Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Targeting USP9X-AMPK Axis in ARID1A-Deficient Hepatocellular Carcinoma.

doi: 10.1016/j.jcmgh.2022.03.009

Figure Lengend Snippet: Figure 4. ARID1A deletion renders HCC cells resistant to glucose deprivation via activation of the AMPK pathway. The effect of ARID1A knockout on Huh7 and YY-8103 cells upon glucose starvation is investigated by (A) Annexin V–fluorescein isothiocyanate (FITC)/PI apoptosis kit, and (B) the result of quantitative analysis is shown. (C) The expression of AMPK signaling proteins in liver tissues from control and Arid1a liver-specific knockout mice. (D) The expression of the indicated proteins in AMPK signaling in primary hepatocytes from control and Arid1a liver-specific KO mice. The expression of (E) PRKAA2 in control, ARID1A knockout YY-8103, Huh7 cells and (F) ARID1A-overexpressing PVTT and SNU-398 cells. The mRNA level of (G) Prkaa1 and (H) Prkaa2 in liver tissues from control and Arid1a liver-specific knockout mice. CTRL, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. *P<0.05, ***P<0.001, ns, not significant.

Article Snippet: Antibodies against acetyl–histone H3 (Lys9) (9649), acetyl–histone H3 (Lys9) (8173), ULK1 (8054), phospho-ULK1 (Ser317) (12753), phospho-ULK1 (Ser555) (5869), phospho-ULK1 (Ser757) (6888), acetyl-CoA carboxylase (3676), phospho-acetyl-CoA carboxylase (Ser79) (11818), AMPKa (2532), phosphoAMPKa (Thr172) (2535), LC3B (3868), and HDAC1 (34589) were purchased from Cell Signaling Technology (Danvers, MA); antibodies against PRKAA1 (10929), PRKAA2 (18167), USP9X (55054), glyceraldehyde-3-phosphate dehydrogenase (10494), and liver kinase B1 (10746) were purchased from Proteintech (Rosemont, IL); antibodies against ARID1A (sc-32761), a-tubulin (sc-32293), b-actin (sc-47778), ubiquitin (sc-8017), and c-myc (sc-764) were purchased from Santa Cruz Biotechnology (Dallas, Texas); and antibodies against Flag and HA were purchased from Sigma-Aldrich (St. Louis, MO).

Techniques: Activation Assay, Knock-Out, Expressing, Control

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Targeting USP9X-AMPK Axis in ARID1A-Deficient Hepatocellular Carcinoma.

doi: 10.1016/j.jcmgh.2022.03.009

Figure Lengend Snippet: Figure 6. ARID1A regulates the ubiquitination of PRKAA2 through USP9X. The influence of ARID1A on the ubiquitination of PRKAA2 in (A) HEK293T, (B and C) SNU-398, PVTT, and (D) Huh7 cells. (E) The mRNA level of proteins involved in PRKAA2 ubiquitination or deubiquitination in liver tissues from control and Arid1a liver-specific knockout mice. (F) The mRNA level of USP9X in control and ARID1A KO Huh7 (left) and YY-8103 (right) cells is examined by real-time PCR. (G) Usp9x expression in liver tissues from control and Arid1a liver-specific knockout mice is examined by Western blot. (H) USP9X expression in control and ARID1A KO Huh7 and YY-8103 cell is examined by Western blot. (I) USP9X expression in control and ARID1A- overexpressing SNU-398 cells is examined by Western blot. (J) USP9X and PRKAA2 expressions in control and ARID1A KO MHCC97H cells are examined by Western blot. CTRL, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. *P<0.05,**P<0.01,***P<0.001, ns, not significant.

