CD44 inhibition by miR-138 induces cell cycle arrest through upregulation of cell cycle inhibitor p27. (A) Cell viability of GBM cells decreases by miR-138 overexpression, which is partially rescued by ectopic overexpression of CD44. GBM cells were transfected with miR-138 or miR-Ctrl for 96 days in the presence CD44 expressing cDNA plasmids or empty control vector DNAs. Cell viability was measured by CellTiter-Glo Luminescent Cell Viability Assay (n = 3). (B) Cell cycle analysis by flow cytometry on GBM28 cells after 4 days of transient transfection of miR-138 or miR-Ctrl. Comparison of cell populations between G0/G1 and G2/M phases were expressed as %gated cells to total cell populations. Arrested cell cycle at G0/G1 phase by miR-138 was partially reversed by ectopic overexpression of CD44. Cell cycle data from GBM12 and GBM43 cells were shown in Supplementary material Fig. S7. Representative cytograms were shown in Supplementary material Fig. S8. (C) CD44 inhibition by miR-138 induces cell cycle arrest through Akt/p27 signaling axis. Transiently transfected GBM cell lysates with miR-138 or miR-Ctrl were analyzed by western blotting. miR-138 mediated CD44/Akt/p27 modulation was partially rescued by ectopic overexpression of CD44. (D) Western blotting showing activation and nuclear translocation of p27 in GBM cells after miR-138 overexpression. Total GBM cell lysates harvested after 4 days of transient transfection with miR-138 or miR-Ctrl were separated into nuclear and cytoplasmic fractions and analyzed by western blotting. HDAC1 was used as loading control for total cell and nucleus fractions, while Vinculin was used for cytoplasmic fractions. (E) Immunofluorescence staining of GBM cells that were transfected with miR-138 or miR-Ctrl for 4 days. Nuclear translocation of p27 was observed by overlapped fluorescence intensity of FITC-labeled anti-p27 antibody with DAPI staining. Scale bars indicate 100 µm.