SUPPLEMENTARY FIGURE LEGENDS
Supplementary Figure S1. Effect of the changes in miR-26a-2 expression in three human LPS cell lines (SW872, LPS141, LP6) in vitro. (A) Endogenous expression level of miR-26a-2 gene in each LPS cell line. For panels (B)-(F), LPS cells were transduced with either pMSCV-miR-26a-2 expression vector (26A2) or empty vector control (EV). Stable clones were selected with puromycin. (B) Validation of miR-26a-2 overexpression clones by qRT-PCR. miR-26a-2 expression level in empty vector control was set to 1. GAPDH was used as a loading control. (C) Migration assay results showing the relative gap width 12 hrs after the scratch was made on the cell monolayer. Initial gap width at 0 hr was set at 100%. (D) Soft agar colony formation assay results. Cells were mixed in soft agar, cultured for 2-3 weeks, and the number of colonies were counted. (E) MTT proliferation assay results. Cells were seeded in 96-well plates and cell growth was measured every 24 hrs up to 96 hrs. (F) Cell cycle analysis results. Cells were exposed to nocodazole for 12 hrs, stained with PI, and subjected to FACS analysis. For panels (G)-(K), LPS cells were transfected with either scrambled control oligos or anti-miR-26a-2 oligos. Cells were harvested 24 hrs after transfection. (G) Validation of inhibition of expression of miR-26a-2 by anti-miR-26a-2 oligos by qRT-PCR. miR-26a-2 expression level in scrambled oligo control was set to 1.0. GAPDH was used as a loading control. (H) Boyden chamber migration assay results. Number of migrated cells in scrambled oligo control was set at 100%. (I) Soft agar colony formation assay results. (J) MTT proliferation assay results. (K) Cell cycle analysis results. Experiments were repeated three times to ensure accuracy. Data represent average ± standard deviation (SD, error bars). Asterisks (* and **) indicate p-value less than 0.05 and 0.001 by t-test, respectively. NS = not significant.
Supplementary Figure S2. Expression status of PTEN, RB1, and Akt in LPS cells. (A) Endogenous expression level of PTEN and RB1 by Western blot. HeLa cells were used as a positive control for PTEN expression. GAPDH was used as a loading control. (B) Expression level of PTEN and RB1 in miR-26a-2 overexpression clones (26A2) or empty vector control cells (EV) of SW872 and LP6 cells. (C) Expression level of PTEN and RB1 in LPS141 and LP6 cells treated with either anti-miR-26a-2 oligos or scrambled control oligos. (D) Changes in phosphorylation level (S473) of Akt upon serum stimulation in the miR-26a-2 overexpression clones of U87 (positive control), SW872, and LP6 cells. Cells were transduced with either pMSCV-miR-26a-2 expression vector (26A2) or empty vector control. Stable clones were selected with puromycin. Clones were serum starved for 48 hrs prior to serum stimulation. Experiments were repeated three times to ensure accuracy. Data represent average ± standard deviation (SD, error bars). Asterisks (* and **) indicate p-value less than 0.05 and 0.001 by t-test, respectively.
1