Article Snippet: Antibodies against acetyl–histone H3 (Lys9) (9649), acetyl–histone H3 (Lys9) (8173), ULK1 (8054), phospho-ULK1 (Ser317) (12753), phospho-ULK1 (Ser555) (5869), phospho-ULK1 (Ser757) (6888), acetyl-CoA carboxylase (3676), phospho-acetyl-CoA carboxylase (Ser79) (11818), AMPKa (2532), phosphoAMPKa (Thr172) (2535), LC3B (3868), and HDAC1 (34589) were purchased from Cell Signaling Technology (Danvers, MA); antibodies against PRKAA1 (10929), PRKAA2 (18167), USP9X (55054), glyceraldehyde-3-phosphate dehydrogenase (10494), and liver kinase B1 (10746) were purchased from Proteintech (Rosemont, IL); antibodies against ARID1A (sc-32761), a-tubulin (sc-32293), b-actin (sc-47778), ubiquitin (sc-8017), and c-myc (sc-764) were purchased from Santa Cruz Biotechnology (Dallas, Texas); and antibodies against Flag and HA were purchased from Sigma-Aldrich (St. Louis, MO).

Techniques: Ubiquitin Proteomics, Control, Knock-Out, Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Targeting USP9X-AMPK Axis in ARID1A-Deficient Hepatocellular Carcinoma.

doi: 10.1016/j.jcmgh.2022.03.009

Figure Lengend Snippet: Figure 9. ARID1A regulates the promoter activity of USP9X via HDAC1. (A) Data from the Catalogue of Somatic Mutations in Cancer shows that 1989* is the most frequent mutation of ARID1A. (B) Interaction between ARID1A-WT or ARID1A-1989* mutation with HDAC1. (C) Influence of ARID1A-WT or ARID1A-1989* mutation on the ubiquitination of PRKAA2. (D) Influence of ARID1A-WT or ARID1A-1989* mutation on the promoter activity of USP9X. The promoter activity of USP9X in (E) HEK293T cells overexpressing ARID1A or HDAC1 (OE) or in (F) ARID1A knockout Huh7 and YY-8103 cells is examined by luciferase reporter assay. (G) Influence of ARID1A-WT or ARID1A-1989* mutation on the expression of USP9X and PRKAA2. CTRL, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. **P<0.01, ***P<0.001, ns, not significant.

Article Snippet: Antibodies against acetyl–histone H3 (Lys9) (9649), acetyl–histone H3 (Lys9) (8173), ULK1 (8054), phospho-ULK1 (Ser317) (12753), phospho-ULK1 (Ser555) (5869), phospho-ULK1 (Ser757) (6888), acetyl-CoA carboxylase (3676), phospho-acetyl-CoA carboxylase (Ser79) (11818), AMPKa (2532), phosphoAMPKa (Thr172) (2535), LC3B (3868), and HDAC1 (34589) were purchased from Cell Signaling Technology (Danvers, MA); antibodies against PRKAA1 (10929), PRKAA2 (18167), USP9X (55054), glyceraldehyde-3-phosphate dehydrogenase (10494), and liver kinase B1 (10746) were purchased from Proteintech (Rosemont, IL); antibodies against ARID1A (sc-32761), a-tubulin (sc-32293), b-actin (sc-47778), ubiquitin (sc-8017), and c-myc (sc-764) were purchased from Santa Cruz Biotechnology (Dallas, Texas); and antibodies against Flag and HA were purchased from Sigma-Aldrich (St. Louis, MO).

Techniques: Activity Assay, Mutagenesis, Ubiquitin Proteomics, Knock-Out, Luciferase, Reporter Assay, Expressing, Control

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Targeting USP9X-AMPK Axis in ARID1A-Deficient Hepatocellular Carcinoma.

