anti p axl  (R&D Systems)

Journal: Frontiers in Oncology

Article Title: AXL Promotes Metformin-Induced Apoptosis Through Mediation of Autophagy by Activating ROS-AMPK-ULK1 Signaling in Human Esophageal Adenocarcinoma

doi: 10.3389/fonc.2022.903874

Figure Lengend Snippet: Downregulation of AXL expression impairs autophagic flux in EAC cells. (A , B) SK-GT-4 and FLO-1 cells stably expressing control shRNA or AXL shRNA were cultured in complete medium with 4.5 g/L glucose or without glucose in the presence or absence of the lysosomal inhibitor chloroquine (10 µM) for 4 h. Western blot analysis of LC3B and AXL proteins in whole cell lysates from SK-GT-4-shCtrl and SK-GT-4-shAXL (A) and FLO-1-shCtrl and FLO-1-shAXL (B) cells. Gel loading was normalized for equal β-actin. Autophagic flux was assessed based on LC3B-II expression change induced by chloroquine following glucose starvation. The LC3B-II expression change was calculated by subtracting quantified LC3B-II value of cells treated with chloroquine from that of cells without chloroquine following glucose starvation. (C) FLO-1-shCtrl and FLO-1-shAXL cells stably expressing mRFP-GFP tandem fluorescent-tagged LC3 reporter (tfLC3) were cultured in the presence or absence of glucose for 4 h to induce autophagy. Representative confocal images (60x) of live cells depicting autophagosomes (green fluorescence) and autolysosomes (red fluorescence). Quantification of total number of autolysosomes per cell in FLO-1-shCtrl versus FLO-1-shAXL cells. Data are represented as median ± SD. (D) Western blot analysis of AXL in whole cell lysates from FLO-1-shCtrl and FLO-1-shAXL cells. (E) Representative confocal images (60x) of live FLO-1-tfLC3 stable cells showing autophagosomes (green puncta) and autolysosomes (red puncta) following treatment with vehicle or AXL inhibitor R428 (500 nM) in the absence or presence of glucose for 4 h. Quantification of total number of autolysosomes per cell in vehicle-treated FLO-1 versus R428-treated FLO-1 cells. Data are represented as median ± SD. (F) Western blot analysis of p-AXL (Y779) and AXL proteins in whole cell lysates from FLO-1 cells treated with vehicle (DMSO) or R428 (500 nM). Data are representative of three independent experiments and differences were analyzed by one-way ANOVA followed by the Newman-Keuls post hoc test. NS, statistically not significant.

Article Snippet: The p-AXL (Y779) (#AF2228, 1:500 dilution) Antibody was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Stable Transfection, Control, shRNA, Cell Culture, Western Blot, Fluorescence

Journal: Acta pharmaceutica Sinica. B

Article Title: The FAP α -activated prodrug Z-GP-DAVLBH inhibits the growth and pulmonary metastasis of osteosarcoma cells by suppressing the AXL pathway.

doi: 10.1016/j.apsb.2021.08.015

Figure Lengend Snippet: Figure 5 Z-GP-DAVLBH inhibits the AXL/AKT/GSK-3b/b-catenin pathway in osteosarcoma cells. (A)e(B) Osteosarcoma cells were treated with vehicle (0.1% DMSO) or Z-GP-DAVLBH (6 nmol/L) for 48 h. (A) The levels of p-AXL (Tyr779), AXL, p-AKT (Ser473), AKT, p-GSK-3b (Ser9), and GSK-3b in SJSA-1 and 143B cells treated with Z-GP-DAVLBH were determined by Western blotting analysis. (B) Representative blots of b-catenin, b-catenin (Ser552), and non-phosphorylated (active) b-catenin (Ser33/37/Thr41) in osteosarcoma cells. (C) and (D) Osteosarcoma cells after transfection with either NC siRNA or AXL siRNA were treated vehicle (0.1% DMSO) or Z-GP-DAVLBH (6 nmol/L) for 48 h. (C) Western blotting analysis was conducted to evaluate the effect of Z-GP-DAVLBH on the AXL/AKT/GSK-3b/b-catenin pathway components and (D) EMT- related markers in osteosarcoma cells. (E) and (F) Osteosarcoma cells were treated with Z-GP-DAVLBH (6 nmol/L) for 24 h. Transwell assays were conducted to evaluate the effect of Z-GP-DAVLBH on the (E) migration and (F) invasion capacities of SJSA-1 and 143B cells transfected with the indicated siRNAs. Scale bar: 200 mm. Data are presented as meanSEM, n Z 3; *P<0.05, **P<0.01, and ***P<0.001 vs. the indicated groups.

