Journal: Acta pharmaceutica Sinica. B

Article Title: The E3 ubiquitin ligase NEDD4-1 protects against acetaminophen-induced liver injury by targeting VDAC1 for degradation.

doi: 10.1016/j.apsb.2023.01.019

Figure Lengend Snippet: Figure 2 Hepatocyte-specific deficiency of NEDD4-1 exacerbates APAP induced hepatotoxicity in vivo and in vitro. (AeD) Serum ALT (A), AST (B), LDH (C) activities, and HMGB1 levels (D) of mice. (E) Representative H&E-stained images (left) and necrotic area quantification (right) of mouse liver sections. Scale bar, 200 mm. (F) Survival rate of Flox (n Z 18) or HepKO (n Z 20) mice treated with 600 mg/kg APAP for 72 h. (G, H) RNA-seq was conducted on total RNA isolated from livers of Flox or HepKO mice treated with 300 mg/kg APAP for 24 h; n Z 3 per group. Volcano plot (G) shows the differentially expressed genes (1201 genes were up-regulated and 736 genes were down-regulated in APAP- treated HepKO mice). GSEA (H) shows the gene sets that the differentially expressed genes significantly enriched. (I) Representative images (left) and quantification (right) of Calcein-AM (green) and PI (red) double-stained primary hepatocytes. Scale bar, 200 mm. (J, K) LDH release (J) and HMGB1 level (K) in the culture medium of primary hepatocytes. Experiments AeE were performed on Flox or HepKO mice treated with saline or 300 mg/kg APAP for 24 h; n Z 5e8 per group. Experiments IeK were performed on primary hepatocytes isolated from Flox or HepKO mice, with vehicle or APAP (10 mmol/L for 12 h) treatment. Data are presented as the mean SEM; *P < 0.05, **P < 0.01.

Article Snippet: Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) activities, and high mobility group box 1 (HMGB1) concentration were measured using the ALT activity assay kit (MAK052; SigmaeAldrich, St. Louis, MO, USA), AST activity assay kit (MAK055; SigmaeAldrich), LDH activity assay kit (MAK066; SigmaeAldrich), and mouse HMGB1 ELISA kit (NBP2-62767; Novus Biologicals, Littleton, CO, USA) according to the manufacturer’s protocols, respectively.

Techniques: In Vivo, In Vitro, Staining, RNA Sequencing, Isolation, Saline

Journal: Acta pharmaceutica Sinica. B

Article Title: The E3 ubiquitin ligase NEDD4-1 protects against acetaminophen-induced liver injury by targeting VDAC1 for degradation.

doi: 10.1016/j.apsb.2023.01.019

Figure Lengend Snippet: Figure 3 Hepatocyte NEDD4-1 overexpression alleviates APAP-induced hepatotoxicity in vivo and in vitro. (A) Representative H&E-stained images (left) and necrotic area quantification (right) of mouse liver sections. Scale bar, 200 mm. (BeE) Serum ALT (B), AST (C), LDH (D) activities, and HMGB1 level (E) of mice. (F) Survival rate of AAV8-Control (n Z 15) or AAV8-NEDD4-1 (n Z 13) infected mice, with 600 mg/kg APAP treated for 72 h. (G) Representative images (left) and quantification (right) of Calcein-AM (green) and PI (red) double-stained primary hepatocytes. Scale bar, 200 mm. (H, I) LDH release (H) and HMGB1 level (I) in the culture medium of primary hepatocytes. Experiments AeE were performed on AAV8-Control or AAV8-NEDD4-1 infected mice, with saline or 300 mg/kg APAP treated for 24 h, n Z 5e8 per group. Experiments GeI were performed on primary hepatocytes transfected with Ad-Vector or Ad-NEDD4-1, with vehicle or APAP (10 mmol/L for 12 h) treatment. Data are presented as the mean SEM; *P < 0.05, **P < 0.01.

Article Snippet: Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) activities, and high mobility group box 1 (HMGB1) concentration were measured using the ALT activity assay kit (MAK052; SigmaeAldrich, St. Louis, MO, USA), AST activity assay kit (MAK055; SigmaeAldrich), LDH activity assay kit (MAK066; SigmaeAldrich), and mouse HMGB1 ELISA kit (NBP2-62767; Novus Biologicals, Littleton, CO, USA) according to the manufacturer’s protocols, respectively.

