Journal: World Journal of Diabetes

Article Title: Mechanism of the epidermal growth factor receptor in promoting endothelial cell dysfunction in gestational diabetes mellitus

doi: 10.4239/wjd.v16.i6.105173

Figure Lengend Snippet: High glucose promotes epidermal growth factor receptor expression by reducing ERRFI1 stability. A: Detection of the ubiquitination level of ERRFI1 ; B: ERRFI1 expression detected by Western blotting; C: ERRFI1 stability detected by cycloheximide chase; D: Binding between ERRFI1 and epidermal growth factor receptor (EGFR) detected by coimmunoprecipitation; E: Western blot showing ERRFI1 knockdown efficiency; F: Western blot showing EGFR expression. a P < 0.05. b P < 0.01. c P < 0.001. IP: Immunoprecipitation; IB: Immunoblotting; NC: Normal control; HG: High glucose; CHX: Cycloheximide; NC: Normal control; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; sh-NC: Short hairpin-negative control; sh- ERRFI1 : Short hairpin RNA targeting ERRFI1 ; EGFR: Epidermal growth factor receptor.

Article Snippet: The resulting lysate was subjected to immunoprecipitation with an anti- ERRFI1 antibody (1:300, Novus Biologicals) and was placed on a rotary shaker overnight at 4 °C.

Techniques: Expressing, Ubiquitin Proteomics, Western Blot, Binding Assay, Knockdown, Immunoprecipitation, Control, Negative Control, shRNA

Journal: World Journal of Diabetes

Article Title: Mechanism of the epidermal growth factor receptor in promoting endothelial cell dysfunction in gestational diabetes mellitus

doi: 10.4239/wjd.v16.i6.105173

Figure Lengend Snippet: ERRFI1 binds to epidermal growth factor receptor and inhibits its activity to suppress high glucose-induced human umbilical vein endothelial cells dysfunction. A: Western blot showing ERRFI1 overexpression efficiency; B: Western blot showing epidermal growth factor receptor (EGFR) overexpression efficiency; C: Western blot showing EGFR expression in cells; D: Cell counting kit-8 assay showing cell viability; E: Cell apoptosis as detected by flow cytometry; F: Cell migration as assessed by scratch experiments; G: Cell migration as assessed by Transwell assay; H: Angiogenesis as assessed by tube formation assay; I: Western blot showing the expression of endothelial cell dysfunction markers. a P < 0.05. b P < 0.01. c P < 0.001. EGFR: Epidermal growth factor receptor; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; OE-NC: Overexpression-negative control; OE- ERRFI1 : Overexpression ERRFI1 ; NC: Normal control; OE-EGFR: Overexpression epidermal growth factor receptor; HG: High glucose; PI: Propidium iodide; FITC: Fluorescein isothiocyanate; VCAM-1: Vascular cell adhesion molecule-1; TNF-α: Tumor necrosis factor-α; VEGF-A: Vascular endothelial growth factor-A; ICAM-1: Intercellular cell adhesion molecule-1.

Article Snippet: The resulting lysate was subjected to immunoprecipitation with an anti- ERRFI1 antibody (1:300, Novus Biologicals) and was placed on a rotary shaker overnight at 4 °C.

Techniques: Activity Assay, Western Blot, Over Expression, Expressing, Cell Counting, Flow Cytometry, Migration, Transwell Assay, Tube Formation Assay, Negative Control, Control

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 1 ERRFI1 deficiency ameliorated IR-induced hepatic injury and hepatocellular apoptosis. (A) Schematic diagram showed the hepatocyte-specific ERRFI1 knockout strategy and ischemia reperfusion model. (B, C) Western blot analysis of ERRFI1 protein level in liver tissues from wild type (WT) mice and hepatocyte-specific ERRFI1-knockout (ERRFI1-HKO) mice with sham treatment or ischemia for 90 min followed by reperfusion for 6 h, and quantita tive analysis is shown. (D) Liver function assessed by ALT and AST of mice with different treatment. (E) Liver pathology was determined by H&E staining (scale bar: 100 μm). (F) Suzike’s injury score was used to assess the degree of injury based on H&E staining. (G, H) TUNEL staining of apoptotic cells in liver tissues from WT mice and ERRFI1-HKO mice under different conditions (scale bar: 50 μm), and quantification showing the percentage of apoptotic cells. (I, J) Western blot analysis of Bax, Bcl-2, and cleaved caspase-3 in liver tissues from WT mice and ERRFI1-HKO mice after IR injury. For statistical analysis, one-way ANOVA was used (n = 6)

