ROS are generated in response to LPS in colon cancer cells.

<p>(a) LPS induced a transient, significant rise in ROS activity in SW480, SW620, Ct-26 colon cancer cells. SW480, SW620 and CT-26 cells were treated with LPS (1 µg/ml) at twenty, forty and sixty minutes. DCF fluorescence was measured using FACScan flow cytometer and CellQuest software. These results are compiled in the above histograms. The y-axis represents cell counts and the x-axis measures the fluorescence level. A shift to the right along the x-axis represents a higher level of DCF fluorescence and thus ROS production. All histograms show a control (solid black line) with a colour line representing an increase in DCF fluorescence. The effect of LPS on ROS activity in SW480, SW620 and CT-26 colon cancer cells was quantified using CellQuest to measure the geometric means of the curves and compared to untreated controls tested on the same day, and compared in a bar chart. (b) LPS induces a dose dependent ROS burst at 40 minutes. A flow cytometry histogram demonstrates a dose dependent shift in ROS activity. (Solid black line = untreated, green = 0.1 µg/ml, red = 1 µg/ml, blue = 10 µg/ml. The dose dependent effect was quantified and compared in a bar chart. * P<0.05. These data are representative of 3 independent experiments.</p

Nox1, Nox2, p22phox and p47phox are expressed in SW480 cells.

<p>(a) Using Western Blotting we show that Nox1 expression in SW480 cells increases in response to LPS (1 ug/ml). (b) Western Blotting also shows that LPS increased expression of Nox2 at 40 minutes. (c) p22phox is shown to be expressed and expression increases earlier than Nox1 and Nox2. (d) p47phox is shown to be expressed but expression is stable over the time points. (e) Quantitative PCR analysis of Nox1 and Nox2 mRNA in SW480 cells treated with LPS (1 µg/ml) over one hour. Data is represented as fold-change relative to control untreated cells hours as determined by the 2−ΔΔCt method. Results are expressed as mean±SD and are representative of three independent experiments. * P<0.05. These data are representative of 3 independent experiments.</p

LPS induces a ROS burst via a Nox dependent mechanism in SW480 cells.

<p>(a) A significant attenuation of fluorescence was seen in samples treated with the DPI (2 µM). DCF fluorescence was measured using FACScan flow cytometer and CellQuest software. Solid black line (control). Red line (LPS treated). Green line (LPS+DPI treated). (b,c) Rotenone and diclofenac failed to inhibit LPS (1 µg/ml) induced ROS activity at 40 minutes. * P<0.05. These data are representative of 3 independent experiments.</p

LPS accelaerates SW480 colon cancer cell adhesion via Nox regulation of PI3K/Akt signalling.

<p>(a,b) LPS treatment resulted in a significant increase in SW480 cell adhesion to collagen I after 1 hour. This was inhibited using a recognised Nox inhibitor, DPI (2 µM), and a PI3K inhibitor, LY294002(5 µM). (c) Nox1 siRNA abrogates LPS induced ROS and Nox1 knockdown is confirmed by Western Blotting. (d) The increase in SW480 colon cancer cell adhesion was completely inhibited by Nox1 siRNA. * P<0.05. These data are representative of 3 independent experiments.</p

Pan-cancer <i>INPP4B</i> prognostic significance.

<p>Standard CoxPH SubID output consisting of (A,E,I) SubID plot, (B,F,J) <i>INPP4B</i> expression boxplot with expression distribution outlier status, and (C,G,K) resampled SubID plot with median (blue) and outlier cut-off values (based on expression distribution; red) for (A-D) kidney clear cell carcinoma, (E-H) bladder urothelial carcinoma, and (I-L) liver hepatocellular carcinoma reveals prognostically significant association between <i>INPP4B</i> expression status and patient survival. Specifically, <i>INPP4B</i>^low is associated with shorter overall survival in (D) kidney, (H) bladder and (L) liver cancers. Furthermore, <i>INPP4B</i> expression status defines three prognostically distinct subgroups within liver cancer.</p

H₂O₂ Production Downstream of FLT3 Is Mediated by p22phox in the Endoplasmic Reticulum and Is Required for STAT5 Signalling