Techniques: Over Expression, In Vivo, In Vitro, Staining, Control, Infection, Saline, Transfection, Plasmid Preparation

Journal: Acta pharmaceutica Sinica. B

Article Title: The E3 ubiquitin ligase NEDD4-1 protects against acetaminophen-induced liver injury by targeting VDAC1 for degradation.

doi: 10.1016/j.apsb.2023.01.019

Figure Lengend Snippet: Figure 5 Knockdown of VDAC1 mitigates the enhanced AILI events caused by hepatocyte-specific NEDD4-1 deficiency in vivo. (A) Representative Western blots of indicated proteins in the cytosolic and mitochondrial fractions of liver lysates from Flox or HepKO mice treated with 300 mg/kg APAP for 24 h. (B) Representative Western blots of indicated proteins in the cytosolic and mitochondrial fractions of liver lysates from mice treated with saline or 300 mg/kg APAP for 6, 12 or 24 h. (C) Representative IHC staining (left) and quantification (right) in mice treated with saline or 300 mg/kg APAP for 24 h; n Z 3 per group. Scar bar, 200 mm. (D) The hepatic mRNA expressions of Vdac1 in mice treated with saline or 300 mg/kg APAP for 24 h; n Z 8 per group. (E) Representative H&E-stained images (left) and necrotic area quantification (right) of mouse liver sections. Scale bar, 200 mm. (FeI) Serum ALT (F), AST (G), LDH (H) activities, and HMGB1 levels (I) of mice (J) Survival rate of AAV8-shControl (n Z 11) or AAV8-shVDAC1 (n Z 11) infected HepKO mice in response to a lethal dose of APAP (600 mg/kg) for 72 h. (K, L) ATP (K) and ROS (L) contents in the livers of mice. (M) Representative Western blots of indicated proteins in the cytosolic and mitochondrial fractions of liver lysates from AAV8-shControl or AAV8-shVDAC1 infected HepKO mice, with 300 mg/kg APAP treated for 24 h. Experiments EeI, K and L were performed on AAV8-shControl or AAV8-shVDAC1 infected Flox or HepKO mice, with 300 mg/kg APAP treated for 24 h; n Z 5e6 per group. Data are presented as the mean SEM; *P < 0.05, **P < 0.01, n. s., not significant.

Article Snippet: Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) activities, and high mobility group box 1 (HMGB1) concentration were measured using the ALT activity assay kit (MAK052; SigmaeAldrich, St. Louis, MO, USA), AST activity assay kit (MAK055; SigmaeAldrich), LDH activity assay kit (MAK066; SigmaeAldrich), and mouse HMGB1 ELISA kit (NBP2-62767; Novus Biologicals, Littleton, CO, USA) according to the manufacturer’s protocols, respectively.

Techniques: Knockdown, In Vivo, Western Blot, Saline, Immunohistochemistry, Staining, Infection

Journal: Acta pharmaceutica Sinica. B

Article Title: The E3 ubiquitin ligase NEDD4-1 protects against acetaminophen-induced liver injury by targeting VDAC1 for degradation.

doi: 10.1016/j.apsb.2023.01.019

Figure Lengend Snippet: Figure 6 Knockdown of VDAC1 mitigates the enhanced APAP-induced pathological events triggered by hepatocyte-specific NEDD4-1 deficiency in vitro. (A) Representative images (left) and quantification (right) of Calcein-AM (green) and PI (red) double-stained primary he- patocytes. Scale bar, 200 mm. (B, C) LDH release (B) and HMGB1 level (C) in the cell culture medium. (D) Seahorse assays showed the OCR (top) and corresponding statistical results (bottom) in primary hepatocytes. (E) Representative JC-1-stained images (left) and quantification of mitochondria depolarization (right) in primary hepatocytes. Scale bar, 100 mm. (F) Representative MitoSOX Red-stained images (left) and quantification (right) in primary hepatocytes. Scale bar, 200 mm. Experiments AeF were performed on HepKO hepatocytes transfected with Ad- shControl or Ad-shVDAC1, with 10 mmol/L APAP treated for 12 h. Data are presented as the mean SEM; *P < 0.05, **P < 0.01, n. s., not significant.

Article Snippet: Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) activities, and high mobility group box 1 (HMGB1) concentration were measured using the ALT activity assay kit (MAK052; SigmaeAldrich, St. Louis, MO, USA), AST activity assay kit (MAK055; SigmaeAldrich), LDH activity assay kit (MAK066; SigmaeAldrich), and mouse HMGB1 ELISA kit (NBP2-62767; Novus Biologicals, Littleton, CO, USA) according to the manufacturer’s protocols, respectively.

