Validation of FLT3LG, survivin, and SOCS1 and SOCS2 expression, and STAT pathway overactivation at the translational level, RQ-PCR quantification of SOCS gene family and confirmation of normal transcript of Survivin in MV4-11-R cells. MV4-11 and MV4-11-R cells were washed, then lysed and subjected to 10% to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analyses were detected with the indicated antibodies for the assessment of expression level changes in (A) FLT3LG, survivin, SOCS1, and SOCS2. Densitometric analysis was performed using Amersham Image Scanner with LabScan ImageQuant TL Software (Amersham Biosciences, Piscataway, NJ). The protein levels of SOCS1 and SOCS2 were normalized with each respective actin level. (B) Western blot analyses of STAT, AKT, and MAPK pathway molecules. (C) MV4-11 parental and MV4-11-R cells were seeded at densities of 2 × 10⁵/mL in 10 mL culture medium and treated with PBS control and 3 μM (final concentration) of 5-aza. Fresh medium was changed, and new drug was added every day. After 3 days, cells were harvested, washed with 1× PBS twice. Then the pellets were lysed, followed by RNA extraction, and RQ-PCR. (D) RT-PCR confirmed the overexpression of Survivin transcripts in resistant lines. The size of normal transcript is 431 bp, and 2 other transcript variants, Survivin-2B and Survivin-ΔEx3, are 500 and 329 bp, respectively (top panel). GAPDH was used as internal control (bottom panel).