Fig 2: Overexpression of INSL5 promotes cell cycle progression and suppresses cell apoptosis AThe cell cycle of vector control or INSL5 overexpressing CNE1, CNE2, and HK1 cells were analyzed by flow cytometry assay.BStatistical analysis of cell percentage in each cell cycle phase.CCyclin D, cyclin E, cyclin B, and p27 expression levels were detected by Western blotting in cells with or without INSL5 overexpression.DWestern blotting for c‐myc, BCL2, and BCL‐xL in CNE1, CNE2, and HK1 with or without INSL5 overexpression.ECNE1 stable cell line was treated with DDP or 5‐FU and stained with annexin V/propidium iodide (PI), and measured by flow cytometry. Data shown are representative of three independent experiments.FStatistical analysis of the effects of INSL5 overexpression on cell apoptosis under DDP or 5‐FU treatment.GWestern blotting for apoptosis pathway in CNE1, CNE2, and HK1 with or without INSL5 overexpression under 5‐FU treatment.Data information: In (F), data are presented as mean ± SEM, from three different experiments, and P‐values were determined by unpaired t‐test. *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significance. Exact P‐values are specified in Appendix Table S4.Source data are available online for this figure.

Fig 3: p21 expression is reduced in early-onset PE complicated by the HELLP syndrome. (A–C) Quantification of cell cycle regulators in placental sections of control donors (control, n = 17) and placental tissues from early-onset PE complicated by the HELLP syndrome (ePEH, n = 16) using the H-score method. The results are presented as bar and scatter plots showing the mean value with SD. The percentage of positive stained cells/area is shown under each graph. (A) H-score of p21 (left panel), p–p21 (middle panel), and p27 (right panel) for CTBs. (B) H-score of p21 (left panel), p–p21 (middle panel), and p27 (right panel) for the STB area. (C) H-score of p21 (left panel), p–p21 (second panel), p27 (third panel), and p57 (right panel) for fCTBs. (D) Quantification of p21 positive (left panel), p–p21 positive (second panel), p27 positive (third panel), and p57 positive EVTs (right panel) in %. (E) Western blot analysis with extracts from placental tissues is shown. GAPDH served as the loading control. (F) The relative amount of the gene levels of CDKN1A (p21), CDKN1B (p27) and CDKN1C (p57) was analyzed with placental tissues. The results are presented as relative quantification (RQ) with minimum and maximum range. TBP was used as the endogenous control. Paired Student’s t-test or Wilcoxon-test was used for statistical analysis. CTBs, cytotrophoblasts; fCTBs, cytotrophoblasts ongoing to fuse; STB, syncytiotrophoblast; EVT, extravillous cytotrophoblasts; no, number; MV, mean value.

Fig 4: Effect of IGF2BP3 and p27 on cell proliferation and G1 cell cycle phase arrest. (A) Western blot of IGF2BP3, p27, and phospho-RB proteins. IGF2BP3 or p27 siRNA- transfected cells show the suppression of specific protein expression in ACC-MESO1 and CRL-5915 cells. Cell lines transfected simultaneously with both IGF2BP3 and p27 siRNAs re-diminish p27 protein expression and recover phospho-RB protein expression. (B) Cell proliferation assay. In ACC-MESO1 and CRL-5915, transfection with IGF2BP3 siRNA and p27 siRNA together show a significant increase in cell proliferation at 72 h and 96 h from transfection of IGF2BP3 siRNA alone. Note the cells transfected with p27 siRNA alone show no significant change from those transfected with NC siRNA. (C) Cell cycle assay. ACC-MESO1 and CRL-5915 cells transfected with IGF2BP3 siRNA and p27 siRNA together, show significant reduction in the percentage of cells in G1 phase of the cell cycle. Note the cells transfected with p27 siRNA alone show no significant difference in the percentage of cells in G1 phase compared with those cells transfected with NC siRNA. NC, Negative control; NS, no significance; *p < 0.05; ***p < 0.001.

Fig 5: TobTRIP and MS analysis identifies a network of functionally related RBPs bound to p27(3ʹUTR) reporter mRNAs. (a) Schematic representation of tobTRIP for isolation of tagged mRNAs together with bound proteins: In a first step, poly(A) RNA-protein complexes are isolated using oligo(dT)25 beads. In a second step, tobramycin aptamer-tagged RNAs are captured with tobramycin-coupled magnetic beads. (b) Immunoblot analysis of the indicated proteins across different steps of tobTRIP. 1:600 of the extract (input), and 1:60 of the 1st and 2nd step eluates were loaded. A quantification of band intensities across the 2nd step eluates is indicated for ELAV1 and hnRNPD. (c) Differential binding of the 54 proteins interacting with GFP-T-p27(3ʹUTR) in CP-treated (+CP) versus untreated (-CP) cells. Mean LFQ values are plotted against log2-transformed average foldchanges obtained from normalized intensities. (d) GO terms significantly enriched among the 54 selected proteins. Bars indicate the fractions of proteins annotated with the respective GO term, either across proteins pulled-down with the p27(3ʹUTR) reporter (black bars) or across the entire reference proteome (Uniprot; 20,395 proteins; white bars). Absolute numbers of proteins are shown to the right. Adjusted P-values (Benjamini-Hochberg) for enrichments relative to the reference proteome are indicated. (e) PPI network of 28 proteins with multiple and propagating interactions. Each node corresponds to one RBP, and the physical interaction between RBPs is shown as an edge. The thickness of edges is proportional to the STRING confidence score (between 0–1). Node colour corresponds to average log2 fold-change of RNA binding in treated (+CP) versus untreated (-CP) cells as indicated in the colour bar.