulmonary Circulation

Article Title: High gestational leucine level dampens WDPCP/MAPK signaling to impair the EMT and migration of cardiac microvascular endothelial cells in congenital heart defects

doi: 10.1002/pul2.70013

Figure Lengend Snippet: High‐leucine levels regulate WDPCP/EMCN expression and the MAPK/ERK signaling, and impairs the EMT in HCMECs. HCMECs were cultured in standard medium (control, 0.8 mM) or high‐leucine medium (4 mM) for 48 h. (a) Western blot analysis of WDPCP and EMCN levels in the control and high‐leucine conditions. (b) Phospho‐p38 and (c). phospho‐ERK levels were examined in HCMECs cultured under the control and high‐leucine conditions. (d) Wound healing assay and (e) transwell invasion assay in HCMECs cultured under the control and high‐leucine conditions. (f) Western blot analysis of mesenchymal markers (vimentin and N‐cadherin) and an epithelial marker (E‐cadherin) in HCMECs cultured under the control and high‐leucine conditions. (g) CCK‐8 cell proliferation assay of HCMECs under the control and high‐leucine culturing conditions. Data are the summary of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Phospho‐p38 MAPK and phospho‐ERK levels were measured using the Human/Mouse/Rat Phospho‐ERK1/2, JNK, p38 MAPK Cell‐Based ELISA kit (#CBEL‐ERK‐SK, RayBiotech) and the ERK1/ERK2 (Phospho) Multispecies InstantOneTM ELISA Kit (#85‐86013‐11, ThermoFisher Scientific.

Techniques: Expressing, Cell Culture, Control, Western Blot, Wound Healing Assay, Transwell Invasion Assay, Marker, CCK-8 Assay, Proliferation Assay

Journal: Pulmonary Circulation

Article Title: High gestational leucine level dampens WDPCP/MAPK signaling to impair the EMT and migration of cardiac microvascular endothelial cells in congenital heart defects

doi: 10.1002/pul2.70013

Figure Lengend Snippet: WDPCP and MAPK signaling mediates the suppressive effect of high‐leucine levels on the EMT and cell migration in HCMECs. HCMECs transfected with a WDPCP expression vector or treated with the MAPK activator were cultured in standard medium (control) or high‐leucine medium for 48 h. (a) Western blot analysis of WDPCP and EMCN levels in each experimental group. (b) Phospho‐p38 and (c) phospho‐ERK levels were examined in HCMECs of indicated experimental groups. (d) Wound healing assay and (e) transwell invasion assay in HCMECs of indicated experimental groups. (f) Western blot analysis of mesenchymal markers (vimentin and N‐cadherin) and an epithelial marker (E‐cadherin) in HCMECs of indicated experimental groups. Data are the summary of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Phospho‐p38 MAPK and phospho‐ERK levels were measured using the Human/Mouse/Rat Phospho‐ERK1/2, JNK, p38 MAPK Cell‐Based ELISA kit (#CBEL‐ERK‐SK, RayBiotech) and the ERK1/ERK2 (Phospho) Multispecies InstantOneTM ELISA Kit (#85‐86013‐11, ThermoFisher Scientific.

Techniques: Migration, Transfection, Expressing, Plasmid Preparation, Cell Culture, Control, Western Blot, Wound Healing Assay, Transwell Invasion Assay, Marker

Journal: Pulmonary Circulation

Article Title: High gestational leucine level dampens WDPCP/MAPK signaling to impair the EMT and migration of cardiac microvascular endothelial cells in congenital heart defects

doi: 10.1002/pul2.70013

Figure Lengend Snippet: EMCN is a leucine‐induced effector negativity regulating the EMT and migratory ability of HCMECs. HCMECs transfected with the control siRNA (si‐NC) or an siRNA targeting EMCN (si‐EMCN) were cultured in a standard medium (control) or high‐leucine medium for 48 h. (a) Western blot analysis of WDPCP and EMCN levels in each experimental group. (b) Phospho‐p38 and (c) phospho‐ERK levels were examined in HCMECs of indicated experimental groups. (d) Wound healing assay and (e) transwell invasion assay in HCMECs of indicated experimental groups. (f) Western blot analysis of mesenchymal markers (vimentin and N‐cadherin) and epithelial marker (E‐cadherin) in HCMECs of indicated experimental groups. Data are the summary of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Phospho‐p38 MAPK and phospho‐ERK levels were measured using the Human/Mouse/Rat Phospho‐ERK1/2, JNK, p38 MAPK Cell‐Based ELISA kit (#CBEL‐ERK‐SK, RayBiotech) and the ERK1/ERK2 (Phospho) Multispecies InstantOneTM ELISA Kit (#85‐86013‐11, ThermoFisher Scientific.