doi: 10.1016/j.jcmgh.2022.03.009

Figure Lengend Snippet: Figure 11. ARID1A negatively correlates with USP9X/PRKAA2 and influences HCC patients’ survival. The correlation among ARID1A, USP9X, and PRKAA2 in the clinical samples is examined by (A) Western blot or by (B) immunohistochemical staining in the Human Protein Atlas (HPA) database. (C) Immunohistochemistry staining of ARID1A, USP9X, and PRKAA2 in HCC tissues in TMAs. Scale bar: 100 mm. (D) The correlation between USP9X and PRKAA2 at the protein level (N ¼ 243) is analyzed using H-scores from TMA analysis. (E) The correlation between ARID1A and USP9X at the protein level (N ¼ 243) is analyzed using H-scores from TMA analysis. (F) Comparison of overall survival between HCC patients with different ARID1A/ USP9X expressions. Data are analyzed using the log-rank test. (G) The correlation between ARID1A and PRKAA2 at the protein level (N ¼ 243) is analyzed using H-scores from TMA analysis. (H) Comparison of overall survival between HCC pa- tients with different ARID1A/PRKAA2 expressions. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Antibodies against acetyl–histone H3 (Lys9) (9649), acetyl–histone H3 (Lys9) (8173), ULK1 (8054), phospho-ULK1 (Ser317) (12753), phospho-ULK1 (Ser555) (5869), phospho-ULK1 (Ser757) (6888), acetyl-CoA carboxylase (3676), phospho-acetyl-CoA carboxylase (Ser79) (11818), AMPKa (2532), phosphoAMPKa (Thr172) (2535), LC3B (3868), and HDAC1 (34589) were purchased from Cell Signaling Technology (Danvers, MA); antibodies against PRKAA1 (10929), PRKAA2 (18167), USP9X (55054), glyceraldehyde-3-phosphate dehydrogenase (10494), and liver kinase B1 (10746) were purchased from Proteintech (Rosemont, IL); antibodies against ARID1A (sc-32761), a-tubulin (sc-32293), b-actin (sc-47778), ubiquitin (sc-8017), and c-myc (sc-764) were purchased from Santa Cruz Biotechnology (Dallas, Texas); and antibodies against Flag and HA were purchased from Sigma-Aldrich (St. Louis, MO).

Techniques: Western Blot, Immunohistochemical staining, Staining, Immunohistochemistry, Comparison

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Targeting USP9X-AMPK Axis in ARID1A-Deficient Hepatocellular Carcinoma.

doi: 10.1016/j.jcmgh.2022.03.009

Figure Lengend Snippet: Figure 14. The effects of inactivation of PRKAA2 and USP9X on HCC cell growth. (A) The growth of control (Scramble, SCR) and PRKAA2 knockdown (sh1#, sh2#) cells is measured by crystal violet staining under both normal and glucose-deprived conditions. The influences of (B, D, E) Compound C and (C, F, G) WP1130 on HCC cell growth is measured by crystal violet staining or cell counting kit-8 (CCK8) assay under both normal and glucose- deprived conditions.

Article Snippet: Antibodies against acetyl–histone H3 (Lys9) (9649), acetyl–histone H3 (Lys9) (8173), ULK1 (8054), phospho-ULK1 (Ser317) (12753), phospho-ULK1 (Ser555) (5869), phospho-ULK1 (Ser757) (6888), acetyl-CoA carboxylase (3676), phospho-acetyl-CoA carboxylase (Ser79) (11818), AMPKa (2532), phosphoAMPKa (Thr172) (2535), LC3B (3868), and HDAC1 (34589) were purchased from Cell Signaling Technology (Danvers, MA); antibodies against PRKAA1 (10929), PRKAA2 (18167), USP9X (55054), glyceraldehyde-3-phosphate dehydrogenase (10494), and liver kinase B1 (10746) were purchased from Proteintech (Rosemont, IL); antibodies against ARID1A (sc-32761), a-tubulin (sc-32293), b-actin (sc-47778), ubiquitin (sc-8017), and c-myc (sc-764) were purchased from Santa Cruz Biotechnology (Dallas, Texas); and antibodies against Flag and HA were purchased from Sigma-Aldrich (St. Louis, MO).

