Fig. 2
Shisa3 decreases EGFR-TKI resistance and inhibits a CSC phenotype. a, b. The histograms show the IC50 of PC9, PC9/ER, HCC827 and H1975 cells for gefitinib (a) and osimertinib (b). c. Shisa3 transcription levels and protein expression were analyzed by qRT-PCR (left panel) and Western blot (right panel) in PC9, PC9/ER, HCC827 and H1975 cells. β-actin was used as a loading control. d. The mRNA and protein levels of shisa3 were measured in PC9/ER cells transfected with shisa3 in Tet-on inducible vector (2 μg/ml of doxycycline-induction) by qRT-PCR and western blot. e. The histogram shows the IC50 for gefitinib and osimertinib in PC9/ER cells expressing shisa3 induced by doxycycline (2 mg/ml) treatment for 48 h. f. Representative the primary and secondary sphere images of PC9/ER cells. Scale bars, 100 μm. g. The histogram demonstrates the primary and secondary sphere formation efficiencies in PC9/ER and PC9/ER cells overexpressing shisa3. h. Lower expression levels of CSC-related markers were observed by qRT-PCR in shisa3-overexpressing PC9/ER cells than in control cells. i. The graph demonstrates the number of migrated and invasive PC9/ER and shisa3-overexpressing PC9/ER cells. j. Representative tumorigenic images formed by transplanting 1 × 102 and 1 × 103 PC9/ER or PC9/ER-shisa3 cells into nod-scid mice (upper panel). Tumorigenic frequency was calculated by extreme limiting dilution analysis (ELDA). e, g, h and i: *p < 0.05; **p < 0.001; ***p < 0.0001