## 2 Results
### Knockdown of FAM13A Suppresses Lung Cancer Cells Migration  
To elucidate the influence of _FAM13A_ gene on lung cancer cells migration we applied the wound healing assay. Figures 3 and 4 show representative images taken during A549 and CORL-105 cells monitoring. The wound healing assay revealed that the closing rate of scratch wounds was significantly decreased in hypoxia compared to cells cultured under normal oxygen conditions in both cell lines (\(p<0.05\), Figures 3 and 4). The percentage of wound closure (WC) for control cells in normal atmospheric conditions reached 95-100%, whereas for cells exposed to 1% O\({}_{2}\) for 96 h percentages were significantly decreased. The migration of A549 FAM13A sh1 RNA and sh2 RNA cells was significantly suppressed in both oxygen tensions, especially after 72 and 96 h (\(p<0.001\) in normoxia, \(p<0.01\) after hypoxia). However, stronger reduction in cell migration to the wound area was observed in normoxia (Figure 3B). All results obtained for A549 cells in normal oxygen concentration were confirmed using wound healing monitoring system (JuLI FL) (Figure S2). Figure 2: Silencing of FAM13A decreases lung cancer cell proliferation. Decreased A549 and CORL-105 cell proliferation after _FAM13A_ gene knockdown was assessed using Violet Proliferation Dye (VPD450) and MTS test. After VPD450 staining the cells were cultured for 4–5 days in hypoxia and normal oxygen tension. The fluorescence intensity was measure by flow cytometry. During MTS test the cells were cultured in 96-well plates and cultured for two days in normal and hypoxic atmosphere. After 24/48 h the CellTitter 96\({}^{\text{th}}\) AQueous One Solution Reagent was added and the absorbance was read at 490 nm with a microplate reader. (**A**) Representative images of A549 cells stained with VPD450. Fluorescence intensity was measured from Day 1 (0 time point) to 5 (96 h time point) by flow cytometry (Flow Sight\({}^{\text{th}}\), Amnis, Seattle, WA, USA). Each cell is represented by a row of three images acquired simultaneously in flow, from left to right: channel 1—brightfield (gray), channel 2—fluorescence from GFP (transduced cells green) and channel 7—fluorescence from dye VPD450 applied to viable cells (purple). (**B**) Representative histogram plots displaying changes in VPD450 fluorescence intensity of A549 cells upon FAM13A knockdown (FAM13 Ash1, red) and control (CtrNT2, green), in three time points: 0 h, 48 h, 96 h. (**C**,**D**) Cell growth kinetics was determined by proliferation index (PI) of FAM13 Ash1 (red), FAM13 Ash2 (green) and CtrNT2 (black), CtrSCR (grey) control cells in A549 (**C**) and CORL-105 (**D**). The difference in PI ratios at 24 h, 48 h, 72 h between FAM13A depleted cells and control cells was evaluated under normoxia (**left** panel) and hypoxia (**right** panel). A two-way ANOVA with Bonferroni posttest was used for statistical analysis. *\(p<0.05\) indicates a significant difference, as marked by an asterisk. The experiments were performed in triplicate and repeated three times. (**E**,**F**) Cell proliferation assessed by MTS assay. The results are given for A549 (E) and CORL-105 cells (**F**). Cell proliferation rate of FAM13 Ash1 (red), FAM13 Ash2 (green) cells and control cells (mean combined for CtrNT2 and SCR, black) was determined by CellTitter\({}^{\text{TM}}\) AQueueous assay (MTS). The difference in cell proliferation rate of FAM13A depleted cells compared to control cells was evaluated under normoxia (bars without filling) or hypoxia (bars with texture) conditions. Data are means \(\pm\) SEM (n = 4), *\(p<0.05\), **\(p<0.01\)**, two-way ANOVA. Figure 3: Knockdown of FAM13A suppresses A549 lung cancer cell migration. The migration of A549 cells with FAM13A knockdown and controls was monitored for 96 h under normoxia and hypoxia conditions. The inserts were used to generate a defined gap for measuring the migratory rates. Cell suspension in low serum media was added to the open end at the top of the insert. The cells were incubated overnight to form the monolayer.