Journal: Genes & Development
Article Title: Plagl1 and Lrrc58 control mammalian body size by triggering target-directed microRNA degradation of miR-322 and miR-503
doi: 10.1101/gad.353138.125
Figure Lengend Snippet: Validation of novel TDMD triggers. ( A ) Schematic of trigger site deletion (Δts) strategy to validate putative TDMD triggers. ( B , D ) Genomic organization of TDMD trigger transcripts with conservation and predicted miRNA base-pairing architecture of the trigger sites. Nucleotides predicted to base-pair with the miRNA are shown in bold. ( C , E ) qRT-PCR analysis of indicated miRNAs, relative to miR-16-5p, in WT and Δts MEFs. Parental MEFs or two independent Δts clones for each trigger site were infected with lentivirus expressing Cas9 and a nontargeting CRISPR guide (sgNT) or Zswim8- targeting guide (sg Zswim8 ). Values were normalized to expression level in sg Zswim8 for each condition. n = 3 technical replicates per clone, with individual data points plotted (mean ± SD shown). P -values were calculated by one-tailed Student's t -test. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001. ( F ) qRT-PCR analysis of the indicated miRNAs, relative to U6, in NIH3T3 cells at time points after serum starvation and restimulation. NIH3T3 cells expressing dCas9-KRAB were infected with lentivirus encoding a nontargeting guide (sgNT) or guides targeting Plagl1 or Lrrc58 . Parental NIH3T3 cells were infected with lentivirus expressing Cas9 and a nontargeting guide (sgNT) or Zswim8- targeting guide (sg Zswim8 ). Values were normalized to expression at the 0 h time point for each condition. n = 3 technical replicates per condition, with mean ± SD shown. P -values were calculated by one-tailed Student's t -test comparing sg Plagl1 (dCas9-KRAB) or sg Lrrc58 (dCas9-KRAB) with sgNT (dCas9-KRAB) or comparing sg Zswim8 (Cas9) with sgNT (Cas9). (*) P < 0.05.
Article Snippet: HEK293T and NIH3T3 cells were obtained from ATCC, and immortalized MEFs were generated previously by transfecting primary MEFs with a plasmid expressing SV40 large T antigen ( ).
Techniques: Biomarker Discovery, Quantitative RT-PCR, Clone Assay, Infection, Expressing, CRISPR, One-tailed Test
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