Fig 2: DDX4 expression increased cell motility and metastatic phenotypes.a–c Cell motility analysis of DDX4-OE and -KO lines in H69AR cells. Each cell line was time-lapse-imaged every 30 min for ~48 h. a Each colored line indicates a track of each cellular movement. The longer line suggests the larger movement. b Cell motility analysis of a representative cell from each cell line. A distance of travel at each time point of time-lapse imaging was calculated and compared between DDX4-OE and LUC and DDX4-KO and SC. c Travel distances per 30 min of all cells in the field were determined by summating and averaging the distance value at each time point for each cell in the field. () indicates the total number of cells analyzed. d Immunoblot results of E-cadherin and Galectin-3. The signal intensity of each protein was normalized by that of Actin to obtain the relative value shown in the graph. n = 4 biologically independent experiments. e, f Immunofluorescence of E-cadherin (e) and Galectin-3 (f) (magenta). Tubulin (green) and DNA (blue) were counterstained. The level of Galectin-3 level was highly heterogenic even within a population of DDX4-OE cells (arrows). g RT-qPCR results of snail and twist. The expression level of each gene was normalized by that of 36b4, a housekeeping gene. n = 3 biologically independent experiments. An unpaired t test was used for all graphs. **P < 0.01; ***P < 0.001; ****P < 0.0001. Columns represent means ± SD or SEM. All scale bars = 5 µm.

Fig 3: DDX4 globally changes protein expression in H69AR cells.a Multiplex cytokine profiling shows DDX4-OE has increased cytokine expression compared to the DDX4-KO cell line at a natural state in H69AR cells. Each value was normalized to that of the corresponding control line for each cytokine. b Multiplex cytokine profiling was performed before and after Cisplatin treatment (10 µM Cisplatin treatment for 20 h) for each cell line of H69AR cells. Each value of the “after” treatment group was normalized to that of the “before” treatment group to obtain the relative expression level of each cytokine after Cisplatin treatment. c RT-qPCR results of cytokine-related genes. The expression level of each gene was normalized by that of 36b4, a housekeeping gene to obtain a relative expression level. The graphs are the average of three replicates. **P < 0.01; ***P < 0.001; ****P < 0.0001. Columns represent means ± SD or SEM. d–g Comparative label-free quantitative proteomic profiling of the DDX4-OE and control (LUC) cell lines. d Principal component analysis (PCA) of total protein abundance data collected from DDX4 and LUC samples. Data represents the close clustering of protein abundance of each replicate under the same group, however, showed variability between DDX4 and LUC samples. e Volcano plot of fold change versus q-value of the total of 4740 proteins quantified from DDX4 and LUC cell lines. Red and green circles represent the significant (q < 0.05) up- and downregulated proteins. Gray circles (q = 0.05) are nonsignificant and below the threshold of fold expression. Reactome (f) and KEGG (g) pathway analyses of the significantly increased (red bars) proteins in DDX4-OE vs LUC. h Heatmap analysis of the DNA repair and immune system-associated proteins that were significantly increased in abundance in DDX4-OE cell lines. Expression of the proteins with asterisk (*) was validated by immunoblot and/or by immunofluorescence.

Fig 4: DDX4 promotes resistance to drug treatment and DNA damage.a, b Cell Viability (CTG) assay results of H69AR DDX4-OE (a) and -KO (b) cells. Each cell line was treated with various doses of drugs indicated on the X axis for 3 days and the level of viability was measured. Graphs in (a, b), n = 3 biologically independent experiments. c–e H69AR cells were incubated with the final 10 µM of Cisplatin for 24 hours and then fixed. Phosphorylated ?H2AX (magenta) levels were measured using immunofluorescence with levels being lower and higher in DDX4-OE and -KO, respectively. The graphs in (d) indicate the quantitative statistical analyses of the phospho-?H2AX signal before and after cisplatin treatment and the graphs in (e) indicate the level of signal increase after cisplatin treatment in each cell line. Graphs in (d, e), n = 8 ROIs; n = 3 biologically independent experiments. DNA, blue. Scale bar = 5 µm. Error bars shorter than the symbols in (a, b) are not shown. Two-way ANOVA was used for the graphs in (a) and (b); unpaired t test was used for the graphs in (d) and (e). **P < 0.01; ***P < 0.001; ****P < 0.0001. Columns represent means ± SD or SEM.

Fig 5: DDX4 is expressed in several SCLC cell lines.a RT-PCR results of four SCLC cell lines for three gene products (ddx4, piwil2, and 36b4), with negative controls (lacking RT enzyme). b DDX4 immunoblot of several SCLC cell lines. Actin was used as a loading standard. c DDX4 immunofluorescence results in five SCLC cells counterstained with tubulin and DNA. Arrows indicate the mitotic spindle. Images were taken at a time with the same experimental and laser condition for all cell lines. d Time-lapse images of DDX4-OE cell lines show random transformation into the mesenchymal state (white arrows) and increased cellular projections (black arrows), while DDX4-KO cells stayed more rounded. LUC cells are shown as a control. Scale bar = 5 µm.