Techniques: Knockdown, In Vitro, Staining, Cell Culture, Transfection

Journal:

Article Title:

doi:

Figure Lengend Snippet: A, BAA473 selectively induces secretion of IL-1β but not IL-6 and IL-8. PBMCs were primed with 0.1 ng/ml LPS overnight followed by BAA473 treatment for 16 h and HTRF detection of secreted levels of IL-1β, IL-6, and IL-8. Data presented are mean ± S.E. (error bars) (n = 3). *, p < 0.005 versus vehicle (paired t test). B–D, BAA473 induces secretion of cytokines as well as caspase-1 and LDH activity in THP-1 cells. THP-1 cells were primed with 1 μg/ml LPS overnight followed by compound treatment for 24 h. B, cytokine secretion in supernatant was measured by HTRF (*, p < 0.0007). C, caspase-1 activity was assessed using the Caspase-Glo® 1 assay. Nigericin was used as an NALP3 activator. D, LDH activity (*, p < 0.003) was measured using a colorimetric LDH assay kit. E, BAA473 induces cytokine production via ASC/CASP1. THP-1 cells primed with 1 μg/ml LPS were pretreated with 10 μm BAA473 or 5 mm ATP for 1 h, followed by overnight treatment with caspase-1 inhibitor (VX-765). The level of IL-1β secretion was measured by an HTRF assay. Data presented are mean ± S.E. (n = 3).

Article Snippet: LDH released into the media was measured using the LDH colorimetric assay kit from Abcam (ab102526).

Techniques: Activity Assay, Lactate Dehydrogenase Assay, HTRF Assay

Journal: International Journal of Molecular Sciences

Article Title: 6-Shogaol Overcomes Gefitinib Resistance via ER Stress in Ovarian Cancer Cells

doi: 10.3390/ijms24032639

Figure Lengend Snippet: Anti-cancer effects of 6-shogaol in ovarian cancer cell lines in time-dependent manner. ( A – D ) 6-shogaol is Table 0. 16, and 24 h; 30 μM) and the WST-1 assay, LDH assay, and caspase-3 activity assays were performed. Western blotting analyses of cleaved caspase-3, -8, and -9 for the indicated times in 6-shogaol-treated A2780 and OVCAR-3 cells; *, p < 0.05. β-actin was used as a protein loading control. ( E – H ) The effect of Z-VAD-FMK (50 μM) and 6-shogaol treatment. A2780 and OVCAR-3 cells were pretreated with Z-VAD-FMK for 4 h and were subsequently treated with 6-shogaol (30 μM, 24 h). Cell viability was determined using the WST-1 assay; cell cytotoxicity was monitored using the LDH assay, and caspase-3 activity was assessed using the caspase-3 activity assay; *, p < 0.05. n.s = not significant. To identify caspase-3 cleavage, a Western blot assay was performed. β-actin was used as a protein loading control.

Article Snippet: A2780 and OVCAR-3 human ovarian cancer cell lines were seeded into a 6-well plate with a growth medium and grown for 24 h. Caspase-3 activity assays (the Biovision colorimetric caspase-3 assay kit) were performed according to the manufacturer’s protocols.

Techniques: WST-1 Assay, Lactate Dehydrogenase Assay, Activity Assay, Western Blot, Caspase-3 Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: 6-Shogaol Overcomes Gefitinib Resistance via ER Stress in Ovarian Cancer Cells

doi: 10.3390/ijms24032639

Figure Lengend Snippet: 6-shogaol mediates ER stress response through intracellular Ca 2+ generation. ( A ) A2780 and OVCAR-3 cells were treated with 6-shogaol (30 μM) in a time-dependent manner (0, 8, 16, and 24 h) and intracellular Ca 2+ release was determined using an intracellular Ca 2+ assay. ( B ) A2780 and OVCAR-3 cells were treated with 6-shogaol (30 μM) in a time-dependent manner (0, 8, 16, and 24 h) and mRNA levels of ATF4 and CHOP were investigated using real-time RT-PCR. ( C ) A2780 and OVCAR-3 cells were treated with 6-shogaol (30 μM) for the indicated times (0, 8, 16, and 24 h) and the activation of ER stress signaling, including GRP78, p-PERK, PERK, p-eIF2α, eIF2α, ATF4, and CHOP, was assessed using the Western blot assay. β-actin was used as a protein loading control. ( D ) A2780 and OVCAR-3 cells were treated with 6-shogaol (30 μM) for the indicated times (0, 8, 16, and 24 h) and exosomes were extracted from the cell culture media. Protein samples extracted from cell lysates and exosomes were quantified by Ponceau S staining. These samples were conducted by Western blotting using the ER stress marker, GRP78, and the exosome marker, CD63. ( E – H ) Cell viability, LDH cytotoxicity, and expression levels of GRP78, p-PERK, PERK, ATF4, CHOP, and cleaved caspase-3 levels were determined using WST-1 assay, LDH assay, and Western blotting analyses in thapsigargin (TG; 3 μM, 24 h) and 6-shogaol (30 μM, 24 h)-treated A2780 and OVCAR-3 cells; *, p <0.05.

