Journal: The Journal of Pathology

Article Title: Lactate‐mediated activation of GPR81 regulates BCR /Abl protein expression in chronic myeloid leukemia cells selected under low oxygen tension

doi: 10.1002/path.6492

Figure Lengend Snippet: GPR81 activation drives lactate‐mediated suppression of BCR/Abl protein expression and signaling and its effects on the maintenance of stem cell potential in low oxygen. K562 or KCL22 cells were incubated at 3 × 10 5 cells/ml in atmosphere at 0.1% O 2 (LC1) for 4 or 7 days, respectively, in the presence or absence of different concentrations of (A) the GPR81 antagonist 3‐OBA (1–10 m m ) or (B) the lactate transporter inhibitor syrosingopine (10 μ m ) and/or the GPR81 agonist 3‐chloro‐5‐hydroxybenzoic acid (GPR81ago). (A and B) Total cell lysates were subjected to SDS‐PAGE and immunoblotting with anti‐c‐Abl or anti‐p‐Crkl Ab; anti‐vinculin Ab was used to verify equal protein loading. One representative experiment out of three with similar results is shown. (C) Cells rescued at end of LC1 were replated at 3 × 10 4 cells/ml and incubated at 21% O 2 (LC2). Trypan blue‐negative cells were counted at the times of incubation in LC2 indicated in abscissa. Values are mean ± SD from three independent experiments; * p < 0.05 versus control; † p < 0.05 versus syrosingopine.

Article Snippet: The primary antibodies used were c‐Abl (K‐12), rabbit polyclonal; phospho‐Crkl (Tyr207), rabbit polyclonal (Cell Signaling Technology, Danvers, MA, USA); Gpr81 (Catalogue No.: 58‐342, ProSci LLC, California, USA), rabbit polyclonal; vinculin (clone hVIN‐1), mouse monoclonal (Sigma‐Aldrich).

Techniques: Activation Assay, Expressing, Incubation, SDS Page, Western Blot, Control