Figure 1
Altering REST-003 ncRNA expression in si-REST-treated MCF-7 and REST-overexpressed MDA-MB-231 cells.
REST expression was calculated as the number of reads normalized to the median number of reads for all genes in each sample. (A) Expression of REST in siRNA-treated MCF-7 and REST-overexpressed MDA-MB-231 cells. (B) Relative expression of each REST splice transcript was normalized to total REST expression and transcript length in siRNA-treated MCF-7 and REST-overexpressed MDA-MB-231 cells. Some exons in the gene model are shared between multiple transcripts (Fig. S2A). For these, relative expression was calculated as the percentage of REST-aligned reads assigned to a specific transcript/transcript-group normalized by the mappable length of the transcript or transcript-group. (C) Schematic diagram of the REST gene and its splice variant transcripts, including illustrations of annotated REST exons and locations of primers employed for the identification of REST splice variants. The constitutive transcript (REST-001) and alternative spliced variants are shown in red, REST-002 in green, REST-003 in yellow and REST-004 in blue. REST-001 and/or REST-002 can produce the REST protein (wt-REST) that has the complete coding region containing 9 zinc fingers with DNA binding activity (purple) and two repressor domains (pink) necessary for recognizing the RE1 elements and exhibiting repressor function. Forward and reverse primers are indicated by right (numbers) and left (letters) arrows, respectively. (D) (E) Detection (D) and expression levels (E) of ncRNA REST-003 (3-B primer pair) and coding REST RNA (R-N or R-M primer pair) in MCF-7 and MDA-MB-231 cells by qRT-PCR. (D) Following PCR amplification, samples were loaded on 4% agarose gel with a 100-bp marker. (E) Expression levels were normalized to GAPDH, CyclophilinA, and/or Actin, converted to MNE and presented as Relative Expression (Rel. Exp.) after normalization to control samples. Biological replicates are shown on the bar graph as mean plus SEM (paired t-test).
