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. 2014 Sep 30;42(19):11952–11964. doi: 10.1093/nar/gku874

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© The Author(s) 2014. 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/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 2. — Effect of PTCAF-1 knockdown on cell survival, IES and transposon retention. (A) Effect of PTCAF-1 knockdown on cell survival. After going through autogamy in silencing medium the cells where recovered in WGP medium. The graph shows the survival after three days. Black: percentage of dead cells; grey: percentage of sick cells showing an altered number of divisions or behavior; white: percentage of cell growing at a normal rate. NOWA1 KD was used as a positive control. Silencing vector without insert was used as negative control. (B) Effect of PTCAF-1 knockdown on IES excision. IES retention was tested by PCR using primers flanking specific IESs sequences within the 51G and 51A genes as well as the mating-type locus (MT Locus). The excised form (IES−) is always detectable due to its presence in the fragments of the parental MAC. The unexcised form (IES+) is only detectable in case of retention in the newly developing macronuclei. Maternally controlled IESs: 51A712, 51A2591, 51A6649, 51G11, 51G2832, 51G4404, MT-Locus; non-maternally controlled IESs: 51A1835, 51A4404, 51A4578, 51A6435, 51G1413, 51G1832, 51G6447. (C) Effect of PTCAF-1 KD on Thon and Sardine transposons. Total DNA was extracted from vegetative (V), control and PTCAF-1 KD cultures at specific time points during sexual reproduction. E, M, L as in Figure 1C; 5, 15, 25 and 45 h after L. After running the DNA on a 1% agarose-gel and transfer to nylon membrane the samples were hybridized with Sardine and Thon transposon-specific probes. A mitochondrial DNA-specific probe was used as loading control. The graph shows the Thon signal quantification normalized to the loading control.