Article Snippet: Immunoprecipitates were subjected to SDS-PAGE, and coimmunoprecipitated proteins were detected by immunoblotting with GRB2 antibody (ab32037, Abcam) or ERRFI1 (sc-137,154, Santa Cruz Biotechnology).

Techniques: Knock-Out, Western Blot, Staining, TUNEL Assay

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 2 ERRFI1 deficiency protected against hepatic IR-induced ferroptosis. (A) Intracellular ROS level was determined by DCFH-DA staining after hepatic IR. (B) Immunohistochemical staining of 8-OHdG in liver tissues (scale bar: 50 μm). (C, D) The content of MDA and the level of GSH in the livers of mice subjected to sham treatment or to an induction of IR. (E) Hepatic Fe2+ content in each group. (F) The mRNA levels of ACSL4, SLC7A11, and GPX4 in liver tissues of mice with different treatments. (G) Representative immunohistochemical images of ACSL4, SLC7A11, and GPX4 in liver tissues (scale bar: 50 μm). For statistical analysis, one-way ANOVA was used (n = 6)

Article Snippet: Immunoprecipitates were subjected to SDS-PAGE, and coimmunoprecipitated proteins were detected by immunoblotting with GRB2 antibody (ab32037, Abcam) or ERRFI1 (sc-137,154, Santa Cruz Biotechnology).

Techniques: Staining, Immunohistochemical staining

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 3 Knockdown of ERRFI1 inhibited apoptosis of hepatocytes induced by hypoxic-reoxygenation. (A) Expression of ERRFI1 in L-02 cells following hy poxia/reoxygenation (H/R) and ERRFI1 knockdown at the mRNA level was determined by real-time PCR. (B) Cell viability of ERRFI1-silenced L-02 cells after OGD/R exposure. (C, D) Apoptosis-positive cells were detected by TUNEL staining (scale bar: 50 μm). (E, F) Western blot analysis of Bax, Bcl-2, and cleaved caspase-3 in L-02 cells under indicated conditions. For statistical analysis, one-way ANOVA was used (n = 3)

Article Snippet: Immunoprecipitates were subjected to SDS-PAGE, and coimmunoprecipitated proteins were detected by immunoblotting with GRB2 antibody (ab32037, Abcam) or ERRFI1 (sc-137,154, Santa Cruz Biotechnology).

Techniques: Knockdown, Expressing, Real-time Polymerase Chain Reaction, TUNEL Assay, Staining, Western Blot

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 4 Knockdown of ERRFI1 suppressed OGD/R-induced ferroptosis in hepatocytes. (A) ROS level in ERRFI1-silenced L-02 cells exposed to hypoxia/ reoxygenation. (B) Flow cytometry analysis of lipid peroxidation using C11-BODIPY 581/591 in L-02 cells under indicated conditions. (C, D) MDA content and GSH level in L-02 cells cultured under indicated conditions. (E) Fe2+ content in ERRFI1-silenced L-02 cells after OGD/R exposure was determined. (F) Real-time PCR showed the mRNA levels of ACSL4, SLC7A11, and GPX4 in response to ERRFI1 knockdown under H/R conditions. (G, H) Fluorescence im munostaining of ACSL4, SLC7A11, and GPX4 in L-02 cells transfected with sh-ERRFI1 during H/R injury (scale bar: 20 μm). For statistical analysis, one-way ANOVA was used (n = 3)

Article Snippet: Immunoprecipitates were subjected to SDS-PAGE, and coimmunoprecipitated proteins were detected by immunoblotting with GRB2 antibody (ab32037, Abcam) or ERRFI1 (sc-137,154, Santa Cruz Biotechnology).

