Journal: Leukemia

Article Title: MAPK/ERK2 phosphorylates ERG at serine 283 in leukemic cells and promotes stem cell signatures and cell proliferation

doi: 10.1038/leu.2016.55

Figure Lengend Snippet: ERG S283 is phosphorylated in human leukemic cells. (a) A schematic of hERG3 scaled to size showing sites of serine phosphorylation in MOLT4 cells relative to its two functional domains. The numbers correspond to the amino acid positions. ETS, E-twenty six; PNT, Pointed. (b) A representative tandem mass spectrum (MS) showing phosphorylated ERG tryptic peptide containing S283. The b- and y-ions are annotated and labeled on the spectrum with the MS1 accurate spectrum shown in the inset. Note that for clarity, some regions of the extracted ion chromatogram have been amplified 10-fold. (c) ERG S283 phosphorylation (pS283) level in MOLT4, KG1 and healthy CD34+ human HSPCs was quantified using MS and expressed as a ratio to the level of pS88 on a log-10 scale. (d) A table summarizing the presence (Y) or absence (N) of ERG phosphorylation. Detection of S55 requires chymotrypsin digestion and was not available for all studies. (e) Lysates of 24 primary leukemic xenografts including 5 ETP ALL, 14 non-ETP T-ALL and 5 primary AML xenografts were probed for pS283 in ERG using a custom anti-pS283 ERG monoclonal antibody. A lysate of healthy CD34+ HSPCs is shown for comparison. Numbers above blots are densitometry reading for pS283 ERG normalized to β-actin. Given variations in exposure time, these numerical values are for within blot interpretation and not for interpretation across individual blots. Expression levels of total ERG and β-actin are also shown. The patient characteristics relating to the xenografts are detailed in previous studies.32–35

Article Snippet: Generation of ERG-pS283 specific antiserum Mouse monoclonal phospho-specific antibody for ERG pS283 was produced by ProMab Biotechnologies, Inc. (Richmond, CA, USA) using the artificially synthesized phosphopeptide PTPQSKAAQP(p)*S*PSTVPKTEDQ, S283 highlighted by asterisk.

Techniques: Phospho-proteomics, Functional Assay, Labeling, Amplification, Comparison, Expressing

Journal: Leukemia

Article Title: MAPK/ERK2 phosphorylates ERG at serine 283 in leukemic cells and promotes stem cell signatures and cell proliferation

doi: 10.1038/leu.2016.55

Figure Lengend Snippet: ERG S283 phosphorylation is mediated by the MAPK/ERK2 pathway. (a) A schematic showing the putative MAPK docking motif, DEF domain, located within ERG exon 12. The amino acid numbers and peptide sequence are shown. (b) A bar graph showing percent phosphorylation of serine residues in WT ERG and in an exon 12 deletion mutant of ERG (ERG-ΔEx12) measured using an in vitro ERK2 kinase assay. Peptide signal intensities of the phosphorylated and unphosphorylated versions were quantified by mass spectrometry (MS). Error bars represent s.d. for all peptide spectral matches quantified in this experiment (n = 3). ****P<0.0001. (c) A bar graph showing changes in phosphorylation in MOLT4 cells in response to treatment with U0126 (10 μM) and/or PMA (100 nM). The levels of phosphorylation were quantified by MS and plotted as a ratio of treated/vehicle on a log-10 scale. (d) Western blots showing the responses of two ETP ALL xenografts with high-level ERG pS283 following treatment with U0126 (10 μM) and/or PMA (100 nM). The levels of ERG pS283, total ERG, active ERK1/2 (pERK1/2), total ERK1/2 and β-actin are shown. Numbers above blots are densitometry readings for pS283 ERG normalized to β-actin. Given variations in exposure time, these numerical values are for within blot interpretation and not for interpretation across individual blots. The upper band in the ERK1/2 panel represents ERK1 and the lower band represents ERK2. (e) Western blots showing levels of endogenous ERG pS283, total ERG and active ERK1/2 (pERK1/2) in a panel of ETP ALL and AML patient-derived xenografts. Levels of each in healthy CD34+ HSPCs are also shown for comparison. Numbers above blots are densitometry reading for pS283 ERG normalized to β-actin. Given variations in exposure time, these numerical values are for within blot interpretation and not for interpretation across individual blots. (f) CFUs (colony-forming units) derived from vehicle-treated (0.1% dimethyl sulfoxide (DMSO)) or U0126-treated (10 μM) KG1, ME1 and CD34+ HSPCs. Error bars represent s.d. from triplicate measurements, *P<0.05 for the relevant comparisons.