Techniques: Transfection, Control, Cell Culture, Western Blot, Wound Healing Assay, Transwell Invasion Assay, Marker

Journal: Cell Reports

Article Title: Restoration of visual function in advanced disease after transplantation of purified human pluripotent stem cell-derived cone photoreceptors

doi: 10.1016/j.celrep.2021.109022

Figure Lengend Snippet:

Article Snippet: Plasmid: L-MYC and LIN28 episonal insert (pCXLE-hUL) , , Addgene plasmid #27080.

Techniques: Virus, Recombinant, Plasmid Preparation, Software

Journal: Nucleic Acids Research

Article Title: Combining TSS-MPRA and sensitive TSS profile dissimilarity scoring to study the sequence determinants of transcription initiation

doi: 10.1093/nar/gkad562

Figure Lengend Snippet: TSS-MPRA of synthetic regulatory sequences mirrors the vast majority of endogenous initiation patterns and transcription levels. ( A ) Schematic of TSS-MPRA. Transcription activity and location of transcription initiation is determined by 5′ RNA-seq of reporter transcripts initiating within synthetic DNA inserts cloned into reporter plasmids and electroporated into cells. Histograms on the right show cumulative DNA-normalized number of sequence tags aligning to each base position of a 153-bp region surrounding the human HBE1 promoter in K562 cells. Capped reporter transcripts are drawn in purple. RT: reverse transcription primer landing site. BC: barcode sequence. ( B ) Spearman's correlation of DNA-normalized RNA levels of all inserts of two replicate episomal TSS-MPRA experiments. ( C ) Correlation between the transcriptional signal of 250 genomic DNA inserts in epi-short TSS-MPRA and csRNA-seq of the corresponding endogenous loci. TSS-MPRA inserts were chosen randomly from locations exhibiting transcription activity as measured by csRNA-seq. Spearman's correlation of TSS-MPRA and csRNA-seq transcription levels of all regulatory sequences, or of promoters (red) or enhancers (blue, outside of a ± 2-kb window of RefSeq-annotated promoters). Regions were chosen to cover a wide range of transcription levels and initiation patterns. Each dot represents the relative transcript levels observed in each assay as total normalized read counts of all transcripts aligning to each region. TSS-MPRA RNA read counts were normalized by the corresponding plasmid DNA read counts.

Article Snippet: The episomal TSS-MPRA plasmid is based on the background-reduced pGL4.10 luciferase reporter plasmid (Promega).

Techniques: Activity Assay, RNA Sequencing Assay, Clone Assay, Sequencing, Reverse Transcription, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Combining TSS-MPRA and sensitive TSS profile dissimilarity scoring to study the sequence determinants of transcription initiation

doi: 10.1093/nar/gkad562

Figure Lengend Snippet: TSS-MPRA fidelity correlates with genomic TSS pattern width, transcription level, and presence of core promoter elements. ( A ) Schematic of the outlier detection model used to determine whether two TSS distributions are similar or not. Higher WIP scores (see methods for derivation) are indicative of more dissimilar initiation patterns. ( B ) TSS-MPRA preferentially recapitulates focused initiation patterns. Focus ratios (y-axis) of TSS-MPRA initiation patterns that are similar (blue) or dissimilar (red) to the corresponding endogenous initiation patterns as measured by csRNA-seq. Focus ratios of 0 indicate fully dispersed (broad) initiation patterns, while 1 indicates fully focused (sharp) patterns. ( C ) TSS-MPRA better recapitulates initiation patterns of more actively transcribed genomic regions. Endogenous locus transcription levels (csRNA-seq tag counts, y-axis) where TSS-MPRA initiation shape is similar (blue) or dissimilar (red) to the corresponding endogenous initiation pattern. ( D ) Strong transcription in the TSS-MPRA correlates with presence of TATA and Inr core promoter elements. Position-specific nucleotide frequencies (y-axis) (A: blue, C: purple, G: red, and T: orange) relative to each TSS in all TSS-MPRA inserts where: overall TSS-MPRA shapes of the inserts mirror the endogenous initiation patterns (I), or where insert TSS shapes do not mirror endogenous initiation patterns and either the respective TSSs within the overall TSS shape have 3x higher contribution to the overall signal of a given insert in TSS-MPRA data than in csRNA-seq (II), or the respective TSSs have 3× higher contribution to the overall signal of a given insert in csRNA-seq over TSS-MPRA data (III). The x-axis denotes the distance in bp from each TSS (bp 0).

