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. 2019 Jul 1;116(29):14620–14629. doi: 10.1073/pnas.1903432116

Fig. 3.

Fig. 3.

EMS exerts its oncogenic role by increasing E2F1 expression. (A) A549 cells were infected with lentiviruses expressing control (pSin-ctrl) or EMS (pSin-EMS). Forty-eight hours later, total RNA was subjected to real-time RT-PCR analysis to evaluate mRNA levels of E2F1 and its target genes as indicated. (B) A549 cells were infected with lentiviruses expressing control (ctrl) shRNA (shctrl), EMS shRNA#1, EMS shRNA#2, control (pSin), or EMS (pSin-EMS). Forty-eight hours later, cell lysates were analyzed by Western blotting. (C) A549 cells were infected with lentiviruses expressing control, EMS, E2F1 shRNA, or both EMS and E2F1 shRNA. Forty-eight hours later, the cell growth curves were measured. Data shown are mean ± SD (n = 3). *P < 0.05. ns., no significance. The successful EMS overexpression and E2F1 knockdown are shown in SI Appendix, Fig. S4A. (D) A549 cells were infected with lentiviruses expressing control, EMS, E2F1 shRNA, or both EMS and E2F1 shRNA. Forty-eight hours later, cells were subjected to EdU incorporation assay. Data shown are mean ± SD (n = 3). ***P < 0.001. (E) A549 cells were infected with lentiviruses expressing control, EMS, E2F1 shRNA, or both EMS and E2F1 shRNA. Seventy-two hours later, cells were subjected to flow cytometry analysis. Data shown are mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001. (F) A549 cells were infected with lentiviruses expressing control, EMS, E2F1 shRNA, or both EMS and E2F1 shRNA. Forty-eight hours later, cells were assayed for their ability to form colonies in soft agar. Data shown are mean ± SD (n = 3). **P < 0.01. (G–J) Total of 2 × 106 A549 cells transduced with lentiviruses expressing control, EMS, E2F1 shRNA, or both EMS and E2F1shRNA were individually injected into the left flank and right flank of nude mice as indicated (n = 6 for each group). (G) Representative photographs of mice and xenograft tumors were taken 5 wk after injection. (H) Excised tumors were weighed. *P < 0.05. (I) Tumor sizes were measured at the indicated time points. (J) RNA and protein extracts from the excised xenografts were also analyzed by RT-PCR (RT) and Western blotting (WB), respectively.