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. 2019 Jun 17;20(7):e47546. doi: 10.15252/embr.201847546

Figure 4. AML‐EV‐contained miR‐1246 suppresses protein synthesis in LT‐HSC via the mTOR pathway.

Figure 4

  • A, B
    qRT–PCR gene expression analysis showing the fold change of PI3K/mTOR‐associated gene panel in KSL cells sorted from: (A) Molm‐14‐xenografted mice or (B) IF‐injected mice with Molm‐14‐EV relative to their controls and normalized to Gapdh endogenous control. Data are expressed as mean ± SEM from at least three independent experiments, performed in technical replicates. Statistics: One‐way ANOVA with Bonferroni post hoc correction (*P < 0.05, **P < 0.01).
  • C–E
    Flow cytometric analysis showing the histograms (C) and MFI of intracellular pS6KRP in LT‐HSC in: (D) Molm‐14 xenograft (red, n = 9) versus non‐engrafted control (black, n = 7), or (E) IF injection of EV from Molm‐14, U‐937, HL‐60 (red, n = 7,3,3), or human CD34 cells (blue, n = 4) normalized to the control contralateral femur with subtraction of background fluorescence. Data were obtained from at least two independent experiments. Statistics: Student's t‐test (*P < 0.05, ***P < 0.001).
  • F, G
    Flow cytometric assessment of protein synthesis showing the MFI of: (F) OPP or (G) pS6RP in NIH‐3T3 cells 72 h after transfection with the indicated miRNA mimics. The results were calculated relative to control (miR‐scramble) with the background fluorescence subtracted and performed with at least three independent experiments, in technical replicates. Statistics: one‐way ANOVA with Bonferroni post hoc correction (*P < 0.05, **P < 0.01).
  • H
    Flow cytometric analysis showing the MFI of Raptor in NIH‐3T3 cells 72 h after transfection with the indicated miRNA mimics. The data were calculated relative to miR‐scramble and are presented as mean ± SEM, and the background fluorescence was subtracted. Performed with at least three independent experiments, in technical replicates. Statistics: one‐way ANOVA with Bonferroni post hoc correction (*P < 0.05, **P < 0.01).
  • I
    Dual‐luciferase reporter assay. NIH‐3T3 was transfected with the miRNA mimics. Three hours later, the cells were transfected with the Raptor 3′UTR cloned into the psiCheck‐2 vector for a total of 48 h. Data are presented as %RLU (relative luciferase units) of the miR‐scramble control as mean ± SEM from at least three independent experiments, performed in technical replicates. Statistics: one‐way ANOVA with Bonferroni post hoc correction (*P < 0.05).
  • J, K
    Cell‐cycle flow cytometric analysis using Ki67/Hoechst‐33342 staining of the percentage of LT‐HSC in the G0 phase after nucleofection of cKit+ cells using the Amaxa™ P3 Primary Cell 4D‐Nucleofector Kit, (J) Cells were nucleofected with miR‐scramble (CTRL) or miR‐1246 mimic (n = 3) for 72 h, (K) cells were nucleofected with anti‐miR‐scramble (CTRL) or anti‐miR‐1246 (n = 5) and 1 h later co‐treatment with Molm‐14 EV for 72 h. Statistics: Student's t‐test (*P < 0.05, **P < 0.01).