Figure 1.
Depletion of GABPA affects the cytoskeleton and migratory properties of MCF10A cells.
(A) Immunofluorescent images of MCF10A cells transfected with the indicated siRNA species, starved for EGF for 48 hours, stimulated with media containing EGF for 24 hours and stained with phalloidin and with Hoechst dye to visualise the actin cytoskeleton and nuclei, respectively. Red arrows – membrane protrusions, white arrowheads – subcortical actin, dashed lines – enlarged cell bodies. (B) RT-PCR quantification of the effect of siGABPA transfection on GABPA mRNA levels. Chart shows average values from three biological repeats with standard deviation. Statistical significance was determined in Student's t-test (*P<0.01). (C) Western blot analysis showing the effect of siGABPA transfection on GABPA mRNA levels. ERK2 levels are shown as a loading control. Each lane was taken from the same western blot to eliminate irrelevant lanes. (D) Quantification of the percentage of cells located on the edges of clusters exhibiting membrane protrusions upon transfection with siGAPDH (control) or siGABPA. Bars show average values from three biological repeats with standard deviations; in each repeat, three fields were scored. (E) Representative images of wounds created in monolayers of MCF10A cells transfected with the indicated siRNA species, starved for EGF for 48 hours, subsequently stimulated with media containing EGF and imaged for 15 hours. Black line marks borders of cell-free areas. (F) Cell-free areas in the images obtained as described in (E) for the siGABPA transfection were measured at hourly intervals and normalised to siGAPDH-transfected control. Shown are average values from three biological repeats with standard deviations. (G and H) MCF10A cells were transfected with the indicated siRNAs, starved for EGF for 48 hours, stimulated with media containing 20 ng/ml EGF and imaged for 24 hours. (G) Box plots show the distributions of lengths of trajectories travelled by MCF10A cells transfected with the indicated siRNA species between t = 1 h and t = 7 h of imaging. Data was obtained in four biological repeats of the experiment, and in each case ten cells were manually tracked. The green and pale yellow areas correspond to the second and third quartile of the distribution, respectively. The shaded area represents the distribution of distances covered in control siGAPDH-transfected cells. P-values were obtained in a Smirnov-Kolomogorov test (*P<0.05). (H) Distribution of the trajectories travelled by cells plotted in (G).
Figure 2.
GABPA directly regulates gene expression in MCF10A cells.
(A) Summary of the effect of siGABPA transfection on the transcriptome of MCF10A cells. Top – overlap of genes which change expression in cells depleted of GABPA (GABPA KD) with genes associated with GABPA binding regions as determined in a ChIP-seq experiment [12]. Bottom – distributions of up- and down-regulated genes in the whole dataset, or only for the overlap with ChIP-seq (GABPA CS) data. P-values were obtained in a chi square test. (B and C) Results of DAVID gene ontology analysis of lists of all genes showing a change of expression in MCF10A cells depleted of GABPA (B) or only those additionally associated with GABPA ChIP-seq regions (C). Bars indicate statistical significance of arbitrarily summarised clusters of terms (shown on the left). (D) Heatmap showing fold changes in mRNA levels of direct GABPA and ELK1 target genes (indicated on the right) coding for cytoskeleton-, migration- and adhesion-related proteins in MCF10A cells transfected with the indicated mRNA species, normalised to control (siGAPDH transfection).
Figure 3.
GABPA controls the expression of a network of cytoskeleton-related genes.
(A) A STRING-derived network of proteins encoded by all genes that exhibit a statistically significant change of expression in MCF10A cells depleted of GABPA, that are associated with regions bound by GABPA, and that belong to GO terms associated with the cytoskeleton, cell migration or adhesion as determined by DAVID analysis. Proteins are circled whose encoding genes were chosen for further analysis. (B) The effect of siGABPA transfection on the expression of genes encoding proteins highlighted in panel A (green) and two negative controls (not GABPA targets; grey). Bars show average values from three biological repeats with standard deviation. Statistical significance was determined in paired Student's t-tests (*P<0.05, **P<0.01). (C) Charts show the binding levels of GABPA to DNA regions associated with genes encoding proteins highlighted in panel A, as determined in ChIP-qPCR experiments in MCF10A cells transfected with the indicated siRNA species and starved for EGF for 48 hours. IgG immunoprecipitation indicates the level of non-specific binding. (D) ChIP-qPCR of ELK1 occupancy on regions tested in (C) and on two positive control regions (associated with CDKL3 and RFC4).
Figure 4.
Depletion of direct target genes of GABPA slows down MCF10A cell migration.
(A) Graph shows the mRNA levels of four GABPA target genes in cells transfected with the respective siRNA species. Values were normalised to control (siGAPDH transfection) and are presented on one chart for clarity. Bars represent average values from three biological repeats with standard deviation. Statistical significance was determined in Student's paired t-tests (*P<0.001). (B and C) MCF10A cells were transfected with the indicated siRNAs, starved for EGF for 48 hours, stimulated with media containing 20 ng/ml EGF and imaged for 24 hours. (B) Shown are trajectories travelled by cells in the first six hours of live imaging experiments in the presence of the siRNA species indicated above each graph (only three out of the six sets of trajectories are depicted). (C) Box plots show the distributions of lengths of trajectories travelled by MCF10A cells transfected with the indicated siRNA species between t = 1 h and t = 7 h after the addition of EGF (which corresponds to t = 0 to t = 6 h of imaging). Data was obtained in three biological repeats of the experiment, in each case ten cells were manually tracked. The green and pale yellow areas correspond to the second and third quartile of the distribution, respectively. The shaded area represents the distribution of distances covered in control siGAPDH-transfected cells. P-values were obtained in a Smirnov-Kolomogorov test (*P<0.05 ** P<0.001).