Journal: Cell reports

Article Title: ZFP36-mediated mRNA decay regulates metabolism

doi: 10.1016/j.celrep.2023.112411

Figure Lengend Snippet: RT-PCR primers

Article Snippet: PCMV6-Kan/Neo-Mouse Eno2 , Origene , Cat# MC201508.

Techniques:

Journal: Cell reports

Article Title: ZFP36-mediated mRNA decay regulates metabolism

doi: 10.1016/j.celrep.2023.112411

Figure Lengend Snippet: Site-directed mutagenesis PCR primers

Article Snippet: PCMV6-Kan/Neo-Mouse Eno2 , Origene , Cat# MC201508.

Techniques: Mutagenesis

Journal: Cell reports

Article Title: ZFP36-mediated mRNA decay regulates metabolism

doi: 10.1016/j.celrep.2023.112411

Figure Lengend Snippet: (A) RNA-seq volcano plot of adeno-GFP-treated (TFWT) vs. adeno-Cre-treated (TFKO) Zfp36/l1/l2 triple floxed MEFs stimulated for 1 h with 10% FBS following overnight serum deprivation. Dashed lines indicate adjusted p ≤0.01 or log 2 (fold change) ≥1 or ≤−1. Red colored dots represent significantly differentially expressed mRNAs also bound by ZFP36 in eCLIP-seq experiment. (B) Top 25 upregulated transcripts in ZFP36/L1/L2 TKO (TFKO) MEFs identified by RNA-seq from serum-starved and -stimulated culture conditions that are also bound by ZFP36. Genes are ranked by eCLIP-seq cross-link score. (C) Integrative Genomics Viewer generated from eCLIP-seq experiments showing the ZFP36 binding site on Eno2 mRNA within 3′ UTR. (D) Clustal Omega Multiple Sequence Alignment of the ZFP36 target binding sequence within human and mouse Eno2 3′ UTRs. AREs are annotated in overlapping gray bars, identities are indicated by asterisks (*), and predominant ZFP36 cross-link sites within mouse Eno2 mRNA are colored red. (E) Luciferase activity in HeLa cells cotransfected with either full-length Eno2 3′ UTR luciferase reporter or one in which the AREs have been deleted (Δ ARE), CMV-driven WT ZFP36 or ZFP36 C124R mutant deficient in RNA binding, and Renilla luciferase (n = 6). Data are normalized to Renilla luciferase and presented relative to full-length Eno2 3′ UTR in the presence of WT ZFP36. (F and G) Relative transcript (F) or protein (G) levels of indicated genes compared between Zfp36/l1/l2 triple-floxed WT MEFs (TFWT) and clones derived from TFWT MEFs treated with adeno-GFP (TFWT.1,2) or adeno-Cre (TFKO.1–3) and single cell expanded. (H and I) Immunoblot (H) or qRT-PCR (I) of Eno2 or Tuba1b in a ZFP36/L1/L2 TKO MEF clone (TFKO.1) transduced with CMV-driven empty vector (EV) control, human zinc finger mutant ZFP36 (C124R), or WT human ZFP36 (ZFP36). (J) Transcript stability of Eno2, Tuba1b, or Fgf21 measured by qRT-PCR in ZFP36/L1/L2 WT (TFWT) or TKO (TFKO) MEF cell clones. Cells were deprived of serum for 24 h and then stimulated with 10% FBS for 1 h before being harvested (control) or subsequently treated for an additional 4 h with actinomycin D (ActD; 5 mg/mL). Data are presented as fold change of ActD (1-h FBS + 4-h ActD) relative to control (1-h 10% FBS-stimulated conditions) for each respective clone. (K) Model of direct ZFP36-dependent regulation of Eno2 mRNA downstream of growth factor signaling. All experiments were performed with biological replicates. Error bars denote SD (n = 3, unless otherwise indicated). *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: PCMV6-Kan/Neo-Mouse Eno2 , Origene , Cat# MC201508.

Techniques: RNA Sequencing, Generated, Binding Assay, Sequencing, Luciferase, Activity Assay, Mutagenesis, RNA Binding Assay, Clone Assay, Derivative Assay, Western Blot, Quantitative RT-PCR, Transduction, Plasmid Preparation, Control

Journal: Cell reports

Article Title: ZFP36-mediated mRNA decay regulates metabolism

doi: 10.1016/j.celrep.2023.112411

Figure Lengend Snippet: (A) Targeted LC-MS metabolomics measurements comparing ZFP36/L1/L2 WT vs. TKO clones. Data are grouped by genotype, prefiltered (log2 fold change [L2FC] ≥ 0.2 or ≤ −0.2; p < 0.01), and ranked by statistically significant differential metabolite abundance, then visualized as Z scores across rows. (B) Volcano plot comparing glycolytic metabolite levels in ZFP36/L1/L2 triple-floxed KO MEFs (TFKO.1) vs. Zfp36/l1/l2 triple-floxed WT (TFWT) MEFs. (C) Schematic diagramming uniformly labeled [13C] glucose tracing through the enolase enzyme and the relevant isotopologues. (D) LC-MS measurements of relative levels of U-13C 6 -glucose-derived M+3 phosphoenolpyruvate (PEP) over time (0, 0.5, 2, or 5 min) in Zfp36/l1/l2 triple-floxed WT MEFs (TFWT) vs. ZFP36/L1/L2 triple-floxed KO MEFs (TFKO.1) cultured in 10% FBS growth medium. Data are normalized to TFWT cells at 0.5-min time point. All experiments were performed with biological replicates. Error bars denote SD (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: PCMV6-Kan/Neo-Mouse Eno2 , Origene , Cat# MC201508.

