Fig. 2

Cytotoxicity and cell penetration measurement on HUVECs. (A) Representative images of live and dead cells. After treating HUVECs with different concentrations of OCT4-30Kc19 protein for 24 h, cells were stained by live and dead kit. Scale bar, 100 μm. HUVECs treated with 20 and 40 μg/ml of OCT4-30Kc19 showed similar high viability, while higher concentrations lowered viability. Accordingly, 40 μg/ml of OCT4-30Kc19 was used throughout the experiment. (B) Cellular penetration of OCT4-30Kc19 protein after 0, 4, 8, 12, 24 h of a protein treatment. When cells were treated with OCT4-30Kc19, protein effectively penetrated HUVECs from 0 to 24 h. Anti-OCT4-antibody (1:400) was used for OCT4-30Kc19 detection. Blue, green represent the nucleus and OCT4-30Kc19 protein, respectively. Scale bar, 20 μm. (C) Nucleus penetration of OCT4-30Kc19 protein after 0, 24, 48 h of a protein treatment. OCT4-30Kc19 protein was localized in the nucleus and acted as a transcription factor for pluripotent genes in 24 and 48 h samples. Blue, green represent the nucleus and OCT4-30Kc19 protein, respectively. Scale bar, 5 μm. (D) Nucleus penetration of OCT4-30Kc19 protein in a time-dependent manner. OCT4-30Kc19 protein increased over time. Quantitative analysis of fluorescence was performed using image J software (n = 20). (E) Quantitative polymerase chain reaction (qPCR) for pluripotency transcription factors, OCT4, SOX2, NANOG. HUVECs were treated with OCT4-30Kc19 for 24 and 48 h, and then PCR analysis was conducted. Gene expression levels of pluripotency transcription factors were normalized to GAPDH. PCR analysis showed elevation of OCT4, SOX2, NANOG expression as OCT4-30Kc19 treatment time increased (n = 3). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001