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 1. FOXA2 expression is up-regulated in CRC patients. A,B) FOXA2 expression profile in CRC tissues from TCGA cohort. C) Representative IHC images for FOXA2 in CRC tissues and normal tissues (https://www.proteinatlas.org/). D) Kaplan-Meier analysis for relapse-free-survival (RFS) of CRC patients with high (n = 709) or low (n = 633) FOXA2 expression based on the median expression of FOXA2 from the Kaplan-Meier Plotter (https://kmplot.com/analysis/index.php?p = service). E) Images of IHC staining for FOXA2 in CRC tissues and the paired adjacent normal tissues from our cohort. Scale bar = 120 μm. F) IHC scores of FOXA2 expression levels in paired normal and CRC samples were quantified. G) RT-qPCR analysis for FOXA2 gene expression in human CRC tissues and the matched adjacent normal tissues (ANT) from our cohort (n = 60). H) FOXA2 protein expression in eighteen paired CRC tissues was examined using western blot. I) RT-qPCR (n = 5) and J) western blot (n = 4) assays for FOXA2 gene and protein expression levels in six CRC cell lines and non-tumor cell line NCM460. Data are marked as the means ± SD. *p < 0.05, **p < 0.01, ****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, Immunohistochemistry, Quantitative RT-PCR, Gene Expression, Western Blot

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 2. FOXA2 enhances the proliferation, migration and invasion of CRC cells in vitro or in vivo. CCK8 analysis for cell proliferation of HCT-116 and SW480 cells with FOXA2 A) knockdown or B) over-expression (n = 4). C) EdU staining of HCT-116 and SW480 cells transfected with sh-FOXA2 or oe-FOXA2 as shown (n = 4). Scale bar = 50 μm. D) Transwell analysis for CRC cell migration and invasion after FOXA2 knockdown or over-expression (n = 5). Scale bar = 50 μm. E) RT-qPCR analysis for EMT markers in HCT-116 and SW480 cells after FOXA2 knockdown or over-expression (n = 3). F) Tumor samples from the indicated groups of mice expressing the control vector, sh-FOXA2 or oe-FOXA2 plasmids (n = 5 or 4 in each). G) Tumor formation volume and H) tumor weights were measured. 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: Migration, In Vitro, In Vivo, Knockdown, Over Expression, Staining, Transfection, Quantitative RT-PCR, Expressing, Control, Plasmid Preparation

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

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 4. FOXA2 suppression induces ferroptosis in CRC cell lines. A,B) ROS production was measured by DCF-DA staining in CRC cells with FOXA2 knockdown or over-expression. Scale bar = 50 μm. C,D) Lipid ROS generation in HCT-116 and SW480 cells was measured by C11-BODIPY581/591 staining post-sh-FOXA2 or oe-FOXA2 plasmid transfection. Scale bar = 20 μm. E) MDA levels, (F) GSH contents and G) iron currents in the show groups of CRC cells were examined. H) HCT-116 and SW480 cells with FOXA2 knockdown were treated with ferroptosis inhibitor (Fer-1, 1 μM), necroptosis inhibitor (Nec-1, 10 μM), or apoptosis inhibitor (Z-VAD-FMK, 10 μM) for another 24 h. Then, all cells were collected for cell viability examination using CCK-8 analysis. Data are marked as the means ± SD (n = 5 in each). **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, no significant difference.

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: Staining, Knockdown, Over Expression, Plasmid Preparation, Transfection, CCK-8 Assay

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

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

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

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 8. RSL3 promotes the sensitivity of FOXA2-overexpressed and chemoresistant CRC cells to OXA treatment. Drug-sensitive CRC cells with FOXA2 over-expression or chemoresistant CRC cells were subjected to OXA (10 μM), GPX4 inhibitor (RSL3, 100 nM) single or double incubation for another 24 h. Next, all cells were harvested for studies as following. A-D) Western blot and IF staining analysis for GPX4 protein expression levels in cells treated as shown (n = 3 or 4 in each). E,F) MDA levels and G,H) iron contents were examined (n = 4). I,J) Lipid ROS production was measured using C11- BODIPY581/591 staining (n = 4). K,L) Cell viability of the shown CRC cells with or without chemoresistance was examined using CCK-8 analysis (n = 4). Data are marked as the means ± SD. Scale bar = 20 μm. +p < 0.05, ++p < 0.01 versus the Ctrl group; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, no significant difference.

