Fig 2: Schematic diagram of proposed model for MkMP-induced signaling in CD34+ HSPCs and its relationship to miRs in MkMPs. The model is based on the following pieces of evidence. Our data showed that a co-culture of CD34+ HSPCs with MkMPs activates the PI3K/Akt/mTOR and JNK signaling pathway toward cell expansion and megakaryocytic differentiation. Specifically, JNK and mTOR signaling are involved in late Mk differentiation (CD42b expression). A crosstalk between JNK and PI3K/Akt/mTOR was identified via the negative regulator of PI3K/Akt/mTOR signaling, PTEN. PI3K and PTEN were known major targets of miR-486-5p.

Fig 3: MkMPs activate PI3K/Akt/mTOR signaling in CD34+ HSPCs by targeting PTEN and mediate JNK signaling. (a,b) CD34+ HSPCs were co-cultured with MkMPs or vehicle control. Cells were harvested after 24 h of co-culture, and the level of targets including mTOR, p-mTOR, and Akt were examined by immunoblotting with GAPDH as the loading control. (a) Representative immunoblot images out of 4 replicates. (b) Semi-quantification of p-mTOR expression from immunoblot images (n = 4). The expression levels for p-mTOR were normalized to the expression of GAPDH. (c–e) CD34+ HSPCs were pretreated with a signaling inhibitor (JNK, Akt, or mTOR inhibitor), or inhibitor vehicle (without an inhibitor), and were co-cultured with MkMPs or without (vehicle control). Cells were harvested after 24 h of co-culture, and the level of p-mTOR, Akt, and p-Akt were examined and quantified by flow cytometric analysis (n = 3–5). Mean fluorescence intensity (MFI) represents the protein expression/levels averaged over the total cell population. Representative histograms of each target are shown on the right. The Y-axis represents the event (cell) count for each specific fluorescent intensity (f,g) CD34+ HSPCs pretreated with JNK inhibitor or solution without an inhibitor were co-cultured with MkMPs or vehicle control for 24 h. (f) PTEN protein expression was quantified by flow cytometric analysis, while (g) PTEN mRNA levels were quantified by qPCR analysis. (h) Schematic diagram of signaling pathway triggered by MkMPs in CD34+ HSPCs, based on the results from (a–f). Error bars in (b) represent the standard error of mean of 2–4 biological replicates, while error bars in (c–g) represent the standard error of mean of 3–5 biological replicates. Statistical comparison analysis was performed between each experimental group against MkMP control (without inhibitor). * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 4: STK39 mediates oncogenic effects on CCA cells through activating the PI3K/AKT signaling pathway(A) Pathway enrichment analysis of differentially expressed genes in RNA-sequence data.(B) Levels of PI3K, AKT, P-PI3K (p85), and P-AKT (Ser473) were examined by western blotting in STK39 stable knockdown or overexpression cells.(C) Stable knockdown of STK39 in QBC-939 cells by shRNA was treated with or without indicated concentrations of IGF-1. The levels of PI3K, AKT, P-PI3K (p85), and P-AKT (Ser473) were determined by western blotting.

Fig 5: Low PTENS380D level causes AKT hyperactivity and PIN lesion formation.a WB of lysates from 2-month-old prostates (n = 3 individual samples) of indicated genotypes. Blots are probed for PTEN and indicated phosphorylated PTEN. Ponceau S (PonS) staining served as loading control. b Tissue fractionation of prostates of the specified genotypes. Indicated fractions (C, cytoplasm; M, membrane; N, nuclear) were blotted and probed for PTEN (various exposures) and compartment-specific proteins. n = 3 independent fractionation experiments. c Measurements of PTEN and PTENS380D protein turnover rates in P4 MEFs by inhibiting protein synthesis with 20 µg/ml cycloheximide for the indicated times. PTEN levels at each timepoint were assessed by WB for PTEN, with Tubulin as loading control. N = 3 independent experiments. d Cell fractionation of MEFs of the indicated genotypes and fractions as in (b), blotted and probed for PTEN. Blots were overexposed to be able to visualize PTENa and PTENß, n = 2 independent fractionation experiments. e As in (a), but now blots are probed for PTEN, AKT and AKT substrates, and are representative of at least 3 individual samples. f Percentage of prostate tubules with indicated PIN lesions at 6 months. A/–, PtenS380A/–; D/–, PtenS380D/–. N = 5 mice per genotype. Data are presented as mean values ± SEM. Statistical significance was assessed by ordinary one-way ANOVA, followed by Dunnett’s multiple comparisons test compared to +/+. g Immunostaining of consecutive sections of prostate tissue of indicated genotypes stained for PTEN and P-AKTS473, n = 5 prostates per genotype. h Consecutive immunostaining of prostate tissue of indicated genotypes stained for PTEN and P-AKTS473-Alexa Fluor 488 conjugate. N = 2 prostates per genotype, both stained at two different levels in two independent experiments. *Indicates lesions. Scale bars in (g and h) are 100 µm. Source data are provided as a Source Data file.