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Figure 3 | Genome Medicine

Figure 3

From: A genome scale overexpression screen to reveal drug activity in human cells

Figure 3

RHOXF2 overexpression in HEK293_M2 cells conferred resistance to a wide variety of DNA damaging agents. (a) In HEK293_M2 cells, RHOXF2 overexpression rescued cisplatin and mitomycin C toxicity. Stable RHOXF2-expressing cells were cultured in the presence of an increasing concentration of cisplatin or mitomycin C and their growth was compared to stable cells with the empty vector PB-TGcMV-Neo. The effect of RHOXF2 expression on cell viability was measured two days after drug exposure and compared to cells cultured in the absence of drug as a 100% viability control. P < 0.05, TukeyHSD test: *significant difference between RHOXF2 vector with and without doxycycline (dox); **significant difference between RHOXF2 vector with doxycycline and empty vector with doxycycline. Error bars represent standard error of the mean (n = 4). (b) Reduction in the number of γ-H2A.X foci by RHOXF2 overexpression in HEK293_M2 cells. Cells expressing or not RHOXF2 were treated with 50 nM of mitomycin C (approximately IC10) for 2 days and stained for the presence of γ-H2A.X foci. Following RHOXF2 overexpression, the number of γ-H2A.X foci per nucleus dropped from 20.71 ± 1.49 to 15.73 ± 0.92 (24% rescue). Nuclear DNA was stained with DAPI. Quantification of γ-H2A.X foci in >250 cells from triplicate experiments. Scale bar: 10 μm. (c) In HEK293_M2 cells, RHOXF2 overexpression conferred resistance to various DNA damaging agents. RHOXF2 drug suppression capacity was tested using DNA damaging agents with various modes of action as previously described for cisplatin and mitomycin C. (d) Gene Ontology analysis of upregulated genes in HEK293_M2 cells. Cells expressing or not RHOXF2 were treated with 40 nM of mitomycin C for 2 days. Based on their P-values, the most enriched biological processes are shown.

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