Fig. 1: Characterisation of expression levels of glycogen related enzymes, effect of PYGL knockdown, glycogen accumulation and bioenergetics of six glioblastoma (GBM) cell lines.
A Differential expression of glycogen related proteins in GBM cell lines: glycogen phosphorylase liver isoform (PYGL), glycogen phosphorylase brain isoform (PYGB), glycogen synthase 1 (GYS1), phospho-glycogen synthase (pGYS1), glycogen branching enzyme 1 (GBE1) and glycogen debranching enzyme (GDE). Beta-actin was used as loading control. B Effects of PYGL knockdown (siPYGL) and PYGB knockdown (siPYGB) on cell numbers after 5 days in six GBM cell lines (n = 3, error bars are ± SD, ***p < 0.001, repeated measures ANOVA, Tuckey post hoc). C Oxygen consumption rate (OCR) was measured in six GBM cell lines, by a mitochondria stress test using oligomycin (an ATP synthase inhibitor), FCCP (an electron transport chain uncoupler) and rotenone/antimycin (inhibitors of electron transport chain). D Extracellular acidification rate (ECAR) was measured in six GBM cell lines. Cells incubated in 0 mM glucose for 1 h in CO2 free conditions were exposed to 10 mM glucose and rotenone/antimycin. E Glycogen levels in response to hypoxia in GBM cancer cell lines. Cell lines were incubated in 21% (normoxia) and 0.1% (hypoxia) oxygen for 24 h. Glycogen levels were analysed using a colorimetric kit (n = 3, Error bars are ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, repeated measures ANOVA, Tukey post hoc). F Kinetics of the cytosolic ATP/ADP ratio [ATP/ADP]cyt in response to high (20 mM) glucose in control (shControl) U87 cells (red) and PYGL knockdown (shPYGL) U87 cells (blue).
