Fig. 3

Glycogen synthesis kinase 3 (GSK3) signaling is a candidate mediator of B2AR and MOR interaction. A The gene expression changes as determined by the CSC PCR array following B2AR and MOR double knockdown were analyzed by Ingenuity Pathway Analysis (IPA) using a threshold [log2(fold-change)] > 1 or < − 1 as compared to control values. Genes that are part of the PCR array dataset and are upregulated are represented in red, and those that are downregulated are represented in green. The Molecule Activity Predictor (MAP) tool was then used to predict activation or inactivation of downstream genes and principal functions of the cell. Orange indicates predicted activation and blue indicates predicted inactivation. The lines correspond to the relationship(s) between different genes or between gene(s) and cell function(s). Orange lines predict activation of the gene–gene or gene-function relation and blue lines predict inactivation of the gene–gene or gene-function relation. Gray lines indicate no significant prediction. B, C Validation by RT-PCR measurements of genes relevant to GSK3 signaling and predicted from IPA analysis in MDA-MB-231 and MDA-MB-468 cells with B2AR, MOR, or both B2AR and MOR knockdown by CRISPR (B, D) or by pharmacological agents PRO, NTX, or both PRO and NTX (C, E). Normalization of each gene in RT-PCR assay was done using four housekeeping genes (GAPDH, ACTB, B2M, and HPRT1). Data are mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001