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: Over Expression, Incubation, Western Blot, Staining, Expressing, CCK-8 Assay

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 9. Identifying a potent suppressor of FOXA2. A) Working model of the protocols used to identify potent binding proteins and suppressors of FOXA2. B) SDS-PAGE band showing the immunoprecipitated proteins in HCT-116 and HEK293T cells with anti-FOXA2 antibody vs the isotype-controlled IgG. C) Immunoblotting assay to validate the presence of FOXA2 in the immunoprecipitated samples. D) There were a total of 31 overlapping FOXA2 binding proteins identified both in HCT-116 and HEK293T cells, including the listed E3 ligases TRIM36, CHIP, TRIM25, RNF2, and MID1. E) Western blot indicating the FOXA2 expression in HCT-116 and HEK293T cells transfected with the shown plasmids. F) TRIM36 expression profile in CRC tissues from TCGA cohort. G) Correlation between FOXA2 and TRIM36 from TCGA database. H) TRIM36 protein expression in eighteen paired CRC tissues was examined using western blot. I) Spearman’s correlation between TRIM36 and FOXA2 protein expression in the eighteen CRC patients. J) Western blot analysis for TRIM36 and FOXA2 in CRC cells transfected with TRIM36 plasmids. (K) RT-qPCR analysis for FOXA2 in CRC cells with or without TRIM36 over-expression. L) FOXA2 protein expression levels in empty vector and TRIM36-overexpressed HCT-116 or SW480 cells were examined at the shown time after CHX (20 μg ml−1) incubation. M) Western blot indicating the influence of MG132 (10 μM) on FOXA2 expression levels in CRC cells with or without TRIM36 over-expression. Data are marked as the means ± SD. **p < 0.01, ****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: Binding Assay, SDS Page, Immunoprecipitation, Western Blot, Expressing, Transfection, Quantitative RT-PCR, Over Expression, Plasmid Preparation, Incubation

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 10. TRIM36 interacts with FOXA2 and induces its K48-linked polyubiquitination. A) Co-IP analysis of HEK293T cells transfected with Flag-tagged FOXA2 and HA-tagged TRIM36. Anti-Flag and anti-HA antibodies were used for western blot assay. B) GST precipitation indicating the direct interaction of TRIM36 with FOXA2 using purified GST-FOXA2 and His-tagged TRIM36 (left) or purified GST-TRIM36 and His-FOXA2 (right) by western blot analysis. GST was defined as a control. C) IF images of HEK293T cells co-transfected with 24 h of Flag-tagged FOXA2 (red) and HA-tagged TRIM36 (green). Scale bar = 15 μm. D,E) Schematic indicating full-length and truncated TRIM36 (top) and FOXA2 (top) with representative Co-IP assays (bottom) for the mapping analysis of the domains responsible for the TRIM36/FOXA2 interaction in HEK293T cells. F) Lysates of HEK293T cells transfected with plasmids expressing HA-FOXA2, Myc-Ub and increasing amounts of Flag-TIRM36 were immunoprecipitated with anti-HA beads and immunoblotted using an anti-Myc antibody. G) Western blots showing FOXA2 ubiquitination in HEK293T cells transfected with the indicated plasmids in different combinations. ∆CC, deletion of the CC domain. H) Western blot analysis showing K48 ubiquitination of FOXA2 in HEK293T cells co-transfected with Flag-TRIM36 and Myc-K48-Ub or Myc-K63-Ub. I) Western blot analysis showing K48 ubiquitination of FOXA2 in HEK293T cells co-transfected with Flag-TRIM36, Flag-TRIM36 (ΔCC), and Myc-K48-Ub. (J) FOXA2 protein expression levels and its ubiquitination levels in 24 h of OXA-treated HCT-116 and SW480 cells.

