Figure 7: IL1α/IL1β and ROS homeostasis are critical for tumour sphere-forming ability.

(a) Simultaneous neutralization of IL1α and IL1β abolished in vitro tumour sphere formation. Anti-IL1α (1:200) and anti-IL1β (1:200) neutralizing antibodies were added to medium on day 1 and day 4 after seeding Hep3B cells for in vitro tumour sphere-forming assay. Data were presented as the mean number of three biological replicates±s.e.m. See Supplementary Fig. 7a and experimental procedures for details. (b) IL1β and NAC co-treatment rescued the defect of NEDD4 knockdown in the in vitro tumour sphere formation. Control and NEDD4 knockdown Hep3B cells were treated with recombinant IL1β and/or NAC (0.5 mM) in the in vitro tumour sphere-forming assay. Data were presented as the mean number of three biological replicates±s.e.m. See Supplementary Fig. 7b and experimental procedures for details. (c,d) NEDD4 and H3 ubiquitination are required for the maintenance of cellular ROS. Control and NEDD4 knockdown or H3.3 WT or K23/36/37R restored Hep3B cells were stained by DCFDA for cellular ROS and subjected to flow cytometry analysis. Data were presented as the mean DCFDA signals of three biological replicates±s.e.m. See experimental procedures for details. (e) GSH level is reduced in NEDD4 knockdown cells. Control and NEDD4 knockdown Hep3B cells were collected and GSH levels were measured by a colorimetric enzymatic reaction. Data were presented as the mean value of three biological replicates±s.e.m. See experimental procedures for details. (f) Model of glucose-induced H3 mono-ubiquitination by NEDD4 and subsequent GCN5-mediated H3 acetylation, which regulates tumour sphere forming and tumour engraftment through transcription activation of genes, such as IL1A, IL1B and GCLM. N-tail represents histone H3 N-terminal tail. All asterisks (*) represent P<0.05, using Student’s T-test.