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. 2021 Jun 3;22(11):6034. doi: 10.3390/ijms22116034

Figure 1.

Figure 1

Temsirolimus treatment leads to widespread gene expression changes in primary neurons. Primary cortical neurons at days in vitro (DIV) 6 were treated with temsirolimus (10 µM) or vehicle (DMSO) and subjected to Western blot analysis (A) or RNA-Seq (B,C). (A) The cells were lysed at the indicated time points and subjected to immunoblot analysis with pS6 and S6 specific antibodies. Bands were quantified using ChemiDoc software (Image Lab V5. Version 5.2.1.). (B–D) For RNA-Seq analysis, total RNA was extracted from DMSO and temsirolimus-treated neurons from three different biological replicates each and converted into cDNA using a QuantSeq 3′mRNA-Seq Reverse (REV) Library Prep Kit (Lexogen, Vienna, Austria). (B) Heat maps indicating differentially expressed genes (DEGs) between neurons treated with DMSO or temsirolimus for 5 or 24 h. (C) Venn diagram of genes downregulated after 5 or 24 h of temsirolimus treatment. (D) Venn diagram showing the overlap between genes downregulated in neurons after 5 and 24 h of temsirolimus treatment and upregulated in mTOR hyperactive MEFs [1].