Roles of IGFBP-3 in cell migration and growth in an endophytic tongue squamous cell carcinoma cell line

Figures

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  Figure 1. Changes in gene expression profile following IGFBP-3 knockdown in SAS-Fucci cells. (a) TCGA data-based Kaplan–Meier analyses for overall survival in patients with head and neck squamous cell carcinoma with either high (n = 25) or low (n = 495) levels of IGFBP3 mRNA. A cohort is separated according to Z-scores relative to diploid samples. p-value; log-rank test. (b, c) IGFBP3 mRNA levels by qPCR (b) and IGFBP-3 and β-actin protein levels by western blotting (c). Cells were transfected with either non-targeted control (Cont) or IGFBP3 siRNA #1 (48 h or 72 h). Total mRNA or cell lysate was extracted either 48 h or 72 h after siRNA treatment. Means ± S.D. (n = 3 independent experiments). Uncropped blots for (c) are presented in Supplementary Fig. S7. (d) Hierarchical cluster analysis of differentially expressed genes between control (Cont) and IGFBP-3-knockdown (IGFBP3 siRNA #1, 48 or 72 h) cells using the RNA-seq data (n = 2 per each group). Red and blue colors on the heat map represent up- and downregulation, respectively, of the indicated gene. (e, f) GO analysis using the gene set significantly downregulated by IGFBP-3 knockdown in RNA-Seq results from control and IGFBP3…

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  Figure 2. Reduced cell migration by IGFBP-3 knockdown in multiple assays. (a) Experimental flow to analyze cell migration. Twenty-four hours after cells were seeded, they were transfected with siRNA for either 48 or 72 h before the indicated experiments were performed. (b) Representative images of single-cell tracking during 10 h-observation (left) and quantification of average distance (right) during 10 h using SAS-Fucci cells 48 and 72 h after transfection with either non-targeted control (Cont) or IGFBP3 siRNA #1. Individual colored lines indicate each cell track traced by the center of the nucleus during the observation period. Each average distance is represented as a box and whisker plot showing outliers, distribution intervals, interquartile range (box), and median. Three independent experiments were performed and representatives are shown. (c) Representative images (left) and quantitative analysis (right) of wound healing assays using non-target control (Cont) and IGFBP-3 knockdown (IGFBP3 #1) cells. Images were acquired by time-lapse imaging and time after wounding is shown. Means ± S.D. (n = 3 independent experiments). Significant difference was detected at all the indicated…

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  Figure 3. Non-significant effect of cell cycle on cell migration by IGFBP-3 knockdown. (a) Representatives of single-cell tracking during 10-h observation merged with fluorescent images in non-target control (Cont) and IGFBP-3 knockdown (IGFBP3 siRNA #1) cells. Red, orange, and green cells are in G1, early S, and S/G2/M phases, respectively. Rounded green cells are in mitosis. White arrows and lines indicate the position of the traced cell at the indicated timepoints and a cell track from the start of the observation to the indicated timepoints, respectively. The cell traced at each timepoint is cropped and presented in the panel on the right. The cell track during the observation is shown as a blue line. Time elapsed after the start of observation is shown (hours: minutes). (b) Quantification of the change in velocity of each cell in G1, early S, and S/G2 phases using non-target control (Cont) and IGFBP-3 knockdown (IGFBP3 siRNA #1) cells. Each line indicates the change in velocity of a single cell. Velocity was calculated based on distance traveled over 30 min. Individual lines for 62–73 cells are shown as representatives. (c) Quantification of the change in velocity of each cell during…

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  Figure 4. Minimal impact of secreted IGFBP-3 on cell migration. (a) Concentration of secreted IGFBP-3 determined by ELISA. SAS-Fucci cells were transfected with non-targeted control (Cont) or IGFBP3 siRNA #1 for 48 h (left) and treated under either normoxia or hypoxia (right) for 24 h. After each treatment, conditioned media were collected and applied for ELISA. Means ± S.D. (n = 3 independent experiments). (b) Representative anti-IGFBP-3 immunofluorescence images of control (Cont) and IGFBP-3 knockdown (IGFBP3 siRNA #1) cells. Scale bar: 10 μm. (c) Representative images of individual cell tracks (top) and average distance traveled (bottom) by IGFBP-3 knockdown (IGFBP3 siRNA #1) and control (Cont) cells with or without recombinant human IGFBP-3 (rhIGFBP-3) derived from mouse or human cells and normoxic- or hypoxic-conditioned medium (CM). rhIGFBP-3 or CM was added 48 h after siRNA treatment, with which cells were pre-treated for 90 min before time-lapse imaging. Each colored line indicates individual cell tracks during the observation period. Each average distance is represented as a box-whisker plot showing outliers, distribution intervals, interquartile range (box), and median. Cell…

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  Figure 5. Association of ERK and AKT signaling modified by IGFBP-3 depletion with cell migration. (a, b) Western blotting (top) for active MAPK/ERK, phosphorylated AKT (p-AKT), AKT, and β-actin, and relative levels of active MAPK/ERK and p-AKT in control (Cont) and IGFBP-3 knockdown (IGFBP3 #1) cells either treated with MEK (MEKi; PD98059, 20 μM, (a) and AKT (AKTi; MK-2206, 5 μM, (b) inhibitors or left untreated. Cells were incubated with siRNA for 48 h. MEK and AKT inhibitors were added 90 and 30 min before cell lysates were extracted, respectively. Uncropped blots are presented in Supplementary Fig. S7. Active MAPK/ERK and p-AKT protein levels were normalized to β-actin and total AKT protein levels, respectively. Means ± S.D. (n = 3 independent experiments). (c) Average distance traveled by control (Cont) and IGFBP-3 knockdown (IGFBP3) cells with or without MEK (MEKi) or AKT (AKTi) inhibitor. Each average distance is represented as a box and whisker plot showing outliers, distribution intervals, interquartile range (box), and median. Each group contains 80 cells. Two or three independent experiments were performed. *p < 0.05, **p < 0.01; one-way ANOVA with Tukey’s multiple comparison…

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  Figure 6. Inhibition of cell growth with prolongation of G1 phase by IGFBP-3 knockdown. (a) Quantitative analysis of cell proliferation assay using non-target control (Cont) and IGFBP-3 knockdown (IGFBP3 siRNA #1) cells. Cell Counting Kit-8 assay was performed each time after siRNA treatment and relative absorbance was plotted. Means ± S.D. (n = 3 independent experiments). (b, c) Representative images and quantification of Fucci fluorescence in SAS-Fucci cells transfected with non-targeted control (Cont) and IGFBP3 siRNA #1 at 48 (b: top) and 72 h (b: bottom). Means ± S.D. (n = 3 independent experiments). (d) Pedigrees (top) and duration of red phase (bottom) for SAS-Fucci cells transfected with either IGFBP3 siRNA #1 or non-targeted control siRNA (Cont). Cells were traced between 0 and 48 h after siRNA treatment for 48 h. Red cells were sorted from the result of pedigree assay, and the duration of the red phase was measured from the start of the red phase to its end. Each duration is represented as a box and whisker plot showing outliers, distribution intervals, interquartile range, and median. (e, f) Representative histograms (upper) and quantification (bottom) of DNA content (e) and EdU…