Figure 2. CAFs promote mobilization of glycogen stores in cancer cells.

(A) Western blot detecting phosphorylated phosphoglucomutase 1 (PGM1) at Y353 in SKOV3ip1 and TYK-nu cells co-cultured with primary human CAFs separated by a transwell cell culture insert. Values below p-PGM1 indicate the relative band intensity normalized to total PGM1 and fold change compared to the control. Images are representative of three biological repeats. (B) PGM1 enzyme activity assay of PGM1 immunoprecipitated from TYK-nu cells stably expressing the WT or Y353F mutant constructs (inset) and analyzed for PGM1 activity using glucose-1-phosphate as the substrate. Right panel, activity of PGM1 (***p< 0.001). Background (BKGD). (C) PGM1 enzyme activity assay and (D) glycogen phosphorylase (GP) activity in TYK-nu cells co-cultured with primary human CAFs (3 hr) separated by a transwell cell culture insert. Values are normalized to enzyme activity in TYK-nu cells cultured alone as indicated by the dotted line. Values are mean + SEM from 4 independent experiments (n=6/group/experiment) *p<0.05, **p< 0.01. (E) Glycogen stores were visualized by 2-NBDG fluorescence, PAS staining and transmission electron microscopy (TEM) in SKOV3ip1 cells co-cultured with or without CAFs (4 hr). In the TEM images, glycogen pools are identified by a “G”. (F) Glycogen assay on SKOV3ip1, TYK-nu, and SNU119 cells with and without CAF conditioned media (CM) after 4 hr. *p<0.05, **p< 0.01. Values are mean + SEM n=3. Data is representative of two biological repeats with CAF CM from two different patient-derived CAFs. (G) Glycogen content in SKOV3ip1 cells transfected with PGM1 siRNA or scramble control (siScr) visualized by 2-NBDG fluorescence. Values are mean + SEM from 3 independent experiments (n=50 cells/group). (H) Glycogen assay on TYK-nu cells expressing either wild-type (WT) PGM1 or the Y353F PGM1 mutant and co-cultured with primary human CAFs in a transwell cell culture insert. Values are mean + SEM from 3 independent experiments. **p<0.01.