Ab203345

To test the interaction between the lncRNAs and either AKT or WDR26, 1 × 10⁷ CA1D were transfected with siRNAs for WDR26 or with the non-targeting siRNA as a negative control. In all, 48 h after the transfection, the cells were incubated overnight with 100 µM 4-thiouridine to allow for the UV-driven crosslinking between RNAs and interacting proteins. The cells were then lysed in 20 mM Tris–HCl at pH 7.5, 100 mM KCl, 5 mM MgCl2, and 0.5% NP-40^{50 (link),51 (link)} and the proteins of interest were immunoprecipitated using 1 µg of each of the following primary antibodies: AKT (9272 S, Cell Signaling), WDR26 (ab203345, Abcam), and normal rabbit IgG (sc-2027, Santa Cruz) as negative control. The presence of the lncRNAs in the CLIP-ed samples was assessed via qRT-PCR using the Taqman probes listed in Supplementary Table 1. To map the regions of TROLL-2 and TROLL-3 interacting with WDR26, 1 × 10⁷ CA1D were utilized. The CLIP assay was performed as above and including an RNAse T1 treatment step (1 U/µL at 22 C for 10 min)^{50 (link),51 (link)} and using 1 µg of each of the following primary antibodies: WDR26 (ab203345, Abcam) and normal rabbit IgG (sc-2027, Santa Cruz) as negative control. The presence of the lncRNA fragments in the CLIP-ed samples was assessed via qRT-PCR using the primers listed in Supplementary Table 5.

In all, 1 × 10⁷ CA1D were transfected with siRNAs for TROLL-2, TROLL-3, or with the non-targeting siRNA as a negative control. To test the interaction between WDR26 and AKT, 24 h after the transfection the cells were serum-starved for 24 h and subsequently treated for 10 min with 10 µM lysophosphatidic acid (LPA). To test the interaction between WDR26 and NOLC1, the cells were kept in their growth media for 48 h. The cells were then lysed and the CoIP assay was performed in 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP-40^{27 (link)}, and 1 µg of each of following primary antibodies was utilized per sample: AKT (9272 S, Cell Signaling), NOLC1 (ab184550, Abcam), WDR26 (ab203345, Abcam), and normal rabbit IgG (sc-2027, Santa Cruz) as negative control. The interaction was then detected via western blot using the following primary antibodies: pAKT (S473) (4060 S, Cell Signaling, 1:100), AKT (ab8805, Abcam, 1:1000), and WDR26 (ab85961, Abcam, 1:2000).

CA1D-derived xenograft tumours, TMAs of breast cancer progression (BR480a, US Biomax), colon cancer progression (CO961, US Biomax), lung cancer progression (BC04002a, US Biomax), ovarian cancer progression (OV1005b, US Biomax), two TMAs of melanoma progression (ME1004f, US Biomax; and the Moffitt TMA-4, Moffitt Cancer Center), and three TMAs of invasive breast cancers (BR20837a, US Biomax; the Dundee TMA^{27 (link)}, Tayside Tissue Bank; and the Moffitt TMA-5, Moffitt Cancer Center) were used for immunohistochemistry (IHC). IHC was performed overnight keeping the slides in humified chambers^{61 (link)}, and incubating them with the following primary antibodies: NCOA5 (ab70831, Abcam, 1:200), WDR26 (ab203345, Abcam, 1:200), TAp63 (618902, Biolegend, 1:200) and pAKT (S473) (4060S, Cell Signaling, 1:100). In the case of the TMAs, the signal intensity (continuous variable, 0 to 1) and the proportion of positive tissue (continuous variable, 0–100%) were measured using the Oncotopix® software (Visiopharm). The IHC score was then quantified by multiplying the signal intensity by the proportion of positive tissue, giving a value comprised between 0 and 100, and visualized using the Circos software^{69 (link)}.