<div><p>The internal tandem duplication (ITD) of the juxtamembrane region of the FLT3 receptor has been associated with increased reactive oxygen species (ROS) generation in acute myeloid leukemia (AML). How this elevated level of ROS contributes to the leukemic phenotype, however, remains poorly understood. In this work we show that ROS in the FLT3-ITD expressing AML cell line MV4-11 is reduced by treatment with PKC412, an inhibitor of FLT3, DPI, a flavoprotein inhibitor, and VAS2870, a Nox specific inhibitor, suggesting that ROS production is both FLT3 and NADPH oxidase dependent. The majority of these ROS co-localize to the endoplasmic reticulum (ER), as determined with the H₂O₂-specific aryl-boronate dye Peroxyorange 1, which also corresponds to co-localization of p22phox. Moreover, knocking down p22phox dramatically reduces H₂O₂ after 24 hours in the ER, without affecting mitochondrial ROS. Significantly, the FLT3 inhibitor PKC412 reduces H₂O₂ in FLT3-ITD expressing cell lines (MV4-11, MOLM-13) through reduction of p22phox over 24 hours. Reduced p22phox is achieved by proteasomal degradation and is prevented upon GSK3-β inhibition. Knockdown of p22phox resulted in reduced STAT5 signalling and reduced Pim-1 levels in the cells after 24 hours. Thus, we have shown that FLT3 driven H₂O₂ production in AML cells is mediated by p22phox and is critical for STAT5 signalling.</p> </div

SubID application to <i>INPP4B</i> across AML datasets.

<p>Standard CoxPH SubID output consisting of a (A,E,I) SubID plot, (B,F,J) <i>INPP4B</i> expression boxplot with expression distribution outlier status, and (C,G,K) resampled SubID plot with median (blue) and outlier cut-off values (based on expression distribution; red) for the Verhaak, TCGA and OCI AML datasets, respectively. (D,H,L) Kaplan-Meier plot of patients dichotomized at the SubID optimal cut-off demonstrates a significant survival difference between <i>INPP4B</i>^low and <i>INPP4B</i>^high subgroups.</p

Transcriptional regulators of <i>INPP4B</i> (Verhaak dataset).

<p>(A-F) resampled Fisher’s exact SubID plot for the top six DNA-binding transcription factors co-expressed with <i>INPP4B</i> in Verhaak dataset. (G) Color and status (based on Verhaak dataset-derived maximal co-expression analysis) heatmaps of the top potential transcriptional regulators of <i>INPP4B</i> expression in AML (Verhaak dataset).</p

EVI1 regulates <i>INPP4B</i> expression in AML.

<p>(A) <i>INPP4B</i> expression in <i>EVI1</i>^high compared to <i>EVI1</i>^low AML patients both in TCGA-LAML and (B) Verhaak datasets. Data represent mean <i>INPP4B</i> expression in <i>EVI1</i> high vs. low groups; cutoffs were determined using Fisher’s exact optimized SubID. (C) <i>EVI1</i> and <i>INPP4B</i> expression in a panel of AML cell lines. (D,E) <i>EVI1</i> and <i>INPP4B</i> transcript expression in U937 and OCI-AML-3 cells transduced with MSCV-puro-IRES-GFP (PIG) and PIG-EVI1 retroviruses. (F) Illustration of predicted EVI1 binding site 1379bp downstream of the <i>INPP4B</i> transcription start site. (G) Anti-EVI1 ChIP in <i>EVI1</i>^<i>hig</i>h OCI-AML -4 and OCI-AML -6 cells demonstrated enrichment at the +1379bp EVI1 binding site of the <i>INPP4B</i> promoter region.</p

FLT3-ITD Inhibition results in proteasomal degradation of p22phox.

<p>(a) Products of quantitative PCR for p22phox mRNA in untreated MV4-11 cells (Unt) and cells treated with PKC412 (250nM) for 24 hours were visualised by agarose gel separation, loading control is β-Actin. Histogram shows relative expression of p22phox mRNA in untreated MV4-11 cells and cells treated with PKC412 (250nM) for 24 hours as determined by quantitative PCR ΔΔCt-method. Results are expressed as mean ± SD and are representative of three independent experiments. Statistical analysis was performed using student t-test (*p<0.005) (b) Western blot analysis of p22phox protein expression in untreated MV4-11 cells (UT), vehicle controls (Veh), and cells treated with PKC412 (250nM) and lactacystin (5μM; Lac) for 8 hours using β-Actin as a loading control. Histograms represent the ratio of the intensity of target bands quantified by densitometry factored by the densitometric measurement of loading control band. The data are expressed as percentage of control, where the ratio in the control was defined as 1. Values are the mean ± SD and are representative of three independent experiments (c) Immunoprecipitation of p22phox from whole-cell lysates of untreated MV4-11 cells (Unt), cells treated with PKC412 (250nM) and lactacystin (5 μM; Lac) for 8 hours. Nitrocellulose membranes were probed for the presence of ubiquitin. Values are the mean ± SD and are representative of three independent experiments.</p