YB‐1 overexpression induces cell cycle alterations, scattering and morphology changes. (A) Cell growth of SPC212 cells stably overexpressing YB‐1 (SPC212^YB1‐s) or the empty vector (SPC212^VC‐s) after 96 h. Data are shown as mean ± SEM of 3 biological replicates performed in triplicate. (B) Cells were subjected to videomicroscopy over 48 h with 10 min intervals. Cell fate maps indicate interphase (gray) and M‐phase duration (black). Cells that died over the 48‐h period are indicated by the bar not reaching the end of the graph. Each bar represents one single cell. (C) M‐phase lengths and (D) doubling times of single cells from videos in (B). Each dot represents one single cell. The horizontal lines indicate the mean. (E) Quantification of colony formation assays. (F) Representative pictures of colony formation assays. Scale bar: 100 μm. (G) Distance to the nearest neighbor of > 160 cells and (H) aspect ratio of > 20 single cells, derived from colony formation assays. Bars show mean ± SEM. Student's T‐test, *P < 0.05, **P < 0.01, ***P < 0.001.

YB‐1 overexpression increases cell migration. (A) Migrated distance of SPC212 cells stably overexpressing YB‐1 (SPC212^YB1‐s) or the empty vector (SPC212^VC‐s) in wound healing assays. Data is shown as mean ± SEM of three biological replicates. (B) Cumulative migrated distance of single cells over 24 h, quantified by manual single cell tracking. Each dot represents one single cell. The horizontal lines indicate the mean. (C) Average speed, (D) mean squared displacement (MSD) and (E) origin plots of tracked cells (N = 70) were calculated by DiPer and are shown as mean ± SEM. Student's T‐test (with Holm‐Sidak correction in A), ***P < 0.001.

Doxycycline‐inducible YB‐1 overexpression increases cell migration. (A) Representative pictures of SPC212^YB1‐i cells in a colony formation assay, with or without 100 ng·mL⁻¹ doxycycline (dox, Co). Scale bar: 100 μm. Experiments were performed twice in triplicates. (B) Distance to the nearest neighbor of > 120 cells derived from colony formation assays. Data are shown as mean ± SEM. (C) Percentage of wound closure in a wound healing assay with or without 100 ng·mL⁻¹ doxycycline (dox). Data are shown as mean ± SEM of 4 biological replicates. (D) Representative pictures of fli1a:EGFP transgenic zebrafish without (Co) and with 100 μg·mL⁻¹ doxycycline (+dox) 2 days post injection (dpi) with SPC212^YB1‐I,RFP cells. The injection site is indicated by the white arrow. Scale bar: 500 μm. Experiments were performed twice. (E) Quantification of tumor cells present in the tail treated as indicated after 1 and 2 dpi. Each dot represents one fish. The horizontal lines indicate the mean. (F) Percentage of fish (N = 73 for Co and N = 78 for dox) with no cells (gray), no extravascular cells (blue), < 20% extravascular cells (orange) and > 20% extravascular cells (red) in the tail at 2 dpi. (G) Representative pictures of SPC212^YB1‐i spheroids and GFP‐expressing LEC^GFP cells and quantification (mean ± SEM) of the spheroid/gap ratio with 100 ng·mL⁻¹ doxycycline (+dox) in relation to the normalized control (Co) after 2 and 4 h. The dashed line marks the value of the normalized control. 10–15 spheroids per group were analyzed. The white dotted circles in the pictures represent the outline of the respective spheroid. Scale bar: 200 μm. Student's T‐test (with Holm‐Sidak correction in C), *P < 0.05, **P < 0.01, ***P < 0.001.

YB‐1 levels directly affect PM cell migration. Cells were transfected with 5 nM of si‐YB1^CDS (si‐CDS), si‐YB1^UTR (si‐UTR) or control siRNA (Co) and on the next day treated with 100 ng·mL⁻¹ doxycycline (+dox). (A) Log2 fold change of YBX1 mRNA levels after 48 h. Each dot represents the mean of one cell line, derived from 3 biological replicates performed in duplicates. Data are shown as mean ± SEM. (B) Representative pictures and YB‐1 protein levels relative to control‐siRNA‐transfected cells (Co) derived from densitometric analysis of western blots (N = 3). Data are shown as mean ± SEM. (C) Cumulative migrated distance of single cells after siRNA transfection and treatment with doxycycline (+dox) as indicated within 72 h. Quantification was performed using manual single cell tracking in imagej. Each dot represents one cell and the horizontal lines indicate the mean. ANOVA and Tukey's multiple comparisons test. **P < 0.01, ***P < 0.001.

YB‐1 regulates the expression of EGFR and snail. Expression levels of target gene mRNA 48 h after transfection with 5 nm of (A) si‐YB1^CDS and (B) si‐YB1^UTR, relative to control siRNA transfected cells (Co). Each dot represents the mean of one cell line, derived from three biological replicates performed in duplicates. Data are shown as mean ± SEM. Blue bars indicate a mean log2 fold change > 1. (C) Expression levels of target gene mRNA 48 h after transfection with 5 nm of si‐YB1^CDS (si‐CDS) or si‐YB1^UTR (si‐UTR) and treatment with 100 ng·mL⁻¹ doxycycline (+dox), relative to control siRNA‐transfected cells (Co). Each dot represents the mean of one cell line, derived from three biological replicates performed in duplicates. Data are shown as mean ± SEM. (D) EGFR and (E) snail protein levels relative to control‐siRNA‐transfected cells (Co) derived from densitometric analysis of western blots (N = 3), 48 h after transfection with 5 nm siRNA and treatment with or without 100 ng·mL⁻¹ doxycycline at the time of transfection as indicated. Each dot represents the mean of one cell line. Data is shown as mean ± SEM. (F) Representative pictures of the western blot.

Snail but not EGFR inhibition prevents YB‐1‐induced PM cell migration. (A) Cells were exposed to 10 μm erlotinib (Erlo) or DMSO (Co) and after 24 h treated with 100 ng·mL⁻¹ doxycycline (+dox) as indicated. Cells were transfected with 10 nm (15 nm for Ren^YB1‐i) of (B) EGFR‐specific (si‐EGFR), (C) SNAI1‐specific (si‐SNAI1) or control (si‐Co) siRNA and on the next day treated with 100 ng·mL⁻¹ doxycycline. Cumulative migrated distance was measured via live cell videomicroscopy for 48 h. Experiments were performed two times in quadruplicates. Quantification was performed using manual single cell tracking of > 20 cells per group in imagej. Each dot represents one cell, and the horizontal lines indicate the mean. ANOVA and Tukey's multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant.