## 2 Results
### Silencing of FAM13A Inhibits Lung Cancer Cells Proliferation  
We performed a proliferation assay using two tests: Violet Proliferation Assay (VPD) and MTS. These assays measure cell proliferation indirectly. The first is based on labeling viable cells with cell tracking VPD450 dye (Figure 2A,B), and the second is based on the measurement of cellular metabolic activity. The results of VPD showed a significant reduction in A549 cells proliferation in FAM13 Ash1 (PI = 3.05 \(\pm\) 0.16) and FAM13 Ash2 (PI = 3.73 \(\pm\) 0.4) silenced cells compared to controls (CtrNT2 PI = 4.02 \(\pm\) 0.71; CtrSCR PI = 4.41 \(\pm\) 0.42), in hypoxia conditions (_p_ < 0.05) (Figure 2C). Knockdown of _FAMI3A_ significantly reduced A549 cells proliferation also in normal oxygen tension, especially measured after 96 h (FAM13 Ash1: PI = 4.09 \(\pm\) 0.29; FAM13 Ash2: PI = 4.34 \(\pm\) 0.38; CtrNT2 PI = 5.95 \(\pm\) 0.72; CtrSCR PI = 4.93 \(\pm\) 0.34). In CORL-105 cells, FAM13A silencing significantly reduced cell proliferation (FAM13 Ash1: PI = 2.4 \(\pm\) 0.04; FAM13 Ash2: PI = 2.54 \(\pm\) 0.09; CtrNT2 PI = 3.13 \(\pm\) 0.15; CtrSCR PI = 2.95 \(\pm\) 0.02), after 96 h of hypoxic cultivation. It is worth mentioning that further incubation in hypoxia conditions, decreased CORL-105 proliferation rates (measured after 120 h) and had strong impact on cells viability (data not shown). The VPD assay showed that FAM13A silencing did not affect the CORL-105 cell viability in normoxia (Figure 2D). In summary, in both cell lines decreased cell proliferation under hypoxia conditions was observed. The tendency of decreased proliferation in _FAMI3A_ depleted cancer cells was also observed in MTS assay (Figure 2E,F). Cell proliferation rates significantly decreased under hypoxia compared to normal oxygen tension (_p_ < 0.05) in both A549 and CORL-105 cells (Figure 2E,F, respectively). Thus, we demonstrated that _FAMI3A_ silencing modulates the growth of cancer cells in hypoxia conditions. Figure 1: _Cont._  
Figure 1: Generating lung cancer cell lines with FAM13A knockdown. FAM13A shRNAs (FAM13 Ash1, FAM13 Ash2) and control shRNAs (CtrNT2, CtrSCR) lentiviral particles were used to generate the stable transduction of A549 and CORL-105 cell lines. Transduction efficiency was assessed with the GFP marker. Knockdown of FAM13A mRNA and FAM13A protein was confirmed by real-time quantitative PCR and western-plot analysis. (**A**) Schematic indicating the binding site for the shRNA in the FAM13A mRNA. Two shRNA transcripts were designed using UCSC Broad Institute GPP Web Portal, against a coding sequence common to the most FAM13A isoforms with the RhoGAP functional domain. (**B**) RT-qPCR analysis of _FAM13A_ expression in A549 and CORL-105 cell lines cultured under normoxia and hypoxia conditions for 72 h. Relative expression of FAM13A mRNA was determined as the mean normalized expression of FAM13A mRNA/GUSB mRNA. The experiments were performed in triplicate and repeated three times. The data are presented as the mean \(\pm\) SD (* \(p\) < 0.05, ** \(p\) < 0.01, unpaired one-tailed _t_-test). (**C**) Western blot (WB) analysis of FAM13A expression in A549 and CORL-105 cell lines cultured under normoxia and hypoxia for 72 h. Relative FAM13A protein (117 kDa) amount was significantly decreased in FAM13A sh1/sh2 RNA cells. Strong FAM13A expression was induced in controls (CtrNT2, CtrSCR) A549 or CORL105 cells after 72 h of hypoxia (+) compared to normoxia (-). Cropped images are displayed, full-length blots are presented in Supplementary Materials Figure S4. Total protein was used for normalization. Representative stain-free total protein blots are presented. The normalized protein factors are given at bar charts (N-normoxia, H-hypoxia, bars with texture). The data are presented as the mean \(\pm\) SD (* \(p\) < 0.05, ** \(p\) < 0.01, unpaired one-tailed _t_-test). exhibited a slight decrease in the percentage of the cellular population in the S phase in normoxia compared to control cells (Figure S5).