Techniques: Knockdown, Flow Cytometry, Cell Culture, Real-time Polymerase Chain Reaction, Fluorescence, Transfection

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 5 ERRFI1 directly interacted with GRB2 and maintained its stability by hindering its proteasomal degradation. (A-C) Transcript and protein levels of GRB2 in ERRFI1-silenced L-02 cells were detected. (D) Co-immunoprecipitation of ERRFI1 and GRB2. L-02 cells were subjected to GRB2 immunoprecipita tion and subsequent immunoblotting of ERRFI1 and GRB2. (E, F) L-02 cells were transfected with sh-ERRFI1 and treated with CHX for the indicated times. Western blot analysis showed the expression of GRB2. (G, H) The expression of GRB2 in sh-ERRFI1-transfected L-02 cells with or without MG132 treatment. For statistical analysis, Student’s t test was used (n = 3)

Article Snippet: Immunoprecipitates were subjected to SDS-PAGE, and coimmunoprecipitated proteins were detected by immunoblotting with GRB2 antibody (ab32037, Abcam) or ERRFI1 (sc-137,154, Santa Cruz Biotechnology).

Techniques: Immunoprecipitation, Western Blot, Transfection, Expressing

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 6 ERRFI1 facilitated OGD/R-induced injury of hepatocytes in a GRB2-dependent manner. (A-D) L-02 cells were transfected with GRB2 overexpression plasmid or vector plasmid, followed by 4 h of hypoxia and 12 h of reoxygenation. Cell apoptosis was detected by TUNEL staining. Scale bar: 20 μm (A, B). Lipid peroxidation was measured using C11-BODIPY 581/591 (C). Fe2+ content was determined (D). (E, F) L-02 cells cotransfected with sh-ERRFI1 and GRB2 overexpression plasmid were subjected to OGD/R stimulation in the presence or absence of 20 µM Z-VAD-FMK (ZVF, an apoptosis inhibitor). L-02 cells were treated with 5 µM camptothecin (CPT, an apoptosis inducer) as positive control at the same time as OGD/R stimulation. TUNEL staining of L-02 cells under indicated conditions was performed (scale bar: 20 μm). (G-L) L-02 cells cotransfected with sh-ERRFI1 and GRB2 overexpression plasmid were subjected to OGD/R stimulation in the presence or absence of 5 µM ferrostatin-1 (Fer-1, a ferroptotic inhibitor). L-02 cells were treated with 10 µM erastin (a ferroptotic inducer) as positive control at the same time as OGD/R stimulation. DCFH-DA staining was used to detect ROS production in cells (G). Fe2+ content in cells was measured by a commercial kit (H). The protein expression of GPX4 in cells was analyzed by immunofluorescence. Scale bar: 20 μm (I, K). Cell death was detected by propidium iodide (PI) staining. Scale bar: 50 μm (J, L). For statistical analysis, student’s t test and one-way ANOVA were used (n = 3)

Article Snippet: Immunoprecipitates were subjected to SDS-PAGE, and coimmunoprecipitated proteins were detected by immunoblotting with GRB2 antibody (ab32037, Abcam) or ERRFI1 (sc-137,154, Santa Cruz Biotechnology).

Techniques: Transfection, Over Expression, Plasmid Preparation, TUNEL Assay, Staining, Positive Control, Expressing, Immunofluorescence

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 1 ERRFI1 deficiency ameliorated IR-induced hepatic injury and hepatocellular apoptosis. (A) Schematic diagram showed the hepatocyte-specific ERRFI1 knockout strategy and ischemia reperfusion model. (B, C) Western blot analysis of ERRFI1 protein level in liver tissues from wild type (WT) mice and hepatocyte-specific ERRFI1-knockout (ERRFI1-HKO) mice with sham treatment or ischemia for 90 min followed by reperfusion for 6 h, and quantita tive analysis is shown. (D) Liver function assessed by ALT and AST of mice with different treatment. (E) Liver pathology was determined by H&E staining (scale bar: 100 μm). (F) Suzike’s injury score was used to assess the degree of injury based on H&E staining. (G, H) TUNEL staining of apoptotic cells in liver tissues from WT mice and ERRFI1-HKO mice under different conditions (scale bar: 50 μm), and quantification showing the percentage of apoptotic cells. (I, J) Western blot analysis of Bax, Bcl-2, and cleaved caspase-3 in liver tissues from WT mice and ERRFI1-HKO mice after IR injury. For statistical analysis, one-way ANOVA was used (n = 6)

