Figure 3: SNHG5 targets mRNAs in the cytoplasm.

(a) Schematic representation of biotinylated antisense DNA probes complementary to the SNHG5 transcript Even (green) and Odd (red) used for the RNA pool-down. (b) qRT–PCR was performed to assess the % of SNHG5 RNA retrieved from HCT116 cell lysates following pull-down with streptavidin beads. Data was normalized to input (1:100) and GAPDH mRNA included as negative control. Error bars indicate±s.d. for the mean of three independent experiments. (c) Venn diagram representing the number of SNHG5 pull-down peaks commonly enriched with both the Even and Odd probe sets. (d) Validation of the RIA-seq results by qRT–PCR. Data for each target transcript is normalized on input (1:100). Error bars indicate±s.d. for the mean of three independent experiments. (e) Left, qRT–PCR. SNHG5 expression levels were measured 48 h after transfection of HCT116 cells. Data was normalized to the GAPDH housekeeping gene and plotted relative to the siRNA control. Error bars indicate the mean±s.e. of three independent experiments. Right, western blot. HCT116 cells 48 h after transfection with the indicated siRNAs. The optical density (OD) of protein bands is indicated relative to corresponding loading control GAPDH and normalized relative the non-targeting siControl. (f) Transcript stability of SNHG5 targets were measured by qRT–PCR in HCT116 cells 36 h after transfection with the indicated siRNAs. The cells were treated with Triptolide at a final concentration of 10 μM for the last 5 h. The mRNA levels are plotted relative to the corresponding expression levels in the untreated cells. Error bars indicate the mean±s.e. of three independent experiments (Student’s t-test paired).