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Figure 3. Positive correlation between FOXA2 and Nrf2/GPX4 signaling in CRC cell lines. A) Positive correlation between FOXA2 expression and GPX4, NFE2L2 (Nrf2), <t>NQO1,</t> G6PD, and SLC7A11 in CRC patients from TCGA database. B) RT-qPCR analysis for genes including Nrf2, GCLC, NQO1, SOD1, GPX4, SLC7A11 and G6PD in HCT-116 and SW480 cells with FOXA2 knockdown or over-expression (n = 3). C,D) IF staining for GPX4 expression in CRC cell lines transfected with sh-FOXA2 or oe-FOXA2 (n = 4). Scale bar = 20 μm. E) Western blot analysis for GPX4, Nrf2, and NQO1 protein expression levels in FOXA2-diminished or -over-expressed HCT-116 and SW480 cells (n = 3). Data are marked as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
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Figure 3. Positive correlation between FOXA2 and Nrf2/GPX4 signaling in CRC cell lines. A) Positive correlation between FOXA2 expression and GPX4, NFE2L2 (Nrf2), <t>NQO1,</t> G6PD, and SLC7A11 in CRC patients from TCGA database. B) RT-qPCR analysis for genes including Nrf2, GCLC, NQO1, SOD1, GPX4, SLC7A11 and G6PD in HCT-116 and SW480 cells with FOXA2 knockdown or over-expression (n = 3). C,D) IF staining for GPX4 expression in CRC cell lines transfected with sh-FOXA2 or oe-FOXA2 (n = 4). Scale bar = 20 μm. E) Western blot analysis for GPX4, Nrf2, and NQO1 protein expression levels in FOXA2-diminished or -over-expressed HCT-116 and SW480 cells (n = 3). Data are marked as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
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Figure 3. Positive correlation between FOXA2 and Nrf2/GPX4 signaling in CRC cell lines. A) Positive correlation between FOXA2 expression and GPX4, NFE2L2 (Nrf2), <t>NQO1,</t> G6PD, and SLC7A11 in CRC patients from TCGA database. B) RT-qPCR analysis for genes including Nrf2, GCLC, NQO1, SOD1, GPX4, SLC7A11 and G6PD in HCT-116 and SW480 cells with FOXA2 knockdown or over-expression (n = 3). C,D) IF staining for GPX4 expression in CRC cell lines transfected with sh-FOXA2 or oe-FOXA2 (n = 4). Scale bar = 20 μm. E) Western blot analysis for GPX4, Nrf2, and NQO1 protein expression levels in FOXA2-diminished or -over-expressed HCT-116 and SW480 cells (n = 3). Data are marked as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
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Figure 3. Positive correlation between FOXA2 and Nrf2/GPX4 signaling in CRC cell lines. A) Positive correlation between FOXA2 expression and GPX4, NFE2L2 (Nrf2), <t>NQO1,</t> G6PD, and SLC7A11 in CRC patients from TCGA database. B) RT-qPCR analysis for genes including Nrf2, GCLC, NQO1, SOD1, GPX4, SLC7A11 and G6PD in HCT-116 and SW480 cells with FOXA2 knockdown or over-expression (n = 3). C,D) IF staining for GPX4 expression in CRC cell lines transfected with sh-FOXA2 or oe-FOXA2 (n = 4). Scale bar = 20 μm. E) Western blot analysis for GPX4, Nrf2, and NQO1 protein expression levels in FOXA2-diminished or -over-expressed HCT-116 and SW480 cells (n = 3). Data are marked as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
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Figure 3. Positive correlation between FOXA2 and Nrf2/GPX4 signaling in CRC cell lines. A) Positive correlation between FOXA2 expression and GPX4, NFE2L2 (Nrf2), NQO1, G6PD, and SLC7A11 in CRC patients from TCGA database. B) RT-qPCR analysis for genes including Nrf2, GCLC, NQO1, SOD1, GPX4, SLC7A11 and G6PD in HCT-116 and SW480 cells with FOXA2 knockdown or over-expression (n = 3). C,D) IF staining for GPX4 expression in CRC cell lines transfected with sh-FOXA2 or oe-FOXA2 (n = 4). Scale bar = 20 μm. E) Western blot analysis for GPX4, Nrf2, and NQO1 protein expression levels in FOXA2-diminished or -over-expressed HCT-116 and SW480 cells (n = 3). Data are marked as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FOXA2 Suppression by TRIM36 Exerts Anti-Tumor Role in Colorectal Cancer Via Inducing NRF2/GPX4-Regulated Ferroptosis.