Article Snippet: Hepatocyte-specific ERRFI1 knockout (ERRFI1-HKO) mice were generated by mating ERRFI1-flox mice (Cyagen Biosciences) with Albumin (Alb)-enhancer/promoter-driven Cre transgenic mice (GemPharmatech Co., Ltd.).

Techniques: Knock-Out, Western Blot, Staining, TUNEL Assay

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 2 ERRFI1 deficiency protected against hepatic IR-induced ferroptosis. (A) Intracellular ROS level was determined by DCFH-DA staining after hepatic IR. (B) Immunohistochemical staining of 8-OHdG in liver tissues (scale bar: 50 μm). (C, D) The content of MDA and the level of GSH in the livers of mice subjected to sham treatment or to an induction of IR. (E) Hepatic Fe2+ content in each group. (F) The mRNA levels of ACSL4, SLC7A11, and GPX4 in liver tissues of mice with different treatments. (G) Representative immunohistochemical images of ACSL4, SLC7A11, and GPX4 in liver tissues (scale bar: 50 μm). For statistical analysis, one-way ANOVA was used (n = 6)

Article Snippet: Hepatocyte-specific ERRFI1 knockout (ERRFI1-HKO) mice were generated by mating ERRFI1-flox mice (Cyagen Biosciences) with Albumin (Alb)-enhancer/promoter-driven Cre transgenic mice (GemPharmatech Co., Ltd.).

Techniques: Staining, Immunohistochemical staining

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 3 Knockdown of ERRFI1 inhibited apoptosis of hepatocytes induced by hypoxic-reoxygenation. (A) Expression of ERRFI1 in L-02 cells following hy poxia/reoxygenation (H/R) and ERRFI1 knockdown at the mRNA level was determined by real-time PCR. (B) Cell viability of ERRFI1-silenced L-02 cells after OGD/R exposure. (C, D) Apoptosis-positive cells were detected by TUNEL staining (scale bar: 50 μm). (E, F) Western blot analysis of Bax, Bcl-2, and cleaved caspase-3 in L-02 cells under indicated conditions. For statistical analysis, one-way ANOVA was used (n = 3)

Article Snippet: Hepatocyte-specific ERRFI1 knockout (ERRFI1-HKO) mice were generated by mating ERRFI1-flox mice (Cyagen Biosciences) with Albumin (Alb)-enhancer/promoter-driven Cre transgenic mice (GemPharmatech Co., Ltd.).

Techniques: Knockdown, Expressing, Real-time Polymerase Chain Reaction, TUNEL Assay, Staining, Western Blot

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 4 Knockdown of ERRFI1 suppressed OGD/R-induced ferroptosis in hepatocytes. (A) ROS level in ERRFI1-silenced L-02 cells exposed to hypoxia/ reoxygenation. (B) Flow cytometry analysis of lipid peroxidation using C11-BODIPY 581/591 in L-02 cells under indicated conditions. (C, D) MDA content and GSH level in L-02 cells cultured under indicated conditions. (E) Fe2+ content in ERRFI1-silenced L-02 cells after OGD/R exposure was determined. (F) Real-time PCR showed the mRNA levels of ACSL4, SLC7A11, and GPX4 in response to ERRFI1 knockdown under H/R conditions. (G, H) Fluorescence im munostaining of ACSL4, SLC7A11, and GPX4 in L-02 cells transfected with sh-ERRFI1 during H/R injury (scale bar: 20 μm). For statistical analysis, one-way ANOVA was used (n = 3)

Article Snippet: Hepatocyte-specific ERRFI1 knockout (ERRFI1-HKO) mice were generated by mating ERRFI1-flox mice (Cyagen Biosciences) with Albumin (Alb)-enhancer/promoter-driven Cre transgenic mice (GemPharmatech Co., Ltd.).

