Figure 6. LncRNA-PNUTS promotes tumor initiation/growth and metastasis in vivo.

(a) Bright-field microscopy pictures of in vitro mammosphere/oncosphere formation assay in NMuMG and A549 cells overexpressing empty vector (CTRL), lncRNA-PNUTS (lncRNA) or mutated lncRNA-PNUTS (lncRNAS1-6M). Scale bar: 20µM

(b) Absolute quantification of the sphere numbers obtained in (a). (mean ± s.d., n=5 independent experiments, two-tailed Student t test, ***p<0.001 NS, not significant). Source data are available in Supplementary table 2.

(c) Number of tumor formed upon limiting dilution injection of control and lncRNA-PNUTS overexpressing MDA-468 cells. MDA-MB-468 cells were injected into the mammary fat pads of 6–8 week-old female mice in limiting dilution. TIC, tumor-initiating cells number was determined using ELDA software⁴⁶. Number of mice used for each condition is indicated.

(d) Flow cytometry analysis of CD24/CD44 cell surface expression levels in the epithelial (CD44⁻/CD24⁺ sorted cells) and mesenchymal (CD44⁺/CD24⁻ sorted cells) HMLE subpopulations.

(e) Cell morphology observed by phase-contrast microscopy. Scale bar: 50µM.

(f) RT-PCR analysis of lncRNA-PNUTS expression level in mesenchymal and epithelial sorted HMLE cells.

(g) Flow cytometry analysis of the CD24/CD44 cell surface expression levels in the epithelial (CD44⁻/CD24⁺ sorted cells) HMLE subpopulation expressing empty vector (control) or overexpressing lncRNA-PNUTS.

(h)Tumor weight of primary tumors obtained following mammary fat pad injection of MDA-231-LM2 expressing scrambled control (SCR) or lncRNA-PNUTS targeting shRNA (shRNA) in NOD/SCID mice. (mean ± s.d., n= 4 mice per condition, two-tailed Mann-Whitney test, p=0.08570). Source data are available in Supplementary table 2.

(i) (Left) Histopathological analysis of paraffin-embedded lung serial sections of mice injected in the mammary fat pad with MDA-231-LM2 expressing scrambled or lncRNA-PNUTS targeting shRNA. Haematoxylin and Eosin (H&E) staining and immunostaining of Ki67 protein was performed in serial lung sections to identify macro- and micro-metastases. (Right) Photographs of primary tumors and of a representative lung collected for each condition. Scale bar: 500 µM