Techniques: Control, Knockdown, Staining, Cell Counting, CCK-8 Assay

Journal: iScience

Article Title: Roseburia hominis enriched by baicalin reverses the non-response to metformin via upregulating linolenic acid metabolism

doi: 10.1016/j.isci.2025.113892

Figure Lengend Snippet: Baicalin combined with metformin improved the blood glucose levels in metformin non-responsive mice by activating the AMPK/ACC/CPT1 pathway (A–F) The serum concentrations of (A) TC, (B) LDL-C, (C) HDL-C, (D) IL-1β, (E) IL-6, and (F) IL-10. (G) Hematoxylin-eosin (H&E) staining of the liver, pancreas, epididymal adipose tissues, and oil red O staining of the liver (magnification, 30×; scale bars, 100 μm). (H) Western blot analysis of pAMPK (Thr172), ACC, and CPT1 proteins in the livers of mice after administration. Data are expressed as the mean ± SD (A‒F, n = 6–8; H, n = 3); ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, as determined by one-way ANOVA with Holm-Sidak’s post hoc test (A, B, C, E, F, and H) and Kruskal-Wallis test (D). NCD, normal chow diet; NR, non-response; Met, metformin; BA, baicalin; TC, total cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; IL-1β, interleukin-1β; IL-6, interleukin-6; IL-10, interleukin-10; AMPK, AMP-activated protein kinase; ACC, acetyl-CoA carboxylase; CPT1, carnitine palmitoyl transferase 1.

Article Snippet: These membranes were then incubated at 4°C overnight with primary antibodies against AMP-activated protein kinase (AMPK) (Proteintech, Cat# 10929-2-AP, RRID: AB_2169568 , 1:4,000), insulin receptor substrate 1 (IRS1) (Proteintech, Cat# 17509-1-AP, RRID: AB_10596914 , 1:1,000), phospho-AMPKα (Thr172) (40H9) (Cell Signaling Technology, Cat# 2535, RRID: AB_331250 , 1:1,000), phospho-IRS-1 (Ser636/639) (Cell Signaling Technology, Cat# 2388, RRID: AB_330339 , 1:1,000), phosphoenolpyruvate carboxykinase (PCK) (Proteintech, Cat# 16754-1-AP, RRID: AB_2160031 , 1:30,000), glucose-6-phosphatase (G6PC) (Proteintech, Cat# 66860-1-Ig, RRID: AB_2882199 , 1:5,000), mammalian target of rapamycin (mTOR) (Proteintech, Cat# 66888-1-Ig, RRID: AB_2882219 , 1:30,000), fatty acid desaturase 2 (FADS2) (Proteintech, Cat# 28034-1-AP, RRID: AB_2918142 , 1:4,000), phospholipase A2 group (PLA2G) (Proteintech, Cat# 18088-1-AP, RRID: AB_10859777 , 1:1,000), acetyl-CoA Carboxylase 1 (ACC1) (Proteintech, Cat# 21923-1-AP, RRID: AB_11042445 , 1:4,000), carnitine palmitoyl transferase 1 (CPT1) (Proteintech, Cat# 15184-1-AP, RRID: AB_2084676 , 1:50,000), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Proteintech, Cat# 10494-1-AP, RRID: AB_2263076 , 1:20,000), β-tubulin (Proteintech, Cat# 10094-1-AP, RRID: AB_2210695 , 1:10,000).

Techniques: Staining, Western Blot

Journal: iScience

Article Title: Roseburia hominis enriched by baicalin reverses the non-response to metformin via upregulating linolenic acid metabolism

doi: 10.1016/j.isci.2025.113892

Figure Lengend Snippet: R. hominis treatment reversed the metformin NR phenotype in NR mice (A) Experimental protocol for administration of R. hominis in mice. (B) Oral glucose tolerance test (OGTT) curve and its area under the curve (AUC). (C) Insulin tolerance test (ITT) curve and its AUC. (D) Homeostasis model assessment of insulin resistance (HOMA-IR) after drug R. hominis . (E–J) The serum concentrations of (E) TC, (F) LDL-C, (G) HDL-C, (H) IL-1β, (I) IL-6, and (J) IL-10. (K) Hematoxylin-eosin (H&E) staining of the liver, pancreas, epididymal adipose tissues, and oil red O staining of the liver (magnification, 30×; scale bars, 100 μm). (L) Western blot analysis of AMPK/ACC/CPT1 proteins in the livers of mice after administration. Data are expressed as the mean ± SD ( n = 7–8); ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, as determined by one-way ANOVA with Holm-Sidak’s post hoc test. ANOVA, analysis of variance; ABX, antibiotic mixed; FMT, fecal microbial transplantation; NCD, normal chow diet; R.h, Roseburia hominis ; TC, total cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; IL-1β, interleukin-1β; IL-6, interleukin-6; IL-10, interleukin-10; AMPK, AMP-activated protein kinase; ACC, acetyl-CoA carboxylase; CPT1, carnitine palmitoyl transferase 1.

