Tumor-associated FLCN mutations fail to delay cell cycle progression.

(A) Expression of exogenous FLCN in UOK257 cells infected with retroviruses harboring empty vector control, FLCN WT, FLCN ?F157, FLCN K508R, or FLCN 1–469. (B) Cell cycle profiles of the UOK257 cells and the isogenic cell lines expressing empty vector control, FLCN WT, FLCN ΔF157, FLCN K508R, or FLCN 1–469, demonstrating the amount of cells in the G1, S and G2/M stages as determined by PI staining. The experiment was repeated three times (n = 3), but only one representative image of the cell cycle profile, 12 hours after release from thymidine block, is shown. (C) Tumor-associated FLCN truncated mutant 1–469 fails to delay cell cycle progression. FLCN deficient UOK257 cells (black dashed line) and their isogenic counterpart reconstituted with FLCN WT (red line) or FLCN 1–469 mutant (blue line) were synchronized by double thymidine block and released. Progression through the cell cycle was analyzed by FACS. Vertical axis indicates the percentage of all cells registering in the specific phase of cell cycle. Horizontal axis indicates hours post thymidine release. (D) Tumor-associated FLCN missense mutant K508R fails to delay cell cycle progression. Same as in (C), except that the reconstituted mutant is FLCN K508R (green line). (E) Tumor-associated FLCN deletion mutant ΔF157 fails to delay cell cycle progression. Same as in (C) and (D), except that the reconstituted mutant is FLCN ?F157 (purple line). The average of three experiments is presented (n = 3) in each panel; * indicates a significant difference between UOK257+ vector only compared to UOK257+ FLCN WT; # indicates a difference between UOK257 FLCN WT and the UOK257+ tumor-associated mutant (1–469, K508R, or ?F157); $ indicates a difference between UOK257+ vector only cells and the UOK257 cells expressing a tumor-associated mutant (One way ANOVA, Tukey's post-test, p<0.05). Error bars correspond to SEM.