Techniques: Knockdown, Flow Cytometry, Cell Culture, Real-time Polymerase Chain Reaction, Fluorescence, Transfection

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 5 ERRFI1 directly interacted with GRB2 and maintained its stability by hindering its proteasomal degradation. (A-C) Transcript and protein levels of GRB2 in ERRFI1-silenced L-02 cells were detected. (D) Co-immunoprecipitation of ERRFI1 and GRB2. L-02 cells were subjected to GRB2 immunoprecipita tion and subsequent immunoblotting of ERRFI1 and GRB2. (E, F) L-02 cells were transfected with sh-ERRFI1 and treated with CHX for the indicated times. Western blot analysis showed the expression of GRB2. (G, H) The expression of GRB2 in sh-ERRFI1-transfected L-02 cells with or without MG132 treatment. For statistical analysis, Student’s t test was used (n = 3)

Article Snippet: Hepatocyte-specific ERRFI1 knockout (ERRFI1-HKO) mice were generated by mating ERRFI1-flox mice (Cyagen Biosciences) with Albumin (Alb)-enhancer/promoter-driven Cre transgenic mice (GemPharmatech Co., Ltd.).

Techniques: Immunoprecipitation, Western Blot, Transfection, Expressing

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 6 ERRFI1 facilitated OGD/R-induced injury of hepatocytes in a GRB2-dependent manner. (A-D) L-02 cells were transfected with GRB2 overexpression plasmid or vector plasmid, followed by 4 h of hypoxia and 12 h of reoxygenation. Cell apoptosis was detected by TUNEL staining. Scale bar: 20 μm (A, B). Lipid peroxidation was measured using C11-BODIPY 581/591 (C). Fe2+ content was determined (D). (E, F) L-02 cells cotransfected with sh-ERRFI1 and GRB2 overexpression plasmid were subjected to OGD/R stimulation in the presence or absence of 20 µM Z-VAD-FMK (ZVF, an apoptosis inhibitor). L-02 cells were treated with 5 µM camptothecin (CPT, an apoptosis inducer) as positive control at the same time as OGD/R stimulation. TUNEL staining of L-02 cells under indicated conditions was performed (scale bar: 20 μm). (G-L) L-02 cells cotransfected with sh-ERRFI1 and GRB2 overexpression plasmid were subjected to OGD/R stimulation in the presence or absence of 5 µM ferrostatin-1 (Fer-1, a ferroptotic inhibitor). L-02 cells were treated with 10 µM erastin (a ferroptotic inducer) as positive control at the same time as OGD/R stimulation. DCFH-DA staining was used to detect ROS production in cells (G). Fe2+ content in cells was measured by a commercial kit (H). The protein expression of GPX4 in cells was analyzed by immunofluorescence. Scale bar: 20 μm (I, K). Cell death was detected by propidium iodide (PI) staining. Scale bar: 50 μm (J, L). For statistical analysis, student’s t test and one-way ANOVA were used (n = 3)

Article Snippet: Hepatocyte-specific ERRFI1 knockout (ERRFI1-HKO) mice were generated by mating ERRFI1-flox mice (Cyagen Biosciences) with Albumin (Alb)-enhancer/promoter-driven Cre transgenic mice (GemPharmatech Co., Ltd.).

Techniques: Transfection, Over Expression, Plasmid Preparation, TUNEL Assay, Staining, Positive Control, Expressing, Immunofluorescence

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 1 ERRFI1 deficiency ameliorated IR-induced hepatic injury and hepatocellular apoptosis. (A) Schematic diagram showed the hepatocyte-specific ERRFI1 knockout strategy and ischemia reperfusion model. (B, C) Western blot analysis of ERRFI1 protein level in liver tissues from wild type (WT) mice and hepatocyte-specific ERRFI1-knockout (ERRFI1-HKO) mice with sham treatment or ischemia for 90 min followed by reperfusion for 6 h, and quantita tive analysis is shown. (D) Liver function assessed by ALT and AST of mice with different treatment. (E) Liver pathology was determined by H&E staining (scale bar: 100 μm). (F) Suzike’s injury score was used to assess the degree of injury based on H&E staining. (G, H) TUNEL staining of apoptotic cells in liver tissues from WT mice and ERRFI1-HKO mice under different conditions (scale bar: 50 μm), and quantification showing the percentage of apoptotic cells. (I, J) Western blot analysis of Bax, Bcl-2, and cleaved caspase-3 in liver tissues from WT mice and ERRFI1-HKO mice after IR injury. For statistical analysis, one-way ANOVA was used (n = 6)

