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. 2016 Nov 8;291(52):26922–26933. doi: 10.1074/jbc.M116.752063

FIGURE 5.

FIGURE 5.

Generation of exchange Ewing cell lines. A, EGFP (selection marker) sorting of pInducer21 vector-transduced cells. A673 cells with 3xFLAG, 3xFLAG-EWS-FLI1, or 3xFLAG-K380R mutant were sorted for the same low EGFP expression. After sorting, all cell pools were analyzed by FACS for EGFP and plotted for corresponding mean fluorescence intensity (MFI). B, shEF sequences of shRNA against the 3′-UTR of EWS-FLI1 are sufficient to down-regulate EWS-FLI1 protein. Shown is a Western blot of A673 cells with shEF constructs 1 and 2 after incubation with 0.1 ng/μl doxycycline for 48 h. C, inducible EWS-FLI1 exchange cell lines. Double-transduced A673 cells were incubated with 0.1 ng/μl doxycycline for 48 h. Endogenous and exogenous EWS-FLI1 protein levels were analyzed by Western blotting using anti-FLI1 antibody, and mRNA levels were analyzed by quantitative RT-PCR (D) with n = 4; error bars represent 95% confidence intervals. dox, doxycycline; EF, EWS-FLI1; ctr, control.