Techniques: Liquid Chromatography with Mass Spectroscopy, Clone Assay, Labeling, Derivative Assay, Cell Culture

Journal: Cell reports

Article Title: ZFP36-mediated mRNA decay regulates metabolism

doi: 10.1016/j.celrep.2023.112411

Figure Lengend Snippet: (A) Relative Eno2 expression levels from human fetal single-cell RNA-seq atlas across 15 different tissues. (B) Lack of correlation between Eno2 expression in single cell RNA-seq and Eno2 ATAC-seq data across tissues in human fetuses ranging from 89 to 125 days post-conception. (C) Schematic diagramming tamoxifen (TAM)-inducible endothelial-specific Zfp36 KO in CDH5-Cre Zfp36 fl/fl neonatal mice. (D) Retina immunostaining of Cre(−) or Cre(+) neonates corresponding to endothelial cell-specific ZFP36 WT or KO respectively. Retinas were stained for endothelial-specific CD31 marker or ENO2. (E) Quantification of ENO2 signal comparing Cre(−) and Cre(+) neonatal paired littermates. (F) Model of ZFP36 regulation of ENO2 expression downstream of VEGF signaling during retinal angiogenesis in murine neonates.

Article Snippet: PCMV6-Kan/Neo-Mouse Eno2 , Origene , Cat# MC201508.

Techniques: Expressing, RNA Sequencing, Immunostaining, Staining, Marker

Journal: Cell reports

Article Title: ZFP36-mediated mRNA decay regulates metabolism

doi: 10.1016/j.celrep.2023.112411

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: PCMV6-Kan/Neo-Mouse Eno2 , Origene , Cat# MC201508.

Techniques: Recombinant, Virus, Plasmid Preparation, Transfection, Luciferase, Reverse Transcription, Bicinchoninic Acid Protein Assay, SYBR Green Assay, Sequencing, Mutagenesis, Software, Microscopy

Journal: Nature Communications

Article Title: A tissue-bioengineering strategy for modeling rare human kidney diseases in vivo

doi: 10.1038/s41467-021-26596-y

Figure Lengend Snippet: a Volcano plots showing the distribution of all differentially expressed genes (DEGs) in TSC2 −/− renal organoids compared to TSC2 +/+ (left) and TSC2 +/− (right) renal organoids (FDR < 0.05). Each dot represents a unique gene; red denotes log 2 (fold change) >2, upregulated genes in TSC2 −/− ; blue denotes log 2 (fold change) <-2, downregulated in TSC2 −/− . Selected statistically significant upregulated and downregulated genes (NCBI/Entrez names) are indicated, as determined by a two-sided Chi-Square test. b Principal Component Analysis (PCA) of RNA-Seq data from renal organoids of the three genotypes, n = 3 samples for each genotype, five organoids per sample. c Heatmap showing hierarchical clustering of three different genotypes of kidney organoids using the top 3000 most variable genes. Color scale representative of gene expression level: red denotes log 2 ≤ 3, blue denotes log 2 ≥ -3. d Representative enrichment plots corresponding to gene set enrichment analysis (GSEA) for pairwise comparison of TSC2 −/− vs . TSC2 +/− . e Venn diagrams indicating 187 common differentially expressed genes, including signature AML markers, in TSC2 −/− vs . TSC2 +/+ renal organoids and kidney AML vs . normal kidney. f Comparative mRNA expression levels for AML hallmark genes in TSC2 −/− , TSC2 +/+ , and TSC2 +/− renal organoids ( n = 3 each) compared to human kidney AML ( n = 28) and human kidney ( n = 8). P values for individual comparisons done using a two-sided Mann–Whitney U test are indicated. Gene expression is shown in FPKM values. g Comparative ENO2 mRNA expression levels in TSC2 +/+ and TSC2 +/− , TSC2 −/− renal organoids ( n = 3 each). P values for the indicated individual comparisons done using two-tailed Student’s t test are shown. Gene expression is shown in FPKM values. h , i Box-and-whisker plot showing minimum value, first quartile, median, third quartile and maximum value for ENO2 content ( g ) and for enolase activity ( h ) in whole extracts of TSC2 +/+ and TSC2 −/− renal organoids. P value for the 2-tailed Student’s t test comparing TSC2 −/− versus TSC2 +/+ is shown. n = 4 independent experiments, containing three organoids each.

Article Snippet: Enolase 2 activity was measured in whole organoid extracts using the human ENO2 ELISA kit (Proteintech, #KE00050), following the manufacturer’s instructions.

Techniques: RNA Sequencing, Gene Expression, Comparison, Expressing, MANN-WHITNEY, Two Tailed Test, Whisker Assay, Activity Assay