1. AXL overexpression decreases proliferation in vitro.
AXL overexpression decreases proliferation in vitro. A, AXL expression by Western blot in C42B control Neomycin-resistant cells (Neo), and two AXL overexpression clones (Axl 1 and Axl 2; n = 1). Arrow represents 125 kDa. B, EdU incorporation over 24 hours in C42B Axl 1 and 2 cells compared with Neo control ± GAS6 by IF. Individual cells were counted manually in 3 FOV per sample (n = 2). P values calculated using the t test between cell lines and their control, and within each cell line between treatments. C, Quantification of the number of large colonies (number of colonies in the top 16% of the range of colony size) in soft agar ± GAS6, relative to reference sample Neo -GAS6 (n = 2). D, AXL expression after dead cell exclusion by flow cytometry in an AXL overexpression clone over increasing passage number. Cells were reselected with Neomycin over 2 passages between passages 16 and 19 (n = 1). E, AXL expression by Western blot in conditionally overexpressing AXL clones (Tet-Axl 1 and Tet-Axl 2) compared with empty vector control (Tet-ø) ± doxycycline (n = 1). Arrow represents 125 kDa. F, EdU incorporation over 2 hours in Tet-Axl 1 and 2 cells compared with Tet-ø control ± doxycycline by flow cytometry (n = 3). P values calculated using one-sample t test against the null hypothesis (log fold change = 0) was performed between treated samples. G, EdU incorporation over 2 hours in all cell lines ± doxycycline and AXL inhibitor R428 by flow cytometry; fold change is relative to untreated (Tet-ø and Tet-Axl 2, n = 3; Tet-Axl 1, n = 2). P values calculated using the Dunnett multiple comparison test against +Dox samples for each cell line, excluding −Dox samples. H, Quantification of the number of large colonies (number of colonies in the top 13% of the range of colony size) in soft agar ± doxycycline, relative to reference sample Tet-ø −Dox (n = 2). For each experiment, treatments were initiated at the time of cell seeding; GAS6: 100 ng/mL, doxycycline: 100 ng/mL, R428: 50 nmol/L. Error bars, mean ± SEM. For soft-agar assays, P values were calculated using multiple t tests to assess differences due to treatment.
Haley D. Axelrod et al. Mol Cancer Res 2019;17:
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