Fig. 4
From: Recurrent WNT pathway alterations are frequent in relapsed small cell lung cancer
Loss of APC induces chemotherapy resistance in human SCLC cell lines. a Left: knockdown of APC in H1694 cells with two different shAPC constructs (shAPC#1 and shAPC#2); right: activation of WNT signaling as measured by AXIN2 upregulation and TOPFlash reporter activity (signal fold changes) in these cells. Control cells expressed shRNA with scrambled target sequence (shScr). APC and AXIN2 mRNA levels were measured by quantitative PCR (qPCR). Fold change is reported with respect to control cells, and values were compared using unpaired t tests. Each experiment was performed in biological triplicate (TOPFlash assay results for shAPC#2 are shown from n = 5 experiments). b Percentage of H1694 cells surviving etoposide following 72-h treatment. c Left: fold change in etoposide IC50 following APC knockdown in H1694 cells compared to control cells, and right: fold change in etoposide IC50 in APC knockdown (shAPC#2) cells following overexpression of APC or GFP (control). IC50 values were compared using ratio-paired t tests. d Results from Surveyor assay demonstrating genomic alterations in APC (cleavage products indicated by black bar) following CRISPR–Cas9-guided deletion in H82 sgAPC cells and in-frame deletions in APC that were identified through targeted sequencing of the APC sgRNA site. e WNT activation in H82 sgAPC cells as measured by AXIN2 mRNA levels by qPCR (p < 0.01, unpaired t test; n = 3 biological samples in technical triplicate) and CTNNB1 protein levels by immunoblot (representative of n = 2 independent experiments). Actin serves as loading control for CTNNB1. f Percentage of H82 cells surviving treatment with etoposide following CRISPR-guided deletion of APC (sgAPC) or control sequence (sgControl) (n = 2 experiments). Experiments were performed in biological triplicate, unless specified otherwise. IC50 inhibitory concentration 50, GFP green fluorescent protein, *p < 0.05, **p < 0.01, ****p < 0.0001, NS not significant. Bp base pair, Del deletion, AA amino acid, H1694 NCI-H1694, H82 NCI-H82. Error bars on box plots and dose-response curves are mean ± standard error
