Fig 2: Knockdown of AXL expression decreases peripheral distribution of lysosomes in EAC cells. (A-D) LAMP1 IF analysis. (A) Representative confocal IF images (60×) of LAMP1 (green) in SK-GT-4-shControl versus SK-GT-4-shAXL cells, or (C) FLO-1-shControl versus FLO-1-shAXL cells. Arrows point to cells with peripheral lysosomes, while arrowheads indicate cells with perinuclear lysosomes. (B) Quantification of the percentage of SK-GT-4-shControl versus SK-GT-4-shAXL cells, or (D) FLO-1-shControl versus FLO-1-shAXL cells with either peripheral or perinuclear lysosomes. Data are represented as mean ± SE. (E-H) Live-TIRF microscopy analysis of lysosomes peripheral localization. (E) Representative TIRFM images (60X) of Lysotracker (+) acidic vesicles in SK-GT-4-shControl versus SK-GT-4-shAXL cells, or (G) FLO-1-shControl versus FLO-1-shAXL cells. (F) Quantification of total number of tracks per cell present close to the plasma membrane of SK-GT-4-shControl versus SK-GT-4-shAXL cells, or (H) FLO-1-shControl versus FLO-1-shAXL cells. Data are represented as median ± SD.

Fig 3: Axl is activated in osteosarcoma patient biopsy samples. (a and b) Immunohistochemistry was performed on osteosarcoma biopsy samples with an IgG isotype control (a) or phospho-Axl (b) antibody. Staining was assessed under a light microscope and images were captured at × 400 magnification. The intensity score (left number) and percentage of positive cells (right number) are reported in the inset boxes for each sample.

Fig 4: Axl contributes to motility and colony formation by the 143B cell line. (a–e) The first validation strategy compared the effects of the siRNA pool with that of the individual duplexes. Knockdown efficiency was assessed by real-time PCR measurements of Axl mRNA levels (mean±s.e.m. of three PCR well replicates) (a). Effects of the siRNAs on in vitro phenotypes were assessed by measuring motility (b), colony formation (c), invasion (d) and cell growth (e). Results are presented as mean±s.e.m. of the number of replicates indicated in the Materials and methods for each assay. (f–j) The second validation strategy compared the effects of the Axl small molecule inhibitor, BGB324, with that of the siRNA pool. Cells were treated for 1 h with BGB324 or the control 1% dimethyl sulfoxide. A dose response of Axl activity inhibition by BGB324 was assessed by western blotting with a phospho-Axl antibody (f, top panel) or the total Axl antibody (f, middle panel). ß-actin was assessed as a housekeeping protein (f, bottom panel). Western blot results are representative of 10 independent experiments. For expanded views of the blots with protein markers see Supplementary Figure S10. Effects of BGB324 on in vitro phenotypes were assessed by measuring motility (g), colony formation (h), invasion (i) and cell growth (j). Results are presented as mean±s.e.m. of three independent experiments.

Fig 5: AXL expression is positively associated with an EMT expression pattern. Median mRNA expression of EMT-associated genes in human primary EAC tumors from TCGA dataset (88 tumors) according to AXL expression levels (low AXL =2-fold median expression, high AXL >2-fold median expression). Boxplots represent RNA absolute counts. PCC: Pearson’s correlation coefficient.