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. 2021 Mar 25;12(10):1536–1548. doi: 10.1111/1759-7714.13937

FIGURE 4.

FIGURE 4

Knockdown of zinc‐finger E‐box‐binding homeobox 1 (ZEB1) reverses the in vitro self‐renewal and in vivo cancer stem cells (CSCs) phenotype of PC9‐GRPs. (a) Silencing of ZEB1 in PC9‐GRPs reduces the number of spheres than shControl (**p < 0.01). (b) Fluorescence immunohistochemistry analysis of spheres using antibodies against ZEB1, Oct4, or CD133. Cell nuclei are stained with DAPI (blue). Images are obtained on an Axioplan 2 imaging system with AxioVision software, scale bars indicate 200 μm. (c) Tumor volume analysis of PC9‐GRPs transduced with shControl or shZEB1. ZEB1‐silenced gefitinib‐resistant persisters (GRPs) showed significantly reduced tumor growth than shControl GRPs (*p < 0.05). (d) Quantitative real‐time PCR analysis of mRNA expression of ZEB1, BMI1, CD133, ALDH1A1 and vimentin. Data are normalized to beta actin (ACTB) expression and represent mean ± SEM for at least three tumors (*p < 0.05; **p < 0.01). (e) Double‐staining fluorescence immunohistochemistry with thyroid transcription factor 1 (TTF1) to analyze protein expression of ZEB1 and BMI1. Scale bars indicate 200 μm