Figure 6. Tumor sphere formation in Matrigel after GPER1 knockdown in tumor spheres developed from HeLa cervical carcinoma cells. Day 3 after transfection is defined as day 0. The main sphere is the largest tumor sphere forming from day 0. Sprouts start from main sphere to periphery. Subsidiary colonies are smaller colonies formed without contact with a main sphere. Evidence of successful knockdown of GPER1 on day eight using qPCR. Relative gene expression of GPER1 in HeLa cells after GPER1 knockdown. (A). Control spheroid (B): arrow 1 = main sphere, arrow 2 = sprouts, arrow 3 = subsidiary colonies; spheroid after GPER1 knockdown (C). Size of main sphere (D), size of sprouts (E), number of sprouts (F), length of sprouts (G), size of subsidiary colonies (H), number of subsidiary colonies (I). Mean with standard error, n=2, one-tailed t-test, normalized to RPLP0 (A). Median, n=3, Mann-Whitney U test (D, E, G, H). Mean with standard error, n=3, two-tailed t-tests (F, I). *p<0.05, **p<0.01 and ***p<0.001.