Figure 2: Manipulation of SNHG5 expression regulates proliferation and survival both in vitro and in vivo.

(a) Proliferation assay. CRC cell lines were transfected with 25 nM siSNHG5-1-2 or siControl. Cell numbers were assessed at the designated time points post transfection. Data normalized to the first time point are shown for the mean of 3 independent experiments. Error bars indicate±s.d. (*P value<0.05, **P value<0.01, Student’s t-test paired). (b) HCT116, CACO-2 and DLD-1 cells were transfected as in a, pulsed with EdU after 36 h and analysed by flow cytometry to measure the number of EdU-labelled cells representing the S-phase populations. Data represents the mean of three independent experiments. Error bars indicate±s.e. (*P value<0.05, **P value<0.01). (c) Top panel: CRC cell lines were transfected as in (a). Thirty-six hours after transfection, cells were labelled with cleaved Caspase-3 antibody and analysed by flow cytometry. Data are shown for the mean of three independent experiments. Error bars indicate±s.e. (*P value<0.05, **P value<0.01). Bottom panel: western blot analysis of endogenous cleaved PARP-1 levels in HCT116, CACO-2 and DLD-1 cells transfected with siSNHG5-1-2 or siControl. GAPDH is included as loading control. A representative experiment is shown (n=3). (d) Schematic representation of constructs expressing the entire SNHG5 genomic (pSNHG5 locus) or only the cDNA (pSNHG5 cDNA). (e) Left, qRT–PCR. SNHG5 expression levels were measured 48 h after transfection into HT-29 cells. Data was normalized to the RPLP0 housekeeping gene and plotted relative to the empty vector control. Error bars indicate the mean±s.e. of three independent experiments. Right, HT-29 cells were transfected with plasmids as described above. Forty-eight hours after transfection the cells were treated with 25 μg ml⁻¹ of oxaliplatin. Twenty-four hours later the cells were stained with cleaved Caspase-3 antibody and analysed by flow cytometry. Error bars indicate the mean±s.e. of three independent experiments. (*P value<0.05, **P value<0.01, ***P value<0.01). (f) Schematic representation of the SNHG5 cDNA expressing mutant plasmids. (g) Overall, 2 × 10⁵ HT-29 cells were transfected with mutant plasmids and treated with oxaliplatin as described above. Twenty-four hours later the cells were stained with cleaved Caspase-3 antibody and analysed by flow cytometry. Error bars indicate the mean±s.e. of three independent experiments. (*P value<0.05, **P value<0.01). (h) Xenograft growth of HCT116 pFULT-shGFP and HCT116 pFULT-shNHG5-4 cells. Doxycycline was added to the drinking water after 13 days. Mean±s.d. of tumor luminescence (photons/seconds/cm2) at the indicated time points (for statistic analysis details see ‘Methods’ section). (i,j) Final volume and weight of tumours from the experiment at day 35, respectively.