Overexpression of PAL31 in C6 did not affect the cell proliferative activity. (A) GFP/bright field-superimposed micrograph in GFP overexpressing cells showing > 50% transfective efficiency (B) GFP-PAL31/bright field-micrograph in PAL31 overexpressing C6 showing correct nuclear localization of GFP-tagged PAL31 and transfection effeciency. (C) Western blot analysis showing the expression of GFP (~27 kDa), GFP-tagged PAL31 (~58 kDa), PCNA (~34 kDa), endogenous PAL31 (~31 kDa) and Actin (~43 kDa) in GFP- or PAL31-C6 cultures. The level of PCNA, a protein expressing in nuclear during DNA synthesis, in cells did not alter after overexpression of GFP or GFP-tagged PAL31. Actin works as a loading control. (D) MTT assay in the transfected cells at 4 different time intervals (from Ctrl to 72 hours). Ctrl represents the cells after subculture and overnight incubation. The data in each time points were analyzed by two-way ANOVA and Bonferroni post hoc test. No significance, compared GFP and GFP tagged PAL31 groups. n = 3. Magnification 100X (A-B).