Fig. 3

ZNF24 induces cell cycle arrest through NF-κB signaling pathways. A Heatmap representation of impact of ZNF24 expression on transcriptome of A549i cells. A549i cells were treated with 1 μg /mL of Dox for 48 h. RNAs were isolated and subjected to RNA-sequencing. Alteration in expression level were presented in Heatmap. B KEGG analysis of upregulated genes in A549 cell in response to ZNF24 knockdown. C, D and E RT-qPCR analysis of impact of ZNF24 on expression of NF-κB target genes. C. A549i was treated with Dox (1 μg/mL) for 48 h. IL-6, IL-1β, TNFα expression was quantified through RT-qPCR analysis. D. data on Hop62i cells. E. RNA was extracted from EKVX-shZNF24 cells (EKVX-shGFP cell served as control). Expression of the IL-6, IL-1β, TNFα was quantified through RT-qPCR. F, G and H Western blot analysis of impact of ZNF24 expression on NF-κB signaling pathway. F. A549i cells were treated with Dox (1 μg/mL) for 48 h. The cells were harvested for immunoblot analysis with indicated antibodies. G. data on Hop62i cells. H. data on EKVX-shZNF24. I Impact of ZNF24 expression on NF-κB reporter in A549i cells. A549i cells were transfected with the NF-κB reporter. Luciferase assays were performed 12, 24 and 48 h after Dox (1 μg/mL) administration. J Knock-down of ZNF24 sensitized EKVX cells to NF-κB inhibition. EKVX-shGFP, EKVX-shZNF24 and EKVX-sh/ + ZNF24 cells (1000) seeded in six-well plates, and treated with DMSO or NF-κB inhibitor (BAY11-7082, 2 μM) for 2 weeks. Cells were fixed and stained with 0.5% crystal violet in methyl alcohol. K Statistics of (J). L Impact of NF-κB inhibitor on cell cycling. EKVX-shGFP, EKVX-shZNF24 and EKVX-sh/ + ZNF24 cells seeded in six-well plates and treated with DMSO or NF-κB inhibitor (BAY11-7082, 2 μM) for 48 h. Cells were stained with propidium iodide (PI) and analyzed through FACS. M Statistics of L. 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