FIGURE 1.
α-Amanitin treatment abrogates expression of mRNAs with short half-lives but does not alter steady-state TERRA levels. A, qPCR analysis of c-Myc and SIAH1 mRNA expression in cells expressing a control hairpin (shCtrl) or FEN1-depleted cells (shFEN1), treated with either vehicle or α-amanitin (α-aman). mRNA levels in α-amanitin-treated cells are shown as a fold change relative to the vehicle-treated cells. Fold changes were calculated using the ΔΔCt method; fold changes from two biological replicates were averaged to produce the graph. Error bars indicate standard error of the mean. B, northern dot blot to detect TERRA. RNA was isolated from cells expressing a control hairpin (shCtrl) or FEN1-depleted cells (shFEN1) that were treated with either vehicle or α-amanitin (α-aman). Serial dilutions of RNA were loaded onto a membrane. Samples treated with RNase A to control for genomic DNA contamination were also loaded (+RNase A). A telomere repeat DNA probe was hybridized to the membrane (telomere probe) to detect TERRA; the membrane was stripped and re-probed with a 5S rRNA DNA probe (5S) as a loading control. The membrane was visualized with autoradiography. C, quantification of TERRA in cells treated with α-amanitin. The northern dot blot in B was imaged with a phosphorimager and analyzed by densitometry using Fiji; TERRA levels in α-amanitin-treated cells are shown as a fold change relative to vehicle-treated cells. Two independent experiments were averaged to produce the graph; error bars represent standard error of the mean.