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. 2016 Feb 15;44(10):4665–4683. doi: 10.1093/nar/gkw095

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© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 8. — RISC inhibition and microprocessor knockdown rescues NANOG and OCT4 expression likely through loading of NANOG Alu-derived transcripts into AGO2. (A and B) NTERA-wt cells were left untreated (UT), treated with 25 μM ATA (aurintricarboxylic acid) or with 1 μM RA + 25 μM ATA and the expression of NANOG (A) and OCT4 (B) determined as above. (C and D) NTERA-wt cells were left untreated (UT), treated with 25 μM ATA or with 1 μM RA + 25 μM ATA and the expression of NANOG Alu-T1 (C) and Alu-T2 (D) determined by RT-qPCR. The oligonucleotides used are indicated in Supplementary Table S1. (E and F) The NANOG Alu was transfected in NTERA-wt cells with or without dominant-negative forms of DROSHA or DGCR8 and their effects on NANOG (E) and OCT4 (F) expression analyzed by RT-qPCR. (G) RNA was extracted from NTERA-wt cells and RNA immunoprecipitations (RIP) for NANOG Alu-T1 and Alu-T2 and OCT4 Alu-T transcripts performed using an anti-AGO2 antibody. RIP was quantified by qPCR using the oligonucleotides indicated in Supplementary Table S1. GAPDH mRNA was used to normalize gene expression (ΔCt) and 2^−ΔΔCt to calculate variations with respect to control or untreated conditions. Panels A and B: n = 6, two technical replicates in three biological replicates; panels C and D: n = 6, two technical replicates in three biological replicates; panels E and F: n = 4, two technical replicates in two biological replicates; panel G: n = 4, two technical replicates in two biological replicates. *P < 0.05, **P < 0.01 and ***P < 0.001. Data are shown as mean ± SD.