Fig. 4

Glucagon treatment reduces in vitro cell viability of GCGR-overexpressing SNU398. a Protein assessment of apoptotic markers in SNU398 cells either expressing eGFP or GCGR and treated with 100 nM glucagon. b Cell proliferation assay of SNU398 cells either expressing eGFP or GCGR and treated with 100 nM glucagon (100G). Vehicle, 100 G, and 20F were added daily in fresh media. Data represent a single experiment of 3 biological replicates. Error bars: ±SEM. ns: not significant, ****p < 0.0001, two-way ANOVA with Tukey’s multiple comparisons test. 20F: 20 uM forskolin, 1P: 1 μM palbociclib (cell cycle inhibitor), 10B: 10 μg/ml blasticidin (cell death inducer). c Protein assessment of pCREB in SNU398 cells either expressing eGFP or GCGR and treated with 100 nM glucagon or 20 μM Forskolin. d Crystal violet assay on SNU398 cells either expressing eGFP or GCGR and treated with 20 μM Forksolin and/or 1 μM CREB inhibitor, and/or 5uM PKA inhibitor. e (Upper panel) Histogram of flow cytometry analysis of gated, single cells staining positive for Annexin V/Propidium iodide in SNU398 cells either expressing eGFP or GCGR and treated with 100 nM glucagon (100 G). Data represent a single, independent experiment of 3 biological replicates. Error bars: ±SEM. ns: not significant, ****: p < 0.0001, two-way ANOVA with Sidak’s multiple comparisons test. 10Blast: 10 μg/ml blasticidin (positive control). (Lower panel) Representative PI/Annexin V scatter plots for SNU398 GCGR with or without glucagon treatment. f Cell proliferation assay on SNU398 cells either expressing eGFP or GCGR and treated with glucagon. Data represent a single experiment with 3 biological replicates. Error bars: ± SEM. g Crystal violet assay on SNU398 cells either expressing eGFP or GCGR and treated with 100 nM glucagon (100 G)