Fig 2: Mechanistic gene candidates identified by NNI-STAT3 gene signature. a Winnowed gene list across patient tumors identified candidates uniquely upregulated in STAT3-high tumors. A dose-dependent differential gene expression after signal transducers and activators of transcription 3 (STAT3) inhibitor AZD1480 treatment distinguished cooperative genes responsible in the STAT3-resistant profile. Results are mean of triplicate experiments. b Graphical illustration of responsive and resistant protein tyrosine kinase candidates in STAT3-high and -low cell lines treated with AZD1480 using computational workflow described in Supplementary Fig. 4c. c Treatment of cells with STAT3 inhibitor (AZD1480) demonstrated an increase in pSTAT3 and insulin-like growth factor binding protein 2 (IGFBP2) expression levels in the nuclear fraction of STAT3-low cells but not in STAT3-high. d Fold change differences in secreted proteins demonstrated increased IGFBP2 and insulin-like growth factor 1 receptor (IGF-1R) in resistant cells post-treatment with STAT3 inhibitor. Results are mean of triplicate experiments. e STAT3-high glioblastoma (GBM) cells displayed modest reduction in IGF-1R and IGFBP2 expression levels. In contrast, IGF-1R and IGFBP2 protein expression in STAT3-low cells increased dose-dependently upon AZD1480 treatment, albeit IGF-1R was marginally insignificant. Fold change differences in protein expression of IGFBP2 and IGF-1R were compared between STAT3-high and -low GBM cells. *p < 0.05; **p < 0.01; STAT3-high versus STAT3-low. For statistical analysis, two-sided Student’s t test was used. Error bars represent standard deviation of the mean

Fig 3: GRP94 is required for membrane expression/maturation of IGF-1R.A Immunoblot analysis of GRP94, IGF-1R, IR, p-AKT, AKT, Bim, and β-actin from total lysates of WT and GRP94 KD cells. B Immunoblot analysis of GRP94, IGF-1R, p-AKT, AKT, Bim, and GAPDH from cytosol or membrane of WT and GRP94 KD cells. C Immunoblot analysis of GRP94, IGF-1R, p-AKT, AKT, Bim, and β-actin in islets harvested from 8-weeks old control or GRP94 KO KO mice. D Immunofluorescence analysis of GRP94 (red), IGF-1R (green), and nucleus (blue) in WT or KO mouse islets. Scale bar, 25 µm.

Fig 4: GRP94 interacts with IGF-1R, and is a critical chaperone for the expression/maturation of IGF-1R.A Immunoblot of IGF-1R and GRP94 following immunoprecipitation of GRP94 from lysates of WT or GRP94 KD cells. B Immunoblot of IGF-1R and GRP94 following immunoprecipitation of IGF-1R from lysates of WT or GRP94 KD cells. C Immunoblots of IGF-1R in WT and GRP94 knockdown cells. D WT and GRP94 knockdown cells were treated with actinomycin D (5 μg/ml, D) or cycloheximide (100 μg/ml, E) for indicated periods of time. After stimulation, the lysates were collected and IGF-1R levels were assayed by immunoblot. F The degradation/half-life after CHX treatment was calculated as (value at 1 h - value at 4 h)/Value at 1 h. Degradation rate was calculated based on three independent experiments using ImageJ quantification. Pro-IGF-1R degraded faster than the mature form of IGF-1R. G Immunoblot analysis for IGF-1R expression in WT and GRP94 KD cells after treatment with proteasome inhibitor (MG-132), autophagy inhibitor (CQ), or ERAD inhibitor (kifunensine, KIF).

Fig 5: Treatment with Exendin-4 or overexpression of IGF-1R or GRP94 protects β cells from TG-induced apoptosis.A WT and GRP94 KD cells were treated with 1 μM TG in the absence or presence of 10 nM or 50 nM Exendin-4 for 6 h. Total cell extracts were analyzed by immunoblot for GRP94, IGF-1R, p-AKT, AKT, Bim, c-Cas-3, and β-actin. B WT and GRP94 KD cells were transfected with control plasmid (p.babe plasmid) or IGF-1R overexpression (p.babe-IGF-1R) plasmid and then treated with TG for 6 h or TU for 48 h. Total cell extracts were then analyzed by immunoblot for GRP94, IGF-1R, p-AKT, AKT, Bim, c-Cas-3, and β-actin. C GRP94 KD cells were transfected with GRP94 WT plasmid. Total cell extracts were then analyzed by immunoblot for GRP94, IGF-1R, p-AKT, Bim, and β-actin.