qRT-PCR of EV-containing RNA expression levels.

The expression of Ascl1, Brn2, Myt1l and PTEN mRNAs and miR-128 in EVs was measured using qRT-PCR following the manufacturer’s recommended protocol. Briefly, total RNAs were obtained using TRIzol reagent according to manufacturer’s instructions (Invitrogen). Reverse transcription of equal amounts of RNA was carried out using first-strand cDNA synthesis kit (Invitrogen) with random hexamers as primers. The expression of genes was measured using the CYBR green PCR Master Mix (ABI). All experiments were performed in triplicate. For measuring full-length PTEN mRNA, the annealing temperature was set at 60°C and extension time was set at 72 seconds. Synthesized PTEN mRNA was used to construct the standard curve. The concentrations used were 0.0001, 0.001, 0.01, 0.1, 1, and 10 ng/μl. The primer sequences used were as follows: Ascl1(mouse), forward: 5’-TGGTGTCTGAACCTAAGCCC-3’, and reverse: 5’-GTCCGAGAACTGACGTTGCT-3’; Myt1l(mouse), forward: 5’-CCTATGAGGACCAGTCTCC-3’, and reverse: 5’-GACATGGCTGTCACTGGAT-3’; Brn2 (mouse), forward: 5’-GACACGCCGACCTCAGAC-3’, and reverse: 5’-GATCCGCCTCTGCTTGAAT-3’; GAPDH (mouse), forward: 5’-GGGAAATTCAACGGCACAGT-3’ and reverse: 5’-AGATGGTGATGGGCTTCCC-3’; PTEN, forward: 5’-CAAGATGATGTTTGAAACTATTCCAATG-3’, and reverse: 5’-CCTTTAGCTGGCAGACCACAA-3’; PTEN (full length), forward: 5’-ATGACAGCCATCATCAAAGAGATC-3’, and reverse: 5’-TCAGACTTTTGTAATTTGTGTATGCTG’; and GAPDH, forward: 5’-GACAGTCAGCCGCATCTTCT-3’, and reverse: 5’-TTAAAAGCAGCCCTGGTGAC-3’.