nih3t3  (ATCC)

Journal: Aging and Disease

Article Title: The Flip Side of the Coin: METTL3 Serves as a Novel Cellular Senescence Accelerator via Negative Regulation of ITGA9

doi: 10.14336/AD.2024.1715

Figure Lengend Snippet: M6A modifications within total mRNA were significantly enhanced during the aging process . ( A ) Left: representative photographs of 2-month and 20-month-old male C57BL/6 mouse; Right: representative photographs of SA-β-gal staining in kidney tissues from young and old C57-BL/6 mice. ( B ) Left: representative photographs of SA-β-gal staining of P3 and P9 MEF cells; Right: the p16 expression levels in P3 and P9 MEF cells. ( C ) Left: representative photographs of SA-β-gal staining of H 2 O 2 -induced premature senescence of NIH/3T3 cells; Right: the p16 expression levels in H 2 O 2 treated NIH/3T3 cells. ( D ) Poly(A)+ RNA was extracted from the brain and liver tissue of young and old C57-BL/6 mice and subjected to RNA dot-blot analysis with an antibody recognizing m6A. Methylene blue staining served as the loading control. ( E ) Poly(A)+ RNA was extracted from the P3 and P9 MEF cells and subjected to RNA dot-blot analysis with an antibody recognizing m6A. Methylene blue staining served as the loading control. ( F ) Poly(A)+ RNA was extracted from the P3 and P9 MEF cells and subjected to RNA dot-blot analysis with an antibody recognizing m6A. Methylene blue staining served as the loading control. ( G ) Expression levels of m6A modification associated proteins were measured in P3 and P9 MEF cells, and the quantitative results were shown on the right. Mean (±SEM), n=4, passed normality test: Shapiro-Wilk test; One sample t test (MEF P9 vs 1, METTL3: p =0.0027, t = 9.176).

Article Snippet: NIH/3T3 fibroblasts were procured from the American Type Culture Collection (ATCC).

Techniques: Staining, Expressing, Dot Blot, Control, Modification

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