Article Snippet: A2780 and OVCAR-3 human ovarian cancer cell lines were seeded into a 6-well plate with a growth medium and grown for 24 h. Caspase-3 activity assays (the Biovision colorimetric caspase-3 assay kit) were performed according to the manufacturer’s protocols.

Techniques: Quantitative RT-PCR, Activation Assay, Western Blot, Cell Culture, Staining, Marker, Expressing, WST-1 Assay, Lactate Dehydrogenase Assay

Journal: International Journal of Molecular Sciences

Article Title: 6-Shogaol Overcomes Gefitinib Resistance via ER Stress in Ovarian Cancer Cells

doi: 10.3390/ijms24032639

Figure Lengend Snippet: Inhibition of ER stress sensors suppressed by 6-shogaol caused cell death in ovarian cancer cells. ( A – C ) A2780 and OVCAR-3 cells were transfected with GRP78-specific siRNA in the presence or absence of 6-shogaol (30 μM, 24 h) and WST-1, caspase-3 activity, and intracellular Ca 2+ assay were performed; *, p <0.05. ( D ) Western blot analysis of GRP78, p-PERK, PERK, p-eIF2α, eIF2α, ATF4, CHOP, and cleaved caspase-3 in 6-shogaol (30 μM, 24 h)-treated A2780 and OVCAR-3 cells was performed in the presence or absence of GRP78 siRNA (30 nM, 24 h). β-actin was used as protein loading controls. ( E – H ) After A2780 and OVCAR-3 cells were transfected with PERK (30 nM, 24 h), WST-1, LDH cytotoxicity assay, intracellular Ca 2+ assay, and Western blotting analyses were performed with/without 6-shogaol (30 μM, 24 h) treatment.; *, p < 0.05. n.s = not significant. Western blotting was carried out to identify the ER stress-related genes p-PERK, PERK, p-eIF2α, eIF2α, ATF4, CHOP, and caspase-3 cleavage in 6-shogaol-treated PERK knockdown cells. β-actin was used as a protein loading control.

Article Snippet: A2780 and OVCAR-3 human ovarian cancer cell lines were seeded into a 6-well plate with a growth medium and grown for 24 h. Caspase-3 activity assays (the Biovision colorimetric caspase-3 assay kit) were performed according to the manufacturer’s protocols.

Techniques: Inhibition, Transfection, Activity Assay, Western Blot, LDH Cytotoxicity Assay

Journal: International Journal of Molecular Sciences

Article Title: 6-Shogaol Overcomes Gefitinib Resistance via ER Stress in Ovarian Cancer Cells

doi: 10.3390/ijms24032639

Figure Lengend Snippet: 6-shogaol induces the binding of CHOP on DR5 promoter in ovarian cancer cells. ( A – D ) After A2780 and OVCAR-3 cells were transfected with CHOP siRNA (30 nM, 24 h), WST-1, intracellular Ca 2+ assay, Western blotting analyses, and ChIP assay were performed with/without 6-shogaol (30 μM, 24 h) treatment.; *, p < 0.05. Western blotting analyses were carried out to determine the expression of CHOP, DR4, DR5, and caspase-3 cleavage in 6-shogaol-treated CHOP knockdown cells. β-actin was used as a protein loading control. ( E – H ) After A2780 and OVCAR-3 cells were transfected with DR5 (30 nM, 24 h), WST-1, LDH cytotoxicity assay, intracellular Ca 2+ assay, and Western blotting analyses were performed with/without 6-shogaol (30 μM, 24 h) treatment.; *, p < 0.05. n.s = not significant. Western blotting was carried out to identify the expression of DR5, caspase-3, and caspase-8 cleavage in 6-shogaol-treated DR5 knockdown cells. β-actin was used as a protein loading control.