Article Snippet: Generation of ERG-pS283 specific antiserum Mouse monoclonal phospho-specific antibody for ERG pS283 was produced by ProMab Biotechnologies, Inc. (Richmond, CA, USA) using the artificially synthesized phosphopeptide PTPQSKAAQP(p)*S*PSTVPKTEDQ, S283 highlighted by asterisk.

Techniques: Phospho-proteomics, Sequencing, Mutagenesis, In Vitro, Kinase Assay, Mass Spectrometry, Western Blot, Derivative Assay, Comparison

Journal: Cancer Science

Article Title: Squalene synthase predicts poor prognosis in stage I‐III colon adenocarcinoma and synergizes squalene epoxidase to promote tumor progression

doi: 10.1111/cas.15248

Figure Lengend Snippet: Squalene synthase (FDFT1) was upregulated in stage I‐III colon adenocarcinoma (COAD) tissues and colon cancer cell lines. A, Comparison of FDFT1 expression between 217 COAD and paired adjacent normal (AN) tissues from tissue microarray (TMA). B, Comparison of FDFT1 expression in different TNM stages (I‐III) from TMA. C, Comparison of FDFT1 expression in different T stages (1‐3, 4) from TMA. D, Comparison of FDFT1 expression in different differentiation grades (well/moderate, poor/mucinous) from TMA. Mean ± SD. E, FDFT1 expression in two paired AN and COAD tissues were detected by immunohistochemical analysis. FDFT1 low and FDFT1 high in COAD tissues are shown below. Scale bars, 50 μm. F, FDFT1 mRNA level in normal colon mucosal epithelial cells (NCM460) and colon cancer cell lines. NCM460 was used as control, housekeeping gene ACTB was used loading control. * P < .05, *** P < .001

Article Snippet: Membranes were incubated with primary Abs against FDFT1 (1:1000 dilution; Cat# 13128‐1‐AP; Proteintech), NAT8 (1:500 dilution; Cat# A7759; Abclonal), SQLE (1:1000 dilution; Cat# 12544‐1‐AP; Proteintech), Tubulin (1:5000 dilution; Cat# E7; DSHB), and β‐actin (1:5000 dilution; Cat# AC026; Abclonal) after being blocked at 4°C.

Techniques: Comparison, Expressing, Microarray, Immunohistochemical staining, Control

Journal: Cancer Science

Article Title: Squalene synthase predicts poor prognosis in stage I‐III colon adenocarcinoma and synergizes squalene epoxidase to promote tumor progression

doi: 10.1111/cas.15248

Figure Lengend Snippet: Comparison of clinicopathologic features of patients with stage I‐III colon adenocarcinoma between low and high expression of squalene synthase (FDFT1)

Article Snippet: Membranes were incubated with primary Abs against FDFT1 (1:1000 dilution; Cat# 13128‐1‐AP; Proteintech), NAT8 (1:500 dilution; Cat# A7759; Abclonal), SQLE (1:1000 dilution; Cat# 12544‐1‐AP; Proteintech), Tubulin (1:5000 dilution; Cat# E7; DSHB), and β‐actin (1:5000 dilution; Cat# AC026; Abclonal) after being blocked at 4°C.

Techniques: Comparison, Expressing

Journal: Cancer Science

Article Title: Squalene synthase predicts poor prognosis in stage I‐III colon adenocarcinoma and synergizes squalene epoxidase to promote tumor progression

doi: 10.1111/cas.15248

Figure Lengend Snippet: Univariate and multivariate analyses for overall survival (OS) and relapse‐free survival (RFS) in patients with stage I‐III colon adenocarcinoma

Article Snippet: Membranes were incubated with primary Abs against FDFT1 (1:1000 dilution; Cat# 13128‐1‐AP; Proteintech), NAT8 (1:500 dilution; Cat# A7759; Abclonal), SQLE (1:1000 dilution; Cat# 12544‐1‐AP; Proteintech), Tubulin (1:5000 dilution; Cat# E7; DSHB), and β‐actin (1:5000 dilution; Cat# AC026; Abclonal) after being blocked at 4°C.

Techniques: Expressing

Journal: Cancer Science

Article Title: Squalene synthase predicts poor prognosis in stage I‐III colon adenocarcinoma and synergizes squalene epoxidase to promote tumor progression

