Fig. 4

ZNF24 binds P65 promoter to negatively regulate its expression. A Genome-wide analysis of ZNF24 binding sites in lung cancer cells through ChIP-seq analysis. A549i cells were treated with Dox (1 μg/mL) for 48 h. ChIP-seq was performed on DNA samples enriched for the Flag antibody. B Impact of ZNF24 expression on activity of P65 promoter. P65 promoter region was cloned into pGL3-Luciferase plasmid (designated pGL3-P65-Luciferase). The construct (0.5 μg) was transfected into A549 cells together with increasing amounts of plasmid (0.1, 0.2, 0.5 mg) for ZNF24 expression. Luciferase activity was monitored 24 h later. C Schematic diagram of 4 potential ZNF24-binding sites in P65 promoter region. D ZNF24 binds to motif 3 to inhibit the activity of P65. Deletion mutants were generated as shown in C. Each of the mutant plasmids was respectively transfected into A549i cells by 1 μg/mL of Dox for ZNF24 expression. Luciferase was measured 24 h later. E ZNF24 bound to motif 3 of P65 promoter region. A549i were treated with Dox (1 μg/mL) treatment. The Flag antibody was used to precipitate ZNF24 and DNA was extracted from the precipitants. Primers flanking 295 bp region of motif 3 was used to amplify DNA. F, G Impact of ZNF24 expression on P65 mRNA expression level. A549i and Hop62i cells were treated with Dox (1 μg/mL) for 48 h. RNA was extracted for quantification of expression of designated genes through RT-qPCR. H Impact of ZNF24 expression on P65 expression in A549i and Hop62i cells. A549i and Hop62i cells were treated with Dox (1 μg/mL) for 48 h. The whole lysates were analyzed by western blot with the indicated antibodies. I-J Western blot evaluating the impact of ZNF24 on cellular localization of P65 and P50 proteins. Cytoplasmic and nuclear fractions were separated from Dox (1 μg/mL) treated A549i and Hop62i cells for 48 h, followed by western blot with indicated antibodies. K Impact of P65 expression on the colony forming ability of A549i cells. A549i cells and A549i-P65 (1000 cells) were seeded in 6-well-plates with media supplemented with Dox (1 μg/mL) for 2 weeks. Left: representative pictures. Right: statistics of colony number. A549i-P65 stands for ectopic expression of P65 in A549i cells. L Impact of P65 expression on the growth rate of A549i. A549i and A549i-P65 (1000 cells) were seeded in 96-well-plates and treated with Dox (1 μg/mL) for 4 days. Cell growth was measured by CCK8. M Impact of P65 expression on the colony forming ability in Hop62i cells. Hop62i cells and Hop62i-P65 (1000 cells) were seeded in 6-well-plates by Dox (1 μg/mL) for 2 weeks before quantification for colonies and statistics of colony number. Left: representative pictures. Right: statistics of colony number. Hop62i-P65 for re-overexpression P65 in Hop62i cells. N Impact of P65 expression on the growth rate of ability in Hop62i, Hop62i-P65 cells. Hop62i, Hop62i-P65 (1000 cells) were seeded in 96-well-plates and treated with or without Dox for 4 days. Cell growth was detected by CCK8. O Impact of P65 expression on cell cycle in A549i cells. A549i and A549i-P65 cells treated with Dox (1 μg/mL) for 48 h, followed by staining with propidium iodide (PI). Cell cycle is analyzed by flow cytometry. P Statistics of O. Bars are represented as means ± SEM of the indicated number (n) of repeats. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t-test