Figure 7.

Knockout of IFGBP2 partially recapitulates the phenotype of RHOA loss. (A) Analysis of the supernatant from K562 C8 and KU812 C7 knockout (KO) cells when compared with the respective mock control (MC) cells, showed a dramatic reduction in IGFBP2 protein levels in the KO cell culture medium. (B) In migration and adhesion analysis, K562 KO C8 cells showed a reduction in both phenotypes compared with MC cells. When exogenous, recombinant IGFBP2 (rIGFBP2) was added to the culture medium, there was a significant increase in both phenotypes in the KO C8 cells. (C, D) CRISPR knockout of IGFBP2 in K562 cells generated two clones, C5 and C11 (C), which when subjected to migration and adhesion assays (D) showed reduced levels compared with MC cells. (E) When these cells were xenografted into NSG hosts, mice receiving KO clones C5 and C11 showed an increased survival compared with MC-engrafted mice. (F) This result was consistent with luminescence intensity in the mice after 28 days, which showed that the tumor burden was significantly reduced in the mice grafted with the KO C5 cells compared to that of mice engrafted with MC cells. White blood cell count at the time of sacrifice was also reduced in the mice grafted with KO C5 cells. The scale bars in (B) and (D) represent 400 µm, with the magnification for the images in (B) and (D) being the same. Differences between the KO and MC cells were evaluated using the Student t test. Pairwise comparisons are indicated by the horizontal lines. **P≤0.001, ***P≤0.0001, ****P ≤0.00001. CM: culture medium; WBC: white blood cells.