Extended Data Fig. 9: Characterisation and comparison of cytoLbNOX expressing cells in vitro and in vivo.
a Immunoblot of cytoLbNOX and catalytic mutant expression in clonal population, detected using αFLAG. Representative result of three biological replicates is shown. b Immunoblot of indicative respiratory chain subunits. Representative result of three individual runs is shown. c Basal oxygen consumption rate (OCR) (n = 36, 21, 24 and 9 technical replicates over n = 12, 7, 8 and 3 biological replicates). A significant decrease is observed in HcMel12 cytoLbNOX, akin to the decrease in basal OCR measured in m.12,43680% cells. d mtDNA copy number (n = 9 technical replicates over n = 3 biological replicates). e Proliferation rate of cell lines in permissive growth media (n = 12, 12, 12 and 8 technical replicates over n = 4, 4, 4 and 3 biological replicates). f Energy (adenylate) charge state (n = 24, 25, 25 and 9 technical replicates over n = 9, 9, 9 and 3 biological replicates). g NAD+:NADH ratio (n = 24, 25, 25 and 9 technical replicates over n = 9, 9, 9 and 3 biological replicates). h GSH:GSSG ratio (n = 22, 21, 20 and 9 technical replicates over n = 9, 9, 9 and 3 biological replicates). i Heatmap of unlabelled steady-state abundance of select metabolites in Hcmel12 cells. Succ. cys, succinylcysteine. (n = 9–25 technical replicates over n = 3–9 biological replicates). j Survival of C57/BL6 mice subcutaneously injected with indicated cells (n = 17, 18, 12 and 9 animals). k Untreated tumor weight at endpoint (n = 17, 18, 12 and 9 individual tumours). l Survival of C57/BL6 mice subcutaneously injected with indicated cells (n = 15, 15 and 10 animals) on sustained anti-PD1 therapy. Only tumors that hit endpoint of 15mm shown for cytoLbNOX. m Tumor weight at endpoint for mice on sustained anti-PD1 therapy (n = 15, 15 and 3 individual tumours). Tumor volume changes recorded from injection date for n wild-type and m.1243680% (n = 15 individual tumours) and o cytoLbNOX tumors (n = 10 individual tumours) on sustained anti-PD1. A one-way ANOVA test with Fisher’s LSD Test (C-I), Log-rank (Mantel-Cox) test (J,L) or one-way ANOVA with Sidak multiple comparisons test (K,M) were applied. Tumor volume calculated as 0.5*L*W2 based on calliper measurements. Error bars indicate SD. Measure of centrality is mean. * P = < 0.05, ** P = <0.01, *** P = <0.001, **** P = <0.0001. Number of replicates are described across conditions from left to right as presented. Heatmap representations of data where asterisks are not present report non-significant changes.