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: Co-Immunoprecipitation Assay, Transfection, Western Blot, Control, Expressing, Immunoprecipitation, Ubiquitin Proteomics

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 11. Conditional knockout of FOXA2 in IECs ameliorates colitis-associated tumorigenesis in vivo. A) Scheme protocols of recombination in Villin- Cre; FOXA2f/f mice. B,C) RT-qPCR and western blot assays for FOXA2 gene and protein expression levels in colon crypts from the mice treated with or without AOM/DSS (n = 6). D) H&E and IHC staining of FOXA2 in colon from FOXA2f/f and FOXA2cKO mice (n = 8). Scale bar = 120 μm. E) Inflammation scores were quantified. (F) FOXA2 expression by IHC staining was calculated. G) Body weights of mice were recorded (n = 10–15 in each group). H) Overall survival rates for each group of mice (n = 10–15 in each group). I) Images for colons from all groups of mice. J) Colon length was measured (n = 15). (K) Tumor number on colon was examined (n = 15). L) IHC staining for KI-67 in colon tissues was performed (n = 5). Scale bar = 50 μm. Data are marked as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, no significant difference.

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: Knock-Out, In Vivo, Quantitative RT-PCR, Western Blot, Expressing, Immunohistochemistry

Journal: bioRxiv

Article Title: Celiac disease patient derived iPSC–small intestinal epithelial cells are more persistent under cytokine stimuli than healthy control cells

doi: 10.64898/2026.03.12.710771

Figure Lengend Snippet: Before differentiation, both iPSC lines expressed the as well as the Nanog gene and protein (n = 3), and POU5F1 / Oct3/4 gene and protein (n = 3) (panel A). At the DE stage, cultures expressed the FOXA2 and SOX17 gene and protein (n = 3; panel B, FOXA2 red, SOX17 green, DAPI blue, 100 μm). At the PDE stage, cultures expressed the Cdx2 and SOX17 gene and protein (n = 3; panel C, Cdx2 red, SOX17 green, DAPI blue, scale bar 100 μm). Microscope images (A-C) were taken with an Evident-Olympus IX83 microscope 20x lens using a Hamamatsu ORCA-Fusion C14440-20UP camera and NEO LiveImaging software. Intestinal epithelial cell specific functionality assessed with PEPT1 dipeptide uptake assay (D). On left there is image captured above the cellular level and on the right, there is image taken from the level which is in the middle of nucleus (n=3; dipeptide D-Ala-Leu-Lys-AMCA green, DAPI blue, 20 μm). Confocal images were taken with a Zeiss LSM780 laser scanning microscope with a 40x lens and ZEN Blue 3.6 software.

Article Snippet: The maturation status of the cultures after DE, PDE, or SIEC induction was assessed by analyzing the cells for their expression of NANOG (Hs02387400_g1) and POUF (OCT4 Hs00999632_g1) mRNA from iPSCs, of SOX17 (Hs06634937_s1) and FOXA2 (Hs00936490_m1) after DE and PDE induction, SLC15A ( PEPT1 (Hs00192639_m1), mRNA after SIEC induction, with TaqMan® gene expression assays (Applied Biosystems, Inc., Foster City, CA, USA) using FAM labels.

