Figure 5
Figure 5. Role of PP2A activation in the regulation of Bcr-Abl1 in response to SK-1/S1P2 targeting. (A) Effects of knockdown of S1P2 in K562/IMA-3 cells using siRNA compared with Scr-transfected controls (lanes 2 and 1, respectively) on total and P-PP2A and P-SHP1 (at Y307 and S591, respectively), were assessed by Western blotting (left and right top and middle panels, respectively). Effects of knockdown of S1P2 on the expression of P-Bcr-Abl1 at Y245 were also determined by Western blotting compared with Scr controls (bottom left panel lanes 2 and 1, respectively). β-actin was used as a loading control (bottom right panel). (B) Effects of overexpression of PP2Ac-HA on the levels of P-Bcr-Abl1 (Y245), or total Bcr-Abl1 (first and second panels) were determined by Western blotting, compared with controls (lanes 2-1, respectively). Expression of PP2Ac-HA was also confirmed in these experiments (third panel). (C) Inhibition of growth in response to imatinib (0.5μM for 48 hours) in the absence/presence of PP2Ac-HA overexpression and/or SKI-II in K562/IMA-3 cells was examined using trypan blue exclusion. (D) Effects of targeting SK and S1P2 using small molecule inhibitors SKI-II and JTE-013, respectively, on PP2A activity levels in extracts isolated from K562 and K562/IMA-3 cells in the absence or presence of imatinib compared with untreated controls were measured as described in “Measurement of PP2A activity.” (E) Inhibitory effects of SKI-II on SK-1 and SK-2 enzyme activities in wt MEFs, versus SK-1 activity on SK-2−/− MEFs were measured using C17-Sph labeling followed by measurement of C17-S1P generation in cells by LC/MS/MS. (F) Effects of knockdown of S1P1 or S1P2 using siRNAs on PP2A activity in the absence/presence of exogenous S1P (1μM) in sensitive K562 cell extracts were measured using the PP2A activity assay kit. Scr siRNA was used as a control. These experiments were performed in duplicates, and error bars represent SD. *P < .05 was considered significant. In panels A and B, relative expression levels of proteins (normalized to actin levels) are shown below each lane.

Role of PP2A activation in the regulation of Bcr-Abl1 in response to SK-1/S1P2 targeting. (A) Effects of knockdown of S1P2 in K562/IMA-3 cells using siRNA compared with Scr-transfected controls (lanes 2 and 1, respectively) on total and P-PP2A and P-SHP1 (at Y307 and S591, respectively), were assessed by Western blotting (left and right top and middle panels, respectively). Effects of knockdown of S1P2 on the expression of P-Bcr-Abl1 at Y245 were also determined by Western blotting compared with Scr controls (bottom left panel lanes 2 and 1, respectively). β-actin was used as a loading control (bottom right panel). (B) Effects of overexpression of PP2Ac-HA on the levels of P-Bcr-Abl1 (Y245), or total Bcr-Abl1 (first and second panels) were determined by Western blotting, compared with controls (lanes 2-1, respectively). Expression of PP2Ac-HA was also confirmed in these experiments (third panel). (C) Inhibition of growth in response to imatinib (0.5μM for 48 hours) in the absence/presence of PP2Ac-HA overexpression and/or SKI-II in K562/IMA-3 cells was examined using trypan blue exclusion. (D) Effects of targeting SK and S1P2 using small molecule inhibitors SKI-II and JTE-013, respectively, on PP2A activity levels in extracts isolated from K562 and K562/IMA-3 cells in the absence or presence of imatinib compared with untreated controls were measured as described in “Measurement of PP2A activity.” (E) Inhibitory effects of SKI-II on SK-1 and SK-2 enzyme activities in wt MEFs, versus SK-1 activity on SK-2−/− MEFs were measured using C17-Sph labeling followed by measurement of C17-S1P generation in cells by LC/MS/MS. (F) Effects of knockdown of S1P1 or S1P2 using siRNAs on PP2A activity in the absence/presence of exogenous S1P (1μM) in sensitive K562 cell extracts were measured using the PP2A activity assay kit. Scr siRNA was used as a control. These experiments were performed in duplicates, and error bars represent SD. *P < .05 was considered significant. In panels A and B, relative expression levels of proteins (normalized to actin levels) are shown below each lane.

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