Article Snippet: The episomal TSS-MPRA plasmid is based on the background-reduced pGL4.10 luciferase reporter plasmid (Promega).

Techniques:

Journal: Nucleic Acids Research

Article Title: Combining TSS-MPRA and sensitive TSS profile dissimilarity scoring to study the sequence determinants of transcription initiation

doi: 10.1093/nar/gkad562

Figure Lengend Snippet: Longer inserts initiate transcription at additional non-endogenous TSSs and decrease overall TSS-MPRA transcription initiation fidelity. ( A ) Non-native TSS use in epi-long TSS-MPRA. Frequency of TSS usage between csRNA-seq (top), short TSS-MPRA (middle), and long TSS-MPRA (bottom). The y-axis, TSS usage frequency, is defined as the oligo position-specific cumulative normalized initiation frequencies in TSS-MPRA and csRNA-seq across all native TSS-MPRA inserts and corresponding genomic regions. At the top a schematic representation of the insert-containing oligos: overhang cloning sequence, followed by genomic DNA insert, 11-mer barcode and second overhang cloning sequence. The box and dots in blue marked ‘CORE’ are the positions covered by the 153-bp insert of the epi-short pool. ( B ) Increased insert length increases enhancer but not promoter transcription correlation between TSS-MPRA and csRNA-seq. Spearman's correlation of epi-long TSS-MPRA and csRNA-seq levels between all (purple) 250 regulatory sequences selected to cover a wide range of transcription levels and initiation patterns, or of only enhancers (blue), or only promoters (red). ( C ) Increased correlation of CORE (153-bp region) initiation frequencies within longer inserts of enhancers but not promoters. This analysis is restricted to the 153-bp region marked ‘CORE’ in (A). Color scheme as in (B).

Article Snippet: The episomal TSS-MPRA plasmid is based on the background-reduced pGL4.10 luciferase reporter plasmid (Promega).

Techniques: Clone Assay, Sequencing

Journal: Nucleic Acids Research

Article Title: Combining TSS-MPRA and sensitive TSS profile dissimilarity scoring to study the sequence determinants of transcription initiation

doi: 10.1093/nar/gkad562

Figure Lengend Snippet: Reporter chromatinization has negligible effect on transcription initiation patterns and lowers correlation between TSS-MPRA and csRNA-seq transcription levels. ( A ) Schematic of genomic integration of a synthetic insert using lentiviral integration. ( B ) High reproducibility of Lenti-TSS-MPRA. Spearman's correlation of RNA/DNA normalized levels between all the inserts of two replicate lentiviral TSS-MPRA experiments. ( C ) High reproducibility in TSS profile changes between episomal and lentiviral TSS-MPRA experiments. WIP scores between csRNA-seq and lentiviral TSS-MPRA on the y-axis are highly correlated with the WIP scores between csRNA-seq and episomal TSS-MPRA on the x-axis. Dots are colored by how dissimilar insert TSS profiles are between lentiviral and episomal TSS-MPRA experiments. Higher WIP scores represent greater dissimilarity. ( D ) Spearman's correlation of lenti-short Lenti-TSS-MPRA and csRNA-seq levels between all 250 randomly selected regulatory sequences covering a wide range of transcription levels and initiation patterns (purple), only enhancers (blue), or only promoters (red). ( E ) Spearman's correlation of lenti-long Lenti-TSS-MPRA and csRNA-seq levels between all 250 randomly selected regulatory sequences covering a wide range of transcription levels and initiation patterns (purple), only enhancers (blue) or only promoters (red). ( F ) Spearman's correlation of lenti-long Lenti-TSS-MPRA and csRNA-seq levels between all 250 randomly selected regulatory sequences covering a wide range of transcription levels and initiation patterns (purple), only enhancers (blue), or only promoters (red). This analysis is restricted to the 153-bp region marked ‘CORE’ in Figure .