doi: 10.1111/cas.15248

Figure Lengend Snippet: Upregulation of squalene synthase (FDFT1) was associated with poor prognosis in stage I‐III colon adenocarcinoma (COAD). A, Kaplan‐Meier curve for overall survival (OS) according to FDFT1 expression in 233 cases of stage I‐III COAD. FDFT1 high expression indicated a lower OS rate (log‐rank P < .001). B, C, Kaplan‐Meier curves for OS according to FDFT1 expression in 115 and 118 cases of right‐ and left‐sided COAD, respectively. FDFT1 high expression indicated a lower OS rate (both log‐rank P < .001). D, Nomogram for predicting 5‐year OS of stage I‐III COAD. CEA, carcinoembryonic antigen. E, Receiver operating characteristic (ROC) analysis of the nomogram for predicting 5‐year OS (area under the ROC curve [AUC] = 0.871). F, Kaplan‐Meier curve for relapse‐free survival (RFS) according to FDFT1 expression in 233 cases of stage I‐III COAD. FDFT1 high expression indicated a lower RFS rate (log‐rank P < .001). G, H, Kaplan‐Meier curves for RFS according to FDFT1 expression in 115 and 118 cases of right‐ and left‐sided COAD, respectively. FDFT1 high expression indicated a lower RFS rate (log‐rank P < .001 and P =.001, respectively). I, Nomogram for predicting 5‐year RFS of stage I‐III COAD. J, ROC analysis of the nomogram for predicting 5‐year RFS (AUC = 0.863)

Article Snippet: Membranes were incubated with primary Abs against FDFT1 (1:1000 dilution; Cat# 13128‐1‐AP; Proteintech), NAT8 (1:500 dilution; Cat# A7759; Abclonal), SQLE (1:1000 dilution; Cat# 12544‐1‐AP; Proteintech), Tubulin (1:5000 dilution; Cat# E7; DSHB), and β‐actin (1:5000 dilution; Cat# AC026; Abclonal) after being blocked at 4°C.

Techniques: Expressing

Journal: Cancer Science

Article Title: Squalene synthase predicts poor prognosis in stage I‐III colon adenocarcinoma and synergizes squalene epoxidase to promote tumor progression

doi: 10.1111/cas.15248

Figure Lengend Snippet: Squalene synthase (FDFT1) deficiency attenuated proliferation of HCT116 and HT29 colon cancer cells. A, B, After knockdown (KD) of FDFT1, the protein level of FDFT1 in HCT116 or HT29 cells was detected by western blot analysis; shcontrol (SC) as control group. C, D, After KD of FDFT1, the proliferation of HCT116 or HT29 cells was measured by counting cell numbers. E, After KD of FDFT1, the proliferation of HCT116 or HT29 cells was measured by EdU staining. F, Statistical results of (E). G, Colony formation of HCT116 or HT29 cells was examined by crystal violet staining after KD of FDFT1. H, Statistical results of (G). I, J, After separate overexpression (OE) of FDFT1 in HCT116 or HT29 cells, the protein level of FDFT1 was detected by western blot analysis; vector as control group. K, Colony formation of HCT116 cells or HT29 cells was examined by crystal violet staining after OE of FDFT1. L, Statistical results of (K). Mean ± SEM. * P < .05; ** P < .01; *** P < .001; ns, no significance

Article Snippet: Membranes were incubated with primary Abs against FDFT1 (1:1000 dilution; Cat# 13128‐1‐AP; Proteintech), NAT8 (1:500 dilution; Cat# A7759; Abclonal), SQLE (1:1000 dilution; Cat# 12544‐1‐AP; Proteintech), Tubulin (1:5000 dilution; Cat# E7; DSHB), and β‐actin (1:5000 dilution; Cat# AC026; Abclonal) after being blocked at 4°C.

Techniques: Knockdown, Western Blot, Control, Staining, Over Expression, Plasmid Preparation

Journal: Cancer Science

Article Title: Squalene synthase predicts poor prognosis in stage I‐III colon adenocarcinoma and synergizes squalene epoxidase to promote tumor progression

doi: 10.1111/cas.15248

Figure Lengend Snippet: Knockdown (KD) of squalene synthase (FDFT1) induced accumulation of N ‐acetyltransferase 8 (NAT8) and D‐pantethine to reduce reactive oxygen species level and inhibit proliferation of HT29 cells. A, Volcano plots of RNA sequencing analysis between shControl and FDFT1 KD HT29 cells; 1940 genes were upregulated, and 1828 genes were downregulated. Arrow indicates NAT8. B, Volcano plots of untargeted metabolomics analysis between shControl and FDFT1 KD HT29 cells. Arrow indicates D‐pantethine. C, Fragments per kilobase of transcript per million mapped reads (FPKM) value of NAT8 in shControl and FDFT1 KD HT29 cells from (A). D, Value of D‐pantethine in shControl and FDFT1 KD HT29 cells from (B). E, Schematic diagram of FDFT1 regulating NAT8 and D‐pantethine. Red boxes represent genes; blue boxes represent metabolites. Downregulation of FDFT1 (green) leads to upregulation of NAT8 and D‐pantethine (red). F, shControl and FDFT1 KD HT29 cells were pretreated with 1 mmol L −1 N ‐acetyl‐l‐cysteine NAC for 1 h, then analyzed by flow cytometry through DCFH‐DA staining. G, NAT8 was overexpressed in shControl and FDFT1 KD HT29 cells. Protein levels of FDFT1 and NAT8 (red star) were detected by western blot analysis; β‐actin as a loading control. Green star indicates degradation of NAT8. H‐J, Cell proliferation and colony formation of these cells were measured by counting cell numbers and crystal violet staining. K, L, shControl and FDFT1 KD HT29 cells were treated with D‐pantethine at concentrations of 0, 5, 10 μmol L −1 , respectively, then stained by crystal violet. Mean ± SEM. * P < .05; ** P < .01; *** P < .001; ns, no significance

