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Fig. 1 PHF8 is crucial for MKN28 cell proliferation and migration in vitro and in vivo. (A) Analysis of PHF8 in MKN28 infected with lentivirus carrying control pLKO or shPHF8 constructs (#1 or #2), followed by puromycin selection. PHF8 expression was analyzed by Western blotting. Actin was the internal control. (B) The depletion of PHF8 exhibited a reduced degree of cell proliferation in MKN28. (C and D) Luciferase-expressing MKN28 cells (pLKO [n = 5], shPHF8#1 [n = 5], and shPHF8#2 [n = 5]) were grown on nude mice. Images of control and shPHF8 MKN28-luc xenografts were taken after 6 wk of injection (C). Two weeks after implantation, the tumor volumes were measured every week up to 6 wk (D). Data are presented as mean ± SD, with the SD derived from five mice. **P < 0.01 (one-way ANOVA test). (E) The depletion of PHF8 exhibited a reduced degree of cell migration in MKN28 using Transwell cell migration assay. (F and G) Zebrafish migration assays. MKN28 labeled with CFSE (green fluorescence dye) were ectopically injected into the yolk-sac parts of 2-d-old zebrafish embryos. Fluorescence microscopic analysis was conducted at 1 dpi and 3 dpi. Representative fluorescence images of a zebrafish embryo displaying cell dissemination (Upper) or no migration (Lower) at 3 dpi (F). (Scale bar, 100 µm.) The depletion of PHF8 exhibited a significantly reduced proportion of embryos with migration behavior in MKN28 (G). Total number of embryos (pLKO, shPHF8#1, or shPHF8#2) is shown in the bracket. Data were obtained from three independent studies. *P < 0.05 (two-tailed Student’s t test). In B, E, and G data are presented as the average of three replicates ± SD *P < 0.05, **P < 0.01 (two-tailed Student's t test).