Figure 7

A candidate EGFR-TKI blocks AXL-mediated bypass route to a stable secondary gefitinib resistance in CTD-resistant cells. (a) 2D structure of the diterpenoid yuanhuadine (YD). (b) IC50 values of YD in indicated cell lines and GPs. Cells were treated with or without YD for 72 h with a concentration dilution series and were assayed for SRB. Values represent mean ± SD of three biological replicates. GraphPad Prism 7.01 was used to generate the plot. (c) Characterization of indicated sensitized GPs upon treatment with YD. IC50 values are relative to day 0. Representative of two independent experiments. GraphPad Prism 7.01 was used to generate the plot. (d) Characterization of YD-induced AXL suppression in indicated PTXR-derived GPs. Cells were treated with or without 30 nM YD (in A549-derived GPs) and 45 nM YD (in H1993-derived GPs) for 48 h. Conditioned culture media (CM) were harvested, immunoprecipitated with antibody against N-terminal AXL, and immunoblotted using anti-N-terminal AXL. Cells were also subjected for immunoblotting with anti-C-terminal AXL. GAPDH was used as a loading control. In-gel proteins were visualized with Coomassie blue. Representative of two independent experiments. 40 μg of total cell lysates were loaded per lane for immunoblotting while 50 μg of pulled-down proteins were loaded for immunoprecipitation. Samples from the same cell line were run on the same gel. Paired samples are highlighted in black frame. (e) Cell viability of indicated PTXR-derived GPs upon treatment with 30 nM YD (in A549-derived GPs) and 45 nM YD (in H1993-derived GPs) for 48 h. Prior to YD treatment, cells were treated or transfected with or without the indicated concentrations of Gas6 ligand or pcDNA3.0-AXL plasmid for 24 h. Values are relative to untreated control. Representative of three independent experiments. Python (seaborn; https://seaborn.pydata.org/) was used to generate the plot. (f) ELISA sandwich-based measurement of EGFR tyrosine 1068 phosphorylation and AXL pan tyrosine phosphorylation and concurrent cell viability analysis of indicated GPs. Cells were treated with or without increasing concentrations of YD (5, 10, 20, and 40 nM) for 48 h. Representative of two independent experiments. Python (seaborn; https://seaborn.pydata.org/) was used to generate the plot. (g) 3D RI distributions of indicated fixed A549-derived GPs upon AXL RNAi followed by treatment with or without 20 nM of YD for 48 h. Representative images show snapshots from holotomography imaging. The color legend indicates RI value. TomoStudio to generate the images. (h) qRT-PCR analysis of expression of EMT and CSC markers in indicated GPs with the same conditions as in g. Values are relative to DMSO control and were normalized to GAPDH levels (mean ± SD of two biological replicates). Python (seaborn; https://seaborn.pydata.org/) was used to generate the plot. All IC50s were calculated using TableCurve 2D v5.01.