Article Snippet: The following primary antibodies were used: antibodies for ERRFI1 (sc-137,154; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bax (#2772; Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (ab182858, Abcam), cleaved caspase-3 (AF7022; Affinity Biosciences, Cincinnati, OH, USA), caspase-3 (AF6311, Affinity Biosciences), GRB2 (ab32037, Abcam), and GAPDH (ab181602, Abcam).

Techniques: Knock-Out, Western Blot, Staining, TUNEL Assay

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 2 ERRFI1 deficiency protected against hepatic IR-induced ferroptosis. (A) Intracellular ROS level was determined by DCFH-DA staining after hepatic IR. (B) Immunohistochemical staining of 8-OHdG in liver tissues (scale bar: 50 μm). (C, D) The content of MDA and the level of GSH in the livers of mice subjected to sham treatment or to an induction of IR. (E) Hepatic Fe2+ content in each group. (F) The mRNA levels of ACSL4, SLC7A11, and GPX4 in liver tissues of mice with different treatments. (G) Representative immunohistochemical images of ACSL4, SLC7A11, and GPX4 in liver tissues (scale bar: 50 μm). For statistical analysis, one-way ANOVA was used (n = 6)

Article Snippet: The following primary antibodies were used: antibodies for ERRFI1 (sc-137,154; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bax (#2772; Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (ab182858, Abcam), cleaved caspase-3 (AF7022; Affinity Biosciences, Cincinnati, OH, USA), caspase-3 (AF6311, Affinity Biosciences), GRB2 (ab32037, Abcam), and GAPDH (ab181602, Abcam).

Techniques: Staining, Immunohistochemical staining

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 3 Knockdown of ERRFI1 inhibited apoptosis of hepatocytes induced by hypoxic-reoxygenation. (A) Expression of ERRFI1 in L-02 cells following hy poxia/reoxygenation (H/R) and ERRFI1 knockdown at the mRNA level was determined by real-time PCR. (B) Cell viability of ERRFI1-silenced L-02 cells after OGD/R exposure. (C, D) Apoptosis-positive cells were detected by TUNEL staining (scale bar: 50 μm). (E, F) Western blot analysis of Bax, Bcl-2, and cleaved caspase-3 in L-02 cells under indicated conditions. For statistical analysis, one-way ANOVA was used (n = 3)

Article Snippet: The following primary antibodies were used: antibodies for ERRFI1 (sc-137,154; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bax (#2772; Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (ab182858, Abcam), cleaved caspase-3 (AF7022; Affinity Biosciences, Cincinnati, OH, USA), caspase-3 (AF6311, Affinity Biosciences), GRB2 (ab32037, Abcam), and GAPDH (ab181602, Abcam).

Techniques: Knockdown, Expressing, Real-time Polymerase Chain Reaction, TUNEL Assay, Staining, Western Blot

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 4 Knockdown of ERRFI1 suppressed OGD/R-induced ferroptosis in hepatocytes. (A) ROS level in ERRFI1-silenced L-02 cells exposed to hypoxia/ reoxygenation. (B) Flow cytometry analysis of lipid peroxidation using C11-BODIPY 581/591 in L-02 cells under indicated conditions. (C, D) MDA content and GSH level in L-02 cells cultured under indicated conditions. (E) Fe2+ content in ERRFI1-silenced L-02 cells after OGD/R exposure was determined. (F) Real-time PCR showed the mRNA levels of ACSL4, SLC7A11, and GPX4 in response to ERRFI1 knockdown under H/R conditions. (G, H) Fluorescence im munostaining of ACSL4, SLC7A11, and GPX4 in L-02 cells transfected with sh-ERRFI1 during H/R injury (scale bar: 20 μm). For statistical analysis, one-way ANOVA was used (n = 3)

Article Snippet: The following primary antibodies were used: antibodies for ERRFI1 (sc-137,154; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bax (#2772; Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (ab182858, Abcam), cleaved caspase-3 (AF7022; Affinity Biosciences, Cincinnati, OH, USA), caspase-3 (AF6311, Affinity Biosciences), GRB2 (ab32037, Abcam), and GAPDH (ab181602, Abcam).

