## 3 Discussion  
The function of FAM13A in non-small cell lung cancer (NSCLC) progression under hypoxic condition is unknown. In the present study, we focused on the role of FAM13A in cellular processes such as proliferation, cell cycle, migration, invasion and apoptosis in NSCLC cells under hypoxia. All these features are relevant for tumor progression and manifestation of metastases. To this end we used A549 and CORL-105 cells with stable FAM13A knockdown. In our previous report, induction of FAM13A in lung cancer cells was confirmed in both in vitro and ex vivo models in short-term cultures after 72 h of hypoxia [6]. In the current study, significant increase in FAM13A expression was confirmed after exposure to chronic hypoxia in both cell lines (Figure 1). So far, few data are available on the FAM13A hypoxia-dependent regulation and its contribution to lung cancer progression. Studies on gene expression signature of the hypoxia response derived from several tissues (not comprising the lung) have shown a consistent increase of FAM13A expression [15]. In the literature there is only one study focused on the expression and function of FAM13A in NSCLC cells [14]. Among patients with lung adenocarcinoma and squamous cell carcinoma, increased FAM13A levels were found in tumor area of the lung. Furthermore, FAM13A mRNA level together with HIF1\(\alpha\) subunit were increased in antitumor CD4 + CD25 T effector cells in the hypoxic lung region of NSCLC patients. It was hypothesized that FAM13A together with HIF1\(\alpha\) might be associated with antitumor effector T cells in the control area surrounding the growing tumor, in which oxygen deprivation occurred [14]. Recently, [PERSON] et al. identified gene expression patterns in breast cancer cells that experienced intratumoral hypoxia in vivo compared to cells exposed to deoxygenation in vitro, with the use of unique hypoxia fate-mapping system. Set of 19 genes, including FAM13A, was induced by hypoxia in primary tumor and remained overexpressed at metastatic site in the lung, suggesting occurrence of a 'hypoxic memory'. It was suggested that hypoxia inducible genes can be used as biomarkers to identify cells at metastatic sites that have been exposed to intratumoral hypoxia [16]. We can assume that _FAM13A_ gene is a crucial member of hypoxia-response gene set in NSCLC, however our understanding of its contribution in lung cancer progression is still limited. To examine the impact of _FAM13A_ gene silencing on lung cancer cells proliferation under hypoxia, we performed series of tests, including VPD assay, MTS test and also BrdU assay. The results of VPD assay show a significant reduction in cell proliferation after FAM13A knockdown in A549 cells cultured in normal oxygen tension, as well as hypoxia, while the effect of FAM13A downregulation on CORL-105 cells was observed only after 96 h exposition to hypoxia. It is worth mentioning that hypoxia decreased cell proliferation in both cell lines, regardless of FAM13A knockdown. We also observed that oxygen deprivation had a stronger impact on CORL-105 cells growth (data not shown), compared to A549 cells. In MTS assay, negative effect of FAM13A shRNA on A549 and CORL-105 cell proliferation was observed already after 24 h and 48 h, especially under hypoxia conditions. The observed differences between VPD450 and MTS assays is probably due to their underlying methodology. In the current study, MTS method appeared to be more sensitive for cell proliferation assessment. Moreover, it was confirmed that VPD450 tracking of cancer cell division could be difficult [17]. Cancer cells are less homogeneous in size and protein content and consequently show broader peaks in histograms, which makes the quantitative population analyses more difficult [17]. We also observed thisphenomenon during experiments. The third method used for proliferation assessment in the current study (BrdU assay) confirmed that FAM13A inhibition induced S phase cell cycle arrest in lung cancer tumor cells under hypoxia conditions.