Fig 2: Identification of positive cross-talk between Ras/Raf/MEK/ERK and PI3K/Akt/mTOR pathway via Ras?PIK3IP1?PI3K signaling network. a The PI3K network analysis using gene expression of PI3K regulators in normal (middle) and transformed BJ cells (right) treated with a131 at 5 µM for 24 h. PI3K regulators, including PIK3IP1, were identified to interact with PI3K with experimental support and high confidence from STRING. The per-sample expression profiles of PIK3IP1 are depicted in the heatmap (left). Color: negative log FDR (false discovery rate), coded from white to red in a scale from 0.15 to 5.37. Size: log ratio; Border: upregulation (red) or downregulation (blue); Shape: upstream (square), parallel (diamond) or downstream (circle). b, e qRT-PCR analysis of PIK3IP1 expression in BJ cell lines treated with a131 or a166 at 5 µM for 24 h. Mean values ± S.D. (n = 3). c Immunoblot analysis of normal BJ cells treated with 4-OHT [4HT(+)] for 24 h to activate H-RasV12-ER and subsequently with a131 (left) or transfected with indicated siRNAs for 48 h (right). Representative immunoblots shown (n = 3). d ChIP analysis of normal BJ cells treated with a131 using antibodies against RNA polymerase II (Pol II) for the PIK3IP1 gene promoter (n = 3). e Normal BJ cells treated with a131 were subsequently treated with MEK inhibitor U0126 (10 µM) for additional 2 h. Then, 4-OHT added to activate H-RasV12-ER for various time points. Where indicated, two-tailed unpaired t tests were performed to determine statistical significance

Fig 3: The high MAPK activity suppresses PIK3IP1 in human cancers. a, b BrdU incorporation (n = 3) (a) and immunoblot analysis (b) of normal BJ cells transfected with indicated siRNAs for 48 h and treated with a131 for additional 24 h. c qRT-PCR analysis of PIK3IP1 expression. Various normal and Ras-mutant cancer cell lines were treated with a166 at 0, 2.5, and 5 µM for 24 h. Mean values ± S.D. (n = 3). d qRT-PCR analysis of PIK3IP1 expression (top) and immunoblot analysis (bottom) of Ras-transformed and Ras-mutant cells treated with MEK inhibitor U0126 (10 µM) and ERK inhibitor SCH722984 (1.2 µM) for 24 h. e Immunoblot analysis (top) and caspase-3/7 activation (bottom, n = 3) of HCT116 cells transfected with indicated siRNAs for 48 h and treated with U0126 (10 µM) and SCH722984 (1.2 µM) for additional 24 h. f Schematic model of cross-talk between Ras/Raf/MEK/ERK and PI3K/Akt/mTOR pathway via Ras?PIK3IP1?PI3K signaling network for cancer cell proliferation. Where indicated, relative ratios of phosphorylated/total levels of Akt and p70S6K are shown compared with DMSO. b, d, e Representative immunoblots shown (n = 3). Where indicated, two-tailed unpaired t tests were performed to determine the statistical significance

Fig 4: Generation of RPE-1 RFX7 knock-out cell lines and validation of novel RFX7 targets.a Western blot analysis of RFX7, PDCD4, PIK3IP1, p53, and actin (loading control) levels in parental and RFX7 knock-out (RFX7-/-) RPE-1 cells treated with Nutlin-3a or DMSO solvent control. Uncropped western blot images available in Supplementary Fig. S1b. b RT-qPCR data of the established RFX7 targets PIK3IP1, PDCD4, MXD4, and PNRC1 in parental and RFX7 knock-out RPE-1 cells treated with Nutlin-3a or DMSO solvent control. MDM2 served as a positive control for p53 induction. c RT-qPCR data of the novel RFX7 targets MAFTRR, MIR22HG, and SYNPO in parental and RFX7 knock-out RPE-1 cells treated with Nutlin-3a or DMSO solvent control. b, c Normalized to DMSO treatment and ACTR10 negative control. Mean and standard deviation is displayed. Statistical significance obtained through a one-way ANOVA test, n = 6 (two biological replicates with three technical replicates each).

Fig 5: Transcriptome analysis of RFX7 knock-out U2OS cells.a Volcano plot of differential gene expression data from RFX7 knock-out clone 1 (left panel) and 2 (right panel) compared to parental U2OS cells under Nutlin-3a treatment condition. Data has been obtained using DESeq2 and is available in Supplementary Table S1. Selected known RFX7 targets are highlighted. b Transcripts per kilobase million (TPM) expression values of PDCD4 and PIK3IP1 obtained from RNA-seq analysis from parental and RFX7 knock-out U2OS cells treated with Nutlin-3a or DMSO solvent control. Statistical significance obtained through a one-way ANOVA test, n = 3 biological replicates. c Log2(fold-change) values of 57 established RFX7 targets [23] from RNA-seq analyses of parental and RFX7 knock-out U2OS cells treated with Nutlin-3a or DMSO solvent control. Fold-changes normalized to DMSO-treated parental U2OS cells. Mean and standard deviation is indicated.