Article Snippet: A2780 and OVCAR-3 human ovarian cancer cell lines were seeded into a 6-well plate with a growth medium and grown for 24 h. Caspase-3 activity assays (the Biovision colorimetric caspase-3 assay kit) were performed according to the manufacturer’s protocols.

Techniques: Binding Assay, Transfection, Western Blot, Expressing, LDH Cytotoxicity Assay

Journal: International Journal of Molecular Sciences

Article Title: 6-Shogaol Overcomes Gefitinib Resistance via ER Stress in Ovarian Cancer Cells

doi: 10.3390/ijms24032639

Figure Lengend Snippet: DPI and NAC suppresses cell death in 6-shogaol-treated ovarian cancer cells. ( A ) The fluorescence data indicate intracellular ROS production by DCFDA dye in 6-shogaol (0, 8, 16, and 24 h; 30 μM)-treated A2780 and OVCAR-3 cells. ( B – G ) A2780 and OVCAR-3 cells were pretreated with DPI (1 μM) and NAC (100 μM) for 4 h and subsequently treated with 6-shogaol (30 μM, 24 h). WST-1, LDH cytotoxicity assay, caspase-3 activity assay, ROS assay, intracellular Ca 2+ assay, and Western blotting analyses were performed; *, p < 0.05. n.s = not significant. Western blot showing p-PERK, PERK, ATF4, and CHOP levels was carried out using these samples. β-actin was used as the protein loading control.

Article Snippet: A2780 and OVCAR-3 human ovarian cancer cell lines were seeded into a 6-well plate with a growth medium and grown for 24 h. Caspase-3 activity assays (the Biovision colorimetric caspase-3 assay kit) were performed according to the manufacturer’s protocols.

Techniques: Fluorescence, LDH Cytotoxicity Assay, Caspase-3 Activity Assay, ROS Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: 6-Shogaol Overcomes Gefitinib Resistance via ER Stress in Ovarian Cancer Cells

doi: 10.3390/ijms24032639

Figure Lengend Snippet: Nox4 regulates cell death and ER stress response in 6-shogaol-treated ovarian cancer cells. ( A – F ) A2780 and OVCAR-3 cells were transfected with Nox4 siRNAs and treated with 6-shogaol (30 μM, 24 h). WST-1 assay, LDH cytotoxicity assay, caspase-3 activity assay, intracellular ROS assay, and intracellular Ca 2+ assay was performed along with Western blot analysis for Nox4, p-PERK, PERK, and CHOP; *, p < 0.05. n.s = not significant. β-actin was used as the protein loading control.

Article Snippet: A2780 and OVCAR-3 human ovarian cancer cell lines were seeded into a 6-well plate with a growth medium and grown for 24 h. Caspase-3 activity assays (the Biovision colorimetric caspase-3 assay kit) were performed according to the manufacturer’s protocols.

Techniques: Transfection, WST-1 Assay, LDH Cytotoxicity Assay, Caspase-3 Activity Assay, ROS Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: 6-Shogaol Overcomes Gefitinib Resistance via ER Stress in Ovarian Cancer Cells

doi: 10.3390/ijms24032639

Figure Lengend Snippet: PERK knockdown blocks gefitinib/6-shogaol-mediated sensitivity in gefitinib-resistant A2780R cells. ( A – F ) After A2780R cells were transfected with PERK shRNA, PERK knockdown A2780R stable cell lines were established. These cells were treated with 6-shogaol (30 μM, 24 h) and gefitinib (1 μM, 24 h), and cell viability assays, LDH assays, caspase-3 activity assays, intracellular ROS assay, intracellular Ca 2+ assays, and Western blot analysis were performed to examine p-PERK, PERK, CHOP, and cleaved caspase-3 expression; *, p < 0.05. n.s = not significant. β-actin was used as a protein loading control. ( G ) Schematic representation of ER stress and apoptotic cell death signaling pathways induced by gefitinib plus 6-shogaol in ovarian cancer and gefitinib-resistant ovarian cancer cells.

Article Snippet: A2780 and OVCAR-3 human ovarian cancer cell lines were seeded into a 6-well plate with a growth medium and grown for 24 h. Caspase-3 activity assays (the Biovision colorimetric caspase-3 assay kit) were performed according to the manufacturer’s protocols.

Techniques: Transfection, shRNA, Stable Transfection, Activity Assay, ROS Assay, Western Blot, Expressing