anti paxl y779  (R&D Systems)

Journal: The Febs Journal

Article Title: The AXL inhibitor bemcentinib overcomes microenvironment‐mediated resistance to pioglitazone in acute myeloid leukemia

doi: 10.1111/febs.17263

Figure Lengend Snippet: Pioglitazone incubation and co‐culture with MS‐5 stromal cells enhances AXL activation in OCI‐AML3 cell line and is PPAR‐γ dependent. OCI‐AML3 cell line was co‐cultured with MS‐5 stromal cells and treated with pioglitazone (10 μ m ) alone, bemcentinib (50 n m ) alone or a combination of both. (A) Levels of phosphorylated AXL Y779 in OCI‐AML3 at day 7 determined by flow cytometry with (B) results expressed by stain index ratio compared with vehicle (DMSO). (C) OCI‐AML3 cell line was co‐cultured with PPAR‐γ KO or Cas9 control MS‐5 stromal cells using Transwell ® inserts with 0.4 μm pore and then treated with pioglitazone (10 μ m ). Cell counts were performed by flow cytometry and compared with vehicle (DMSO). (D) Levels of phosphorylated AXL Y779 in OCI‐AML3 at day 7 determined by flow cytometry. (E) Mouse Gas6 protein levels secreted by wild‐type or PPAR‐γ KO MS‐5 stromal cells were examined when cultured alone or co‐cultured with OCI‐AML3 cell line. (F) Mouse Gas6 protein levels secreted by wild‐type stromal cells, co‐cultured with OCI‐AML3 cell line and treated with pioglitazone (10 μ m ) compared with vehicle (DMSO). Results expression: mean ± SEM of at least three independent experiments with *, P < 0.05; **, P < 0.01; ns, non‐significant.

Article Snippet: AXL phosphorylation was analyzed using pAxl Y779 antibody (R&D Systems, Minneapolis, MN, USA) on fixed and permeabilized cells (BD Cytofix/CytopermTM, BD Biosciences, Le Pont de Claix, France).

Techniques: Incubation, Co-Culture Assay, Activation Assay, Cell Culture, Flow Cytometry, Staining, Control, Expressing

Journal: The Febs Journal

Article Title: The AXL inhibitor bemcentinib overcomes microenvironment‐mediated resistance to pioglitazone in acute myeloid leukemia

doi: 10.1111/febs.17263

Figure Lengend Snippet: Exogenous Gas6 addition or MS‐5 conditioned medium mimics stroma‐induced resistance to pioglitazone. (A) OCI‐AML3 cell line was treated with pioglitazone (10 μ m ) alone, exogenous Gas6 (50 ng·mL −1 ) alone or in combination. Cell counts were performed by flow cytometry and compared with vehicle (DMSO). (B) Levels of phosphorylated AXL Y779 in OCI‐AML3 at day 7 determined by flow cytometry with (C) results expressed by stain index ratio compared with vehicle (DMSO). The same color code has been used for (B and C). OCI‐AML3 cell line was cultured in standard medium or MS‐5 conditioned medium (CM) and treated with pioglitazone (10 μ m ) alone, bemcentinib (50 n m ) alone or in combination (D). Cell counts were performed by flow cytometry and compared with vehicle (DMSO). (E) Levels of phosphorylated AXL Y779 in OCI‐AML3 at day 7 determined by flow cytometry with results expressed by stain index ratio compared with vehicle (DMSO). (F) Relative mouse Gas6 protein levels assessed by ELISA in conditioned medium at day 7 compared with untreated conditioned medium. The same color code has been used for (E and F). Results expression: mean ± SEM of at least three independent experiments with *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, non‐significant.

Article Snippet: AXL phosphorylation was analyzed using pAxl Y779 antibody (R&D Systems, Minneapolis, MN, USA) on fixed and permeabilized cells (BD Cytofix/CytopermTM, BD Biosciences, Le Pont de Claix, France).

Techniques: Flow Cytometry, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing

Journal: The Febs Journal

Article Title: The AXL inhibitor bemcentinib overcomes microenvironment‐mediated resistance to pioglitazone in acute myeloid leukemia

doi: 10.1111/febs.17263

Figure Lengend Snippet: Pioglitazone and co‐culture with MS‐5 stromal cells enhance AXL activation in primary AML cells via a PPAR‐γ‐dependent mechanism. Primary AML cells from two different AML patients ( n = 1 for each patient) were co‐cultured with wild type (A, UPN6 and B, UPN7) or PPAR‐γ KO (C, UPN6 and D, UPN7) MS‐5 stromal cells, and treated with pioglitazone (10 μ m ) alone, bemcentinib (50 n m ) alone or a combination of both. First plots (left): reduction in cell growth induced in AML primary cells after drug incubation compared with vehicle. Cell counts were performed by flow cytometry. Second and third plots (middle and right): levels of phosphorylated AXL Y779 in AML cells at day 7 determined by flow cytometry and results expressed by stain index ratio compared to vehicle (DMSO).

Article Snippet: AXL phosphorylation was analyzed using pAxl Y779 antibody (R&D Systems, Minneapolis, MN, USA) on fixed and permeabilized cells (BD Cytofix/CytopermTM, BD Biosciences, Le Pont de Claix, France).

Techniques: Co-Culture Assay, Activation Assay, Cell Culture, Incubation, Flow Cytometry, Staining