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

Journal: Experimental and Therapeutic Medicine

Article Title: Triple‑negative breast cancer cells that survive ionizing radiation exhibit an Axl‑dependent aggressive radioresistant phenotype

doi: 10.3892/etm.2023.12147

Figure Lengend Snippet: Establishment and identification of ionizing radiation-resistant mouse TNBC 4T-1/IRR cells. (A) pAxl, Axl, pPI3K, PI3K, pAkt, Akt, mTOR, and PTEN expression in 4T-1 cells at different times following irradiation. (B) The construction process of 4T-1/IRR cells using a gradient irradiation method. The (C) colony formation rate and (D) viability of 4T-1/IRR cells was increased. (E) pAxl and Axl expression in 4T-1/IRR cells. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. NS, not significant; IRR, ionizing radiation resistance; TNBC, triple-negative breast cancer.

Article Snippet: Subsequently, the membranes were blocked with 5% skimmed milk, incubated with primary antibodies against pPI3K (cat. no. ab182651; 1:200; Abcam), PI3K (cat. no. ab191606; 1:1,000; Abcam), pAxl (cat. no. 96453; 1:1,000; Cell Signaling Technology, Inc.), Axl (cat. no. ab215205; 1:1,000; Abcam), pAkt (cat. no. ab81283; 1:2,000; Abcam), Akt (cat. no. ab38499; 1:1,000; Abcam), mTOR (cat. no. ab2732; 1:2,000; Abcam), PTEN (cat. no. ab32199; 1:5,000; Abcam), or GAPDH (cat. no. ab8245; 1:2,000; Abcam), followed by incubation with the relevant horseradish peroxidase-conjugated secondary antibody (anti-Rabbit; cat. no. ab205718; anti-Mouse: cat. no. ab6789; Abcam).

Techniques: Expressing, Irradiation

Journal: Experimental and Therapeutic Medicine

Article Title: Triple‑negative breast cancer cells that survive ionizing radiation exhibit an Axl‑dependent aggressive radioresistant phenotype

doi: 10.3892/etm.2023.12147

Figure Lengend Snippet: Effects of Axl inhibition and irradiation on 4T-1/IRR cell growth. (A) pAxl and Axl expression was downregulated by siRNA lentivirus infection. (B) Growth curve of 4T-1/IRR cells treated with different concentrations of R428. The effect of combined irradiation with Axl inhibition by R428 treatment and Axl siRNA lentivirus infection on (C) cell viability and (D) colony formation ability of TNBC cells. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. NS, Not Significant; NC, negative control; TNBC, triple-negative breast cancer.

Article Snippet: Subsequently, the membranes were blocked with 5% skimmed milk, incubated with primary antibodies against pPI3K (cat. no. ab182651; 1:200; Abcam), PI3K (cat. no. ab191606; 1:1,000; Abcam), pAxl (cat. no. 96453; 1:1,000; Cell Signaling Technology, Inc.), Axl (cat. no. ab215205; 1:1,000; Abcam), pAkt (cat. no. ab81283; 1:2,000; Abcam), Akt (cat. no. ab38499; 1:1,000; Abcam), mTOR (cat. no. ab2732; 1:2,000; Abcam), PTEN (cat. no. ab32199; 1:5,000; Abcam), or GAPDH (cat. no. ab8245; 1:2,000; Abcam), followed by incubation with the relevant horseradish peroxidase-conjugated secondary antibody (anti-Rabbit; cat. no. ab205718; anti-Mouse: cat. no. ab6789; Abcam).

Techniques: Inhibition, Irradiation, Expressing, Infection, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: Triple‑negative breast cancer cells that survive ionizing radiation exhibit an Axl‑dependent aggressive radioresistant phenotype

doi: 10.3892/etm.2023.12147

Figure Lengend Snippet: The protein expression levels of pAxl, Axl, pPI3K, PI3K, pAkt, Akt, mTOR, and PTEN in TNBC cells with Axl siRNA lentivirus infection and radiotherapy combination therapy. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. TNBC, triple-negative breast cancer.

Article Snippet: Subsequently, the membranes were blocked with 5% skimmed milk, incubated with primary antibodies against pPI3K (cat. no. ab182651; 1:200; Abcam), PI3K (cat. no. ab191606; 1:1,000; Abcam), pAxl (cat. no. 96453; 1:1,000; Cell Signaling Technology, Inc.), Axl (cat. no. ab215205; 1:1,000; Abcam), pAkt (cat. no. ab81283; 1:2,000; Abcam), Akt (cat. no. ab38499; 1:1,000; Abcam), mTOR (cat. no. ab2732; 1:2,000; Abcam), PTEN (cat. no. ab32199; 1:5,000; Abcam), or GAPDH (cat. no. ab8245; 1:2,000; Abcam), followed by incubation with the relevant horseradish peroxidase-conjugated secondary antibody (anti-Rabbit; cat. no. ab205718; anti-Mouse: cat. no. ab6789; Abcam).

Techniques: Expressing, Infection

Journal: Experimental and Therapeutic Medicine

Article Title: Triple‑negative breast cancer cells that survive ionizing radiation exhibit an Axl‑dependent aggressive radioresistant phenotype

doi: 10.3892/etm.2023.12147

Figure Lengend Snippet: The protein expression levels of pAxl, Axl, pPI3K, PI3K, pAkt, Akt, mTOR, and PTEN in TNBC cells following a combination of R428 treatment and radiotherapy. ** P<0.01, *** P<0.001, **** P<0.0001. TNBC, triple-negative breast cancer.

Article Snippet: Subsequently, the membranes were blocked with 5% skimmed milk, incubated with primary antibodies against pPI3K (cat. no. ab182651; 1:200; Abcam), PI3K (cat. no. ab191606; 1:1,000; Abcam), pAxl (cat. no. 96453; 1:1,000; Cell Signaling Technology, Inc.), Axl (cat. no. ab215205; 1:1,000; Abcam), pAkt (cat. no. ab81283; 1:2,000; Abcam), Akt (cat. no. ab38499; 1:1,000; Abcam), mTOR (cat. no. ab2732; 1:2,000; Abcam), PTEN (cat. no. ab32199; 1:5,000; Abcam), or GAPDH (cat. no. ab8245; 1:2,000; Abcam), followed by incubation with the relevant horseradish peroxidase-conjugated secondary antibody (anti-Rabbit; cat. no. ab205718; anti-Mouse: cat. no. ab6789; Abcam).

Techniques: Expressing