Fig 6.

(A) Levels of intracellular ROS were significantly higher in MDA-MB-231 SH-4-54-resistant clones relative to wild-type (WT) cells, while in clones derived from resistant T47D cells, ROS levels were significantly lower than their WT counterpart. (B) Treatment with capsazepine, paclitaxel, or bleomycin resulted in lower cell counts of MDA-MB-231 clones compared to vehicle (DMSO), whereas the number of WT MDA-MB-231 cells was not affected by treatment with capsazepine. (C) Western blotting revealed that levels of phospho-STAT5 (p-STAT5) at 95 kDa increased significantly in MDA-MB-321 clones relative to their WT counterpart (indicated by the arrow), with an overall decrease in the intensity of bands at approximately 130 and 200 kDa, while in T47D clones, the opposite occurred. (D) Western blots of lysates derived from MDA-MB-231 WT and clones probed with SUMO-1, SUMO-2/3, and total STAT5 antibodies revealed similar banding patterns, with common bands observed at approximately 95, 130, and 200 kDa. Lower overall band intensities were observed in SH-4-54-resistant clones compared to their WT counterpart, with decreased levels of phospho-STAT3. (E) A representative set of IPs confirmed that SUMO-2/3 co-migrated with total STAT5 in MDA-MB-231 cells, and that levels of SUMOylated STAT5 were lower in SH-4-54-resistant clones than in WT cells (compare lanes 3 and 6). Data represent the mean of three independent experiments (±SEM) calculated relative to appropriate controls. A star (*) denotes statistically significant differences determined using a t-test (p<0.05).