Fig. 6: Exovesicular-Shh imparts Imatinib resistance.

A Western blot-based analysis of the cell lysate and Exo-fraction isolated from Shh-K562 and K562. Shh, Actin (cytosolic protein), GM130 (a golgi marker), Tsg101, and Flotilin2 (exovesicle markers) analyzed. Data from two to three biological repeats each. B Western blot evaluating relative levels of Shh in Exo and free-fractions derived from Shh-K562 and K562 condition media. Equal total protein loaded in each well for analysis (n = 4). C Western blot representing presence of Shh in extracellular fractions Exo and free-fractions derived from BM sample from CML patient (CML12, CML-variant). Different concentrations of total protein were loaded per well to evaluate relative enrichment of Shh in each fraction, as indicated. Graph represents densitometric estimation of relative protein levels normalized to total amount of protein loaded per well. D’ Schematic represents the assay procedure using conditioned medium. D Bar graph represents viability of K562 cells pretreated with Exo-fraction or free-fraction derived from K562 or Shh-K562 (12-h pre-treatment), followed by treatment with 0.5 µM Imatinib for 72 h. Imatinib-treated K562 cells (black bar) and Shh-K562 cells (green bar) used as negative and positive controls, respectively. Bar represents mean ± SD from three different experiments. E’ Schematic represents the assay procedure using BM plasma. E Bar graph represents viability of K562 cells after 12-hr pre-treatment with Exo or free-fraction derived from CML patients BM (red bar, V-CML; CML-4, 9, 12) (n = 3); gray bar, CML-CP (9:22; CML-1, 3, 8) (n = 3), followed by treatment with 0.5 µM Imatinib for 72 h. Imatinib-treated K562 cells (black bar) and Shh-K562 cells (green bar) used as negative and positive controls, respectively. Data represent mean ± SD from three different CML patient-derived fractions. P values, *<0.05, **<0.01, and ***<0.001. Statistical analysis done using t-test.