Journal: Journal of Cell Science

Article Title: The STEAP4 target NQO1 mediates colon tumorigenesis

doi: 10.1242/jcs.263402

Figure Lengend Snippet: Colon epithelial cell-specific Steap4 knockout suppresses mouse colon tumor development. (A–D) Macroscopic images of colon tumors (A), total tumor count (B), tumor distribution according to different sizes (C) and overall tumor burden (D) are presented for Steap4 wild-type ( N =6) and knockout ( N =15) mice. Images in A are representative of at least two independent experiments. (E,F) Representative images of Ki67 staining (E) and its quantification (F) show differences in proliferative activity between Steap4 wild-type ( N =5) and knockout ( N =5) mice. Images in E are representative of two experiments. (G,H) Representative images (G) and quantification (H) of CC3 staining for tumors in Steap4 wild-type ( N =5) and knockout ( N =5) mice provide insights into apoptotic processes. Images in G are representative of two experiments. * P <0.05, ** P <0.01. Unpaired two-tailed t -tests were applied for data in B, D, F and H; data in C underwent analysis through two-way ANOVA. Scale bars: 200 μm. Graphs show mean±s.e.m. with individual data points.

Article Snippet: Steap4 F/F mice were produced utilizing exon 2–3 of the mouse Steap4 gene as the conditional knockout region at Cyagen (Santa Clara, CA) and crossed with Cdx2 Cre-ERT2 Apc F/+ mice to generate Steap4 F/F Cdx2 Cre-ERT2 Apc F/+ mice.

Techniques: Knock-Out, Staining, Activity Assay, Two Tailed Test

Journal: Journal of Cell Science

Article Title: The STEAP4 target NQO1 mediates colon tumorigenesis

doi: 10.1242/jcs.263402

Figure Lengend Snippet: Steap4 knockdown suppresses mouse colon tumor cell growth in vitro and in vivo . (A) Western blot analysis provides insights into Steap4 knockdown (KD) efficiency in MC38 cells compared to the control empty vector (EV). (B) Impact of Steap4 KD on cell viability, as determined by MTT assay. FC, fold change. (C) Xenograft tumor weights for MC38 cells with Steap4 knockdown or control EV are presented. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. Two-way ANOVA was applied for data in B; unpaired two-tailed t -tests were used for data in C. N =3. Graphs show mean±s.e.m. with individual data points.

Article Snippet: Steap4 F/F mice were produced utilizing exon 2–3 of the mouse Steap4 gene as the conditional knockout region at Cyagen (Santa Clara, CA) and crossed with Cdx2 Cre-ERT2 Apc F/+ mice to generate Steap4 F/F Cdx2 Cre-ERT2 Apc F/+ mice.

Techniques: Knockdown, In Vitro, In Vivo, Western Blot, Control, Plasmid Preparation, MTT Assay, Two Tailed Test

Journal: Journal of Cell Science

Article Title: The STEAP4 target NQO1 mediates colon tumorigenesis

doi: 10.1242/jcs.263402

Figure Lengend Snippet: STEAP4 overexpression in human HCT116 cells activates the NRF2 signaling pathway. (A–C) Results from qPCR analysis of STEAP4 ( N =3) (A), western blot analysis ( N =3) (B) and luciferase assay for NQO1 activity ( N =6) (C) illustrate the impact of STEAP4 overexpression (OE) in HCT116 cells compared to the control EV. * P <0.05, ** P <0.01, **** P <0.0001; ns, not significant. Unpaired two-tailed t -tests were employed for data in A and C. Graphs show mean±s.e.m. with individual data points.

Article Snippet: Steap4 F/F mice were produced utilizing exon 2–3 of the mouse Steap4 gene as the conditional knockout region at Cyagen (Santa Clara, CA) and crossed with Cdx2 Cre-ERT2 Apc F/+ mice to generate Steap4 F/F Cdx2 Cre-ERT2 Apc F/+ mice.