Article Snippet: Membranes were incubated with primary Abs against FDFT1 (1:1000 dilution; Cat# 13128‐1‐AP; Proteintech), NAT8 (1:500 dilution; Cat# A7759; Abclonal), SQLE (1:1000 dilution; Cat# 12544‐1‐AP; Proteintech), Tubulin (1:5000 dilution; Cat# E7; DSHB), and β‐actin (1:5000 dilution; Cat# AC026; Abclonal) after being blocked at 4°C.

Techniques: Knockdown, RNA Sequencing, Flow Cytometry, Staining, Western Blot, Control

Journal: Cancer Science

Article Title: Squalene synthase predicts poor prognosis in stage I‐III colon adenocarcinoma and synergizes squalene epoxidase to promote tumor progression

doi: 10.1111/cas.15248

Figure Lengend Snippet: Combined inhibition of squalene synthase (FDFT1) and squalene epoxidase (SQLE) suppressed growth of colon cancer cells and xenograft tumors. A, After separate or double knockdown (KD) of FDFT1 and SQLE, the protein levels of FDFT1 and SQLE in HT29 cells were detected by western blot analysis; shControl, control group. B, Colony formation of cells in (A) were measured by crystal violet staining. C, Statistical results of (B). D, Intracellular total cholesterol level of shControl, FDFT1 KD, SQLE KD, and FDFT1/SQLE HT29 cells were measured. E, HT29 cells were treated with lapaquistat (La) and terbinafine (Te) at different concentrations for 48 or 72 h, then stained with EdU. F, After combined treatment with La and Te, the colony formation of HCT116 and HT29 cells was measured by crystal violet staining. G, Statistical results of (F). H, Tumor growth of shControl, FDFT1 KD, SQLE KD, and FDFT1/SQLE KD HT29 cells in nude mice after injection for 1 wk. I, J, Images and weights of the tumors in (H) removed from nude mice. K, Immunohistochemical analysis of Ki‐67 in xenograft tumors from (I). Scale bars, 50 μm. L, Statistical results of (K). M, FDFT1 and N ‐acetyltransferase 8 (NAT8) mRNA levels in tumors derived from shControl and FDFT1 KD HT29 cells. Mean ± SEM. * P < .05, ** P < .01, *** P < .001

Article Snippet: Membranes were incubated with primary Abs against FDFT1 (1:1000 dilution; Cat# 13128‐1‐AP; Proteintech), NAT8 (1:500 dilution; Cat# A7759; Abclonal), SQLE (1:1000 dilution; Cat# 12544‐1‐AP; Proteintech), Tubulin (1:5000 dilution; Cat# E7; DSHB), and β‐actin (1:5000 dilution; Cat# AC026; Abclonal) after being blocked at 4°C.

Techniques: Inhibition, Knockdown, Western Blot, Control, Staining, Injection, Immunohistochemical staining, Derivative Assay

Journal: Cancer Science

Article Title: Squalene synthase predicts poor prognosis in stage I‐III colon adenocarcinoma and synergizes squalene epoxidase to promote tumor progression

doi: 10.1111/cas.15248

Figure Lengend Snippet: Schematic diagram showing synergistic function of squalene synthase (FDFT1) and squalene epoxidase (SQLE) in promoting colon adenocarcinoma (COAD). FDFT1 induces accumulation of reactive oxygen species (ROS) level and promotes colon cancer cell proliferation through upregulation of D‐pantethine and N ‐acetyltransferase 8 (NAT8). FDFT1 also synergizes SQLE to facilitate COAD progression

Article Snippet: Membranes were incubated with primary Abs against FDFT1 (1:1000 dilution; Cat# 13128‐1‐AP; Proteintech), NAT8 (1:500 dilution; Cat# A7759; Abclonal), SQLE (1:1000 dilution; Cat# 12544‐1‐AP; Proteintech), Tubulin (1:5000 dilution; Cat# E7; DSHB), and β‐actin (1:5000 dilution; Cat# AC026; Abclonal) after being blocked at 4°C.

Techniques: