Fig 2: 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.

Fig 3: Inhibition of the FLT3 receptor in 32D/FLT3-ITD cells and FLT3-ITD expressing MV4-11 cells reduces NOX4 67 kDa and NOX4D 28 kDa protein expression and total endogenous H2O2Inhibition of the FLT3 receptor following treatment with PKC412, results in a decrease in total endogenous H2O2 in 32D/FLT3-ITD cells but not in 32D/FLT3-WT cells. (A) 32D/FLT3-WT and 32D/FLT3-ITD cells were IL-3 starved overnight and treated for 24 h with PKC412 (50 nM and 200 nM), followed by staining with H2O2 specific probe PO1 for 1 h before FACS reading. Bar charts show relative mean PO1 fluorescence of treated cells expressed as % of control. The results are representative of four independent experiments. Asterisks indicate statistically significant differences (****p<0.0001) as analysed by Student's t-test. Error bars represent SD. (B) Western blot analysis of NOX4 67 kDa and NOX4D 28 kDa protein expression in MV4-11 whole cell lysates following treatment with PKC412 for 24 h at indicated concentrations. ß-actin was used as a loading control. Blots are representative of three independent experiments. (C) (i) Flow cytometric analysis of mean relative PO1 fluorescence in MV4-11 cells treated with PKC412 for 24 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 represent SD. (D and E) Western blot analysis of NOX4D 28 kDa protein expression in the nuclear fraction following treatment with AC220 (10 nM, 20 nM and 30 nM) for 12 h in MV4-11 cells (D) and 32D cells transfected with FLT3-ITD (E). Equal loading of nuclear fractions was demonstrated by probing for nuclear-localised Lamin A/C. Blots are representative of three independent experiments.

Fig 4: ATP directly binds NOX4 and negatively regulates NOX4 activity. a Alignment of the human NOX isoforms; NOX1–5, DUOX 1, and DUOX 2 shows a Walker A, ATP-binding motif (A/GXXXGKT/S) uniquely within the NOX4 isoform. b The Walker A ATP-binding motif is located at amino acids 534–541 conserved among Homo sapiens (hNOX4), Rattus norvegicus (rNOX4), and Mus musculus (mNOX4). c In vitro ATP-binding assay was performed using equal amounts (1 µg) of recombinant WT NOX4341–561 incubated with increasing doses (0.125–1.0 µCi) of 32P-labeled ATP ([a-32P]-ATP) and blotted onto 0.45-micron nitrocellulose, washed, and counted by scintillation as described in Methods. The results are presented as counts (dpm) to background (bkg) and are representative of two independent experiments of an n = 6 per sample. d In vitro ATP-binding assay was performed as in c; however, excess cold ATP was incubated with 0.5 or 1.0 µg recombinant WT NOX4341–561 protein prior to [a-32P]-ATP incubation. The results are presented as counts (dpm) to background (bkg) and are representative of two independent experiments of an n = 6 per sample. e In vitro ATP-binding assay was performed using 1 µg recombinant WT NOX4341–561 and 1 µg MUT NOX4341–561, K540A protein. f Surface plasmon resonance-based technology was employed to evaluate ATP binding to recombinant WTNOX4 and the ATP MUTNOX4 as described in Methods. The results are presented as counts (dpm) to background (bkg) and are representative of two independent experiments of an n = 6 per sample. g Increasing doses of ATP (2–5 mM) were incubated in total homogenates prepared from RCC 786-O cells. NADPH-dependent superoxide generation (NOX activity) was measured by enhanced chemiluminescence as described in Methods. h NOX activity was examined in RCC 786-O or i A498 total homogenates pre-incubated with 5.0 mM ATP, ADP, or GTP on ice for 30 min. Unless otherwise noted, the results are from at least three independent experiments and expressed as the means using one-way ANOVA with Tukey’s post hoc test where ±S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001 compared to control

Fig 5: NOX4 knockdown markedly improves survival and attenuates acute lung injury in CLP-induced septic mice. Sepsis was induced by cecal ligation and puncture (CLP) in wild-type (WT) mice and mice deficient in NOX2 or p47phox, or mice transfected of control siRNA, NOX1 or NOX4 siRNA. Natural death time was recorded by observation every 30 min starting 12 h after CLP, and the death time was presented as a Kaplan-Meier plot using the Prism software, and log-rank test was used to compare survival between groups. Other mice were harvested at 16 h post CLP and lung tissues harvested for analyses of histomorphology, Evans Blue index and wet-to-dry weight ratio were used to assess severity of acute lung injury. (A) Survival curves in sham group, CLP groups treated with control siRNA, NOX1 or NOX4 siRNA, CLP WT group and CLP groups made in NOX2 or p47phox knockout mice. **p < 0.01, ##p < 0.01. (B) Representative images of H&E staining of lung tissue sections and semiquantitative histological scores of lung injury in experimental groups described in panel A. H&E stainings indicate signs of inflammation (red arrows), edema (blue arrows), hemorrhage (black arrows), and alveolar septal thickening (green arrows) from the cross sections of the lung. Bar = 100 µm. Data are presented as Mean±SEM, n = 5. **p < 0.01 vs. CLP group. (C–D) Lung wet-to-dry weight ratio and Evans Blue index were determined in experimental groups as described in panel A. Data are presented as Mean±SEM, n = 5. *p < 0.05, **p < 0.01 vs. control siRNA transfected CLP group. Data indicate NOX4 knockdown in CLP mice is selectively and robustly effective in improving survival and attenuating acute lung injury. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)