Techniques: Microscopy, Software, Laser-Scanning Microscopy

Journal: Cytokine

Article Title: FOXA2 inhibits the TLR4/NF-κB signaling pathway and alleviates inflammatory activation of macrophages in rheumatoid arthritis by repressing LY96 transcription.

doi: 10.1016/j.cyto.2024.156796

Figure Lengend Snippet: Fig. 5. FOXA2, a transcriptional repressor of LY96, is poorly expressed in RA. A, DEGs (adj. p. val < 0.05) between synovial macrophages from RA patients and healthy individuals by analyzing the GEO GSE97779 dataset; B, intersections of the top 1,000 DEGs, transcription factors targeting LY96 predicted using the hTFtarget system, and transcription factors targeting the LY96 gene promoter/enhancer predicted the GeneCards system; C, intersections of all DEGs (adj. p. val < 0.05) from the GSE97779 dataset, transcription factors targeting LY96, and transcription factors targeting the LY96 gene promoter/enhancer; D, a PPI network of the eight intersecting factors; E, putative binding relationship between FOXA2 and LY96 predicted in the hTFtarget system; F, the existence of multiple binding sites between FOXA2 and the LY96 promoter predicted using the Jaspar system; G, positive staining of FOXA2 in the ankle joint of RA or control mice (n = 6); H, mRNA expression of FOXA2 and LY96 in the mouse RAW264.7 cells after OE-FOXA2 transfection determined by RT-qPCR; I, the direct binding relationship between FOXA2 and the LY96 promoter examined using the ChIP-qPCR assay; J, transcription activity of the LY96 promoter in the mouse RAW264.7 cells overexpressing FOXA2 examined by dual luciferase reporter gene assay. Three biological replicates were performed. Differences were compared by the unpaired t-test (G-J). ***p < 0.001; ****p < 0.0001.

Article Snippet: Following further blocking with 3 % bovine serum albumin and digestion with pepsin for antigen retrieval, the sections were incubated with the antibodies against MD2 (LY96) (ab24182, Abcam), NF-κB p65 (phospho T254) (1:50, ab131100, Abcam), FOXA2 (NBP2-02088, Novus Biologicals, Littleton, CA, USA), CD86 (1:100, MA5-48134, Thermo Fisher Scientific), and CD206 antibody (1:100, PA5-101657, Thermo Fisher Scientific) at 4 ◦C for 12 h. Following this, the sections were incubated with the secondary antibodies IgG H&L (HRP) (ab205718 or ab97023, Abcam) at room temperature for 50 min.

Techniques: Binding Assay, Staining, Control, Expressing, Transfection, Quantitative RT-PCR, ChIP-qPCR, Activity Assay, Luciferase, Reporter Gene Assay

Journal: Cytokine

Article Title: FOXA2 inhibits the TLR4/NF-κB signaling pathway and alleviates inflammatory activation of macrophages in rheumatoid arthritis by repressing LY96 transcription.

doi: 10.1016/j.cyto.2024.156796

Figure Lengend Snippet: Fig. 6. LY96 overexpression diminishes the anti-inflammatory role of FOXA2 in RAW264.7 macrophages. Mouse RAW264.7 macrophages stably transfected with sh- LY96 were treated with the TLR4/MD-2 agonist or PBS, followed by LPS stimulation. A, mRNA expression of LY96 in RAW264.7 cells determined using RT-qPCR; B, protein level of Myd88 and the phosphorylation of NF-κB (p65) in RAW264.7 cells assessed using WB analysis; C, concentrations of IL-6, TNF-α, IL-1β, and IL-10 in the RAW264.7 cell culture supernatant determined using ELISA kits; D, populations of M1 (CD86) and M2 (CD206) macrophages analyzed using flow cytometry. Three biological replicates were performed. Differences were compared by the unpaired t-test (A) or one-way ANOVA (B-D). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Following further blocking with 3 % bovine serum albumin and digestion with pepsin for antigen retrieval, the sections were incubated with the antibodies against MD2 (LY96) (ab24182, Abcam), NF-κB p65 (phospho T254) (1:50, ab131100, Abcam), FOXA2 (NBP2-02088, Novus Biologicals, Littleton, CA, USA), CD86 (1:100, MA5-48134, Thermo Fisher Scientific), and CD206 antibody (1:100, PA5-101657, Thermo Fisher Scientific) at 4 ◦C for 12 h. Following this, the sections were incubated with the secondary antibodies IgG H&L (HRP) (ab205718 or ab97023, Abcam) at room temperature for 50 min.