Article Snippet: The episomal TSS-MPRA plasmid is based on the background-reduced pGL4.10 luciferase reporter plasmid (Promega).

Techniques:

Journal: Nucleic Acids Research

Article Title: Combining TSS-MPRA and sensitive TSS profile dissimilarity scoring to study the sequence determinants of transcription initiation

doi: 10.1093/nar/gkad562

Figure Lengend Snippet: TSS-MPRA enables studying the effect of motif mutations on reporter-driven initiation patterns and transcription levels. ( A ) Tracking transcription initiation changes caused by mutations in transcription factor and core promoter element motifs in episomal plasmids. Scatterplot comparing the changes in initiation patterns (WIP score, y-axis) and transcription levels (fold change, x-axis) between control and mutated inserts in episomal plasmids. Red dots signify inserts with significantly changed TSS shapes after mutation. ( B ) TSS shape changes after motif mutation in episomal constructs. The y-axis represents the mean WIP score between all inserts (and their barcode replicates) containing a particular motif and the corresponding insert with the mutated motif. Colors correspond to motif identities. ( C ) Transcription level changes associated with motif mutation in episomal constructs. The y-axis represents the mean fold transcription change of the inserts (and their barcode replicates) containing a particular wild-type or mutated motif (x-axis). ( D ) Tracking transcription initiation changes caused by transcription factor and core promoter element motif mutations after lentiviral integration into the genome. Scatterplot comparing the changes in initiation patterns (WIP score, y-axis) and transcription levels (fold change, x-axis) between control and mutated inserts in lentiviral plasmids. Red dots signify inserts with significantly changed TSS shapes after mutation. ( E ) TSS shape changes after motif mutation in lentiviral constructs. The y-axis represents the mean WIP score between all inserts (and their barcode replicates) containing a particular motif and the corresponding insert with the mutated motif. Colors correspond to motif identities. ( F ) Transcription level changes associated with motif mutation in lentiviral constructs. The y-axis represents the mean fold transcription change of the inserts (and their barcode replicates) containing a particular wild-type or mutated motif (x-axis). ( G ) Example track for the ACTB promoter, showing csRNA-seq (top), TSS-MPRA, and Lenti-TSS-MPRA output without (black) and with (red) TATA-box motif mutation. Blue highlights indicate the positions where motifs were replaced by a constant sequence with no known transcription factor motif. ( H ) Example track for the ENO1 promoter, showing csRNA-seq (top), TSS-MPRA, and Lenti-TSS-MPRA output before (black) and after (red) PU.1 motif mutation. Blue highlights indicate the positions where motifs were replaced.

Article Snippet: The episomal TSS-MPRA plasmid is based on the background-reduced pGL4.10 luciferase reporter plasmid (Promega).

Techniques: Control, Mutagenesis, Construct, Sequencing

Journal: Nucleic Acids Research

Article Title: Combining TSS-MPRA and sensitive TSS profile dissimilarity scoring to study the sequence determinants of transcription initiation

doi: 10.1093/nar/gkad562

Figure Lengend Snippet: Assessing the effects of single nucleotide polymorphisms on reporter-driven initiation patterns and transcription levels. ( A ) Allele-specific transcription initiation differences caused by known GWAS SNPs in episomal plasmids. Scatterplot comparing the changes in initiation patterns (y-axis) and transcription fold change (x-axis) between control and variant inserts in episomal constructs. Red dots signify inserts that had significant changes to their TSS shapes. ( B ) Allele-specific transcription initiation differences caused by known GWAS SNPs in lentiviral constructs. Scatterplot comparing the changes in initiation patterns (y-axis) and transcription fold change (x-axis) between control and variant inserts in lentivirally integrated constructs. Red dots signify inserts that had significant changes to their TSS shapes. ( C ) SNP rs1991401 is associated with TSS shape changes. Track showing csRNA-seq (top), TSS-MPRA, and Lenti-TSS-MPRA output of the T (black) and G (red) allele. A blue ‘^’ symbol indicates the location of the SNP. ( D ) TSS shape differences associated with SNP rs131804. Track showing csRNA-seq (top), TSS-MPRA, and Lenti-TSS-MPRA output of the C (black) and A (red) allele. A blue ‘^’ symbol marks the SNP location.

Article Snippet: The episomal TSS-MPRA plasmid is based on the background-reduced pGL4.10 luciferase reporter plasmid (Promega).

Techniques: Control, Variant Assay, Construct