Fig 2: NOX4 67 kDa- and NOX4D 28 kDa–generated pro-survival ROS require AKT activation(A) Western blot analysis of AKT signalling in FLT3-ITD expressing MV4-11 cells following treatment with LY294002 (20 µM, 30 µM and 50 µM) for 16 h. ß-actin and total protein were used as loading controls. (B) NOX4 67 kDa, NOX4D 28 kDa and p22phox protein expression in membrane and soluble nuclear fractions of MV4-11 cells following treatment with LY294002 for 16 h at indicated concentrations. Equal loading of samples is shown by total protein and verification of subcellular fractions were assessed by probing for nuclear-localised NUP98 and membrane-localised calreticulin. Western blot analysis is representative of three independent experiments. (C) (i) Flow cytometric analysis of mean relative PO1 fluorescence in MV4-11 cells treated with LY294002 for 16 h at indicated concentrations. (ii) Bar chart shows relative mean PO1 fluorescence of treated cells expressed as % of control. Results are representative of three independent experiments. Asterisks indicate statistically significant differences (****p<0.0001) as analysed by Student's t-test. Error bars are representative of SD.

Fig 3: Inhibition of GSK3ß signalling results in elevated NOX4 67 kDa and NOX4D 28 kDa protein expression(A) Western blot analysis of GSK3ß signalling in FLT3-ITD expressing MV4-11 cells following treatment with SB216763 (1 µM, 2 µM and 5 µM) for 16 h. ß-actin and total protein were used as loading controls. (B) NOX4 67 kDa, NOX4D 28 kDa and p22phox protein expression in membrane and soluble nuclear fractions of MV4-11 cells following treatment with SB216763 for 16 h at indicated concentrations. Equal loading of samples is shown by total protein and verification of subcellular fractions were assessed by probing for nuclear-localised NUP98 and membrane-localised calreticulin. Western blot analysis is representative of four independent experiments. (C)(i) Flow cytometric analysis of mean relative PO1 fluorescence in MV4-11 cells treated with SB216763 for 16 h at indicated concentrations. (ii) Bar chart shows relative mean PO1 fluorescence of treated cells expressed as % of control. Results are representative of three independent experiments. Asterisks indicate statistically significant differences (***p<0.001, ****p<0.0001) as analysed by Student's t-test. Error bars are representative of SD. (D) Western blot analysis of GSK3ß signalling in FLT3-ITD expressing MV4-11 cells following treatment with lithium chloride (10 mM, 20 mM and 50 mM) for 16 h. ß-actin and total protein were used as loading controls. (E) NOX4 67 kDa, NOX4D 28 kDa and p22phox protein expression in membrane and soluble nuclear fractions of MV4-11 cells following treatment with lithium chloride for 16 h at indicated concentrations. Equal loading of samples is shown by total protein and verification of subcellular fractions were assessed by probing for nuclear-localised NUP98 and membrane-localised calreticulin. Western blot analysis is representative of three independent experiments. (F)(i) Flow cytometric analysis of mean relative PO1 fluorescence in MV4-11 cells treated with lithium chloride for 16 h at indicated concentrations. (ii) Bar chart shows relative mean PO1 fluorescence of treated cells expressed as % of control. Results are representative of four independent experiments. Asterisks indicate statistically significant differences (**p<0.01, ***p<0.001) as analysed by Student's t-test. Error bars are representative of SD.

Fig 4: NOX4 67 kDa- and NOX4D 28 kDa-generated pro-survival ROS are independent of ERK1/2 signalling. p22phox generated H2O2 requires ERK1/2 activation(A) Western blot analysis of ERK1/2 signalling in FLT3-ITD expressing MV4-11 cells following treatment with U0126 (10 µM, 20 µM, 50 µM and 100 µM) for 16 h. a-tubulin and total protein were used as loading controls. (B) NOX4 67 kDa, NOX4D 28 kDa and p22phox protein expression in membrane and soluble nuclear fractions of MV4-11 cells following treatment with U0126 for 16 h at indicated concentrations. Equal loading of samples is shown by total protein and verification of subcellular fractions was assessed by probing for nuclear-localised NUP98 and membrane-localised calreticulin. Western blot analysis is representative of three independent experiments. (C) (i) Flow cytometric analysis of mean relative PO1 fluorescence in MV4-11 cells treated with U0126 for 16 h at indicated concentrations. (ii) Bar chart shows relative mean PO1 fluorescence of treated cells expressed as % of control. Results are representative of four independent experiments. Asterisks indicate statistically significant differences (*p<0.05, ***p<0.001, **** p<0.0001) as analysed by Student's t-test. Error bars are representative of SD.

Fig 5: FLT3-ITD expressing AML patient samples and cell lines express the NOX4D 28 kDa isoform(A) Western blot analysis of NOX4 67 kDa and NOX4D 28 kDa protein expression in FLT3-ITD- and FLT-WT-expressing AML patient samples. ß-actin and Vinculin were used as loading controls. (B and C) Subcellular fractionation was carried out in FLT3-ITD expressing AML cell line, MV4-11 (B) and 32D cells transfected with FLT3-WT or FLT3-ITD (C). Expression of NOX4 67 kDa, NOX4D 28 kDa and p22phox was assessed by means of western blot analysis. Equal loading of samples and verification of the subcellular fractions were demonstrated by probing for nuclear-localised NUP98, HDAC1 and Histone H3, membrane-localised calreticulin and KDEL and cytosolic-localised GAPDH. Blots are representative of five independent experiments. (D) (i) 32D cells stably transfected with FLT3-WT and FLT3-ITD were IL-3 starved overnight, followed by ROS visualisation with H2O2 specific probe, Peroxy Orange 1 (PO1) for 1 h before flow cytometric analysis. (ii) Bar chart shows relative mean PO1 fluorescence of 32D/FLT3-ITD cells expressed as a % of 32D/FLT3-WT cells. The results are representative of three independent experiments. Asterisks indicate statistically significant differences (****p<0.0001) as analysed by Student's t-test. Error bars represent SD.