Figure 4. Cellular experiments revealed differing proliferation and invasion abilities in the high- and low-score groups. (A–C) Morphology of GBM spheres in the high and low PHQ-9/GAD-7 groups. The diameters of the GBM spheres in both groups increased gradually on days 3, 5 and 7, but those in the high group grew significantly faster (high group, n = 3; low group, n = 3). The number of spheres that developed from single cells was significantly greater in the high group than in the low group (P = 0.0216, n = 5). Data are presented as the mean ± standard error, and Student's t-test was chosen as the statistical method; *, P < 0.05. Scale bar: 200 μm. (D, E) The proliferation capacities of primary GBM cells from the different groups were evaluated with an EdU assay. Primary GBM cells from the low group (top) and high group (bottom) were stained with EdU (red, middle) and counterstained with Hoechst (blue, left). The ratio of EdU-positive cells to the total number of Hoechst-labeled cells is presented as the mean ± standard error, and Student's t-test was chosen as the statistical method; ***, P < 0.001, n = 3. Scale bar: 20 μm. (F) Wound healing assay of primary GBM cells from the high and low PHQ-9/GAD-7 groups. A six-well plate was scratched, and primary cells from the different groups were photographed at 0 and 24 h.