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. 2014 Dec 8;207(5):627–642. doi: 10.1083/jcb.201405105

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Figure 6. — DLC1-4A mutant lacks the Rho-GAP and tumor suppressor activities of DLC1-WT. (A) Anchorage-independent growth of H358 cells stably transfected with GFP, GFP-DLC1-WT, GFP-DLC1-4A, or GFP-DLC1-R718A mutant. Bar, 2 mm. (B) Quantification of agar colonies (>0.4 mm) in the indicated groups from three independent experiments. (C) Relative cell migration rate and wound density (%) were calculated by IncuCyte assay. Multiple scratch wounds were made in confluent cells stably transfected with DLC1 constructs. Scratch wounds were allowed to heal for 24 h, and the cell migration rate was plotted in the graph. (D) RhoGTP (top) and total Rho (bottom) in stably transfected H358 cells expressing the indicated DLC1 constructs. (E) Immunoblots for RhoGTP and total Rho as in D were quantified by densitometry, and the ratio of RhoGTP to total Rho was normalized. Statistical analysis demonstrated a significant decrease (P < 0.001) in RhoGTP/total Rho in DLC1-WT–transfected cells compared to GFP control, DLC1-4A–transfected, or DLC1-R718A–transfected cells. The graph represent means ± SD of three independent experiments. (F) Xenographic tumors from nude mice after injecting H358 cells, which stably expressed GFP, DLC1-WT, DLC1-4A, or DLC1-R718A mutant. Tumors were excised after 6 wk, and representative tumors are shown. (G) Average tumor weight ± SD (g) for each group is shown in graph. Unlike the stable DLC1-WT–positive control in H358 cells, the stable DLC1-4A mutant is as defective as a “GAP-dead” DLC1-R718A mutant for regulating anchorage-independent growth, cell migration rate, RhoGTP, and xenographic tumor growth in nude mice. Error bars indicate ± SD.