Article Snippet: The following antibodies were used at a dilution of 1:500e1:1000: p-AXL (Tyr779) (catalog AF2228) and FAPa (catalog AF3715) were obtained from R&D system (Minneapolis, MN, USA).

Techniques: Western Blot, Transfection, Migration

Journal: Acta pharmaceutica Sinica. B

Article Title: The FAP α -activated prodrug Z-GP-DAVLBH inhibits the growth and pulmonary metastasis of osteosarcoma cells by suppressing the AXL pathway.

doi: 10.1016/j.apsb.2021.08.015

Figure Lengend Snippet: Figure 6 Ectopic expression of AXL attenuates the effect of Z-GP-DAVLBH on the malignant behaviors of osteosarcoma cells. (A) and (B) Osteosarcoma cells were treated with vehicle (0.1% DMSO) or Z-GP-DAVLBH (6 nmol/L) for 48 h. (A) Western blotting analysis of p-AXL (Tyr779), AXL, p-AKT (Ser473), AKT, p-GSK-3b (Ser9), GSK-3b, b-catenin, p-b-catenin (Ser552), and non-phosphorylated (active) b-catenin (Ser33/37/Thr41), and (B) EMT-related markers in SJSA-1 and 143B cells. (C) Osteosarcoma cells were treated with Z-GP-DAVLBH (6 nmol/L) for 24 h. Representative image of migrated (upper) and invaded (lower) osteosarcoma cells. Scale bar: 200 mm. Quantification of the number of migrated and invaded cells is shown. (D) and (E) SJSA-1 and 143B were treated with vehicle (0.1% DMSO) or Z-GP-DAVLBH (50 nmol/L for SJSA-1 cells and 100 nmol/L for 143B cells) for 48 h. (D) An MTT assay was conducted to evaluate the effect of Z-GP-DAVLBH on osteo- sarcoma cell viability. (E) An Annexin V-FITC apoptosis detection kit was used to evaluate the effect of Z-GP-DAVLBH on osteosarcoma cell apoptosis. Data are presented as meanSEM, n Z 3; *P<0.05, **P<0.01, and ***P<0.001 vs. the indicated group.

Article Snippet: The following antibodies were used at a dilution of 1:500e1:1000: p-AXL (Tyr779) (catalog AF2228) and FAPa (catalog AF3715) were obtained from R&D system (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, MTT Assay

Journal: Acta pharmaceutica Sinica. B

Article Title: The FAP α -activated prodrug Z-GP-DAVLBH inhibits the growth and pulmonary metastasis of osteosarcoma cells by suppressing the AXL pathway.

doi: 10.1016/j.apsb.2021.08.015

Figure Lengend Snippet: Figure 8 Z-GP-DAVLBH inhibits epithelialemesenchymal transition and suppresses pulmonary metastasis of osteosarcoma cells in vivo. (A) BALB/c nude mice bearing 143B tumors were treated with vehicle (0.9% NaCl solution containing 1% DMSO) or Z-GP-DAVLBH (2 mg/kg, i.v.) every other day for 22 days. Lung tissues were collected and performed by hematoxylin-eosin (H&E) staining. Scale bar: 500 mm for low magnification images or 100 mm for high magnification images. (B) Quantification of the area and number of lung metastatic foci. (C) IHC staining of EMT-related markers in SJSA-1 and 143B tumor tissues. Quantification of IHC staining of EMT-related markers is shown. (D) IHC staining of p-AXL (Tyr779) and non-phosphorylated (active) b-catenin (Ser33/37/Thr41) in SJSA-1 and 143B tumor tissues. Scale bar: 50 mm. Quantification of IHC staining is shown. Data are presented as meanSEM, n Z 6; *P<0.05, **P<0.01, ***P<0.001 vs. the Vehicle group.

Article Snippet: The following antibodies were used at a dilution of 1:500e1:1000: p-AXL (Tyr779) (catalog AF2228) and FAPa (catalog AF3715) were obtained from R&D system (Minneapolis, MN, USA).

Techniques: In Vivo, Staining, Immunohistochemistry