Fig 2: 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).

Fig 3: Schematic time-course changes in cellular processes in phosphorylase liver isoform knockdown (shPYGL) and control (shControl) U87MG glioblastoma (GBM) cells following high dose ionising radiation (IR).PYGB Glycogen phosphorylase brain isoform, PYGM Glycogen phosphorylase muscle isoform, PYGL Glycogen phosphorylase liver isoform, GAA alpha-acid glucosidase, GYS1 Glycogen Synthase 1, GBE1 Glycogen branching enzyme 1, AMPK AMP-activated protein kinase, ß-gal ß-galactosidase, PPP Pentose phosphate pathway, OCR Oxygen consumption rate, ECAR Extracellular acidification rate. (Figure created with BioRender.com).

Fig 4: Inhibition of glycogen phosphorylase liver isoform (PYGL) sensitises glioblastoma (GBM) cells to ionising radiation (IR).A, B Cell proliferation in response to different doses of IR (n = 3, error bars are ± SD, ***p < 0.001). C, D Cell numbers and phase contrast images in response to 10 and 12 Gy IR in control (shControl) and PYGL knockdown (shPYGL) U87MG cells (n = 3, error bars are ±SD, ***p < 0.001, p-values calculated by unpaired t-test). E Coomassie blue staining was conducted 21 days after a single IR dose of 12 Gy in shControl and shPYGL U87MG cells. (n = 3). F, G Representative spheroid images F and quantification of spheroid volume (G) on day 3 (T3), day 8 (T8), day 13 (T13) and day 21 (T21) of shControl and shPYGL U87MG cells with or without IR on day 0 (n = 3, error bars are ±SD, ***p < 0.001, p values were calculated by Two Way ANOVA - Tuckey post-hoc). H, I. Cell numbers and Coomassie blue staining 21 days after different fractionated schedules of IR (2x12Gy, 3x6Gy, 2x8Gy) in shControl and shPYGL U87MG cells (n = 3, error bars are ±SD, **p < 0.01, ***p < 0.001, p values were calculated by unpaired t-test).

Fig 5: Gene and protein expression of glycogen and glycogen related enzymes in glioblastoma (GBM) patient tumours.A Maximal and minimal protein expression of glycogen, glycogen phosphorylase liver isoform (PYGL) and glycogen phosphorylase brain isoform (PYGB) observed in the tissue cores on the stained tissue microarray. B Kaplan-Meier curves plotted based on the mean protein expression score of three or four GBM tissue cores per patient younger than 70 years of age at date of surgery. Low or high protein expression of glycogen, PYGL and PYGB was determined based on the median score per patient in the complete cohort. C Heatmap showing Spearman’s correlations between protein expression scores of glycogen, glucose transporter 1 (GLUT-1), glycogen synthase 1(GYS1), PYGL and PYGB per glioblastoma tissue core. D t-Distributed Stochastic Neighbour Embedding (t-SNE) [67] plots showing the expression of PYGL and PYGB in FACS-sorted tumor cells of 3 GBM PDXs (P3, P8, P13) and 2 patient derived GBM cultures (NCH644, NCH421k) and subpopulations of NCH644 P2 (CD133 + CD44-A2B5 + CD15 + ) and P6 (CD133 + CD44 + A2B5 + CD15 + ). One sample per tumour is available.