Fig. 5: siRNA and transport inhibitors phenocopy sgRNA-mediated POM121 knockout.

A POM121 knockdown (KD) by siRNA increases mRNA expression of PPARγ target genes but reduces protein levels. Parental HT29 cells were transiently transfected with POM121A/C siRNA or control siRNA for 48 h followed by extraction of RNA or total cell lysate (TCL). Left: Quantitative analyses from RT-qPCRs. Ct-values normalized to B2M are -fold ± S.E.; Right: Quantitative analyses and representative images from Western blots. O.D. values from gels normalized to lamin A/C are -fold ± S.E. (*p < 0.05 vs. control siRNA, 2way-ANOVA with Bonferroni post-tests, n = 3 per method). B Inhibitors of NPC transport mitigate the transcriptional activity of PPARγ. Clonal cells from Fig. 4 were treated with importazol, leptomycin B, vehicle (DMSO) or rosi (1–10 µM) for 48 h, followed by RNA extraction. Ct-values from RT-qPCRs normalized to B2M are -fold ± S.E. (*p < 0.05 vs. vehicle or EV, 2way-ANOVA with Bonferroni post-tests, n = 3 per clone). C Model of POM121-mediated transport of PPARγ. POM121 (FL, 120–150 kDa) as an essential integral transmembrane protein of the NPC is overexpressed in cancers and gives rise to soluble N- and C-terminal truncation variants. It has been identified to bind to PPARγ via amino acids C-terminal to its NLS and to disrupt bi-directional traffic of transcription factors (e.g., NFκB, MYC) across the nuclear pore. POM121 gain of function (GOF) reduced transcription in the nucleus and enhanced translation of PPARγ target genes/proteins in the cytosol, whereas POM121 loss of function (LOF) had the opposite effect. We propose that changes in the availability of POM121 FL protein and/or its soluble variants tip the balance between import and export of PPARγ. This “dysbalance” may be caused by competition for transport factors (importins, exportins) between POM121 and known PPARγ transport motifs: (i) the NLS in the DBD and the adjacent hinge region [73, 74] and (ii) the MEK1-binding NES-like motifs in the LBD [37, 38]. Color legend: Blue circles = ribosome; Blue cyclinders (with TM, NLS and FG domains) = POM121 (FL); Orange full circle = PPARγ-ligand (e.g., rosi); Orange quarter = PPARγ DNA-binding domain (DBD); Green quarter = PPARγ ligand-binding domain (LBD); Blue arrow = Transport nucleus-to-cytosol (mRNA translation); Orange arrow = Transport cytosol-to-nucleus (mRNA transcription).