Transcriptional regulators of Igf1r in iPI3K-δ–resistant tumors. (A) Resistant and sensitive tumors were treated with dimethyl sulfoxide (DMSO), GSK3 inhibitor (CHIR99021; iGSK3), or FOXO1 inhibitor (AS1842856; iFOXO1) for 3 hours, followed by treatment with DMSO or GS-649443 for 12 hours; Igf1r expression was analyzed using RT-qPCR. All P values were calculated using a 2-tailed paired Student t test. (B) Representative images from immunohistochemistry analysis showing FOXO1 localization after treatment of iPI3K-δ–resistant and -sensitive tumors with DMSO, GSK3 inhibitor (CHIR99021), GS-649443, or the combination of GS-649443 and CHIR99021. For every slide, 10 fields were analyzed with ×600 magnification and the localization of FOXO1 in all cells in every field was scored. Green arrows indicate FOXO1 localization in the nucleus (transcriptionally active FOXO1). Red: FOXO1 (Cy3), blue: DAPI. (C) Incidence of FOXO1 in the different compartments (only cytoplasm, nucleus, and no expression) was quantified using the mean from the 10 fields of view and normalized to the total number of cells per field. The P values were calculated using the Mann-Whitney U test. (D) Western blot analysis for expression levels of p-IGF1R, IGF1R, p-FOXO1, FOXO1, p-AKT, AKT, p-ERK, and ERK in cells from the fourth transfer and treatment round. Analysis of LAMIN B was included as a loading control. (E) Quantification of FOXO1, p-FOXO1, and p-ERK1/2 protein levels from the western blot analysis using ImageJ densitometry analysis software. All protein expression data were normalized to that of LAMIN B. p-ERK and p-FOXO1 were further normalized to their respective total protein expression levels. All P values were calculated using the Mann-Whitney U test. ns, not statistically significant. *P ≤ .05; **P ≤ .01; ns, P > .05.