doi: 10.1002/advs.202304521

Figure Lengend Snippet: Figure 3. Positive correlation between FOXA2 and Nrf2/GPX4 signaling in CRC cell lines. A) Positive correlation between FOXA2 expression and GPX4, NFE2L2 (Nrf2), NQO1, G6PD, and SLC7A11 in CRC patients from TCGA database. B) RT-qPCR analysis for genes including Nrf2, GCLC, NQO1, SOD1, GPX4, SLC7A11 and G6PD in HCT-116 and SW480 cells with FOXA2 knockdown or over-expression (n = 3). C,D) IF staining for GPX4 expression in CRC cell lines transfected with sh-FOXA2 or oe-FOXA2 (n = 4). Scale bar = 20 μm. E) Western blot analysis for GPX4, Nrf2, and NQO1 protein expression levels in FOXA2-diminished or -over-expressed HCT-116 and SW480 cells (n = 3). Data are marked as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The following primary antibodies were used: anti-FOXA2 antibody (#NBP202088, 1:1000), anti-NQO1 antibody (#NB200-209, 1:1000), and antiGPX4 antibody (#NBP2-75511, 1:1000) were obtained from Novus Biologicals (Littleton, CO, USA); anti-Nrf2 antibody (#12721, 1:1000), anti-KI-67 antibody (#34330, 1:1000), anti-Flag antibody (#14793), anti-HA antibody (#5017), anti-Myc antibody (#2276) and anti-GAPDH (#5174) were obtained from Cell Signaling Technology (Beverly, MA, USA); anti-Fibronectin antibody (#MA5-11981, 1:1000), and anti-TRIM36 antibody (#PA5-28401, 1:1000) were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Over Expression, Staining, Transfection, Western Blot

Figure 5. FOXA2 suppression sensitizes CRC cells to OXA treatment by facilitating ferroptosis. HCT-116 and SW480 cells were transfected with sh- FOXA2, followed by OXA treatments for another 24 h. Then, all cells were harvested for subsequent assays. A) CCK-8 analysis for cell viability evaluation (n = 4). B,C) DCF-DA staining was used to examine ROS production (n = 4). Scale bar = 50 μm. D,E) C11-BODIPY581/591 staining was conducted for the measurements of lipid ROS production (n = 4). Scale bar = 20 μm. F) MDA contents, G) GSH levels, and H) iron currents were assessed (n = 4). I) Ferroptosis hallmarks including Nrf2, NQO1, SOD1, GPX4 and SLC7A11 were calculated by RT-qPCR (n = 3). Data are marked as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FOXA2 Suppression by TRIM36 Exerts Anti-Tumor Role in Colorectal Cancer Via Inducing NRF2/GPX4-Regulated Ferroptosis.

