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. 2021 Nov 10;12:6486. doi: 10.1038/s41467-021-26536-w

Fig. 4. Liver cell follistatin secretion is controlled by the glucokinase regulatory protein- glucokinase (GCKR-GCK) complex.

Fig. 4

a Human liver carcinoma-derived HepG2 cells were transfected with plasmids as indicated: (i) control (pCMV-XL4, grey round plots); (ii) GCK:GCKR (1:0; no GCKR, orange square plots); (iii) GCK:GCKR (1:3, blue triangle plots). Forty-eight hours after transfection, cells were serum starved in 5.5 mM DMEM for 3 h, and a GCKR-GCK disruptor molecule AMG-3969 (0.7 μM) was added in the medium for 30 min. Cells were then incubated in serum-free low glucose (5.5 mM) DMEM containing glucagon (0.3 µM) and forskolin (20 µM), and AMG-3969 (0.7 μM) was added to respective wells. After 4-hour incubation, the medium was collected for follistatin assay by ELISA. Follistatin levels were normalized to the protein concentration within each sample. b HepG2 cells were treated as described in panel a, but in the presence of insulin (100 mM). Two independent experiments with 3 technical replicates per condition were performed in different days using different plasmid preparations and cell passage numbers (n = 6; *p = 0.03 and **p = 0.005; ANCOVA with “experiment” as covariable and LSD post-hoc test; data are presented as mean values + /− SD).