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:

Journal: American Journal of Cancer Research

Article Title: Differences of ferroptosis-related genes between White and Asian patients with liver cancer

doi:

Figure Lengend Snippet: The roles of FDFT1, ACSL4 and EMC2 in ferroptosis of liver cancer cells. (A) Cell viability was assayed by MTT of HepG2 and SNU-449 cells after FDFT1, ACSL4 or EMC2 knockdown juxtaposed to parental cells. (B) Intracellular ROS, (C) MDA formation, and (D) Gln uptake activities of HepG2 and SNU-449 cells after FDFT1, ACSL4 or EMC2 knockdown juxtaposed to parental cells.

Article Snippet: SNU-449 and HepG2 cells were transfected with shFDFT1 plasmid (sc-61610-SH, Santa Cruz Biotechnology, Shanghai, China), shACSL4 plasmid (sc-60619-SH, Santa Cruz), or shEMC2 plasmid (sc-77588-SH, Santa Cruz) using Lipofectamine 2000 (Invitrogen, Shanghai, China) following manufacturer’s protocol.

Techniques: Knockdown

Journal: American Journal of Cancer Research

Article Title: Differences of ferroptosis-related genes between White and Asian patients with liver cancer

doi:

Figure Lengend Snippet: The molecular mechanisms of FDFT1, ACSL4 and EMC2 in ferroptosis of liver cancer cells. A. SLC7A11, SLC1A5, FANCD2 and GPX4 protein expression in FDFT1 knockdown HepG2, SNU-449, and parental cells by western blotting. B. SLC1A5 and GPX4 protein expression in EMC2 knockdown HepG2, SNU-449, and parental cells by western blotting. C. FANCD2 and GPX4 protein expression in ACSL4 knockdown HepG2, and SNU-449, and parental cells by western blotting.

Article Snippet: SNU-449 and HepG2 cells were transfected with shFDFT1 plasmid (sc-61610-SH, Santa Cruz Biotechnology, Shanghai, China), shACSL4 plasmid (sc-60619-SH, Santa Cruz), or shEMC2 plasmid (sc-77588-SH, Santa Cruz) using Lipofectamine 2000 (Invitrogen, Shanghai, China) following manufacturer’s protocol.

Techniques: Expressing, Knockdown, Western Blot

Journal: American Journal of Cancer Research

Article Title: Differences of ferroptosis-related genes between White and Asian patients with liver cancer

doi:

Figure Lengend Snippet: Differentially ferroptosis-related genes in liver cancer tissues of Asian patients and White patients. Heatmap (A) and column chart (B) of differentially ferroptosis-related genes in Asian patients and White patients with liver cancer. G1: Asian patients; G2: White patients. Kaplan-Meier analysis for the prognostic roles of FDFT1 (C) and EMC2 (D) in the diseases-free survival of patients with liver cancer. Circles represent ferroptosis-related mRNA, the line represents the relationship between genes in Asian patients (E) and White patients (F).

Article Snippet: Primary antibodies included FDFT1 (sc-271602; Santa Cruz Biotechnology, Shanghai, China), ACSL4 (sc-365230; Santa Cruz), EMC2 (sc-166011; Santa Cruz), FANCD2 (sc-20022; Santa Cruz), SLC1A5 (8057; Cell Signaling Technology, Shanghai, China), SLC7A11 (98051; Cell Signaling), and GAPDH (sc-47724; Santa Cruz).

Techniques:

Journal: American Journal of Cancer Research

Article Title: Differences of ferroptosis-related genes between White and Asian patients with liver cancer

doi:

Figure Lengend Snippet: The roles of FDFT1, ACSL4 and EMC2 in ferroptosis of liver cancer cells. (A) Cell viability was assayed by MTT of HepG2 and SNU-449 cells after FDFT1, ACSL4 or EMC2 knockdown juxtaposed to parental cells. (B) Intracellular ROS, (C) MDA formation, and (D) Gln uptake activities of HepG2 and SNU-449 cells after FDFT1, ACSL4 or EMC2 knockdown juxtaposed to parental cells.

Article Snippet: Primary antibodies included FDFT1 (sc-271602; Santa Cruz Biotechnology, Shanghai, China), ACSL4 (sc-365230; Santa Cruz), EMC2 (sc-166011; Santa Cruz), FANCD2 (sc-20022; Santa Cruz), SLC1A5 (8057; Cell Signaling Technology, Shanghai, China), SLC7A11 (98051; Cell Signaling), and GAPDH (sc-47724; Santa Cruz).

Techniques: Knockdown

Journal: American Journal of Cancer Research

Article Title: Differences of ferroptosis-related genes between White and Asian patients with liver cancer

doi:

Figure Lengend Snippet: The molecular mechanisms of FDFT1, ACSL4 and EMC2 in ferroptosis of liver cancer cells. A. SLC7A11, SLC1A5, FANCD2 and GPX4 protein expression in FDFT1 knockdown HepG2, SNU-449, and parental cells by western blotting. B. SLC1A5 and GPX4 protein expression in EMC2 knockdown HepG2, SNU-449, and parental cells by western blotting. C. FANCD2 and GPX4 protein expression in ACSL4 knockdown HepG2, and SNU-449, and parental cells by western blotting.

Article Snippet: Primary antibodies included FDFT1 (sc-271602; Santa Cruz Biotechnology, Shanghai, China), ACSL4 (sc-365230; Santa Cruz), EMC2 (sc-166011; Santa Cruz), FANCD2 (sc-20022; Santa Cruz), SLC1A5 (8057; Cell Signaling Technology, Shanghai, China), SLC7A11 (98051; Cell Signaling), and GAPDH (sc-47724; Santa Cruz).

Techniques: Expressing, Knockdown, Western Blot