doi: 10.1002/advs.202304521

Figure Lengend Snippet: Figure 5. FOXA2 suppression sensitizes CRC cells to OXA treatment by facilitating ferroptosis. HCT-116 and SW480 cells were transfected with sh- FOXA2, followed by OXA treatments for another 24 h. Then, all cells were harvested for subsequent assays. A) CCK-8 analysis for cell viability evaluation (n = 4). B,C) DCF-DA staining was used to examine ROS production (n = 4). Scale bar = 50 μm. D,E) C11-BODIPY581/591 staining was conducted for the measurements of lipid ROS production (n = 4). Scale bar = 20 μm. F) MDA contents, G) GSH levels, and H) iron currents were assessed (n = 4). I) Ferroptosis hallmarks including Nrf2, NQO1, SOD1, GPX4 and SLC7A11 were calculated by RT-qPCR (n = 3). Data are marked as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The following primary antibodies were used: anti-FOXA2 antibody (#NBP202088, 1:1000), anti-NQO1 antibody (#NB200-209, 1:1000), and antiGPX4 antibody (#NBP2-75511, 1:1000) were obtained from Novus Biologicals (Littleton, CO, USA); anti-Nrf2 antibody (#12721, 1:1000), anti-KI-67 antibody (#34330, 1:1000), anti-Flag antibody (#14793), anti-HA antibody (#5017), anti-Myc antibody (#2276) and anti-GAPDH (#5174) were obtained from Cell Signaling Technology (Beverly, MA, USA); anti-Fibronectin antibody (#MA5-11981, 1:1000), and anti-TRIM36 antibody (#PA5-28401, 1:1000) were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: Transfection, CCK-8 Assay, Staining, Quantitative RT-PCR

Figure 6. FOXA2 knockdown induces ferroptosis in chemoresistant CRC cell lines. A) RT-qPCR analysis for Nrf2, NQO1, SOD1, GPX4 and SLC7A11 gene expression levels in drug-sensitive or -resistant HCT-116 and SW480 cells (n = 3). B) Western blot assay for GPX4, Nrf2, and NQO1 protein expression levels in HCT-116 and SW480 cells with or without chemoresistance (n = 4). C) Western blot assay for FOXA2, GPX4, Nrf2, and NQO1 protein expression levels in chemoresistant HCT-116 and SW480 cells with or without FOXA2 knockdown (n = 4). (D) GPX4 expression by IF staining in drug-resistant CRC cells after transfection with sh-FOXA2 (n = 4). Scale bar = 20 μm. E) ROS and (F) lipid ROS production by DCF-DA (Scale bar = 50 μm) and C11-BODIPY581/591 (Scale bar = 20 μm) staining, respectively, in chemoresistant HCT-116 and SW480 cells with FOXA2 knockdown (n = 4). G) MDA levels, H) GSH contents, and I) iron currents in drug-resistant HCT-116 and SW480 cells after FOXA2 knockdown (n = 4). Data are marked as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FOXA2 Suppression by TRIM36 Exerts Anti-Tumor Role in Colorectal Cancer Via Inducing NRF2/GPX4-Regulated Ferroptosis.

doi: 10.1002/advs.202304521

Figure Lengend Snippet: Figure 6. FOXA2 knockdown induces ferroptosis in chemoresistant CRC cell lines. A) RT-qPCR analysis for Nrf2, NQO1, SOD1, GPX4 and SLC7A11 gene expression levels in drug-sensitive or -resistant HCT-116 and SW480 cells (n = 3). B) Western blot assay for GPX4, Nrf2, and NQO1 protein expression levels in HCT-116 and SW480 cells with or without chemoresistance (n = 4). C) Western blot assay for FOXA2, GPX4, Nrf2, and NQO1 protein expression levels in chemoresistant HCT-116 and SW480 cells with or without FOXA2 knockdown (n = 4). (D) GPX4 expression by IF staining in drug-resistant CRC cells after transfection with sh-FOXA2 (n = 4). Scale bar = 20 μm. E) ROS and (F) lipid ROS production by DCF-DA (Scale bar = 50 μm) and C11-BODIPY581/591 (Scale bar = 20 μm) staining, respectively, in chemoresistant HCT-116 and SW480 cells with FOXA2 knockdown (n = 4). G) MDA levels, H) GSH contents, and I) iron currents in drug-resistant HCT-116 and SW480 cells after FOXA2 knockdown (n = 4). Data are marked as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The following primary antibodies were used: anti-FOXA2 antibody (#NBP202088, 1:1000), anti-NQO1 antibody (#NB200-209, 1:1000), and antiGPX4 antibody (#NBP2-75511, 1:1000) were obtained from Novus Biologicals (Littleton, CO, USA); anti-Nrf2 antibody (#12721, 1:1000), anti-KI-67 antibody (#34330, 1:1000), anti-Flag antibody (#14793), anti-HA antibody (#5017), anti-Myc antibody (#2276) and anti-GAPDH (#5174) were obtained from Cell Signaling Technology (Beverly, MA, USA); anti-Fibronectin antibody (#MA5-11981, 1:1000), and anti-TRIM36 antibody (#PA5-28401, 1:1000) were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: Knockdown, Quantitative RT-PCR, Gene Expression, Western Blot, Expressing, Staining, Transfection