Techniques: Over Expression, Stable Transfection, Transfection, Expressing, Quantitative RT-PCR, Phospho-proteomics, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Cytokine

Article Title: FOXA2 inhibits the TLR4/NF-κB signaling pathway and alleviates inflammatory activation of macrophages in rheumatoid arthritis by repressing LY96 transcription.

doi: 10.1016/j.cyto.2024.156796

Figure Lengend Snippet: Fig. 7. LY96 reactivates inflammatory responses in RA mice suppressed by FOXA2 overexpression. Lentiviral vectors-encapsulated OE-NC or OE-FOXA2, or OE- FOXA2 + OE-NC and OE-FOXA2 + OE-LY96 were injected into the joint cavity of mice during the RA modeling. A, mRNA expression of FOXA2 and LY96 in the mouse ankle joint tissue examined using RT-qPCR; B, positive staining of Myd88 in the mouse ankle joint tissues examined using immunofluorescence staining; C, positive staining of p-NF-κB in the mouse ankle joint tissues examined using IHC; D, arthritis score of mice after the second immunization; E, pathological changes in the mouse ankle joint tissues examined by HE, safranin O-fast green, and toluidine blue staining; F, positive staining of CD86 and CD206 in the mouse ankle joint tissues examined using IHC; G, concentrations of IL-6, TNF-α, IL-1β, and IL-10 in the mouse serum determined using ELISA kits. Each group contained 6 mice. Differences were compared by the one-way (A-C, F-G) or two-way (D) ANOVA. **p < 0.01; ***p < 0.001; ****p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Following further blocking with 3 % bovine serum albumin and digestion with pepsin for antigen retrieval, the sections were incubated with the antibodies against MD2 (LY96) (ab24182, Abcam), NF-κB p65 (phospho T254) (1:50, ab131100, Abcam), FOXA2 (NBP2-02088, Novus Biologicals, Littleton, CA, USA), CD86 (1:100, MA5-48134, Thermo Fisher Scientific), and CD206 antibody (1:100, PA5-101657, Thermo Fisher Scientific) at 4 ◦C for 12 h. Following this, the sections were incubated with the secondary antibodies IgG H&L (HRP) (ab205718 or ab97023, Abcam) at room temperature for 50 min.

Techniques: Over Expression, Injection, Expressing, Quantitative RT-PCR, Staining, Immunofluorescence, Enzyme-linked Immunosorbent Assay

Journal: Cytokine

Article Title: FOXA2 inhibits the TLR4/NF-κB signaling pathway and alleviates inflammatory activation of macrophages in rheumatoid arthritis by repressing LY96 transcription.

doi: 10.1016/j.cyto.2024.156796

Figure Lengend Snippet: Fig. 8. Proposed model. FOXA2-mediated transcriptional repression of LY96 blocks the TLR4/NF-κB signaling pathway to limit the inflammatory response of sy novial macrophages to alleviate rheumatoid arthritis.

Article Snippet: Following further blocking with 3 % bovine serum albumin and digestion with pepsin for antigen retrieval, the sections were incubated with the antibodies against MD2 (LY96) (ab24182, Abcam), NF-κB p65 (phospho T254) (1:50, ab131100, Abcam), FOXA2 (NBP2-02088, Novus Biologicals, Littleton, CA, USA), CD86 (1:100, MA5-48134, Thermo Fisher Scientific), and CD206 antibody (1:100, PA5-101657, Thermo Fisher Scientific) at 4 ◦C for 12 h. Following this, the sections were incubated with the secondary antibodies IgG H&L (HRP) (ab205718 or ab97023, Abcam) at room temperature for 50 min.

Techniques: