Page 131 - Haematologica May 2020

BCR-ABL suppresses autophagy via BECLIN-1
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Figure 4. BCR-ABL mediated BECLIN-1 phosphorylation leads to suppression of autophagy and altered BECLIN-1 complex formation. (A) Confocal microscopy of K562 cells reveals significant induction of autophagy in BECLIN-1 Y233F/Y352F-expressing cells indicated by increased number of LC3 puncta. (B) Statistical analy- sis of LC3 puncta count in Beclin-1-mutant-transduced K562 cells (3.3, 4.1, 1.9, and 18 units respectively; ****P<0.0001 Student's t-test; Scale bar 10 mm). (C) Upregulation of LC3-II in K562 cells transduced with the phosphorylation-deficient Beclin-1 Y233F/Y352F mutant. (D) Immunoblot analyses were used for the detec- tion of ATG14, UVRAG, VPS34 and RUBICON expression levels in Beclin-1 knockout MEF transduced with BCR-ABL and the indicated Beclin-1 construct. (E) Immunoprecipitation of FLAG-tagged phosphorylation-deficient BECLIN-1 Y233F/Y352F mutant (BecFF) in K562 cells indicates altered autophagy complex formation compared to the phosphorylation-mimic BECLIN-1 mutant (BecEE). (F) Beclin-1 tyrosine phosphorylation-mimic mutation blocks nilotinib-induced autophagy seen by reduced LC3-II levels compared to empty vector, Beclin-1 wt, and Beclin-1 FF. LC3 levels were evaluated by immunoblotting in the indicated Beclin-1 mutant-trans- duced K562 cells. (G) LC3 puncta formation was measured using confocal microscopy in K562 cells transduced with mCherry-EGFP-LC3 and the indicated Beclin-1 construct upon nilotinib treatment. Cells were stained with anti-LC3 antibody and nuclei were counterstained with DAPI. Scale bar, 10 mm. (H) Quantitation of LC3 puncta (16.5, 17.5, 3.4, and 16.9 dots/cell, respectively; P<0.0001 for comparison of Beclin-1 EE to empty vector [EV], Beclin-1 wt and Beclin-1 FF). K562 cells trans- duced with mCherry-EGFP-LC3 and the indicated Beclin-1 constructs were used for LC3 puncta measurement via Olympus ScanR screening station. Bars represent the mean±SD. ****P<0.0001 by Student's t-test. EV: empty vector; wt: wild-type FLAG-Beclin-1; EE: FLAG-Beclin-1 Y233E/Y352E; FF: FLAG-Beclin-1 Y233F/Y352F.
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