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. 2019 Jan 23;9:351. doi: 10.1038/s41598-018-37064-x

Figure 3.

Figure 3

TREX1 silencing causes accumulation of sub-G1 cells and p53 upregulation. TREX1 silencing in SiHa, HeLa, C33A and PHK was performed as described in Fig. 1. (A) For cell cycle study, monolayers cultures of the different cell lines silenced for TREX1 expression were seeded in triplicate in 24 well plates (5000 cells/well). After 5 days cells and supernatants were harvested and analyzed in a FACSCalibur. At least 10,000 events were acquired for each condition. The data obtained were analyzed with the FlowJo software. (B) To analyze the effect of TREX1 silencing on the levels of regulators of the cell cycle 30 μg of total protein extracts from monolayer cultures of each cell type were analyzed using antibodies against cyclin A, PCNA, p53 and TREX1. (C) To determine the effect of TREX1 superexpression on the levels of regulators of the cell cycle in PHK expressing HPV16 E6 and/or E7 the cells were transfected with pcDNA5 expression vector harboring the TREX1 sequence. The analysis of the expression of PCNA, p53 and TREX1 was performed as described in (B). Western blot signals were quantified using ImageJ software using housekeeping genes actin or tubulin as normalizers and presented as expression relative to normal keratinocytes.