Fig. 4

SERPINE1 is a key transcriptional target of NONO and PKM2 in TNBC. (A) Venn diagram depicting the overlap of NONO- and PKM2-regulated differentially expressed genes (DEGs) in MDA-MB-231 cells obtained from RNA-seq analysis. (B) Gene ontology (GO) analysis to show the top 10 pathways with the smallest p.adjust values, pathway shown as the ordinate and gene ratio shown as the abscissa. The size indicated the number; the redder the color, the smaller the p.adjust value. (C) Heatmap showing the top 20 most differentially expressed genes whose expression was coupregulated or codownregulated by NONO or PKM2. NC: negative control; KD: knockdown. (D) Relative mRNA levels of SERPINE1, IL11, CCN2, RCN1, VDAC1, CDKN1A, ADAMTSL4, MATN2, FERMT2 and MCAM normalized to those of GAPDH were examined by RT‒qPCR in negative control (NC) or NONO-silenced MDA-MB-231 cells. The data are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to NC. (E) The protein expression of PAI-1 in NC and NONO-KD MDA-MB-231 cells was assessed by western blot analyses with the indicated antibodies. (F) Western blot analysis of the indicated proteins in MDA-MB-231 cells treated with NC, NONO-KD, or NONO-KD + PAI-1. GAPDH served as a loading control. (G) Cell proliferation was examined by a CCK-8 assay in MDA-MB-231 cells treated with NC, NONO-KD, or NONO-KD + PAI-1. The data are presented as the mean ± SD (n = 5). ****P < 0.0001 compared to the NONO-KD group. (H) Colony formation was determined in MDA-MB-231 cells treated with NC, NONO-KD, or NONO-KD + PAI-1. The data are presented as the mean ± SD (n = 3). **P < 0.01, ***P < 0.001 compared to the corresponding control. (I) Representative images of the migration (top panels) and invasion (bottom panels) of MDA-MB-231 cells treated with NC, NONO-KD, or NONO-KD + PAI-1. The data are presented as the mean ± SD (n = 5). ***P < 0.001, ****P < 0.0001 compared to the corresponding control. J-L. Photographs (J), tumor growth curves (K), and tumor weights (L) of MDA-MB-231 xenograft tumors from the scramble (Scr), NONO-KD, and NONO-KD + PAI-1 groups. The data are presented as the mean ± SD (n = 6). ***P < 0.001, ****P < 0.0001 compared to the corresponding control. M. Relative mRNA levels of SERPINE1, IL11, CCN2, RCN1, VDAC1, CDKN1A, ADAMTSL4, MATN2, FERMT2 and MCAM (normalized to GAPDH) were examined by RT‒qPCR in NC and PKM2-KD MDA-MB-231 cells. The data are presented as the mean ± SD (n = 3). **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to NC. N. The protein expression of PAI-1 in NC and PKM2-KD MDA-MB-231 cells was assessed by western blot analyses with the indicated antibodies. O. Western blot analysis of the indicated proteins in MDA-MB-231 cells treated with NC, PKM2-KD, or PKM2-KD + PAI-1. GAPDH served as a loading control. P. Cell proliferation was examined by a CCK-8 assay in MDA-MB-231 cells treated with NC, PKM2-KD, or PKM2-KD + PAI-1. The data are presented as the mean ± SD (n = 5). ***P < 0.001 compared to the PKM2-KD group. Q. Colony formation was determined in MDA-MB-231 cells treated with NC, PKM2-KD, or PKM2-KD + PAI-1. The data are presented as the mean ± SD (n = 3). **P < 0.01, ***P < 0.001 compared with the PKM2-KD group. R. Representative images of the migration (top panels) and invasion (bottom panels) of MDA-MB-231 cells treated with NC, PKM2-KD, or PKM2-KD + PAI-1. The data are presented as the mean ± SD (n = 5). **P < 0.01, ****P < 0.0001 compared to the corresponding control. S. Pearson correlation scatter plot of H scores for NONO, PKM2, and PAI-1 in human TNBC tissues (n = 60)