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. 2019 Apr 12;43:270–281. doi: 10.1016/j.ebiom.2019.04.010

Fig. 4.

Fig. 4

Involvement of PIK3C3 in miR-338-5p mediated cell migration and invasion. (a) miR-338-5p or negative control (N.C.) (100 nM) was transiently transfected into HCT116 cells, put into 96-well plates (4000/well) and cultured for 24 h. The number of cells was calculated every 24 h using MTT assays for four consecutive days. Data are expressed as means (n = 6) (P values were analysed using ANOVA). (b, c) Cell migration was analysed using transwell migration assays 48 h after transfection with miR-338-5p or anti-miR-338-5p into HCT116 or SW480. The transfected cells were seeded on transwell plates, and migrated cells at the bottom of membrane were calculated. The scale bar represents 50 μm. The number of migrated HCT116 cells are shown as means ± SEM (n = 8) (P values were analysed using t tests).The number of migrated SW480 cells are shown as median (IQR) (n = 5) (P values were analysed using Mann-Whitney tests). (d) miR-338-5p or pCMV-Vps34 plasmid was transiently transfected into SW480 cells and analysed for PIK3C3 protein expression 48 h later. Cell migration was evaluated using transwell assay. Forty-eight hours after transfection, SW480 cells were seeded on transwell plates, and those cells appeared at the bottom of membrane were counted. Data are expressed as means ± SEM (n = 8) (P value was analysed using t tests). (e) Both miR-338-5p and pCMV-Vps34 plasmids were transiently transfected into SW480 cells. Transwell columns were coated with a Matrigel membrane and seeded with transfected cells 48 h later. The invaded cells at the bottom of membrane were counted after 96 h. Data are expressed as means ± SEM (n = 8) (P value was analysed using t tests). (f) SW480 cells were transiently transfected with anti-miR-338-5p (100 nM) and shVps34 lentivirus and assayed for PIK3C3 protein expression. Migration of SW480 cells was assayed using transwell assay. Number of migrated CRC cells was counted after 48 h. Data are expressed as means ± SEM (n = 8) (P value was analysed using t tests). (g) Transwell invasion assays were used to analyse SW480 cells transfected with anti-miR-338-5p or shVps34 lentivirus and counted for invaded cells after 96 h. Data are expressed as means ± SEM (n = 8) (P value was analysed using t tests). The scale bar represents 50 μm.