Techniques: Knockdown, Flow Cytometry, Cell Culture, Real-time Polymerase Chain Reaction, Fluorescence, Transfection

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 5 ERRFI1 directly interacted with GRB2 and maintained its stability by hindering its proteasomal degradation. (A-C) Transcript and protein levels of GRB2 in ERRFI1-silenced L-02 cells were detected. (D) Co-immunoprecipitation of ERRFI1 and GRB2. L-02 cells were subjected to GRB2 immunoprecipita tion and subsequent immunoblotting of ERRFI1 and GRB2. (E, F) L-02 cells were transfected with sh-ERRFI1 and treated with CHX for the indicated times. Western blot analysis showed the expression of GRB2. (G, H) The expression of GRB2 in sh-ERRFI1-transfected L-02 cells with or without MG132 treatment. For statistical analysis, Student’s t test was used (n = 3)

Article Snippet: The following primary antibodies were used: antibodies for ERRFI1 (sc-137,154; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bax (#2772; Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (ab182858, Abcam), cleaved caspase-3 (AF7022; Affinity Biosciences, Cincinnati, OH, USA), caspase-3 (AF6311, Affinity Biosciences), GRB2 (ab32037, Abcam), and GAPDH (ab181602, Abcam).

Techniques: Immunoprecipitation, Western Blot, Transfection, Expressing

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 6 ERRFI1 facilitated OGD/R-induced injury of hepatocytes in a GRB2-dependent manner. (A-D) L-02 cells were transfected with GRB2 overexpression plasmid or vector plasmid, followed by 4 h of hypoxia and 12 h of reoxygenation. Cell apoptosis was detected by TUNEL staining. Scale bar: 20 μm (A, B). Lipid peroxidation was measured using C11-BODIPY 581/591 (C). Fe2+ content was determined (D). (E, F) L-02 cells cotransfected with sh-ERRFI1 and GRB2 overexpression plasmid were subjected to OGD/R stimulation in the presence or absence of 20 µM Z-VAD-FMK (ZVF, an apoptosis inhibitor). L-02 cells were treated with 5 µM camptothecin (CPT, an apoptosis inducer) as positive control at the same time as OGD/R stimulation. TUNEL staining of L-02 cells under indicated conditions was performed (scale bar: 20 μm). (G-L) L-02 cells cotransfected with sh-ERRFI1 and GRB2 overexpression plasmid were subjected to OGD/R stimulation in the presence or absence of 5 µM ferrostatin-1 (Fer-1, a ferroptotic inhibitor). L-02 cells were treated with 10 µM erastin (a ferroptotic inducer) as positive control at the same time as OGD/R stimulation. DCFH-DA staining was used to detect ROS production in cells (G). Fe2+ content in cells was measured by a commercial kit (H). The protein expression of GPX4 in cells was analyzed by immunofluorescence. Scale bar: 20 μm (I, K). Cell death was detected by propidium iodide (PI) staining. Scale bar: 50 μm (J, L). For statistical analysis, student’s t test and one-way ANOVA were used (n = 3)

Article Snippet: The following primary antibodies were used: antibodies for ERRFI1 (sc-137,154; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bax (#2772; Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (ab182858, Abcam), cleaved caspase-3 (AF7022; Affinity Biosciences, Cincinnati, OH, USA), caspase-3 (AF6311, Affinity Biosciences), GRB2 (ab32037, Abcam), and GAPDH (ab181602, Abcam).

Techniques: Transfection, Over Expression, Plasmid Preparation, TUNEL Assay, Staining, Positive Control, Expressing, Immunofluorescence