![Figure 3. Loss-of-function Src mutants or shRNA knock-down prevent ERK1/2 activation and sensitize MM cells to UCN-01, whereas constitutively active Ras or MEK1 diminishes potentiation of UCN-01 lethality by SKI-606. (A) U266 cells were stably transfected with wt or loss-of-function mutants (K297R, kinase-inactive; K296R/Y528F, dominant-negative) of human c-Src. Western blot analysis indicated ectopic expression of these proteins and ERK1/2 phosphorylation. EV, Empty-vector controls. (B-D) U266 cells bearing either wt or mutant c-Src were incubated with the indicated concentrations of UCN-01 for 24 or 48 hours (for blots of PARP [C]), Western blot analysis was performed to monitor phosphorylation of Src (Y418 and Y215) and its downstream protein paxillin (Y118), ERK1/2 phosphorylation, PARP cleavage, BimEL phosphorylation/expression (Calbiochem), and γH2A.X expression. CF, Cleavage fragment; p, phosphorylated; up, unphosphorylated. (E) U266 cells were stably transfected with 2 plasmids (#1 and #2) encoding shRNA targeting different sequences of the human c-Src gene. Western blot analysis demonstrates down-regulation of Src protein expression as well as diminished basal levels of ERK1/2 phosphorylation compared with cells transfected with a plasmid encoding a negative control sequence (left panels). Cells were then exposed to the indicated concentrations of UCN-01 for 24 hours, after which cells were lysed and subjected to Western blot analysis to monitor phosphorylation of ERK1/2, expression of Bim (ProSci) and γH2A.X, and cleavage of PARP (right panels). (F) U266 cells were stably transfected with constitutively active human H-Ras (Q61L) or MEK1 (CA-MEK1). Western blot analysis demonstrated ectopic expression of the mutant proteins and resulting phosphorylation/activation of downstream targets (eg, MEK1/2 and ERK1/2, respectively). These cells were then exposed to 150nM UCN-01 ± 2μM SKI-606 for 48 hours, after which the percentage of apoptotic (annexin V+) cells was determined by flow cytometry (means ± SD, **P < .001 vs EV controls). For blots in panels A, C, E and F, vertical lines have been inserted to indicate repositioned gel lanes.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/117/6/10.1182_blood-2010-06-291146/4/zh89991066390003.jpeg?Expires=1722438207&Signature=wo7wQZR1OBekxHZ71DecLTaBIAGHztuGXAcRsfxmfr-ViHeolpr1HLwDkiRxfY8ue9KZpNPizCEGqIu9HvDyR2~7TPOzPChI94ivVHYm8UIocZZtgQL0qPkpDtplo8yWP6FPUnVtJRtQ1mk7grgGDN3FCS9U1CJlgTKiq9I9uJL7DHd~KJliaIYwP4~w2qJJ2wGfoYnEBAIFNz8BKNLe~YDO2W3SeJ9ySYKXm5FoCriERfmy1Pvwg~Pk2suwFXMHiULcoEr22f8Sr0YEVd9QnD~F-Ej9uNTdzUcl2miCuB2ZnmUHcYSA2RKdsWdJt7~FrX-ESQO48XJWsHAWeKW5Ug__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Loss-of-function Src mutants or shRNA knock-down prevent ERK1/2 activation and sensitize MM cells to UCN-01, whereas constitutively active Ras or MEK1 diminishes potentiation of UCN-01 lethality by SKI-606. (A) U266 cells were stably transfected with wt or loss-of-function mutants (K297R, kinase-inactive; K296R/Y528F, dominant-negative) of human c-Src. Western blot analysis indicated ectopic expression of these proteins and ERK1/2 phosphorylation. EV, Empty-vector controls. (B-D) U266 cells bearing either wt or mutant c-Src were incubated with the indicated concentrations of UCN-01 for 24 or 48 hours (for blots of PARP [C]), Western blot analysis was performed to monitor phosphorylation of Src (Y418 and Y215) and its downstream protein paxillin (Y118), ERK1/2 phosphorylation, PARP cleavage, BimEL phosphorylation/expression (Calbiochem), and γH2A.X expression. CF, Cleavage fragment; p, phosphorylated; up, unphosphorylated. (E) U266 cells were stably transfected with 2 plasmids (#1 and #2) encoding shRNA targeting different sequences of the human c-Src gene. Western blot analysis demonstrates down-regulation of Src protein expression as well as diminished basal levels of ERK1/2 phosphorylation compared with cells transfected with a plasmid encoding a negative control sequence (left panels). Cells were then exposed to the indicated concentrations of UCN-01 for 24 hours, after which cells were lysed and subjected to Western blot analysis to monitor phosphorylation of ERK1/2, expression of Bim (ProSci) and γH2A.X, and cleavage of PARP (right panels). (F) U266 cells were stably transfected with constitutively active human H-Ras (Q61L) or MEK1 (CA-MEK1). Western blot analysis demonstrated ectopic expression of the mutant proteins and resulting phosphorylation/activation of downstream targets (eg, MEK1/2 and ERK1/2, respectively). These cells were then exposed to 150nM UCN-01 ± 2μM SKI-606 for 48 hours, after which the percentage of apoptotic (annexin V+) cells was determined by flow cytometry (means ± SD, **P < .001 vs EV controls). For blots in panels A, C, E and F, vertical lines have been inserted to indicate repositioned gel lanes.