Figure 7. FOXA2 knockdown CRC cells are sensitive to OXA-induced ferroptosis via the Nrf2 activation. HCT-116 cells with FOXA2 knockdown or over- expression were incubated with OXA (10 μM) alone or combination with Nrf2 activator (ML334, 20 μM) or inhibitor (ML385, 5 μM) for an additional 24 h. SW480 cells co-transfected with sh-FOXA2 and Nrf2 plasmids, or oe-FOXA2 and si-Nrf2 were exposed to OXA (10 μM) treatment for another 24 h. Then, all HCT-116 and SW480 cells were harvested for studies as follows. A-H) DCF-DA (Scale bar = 50 μm) and C11-BODIPY581/591 (Scale bar = 20 μm) staining were performed to examine ROS and lipid ROS production in CRC cells treated as shown (n = 4). I,J) MDA levels in HCT-116 and SW480 cells were examined. K,L) Examination of iron currents in CRC cells. (M,N) Calculation for cellular MDA levels. O,P) Iron currents in CRC cells were assessed (n = 5). Q-T) Western blot analysis for Nrf2, NQO1, and GPX4 protein expression levels in CRC cells were conducted (n = 3). Data are marked as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FOXA2 Suppression by TRIM36 Exerts Anti-Tumor Role in Colorectal Cancer Via Inducing NRF2/GPX4-Regulated Ferroptosis.

doi: 10.1002/advs.202304521

Figure Lengend Snippet: Figure 7. FOXA2 knockdown CRC cells are sensitive to OXA-induced ferroptosis via the Nrf2 activation. HCT-116 cells with FOXA2 knockdown or over- expression were incubated with OXA (10 μM) alone or combination with Nrf2 activator (ML334, 20 μM) or inhibitor (ML385, 5 μM) for an additional 24 h. SW480 cells co-transfected with sh-FOXA2 and Nrf2 plasmids, or oe-FOXA2 and si-Nrf2 were exposed to OXA (10 μM) treatment for another 24 h. Then, all HCT-116 and SW480 cells were harvested for studies as follows. A-H) DCF-DA (Scale bar = 50 μm) and C11-BODIPY581/591 (Scale bar = 20 μm) staining were performed to examine ROS and lipid ROS production in CRC cells treated as shown (n = 4). I,J) MDA levels in HCT-116 and SW480 cells were examined. K,L) Examination of iron currents in CRC cells. (M,N) Calculation for cellular MDA levels. O,P) Iron currents in CRC cells were assessed (n = 5). Q-T) Western blot analysis for Nrf2, NQO1, and GPX4 protein expression levels in CRC cells were conducted (n = 3). Data are marked as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The following primary antibodies were used: anti-FOXA2 antibody (#NBP202088, 1:1000), anti-NQO1 antibody (#NB200-209, 1:1000), and antiGPX4 antibody (#NBP2-75511, 1:1000) were obtained from Novus Biologicals (Littleton, CO, USA); anti-Nrf2 antibody (#12721, 1:1000), anti-KI-67 antibody (#34330, 1:1000), anti-Flag antibody (#14793), anti-HA antibody (#5017), anti-Myc antibody (#2276) and anti-GAPDH (#5174) were obtained from Cell Signaling Technology (Beverly, MA, USA); anti-Fibronectin antibody (#MA5-11981, 1:1000), and anti-TRIM36 antibody (#PA5-28401, 1:1000) were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: Knockdown, Activation Assay, Over Expression, Incubation, Transfection, Staining, Western Blot, Expressing