Fig 2: The TGFß/E1KD/Platr18 axis controls axonogenesis in vitro.(A) Schematic of the PC12 axonogenesis assay in vitro (top panel). PC12 cells derived from pheochromocytoma of the rat adrenal medulla are exposed to culture supernatants of tumor cells (lower panels). Micrographs of PC12 cells complemented with cultured media from NMuMG cells (NMuMG), overexpressing Platr18 (-Platr18) silenced for Sema4F expression (Sema4F-KD), silenced for hnRNP E1 (-E1KD), silenced for Platr18 (Platr18 KD) and treated with TGFß (+TGFß). (B) Schematic of the Neuro2A axonogenesis co-culture assay in vitro (top panel). Mouse Neuro2A neuroblastoma cells are transduced with a lentivirus carrying the eGFP gene, FACS-sorted, and then cultured in the presence of tumor cells. The neurite outgrowth is tracked by fluorescence microscopy that allows distinguishing the neuronal component of the co-culture through the GFP expression (bottom panels). DAPI+/eGFP+ = Neuro2A; DAPI+/eGFP- = tumor cells. Neuro2A confocal microscopy pictures of the Neuro-2A cells cultivated alone (N2a Alone) or in the presence of NMuMG cells overexpressing Platr18 (Platr18), silenced for hnRNP E1 (E1KD), silenced for Platr18 (Platr18 KD), or pretreated with TGFß prior (not during the co-culture) their addition to the Neuro2AGFP+ culture. (C) Scatter plot of the neurite outgrowth absolute quantification of the PC12 axonogenesis assay in vitro. For each culture condition (bottom table) the number of dendrites per cell is reported. (n=) number of PC12 cells per condition. The histogram displays the median ± interquartile range. *P < 0.05; **P < 0.01; ***P < 0.001. Experiments have been repeated three times or as specified in the legend. Scale bars: 50 µm.

Fig 3: The TGFß/E1KD/Platr18 axis controls semaphorin-4F expression in vitro and in vivo.(A) Single-molecule RNA-FISH labeling of NMuMG cells overexpressing Platr18 LincRNA or treated with TGFß (bottom). Scale bar: 10 µm. (B) Volcano plot of the genome-wide transcriptome analysis of NMuMG overexpressing Platr18 (NMuMG-Platr18), silenced for hnRNP E1 (NMuMG-E1KD), treated with TGFß for 4 d (NMuMG-TGFß), and silenced for Platr18 in NMuMG-E1KD (NMuMG-E1KD-Platr18 KD). All genes are plotted. (C) (Left) Sema4F transcript expression in NMUMG cells overexpressing (+Platr18) or silenced for hnRNP E1(E1KD). (Right) Sema4F transcript expression after Platr18 silencing (Platr18 KD) in NMuMG or NMuMG silenced for hnRNP-E1 expression (NMuMG E1KD). Data are expressed in fold changes compared to the control with mean ± SD; *P < 0.05; ***P < 0.001 (n = 3). (D) Time course of Sema4F transcript expression under TGFß exposure in (left) NMuMG cells or (right) NMuMG cells silenced for Platr18 expression (SCR versus Platr18 KD cells). Data are expressed in fold changes compared to the control with mean ± SD. TGFß treatment: ANOVA P < 0.0001; ***P < 0.001; NS, not significant (n = 3). (E) Immunofluorescence of Sema4F protein expression and localization in NMUMG cells overexpressing Platr18 (NMUMG-Platr18), silenced for E1KD (NMUMG-E1KD), or treated with TGFß (NMUMG + TGFß) for 3 d. (F) Time course of IGSF11/VSIG3 transcript expression under TGFß exposure, hnRNP E1 silencing (E1KD), or Platr18 expression. (n = 3) Scale bar: 20 µm. (G) Organoid cultures derived from mouse mammary tumor virus (MMTV)-Cre- PCBP1fl/fl or MMTV-Cre+ PCBP1fl/fl. Phase-contrast micrograph of organoid morphology and E-cadherin immunofluorescence (green) with nuclei counterstained by DAPI (blue). 60X magnification. Scale bar: 20 µm. (H) Immunofluorescence analysis of E-Cadherin and vimentin markers expression in MMTV-Cre- PCBP1fl/fl or MMTV-Cre+ PCBP1fl/fl hTert-immortalized cells in culture in vitro. Scale bar: 20 µm. (I) Quantitative PCR analysis of PCBP1 (hnRNP E1), Platr18, and Sema4F expression in 2D cultures derived from MMTV-Cre- PCBP1fl/fl and MMTV-Cre+ PCBP1fl/fl mice after exposure to TGFß (n = 3). Data are expressed in fold changes compared to the control with mean ± SD; ***P < 0.001; ANOVA P < 0.0001. Experiments have been repeated three times or as specified in the legend.

Fig 4: Platr18 silencing.(A) Characterization of Platr18 silencing. Quantitative PCR analysis of Platr18 relative expression in NMuMG cells expressing scrambled (SCR) or Platr18 specific (Platr18 KD) shRNAs and treated with TGFß (0–5 d; 5 ng.mL-1) (left) or silenced for hnRNP E1 (E1KD) (right). (n = 3) Data are mean ± SD. (B) Platr18 lincRNA silencing does not prevent TGFß-induced epithelial–mesenchymal transition. Western-blot analysis of epithelial (E-cadherin; occludin) and mesenchymal (vimentin; N-cadherin) markers expression in NMuMG cells expressing scrambled (SCR) or Platr18 specific (Platr18 KD) shRNAs and treated with TGFß for up to 5 d. (C) Immunofluorescence staining of vimentin/E-cadherin expression in NMuMG cells overexpressing Platr18 (Platr18), silenced for Platr18 (shPlatr18), and treated TGFß (+beta 3 d) or after silencing of hnRNP E1 (E1KD). Experiments have been repeated three times or as specified in the legend. Scale bar: 20 µm.

Fig 5: Supplementary figure 4.(A) Gene ontology term enrichment classification of transcripts up-regulated in human liver carcinoma HePG2 cells silenced for hnRNP E1. RNA-Seq data extracted from the ENCODE project ENCSR603TCV & ENCSR635FRH. (B) Gene ontology term enrichment classification of transcripts up-regulated in 4T1 cells treated with TGFß for 24 h. RNA-seq data extracted from GSE110912. (C) Validation of Sema4F silencing. Immunofluorescence staining of E1KD cells expressing scrambled (E1KD SCR) or Sema4F-specific shRNAs (E1KD S4F KD). Nuclei are counterstained with DAPI. Scale bar: 20 µm. (D) Sema4F silencing does not impair TGFß-mediated changes in cell morphology. Phase contrast micrographs of NMuMG cells expressing scrambled (NMuMG SCR) or Sema4F specific (NMuMG S4F KD) shRNAs and treated with TGFß for up to 2 d (0–2 d; 5 ng.mL-1). Scale bar: 100 µm. (E) PC12 cells do not differentiate after TGFß exposure. Bright-field micrographs of PC12 cultures exposed to TGFß (1–50 ng.mL-1) for 5 d. Nerve growth factor (NGF) serves as a positive control. Scale bar: 50 µm. (F) NMuMG-derived models do not produce significant amount of NGF. Western blot analysis of NGF production from whole cell lysates of NMuMG cells (WT), NMuMG cells overexpressing Platr18 (OE Platr18), silenced for hnRNP E1 (E1KD), or treated with TGFß for 3 d. Mouse whole brain (Whole Brain) and N2A cell line (Neuro2A) lysates are used as positive controls. HSP90 is used as a loading control. Experiments have been repeated three times or as specified in the legend.