Fig. 8.

Disruption of c-Jun activity does not prevent MOMIPP-induced methuosis. A stable U251 cell line capable of doxycycline-inducible expression of c-JunDN was developed as described in the Methods. a The expression of HA-tagged c-JunDN was verified by immunoblot analysis of cells collected after 48 h in standard culture medium or medium with 1 μg/ml doxycycline (Dox). α-Tubulin served as a loading control. b U251-c-JunDN cells were cultured for 24 h in medium with or without Dox, and expression of the indicated genes was quantified by RT-PCR as described in the Methods. The expression fold change is the value: 2^-ΔΔCt, calculated from three cultures (mean ± SD). c U251-c-JunDN cells were cultured for 24 h in the presence or absence of Dox. 10 μM MOMIPP was then added and cell morphology was assessed after 48 h. d U251-c-JunDN cells were cultured for 24 h in the presence or absence of Dox and then treated for 48 h with indicated concentrations of MOMIPP (in the presence of absence of Dox). Cell viability was determined with the CellTiterGlo® assay. e U251-c-JunDN cells were cultured for 24 h in the presence or absence of Dox. Incubation was then continued for an additional 24 h with or without 10 μM MOMIPP, as indicated. Immunoblot analysis was performed as described in the Methods to detect phosphorylated and total JNK1/2, Bcl-2 and Bcl-xL. Similar results were obtained in three separate experiments