Involvement of S1P2 signaling in SK-1/S1P-induced stability of Bcr-Abl1 in drug resistant compared with sensitive K562 cells. (A) Effects of the knockdown of S1P1, S1P2, S1P3 and S1P4 using siRNAs on Bcr-Abl1 protein in K562/IMA-3 cells were examined using Western blotting compared with nontargeting scrambled (Scr) controls. Actin was used as a loading control. (B) Effects of knockdown of S1P2 using siRNA on the stability of Bcr-Abl1 protein compared with controls (Scr siRNA transfected cells) in the K562/IMA-3 drug resistant cells was measured at 0, 6, 12, and 24 hours after CHX treatments using Western blotting (first and third panels lanes 1-4, respectively). GAPDH was used as a loading control (second and fourth panels). Relative expression levels of Bcr-Abl1 (normalized to GAPDH levels) at 0-24 hours post-CHX treatment in cells transfected with Scr or S1P2 siRNAs are shown below each lane. (C) Effects of knockdown of S1P1, or S1P2 using siRNAs in the absence/presence of exogenous BSA-conjugated S1P (1.0μM, added every 3 hours for 24 hours) on P-Bcr-Abl1 were assessed in K562/IMA-3 cells using Western blotting. Actin was used as a loading control. (D-E) Effects of overexpression of human S1P2-GFP using a lentiviral expression vector, as confirmed by Q-PCR (D) on imatinib-induced growth inhibition in sensitive K562 cells at 0.3, 0.5, and 1.0μM for 48 hours in the absence/presence of exogenous S1P (1μM) were examined using trypan blue exclusion (E). Vector-transduced cells were used as controls. (F) Regulation of P-Bcr-Abl1 (Y245) by S1P/S1P2 signaling was determined after treatment of K562 cells with 0.5μM BSA-conjugated S1P for 24 hours in the absence/presence of JTE-013 compared with untreated controls or JTE-013–treated cells using Western blotting. Actin was used as a loading control. These experiments were performed in duplicates, and error bars represent SD. *P < .05 was considered significant.