Techniques: Over Expression, Western Blot, Luciferase, Activity Assay, Control, Two Tailed Test

Journal: Journal of Cell Science

Article Title: The STEAP4 target NQO1 mediates colon tumorigenesis

doi: 10.1242/jcs.263402

Figure Lengend Snippet: Oxidative stress response during STEAP4-catalyzed Fe 3+ reduction in vitro . (A,B) Representative images for MitoSOX assay (A) and dihydroethidium (DHE) staining (B) in mouse colon enteroid cultures derived from STEAP4 OE mice and their littermates ( STEAP4 +/+ ). Images are representative of two experiments. (C,D) Representative images (C) and quantitative analysis (D) of 2′,7′-dichlorofluorescein diacetate (DCFH-DA) staining in human colon-derived HCT116 cancer cells with ( N =6) or without ( N =3) STEAP4 overexpression. Images in C are representative of two experiments. (E,F) Immunoblot analysis (E) and quantification (F) for HCT116 cells treated with Fe 2+ ( N =3). (G,H) Immunoblot analysis (G) and quantification (H) for HCT116 cells treated with Fe 3+ ( N =3). Enteroids or HCT116 cells were treated with 10 μM doxorubicin (Dox), 100 μM ferrous sulfate (Fe 2+ ) or 100 μM ferric chloride (Fe 3+ ) overnight. ns, not significant; * P <0.05, **** P <0.0001. Data in D, F and H were analyzed with two-way ANOVA. Scale bars: 200 μm. Graphs show mean±s.e.m. with individual data points.

Article Snippet: Steap4 F/F mice were produced utilizing exon 2–3 of the mouse Steap4 gene as the conditional knockout region at Cyagen (Santa Clara, CA) and crossed with Cdx2 Cre-ERT2 Apc F/+ mice to generate Steap4 F/F Cdx2 Cre-ERT2 Apc F/+ mice.

Techniques: In Vitro, Mitosox Assay, Staining, Derivative Assay, Over Expression, Western Blot

Journal: Journal of Cell Science

Article Title: The STEAP4 target NQO1 mediates colon tumorigenesis

doi: 10.1242/jcs.263402

Figure Lengend Snippet: STEAP4 overexpression increases oxidative stress and susceptibility to NQO1 bioactivatable drugs in colorectal cancer. (A–D) Cell survival, determined by MTT assay 24 h following treatment with NQO1 bioactivatable drug β-lapachone (β-LPC) ( N =3) (A), NQO1 bioactivatable drug KP-372 ( N =3) (B), β-LPC and NQO1 inhibitor dicoumarol (DIC) ( N =6) (C), or KP-372 and DIC ( N =9) (D) for 4 h in HCT116 cells with stable STEAP4 OE or control EV. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. Two-way ANOVA was applied. Graphs show mean±s.e.m. with individual data points.

Article Snippet: Steap4 F/F mice were produced utilizing exon 2–3 of the mouse Steap4 gene as the conditional knockout region at Cyagen (Santa Clara, CA) and crossed with Cdx2 Cre-ERT2 Apc F/+ mice to generate Steap4 F/F Cdx2 Cre-ERT2 Apc F/+ mice.

Techniques: Over Expression, MTT Assay, Control

Journal: Journal of Cell Science

Article Title: The STEAP4 target NQO1 mediates colon tumorigenesis

doi: 10.1242/jcs.263402

Figure Lengend Snippet: A proposed working model. (A) STEAP4, a mitochondrial protein, functions as a catalyst for the reduction of Fe 3+ into Fe 2+ while generating H 2 O 2 . Both Fe 2+ and H 2 O 2 contribute to STEAP4-mediated colon cancer cell proliferation. Antioxidant proteins including NQO1 are induced in STEAP4-overexpressing cells through NRF2 transcriptional regulation, acting to counteract the cytotoxic effects of H 2 O 2 . (B) Capitalizing on the characteristic of tumors with low catalase expression, NQO1 bioactivatable drugs (β-LPC and KP372-1) are strategically employed to generate an overwhelming amount of H 2 O 2 . This approach aims to selectively target and eliminate colon cancer cells characterized by high expression of STEAP4. Figure made using public domain icons from https://bioart.niaid.nih.gov/ .

Article Snippet: Steap4 F/F mice were produced utilizing exon 2–3 of the mouse Steap4 gene as the conditional knockout region at Cyagen (Santa Clara, CA) and crossed with Cdx2 Cre-ERT2 Apc F/+ mice to generate Steap4 F/F Cdx2 Cre-ERT2 Apc F/+ mice.

Techniques: Expressing