Article Snippet: These membranes were then incubated at 4°C overnight with primary antibodies against AMP-activated protein kinase (AMPK) (Proteintech, Cat# 10929-2-AP, RRID: AB_2169568 , 1:4,000), insulin receptor substrate 1 (IRS1) (Proteintech, Cat# 17509-1-AP, RRID: AB_10596914 , 1:1,000), phospho-AMPKα (Thr172) (40H9) (Cell Signaling Technology, Cat# 2535, RRID: AB_331250 , 1:1,000), phospho-IRS-1 (Ser636/639) (Cell Signaling Technology, Cat# 2388, RRID: AB_330339 , 1:1,000), phosphoenolpyruvate carboxykinase (PCK) (Proteintech, Cat# 16754-1-AP, RRID: AB_2160031 , 1:30,000), glucose-6-phosphatase (G6PC) (Proteintech, Cat# 66860-1-Ig, RRID: AB_2882199 , 1:5,000), mammalian target of rapamycin (mTOR) (Proteintech, Cat# 66888-1-Ig, RRID: AB_2882219 , 1:30,000), fatty acid desaturase 2 (FADS2) (Proteintech, Cat# 28034-1-AP, RRID: AB_2918142 , 1:4,000), phospholipase A2 group (PLA2G) (Proteintech, Cat# 18088-1-AP, RRID: AB_10859777 , 1:1,000), acetyl-CoA Carboxylase 1 (ACC1) (Proteintech, Cat# 21923-1-AP, RRID: AB_11042445 , 1:4,000), carnitine palmitoyl transferase 1 (CPT1) (Proteintech, Cat# 15184-1-AP, RRID: AB_2084676 , 1:50,000), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Proteintech, Cat# 10494-1-AP, RRID: AB_2263076 , 1:20,000), β-tubulin (Proteintech, Cat# 10094-1-AP, RRID: AB_2210695 , 1:10,000).

Techniques: Staining, Western Blot, Transplantation Assay

Journal: Cell death discovery

Article Title: Targeting oncogenic MAGEA6 sensitizes triple negative breast cancer to doxorubicin through its autophagy and ferroptosis by stabling AMPKα1.

doi: 10.1038/s41420-024-02196-9

Figure Lengend Snippet: Fig. 4 Inhibition of AMPK by MAGEA6 impacted the autophagy of TNBC. A, B In vitro ubiquitination assay displayed that AMPKα1 ubiquitination requires MAGEA6. C Co-immunoprecipitation assay stated the association between MAGEA6 and AMPKα1. D Western blotting showed that knockdown of MAGEA6 increased the levels of AMPKα1 and p-AMPKα (Thr-172). E Whether sh-MAGEA6 influenced autophagy via activating AMPK signaling was verified by Western blotting assay. *P < 0.05, **P < 0.01, ***P < 0.001; Data were obtained from three independent experiments. TNBC triple-negative breast cancer.

Article Snippet: The membranes were blocked with 5% skimmed milk, followed by incubating overnight with antibodies against MAGEA6 (PA575647, 1:500, Invitrogen, Carlsbad, CA, USA), LC3 (14600-1-AP, 1:3000), Cleaved caspase 3 (25128-1-AP, 1:1500), Cleaved PARP (13371-1-AP, 1:5000), AMPKα1 (66536-1-Ig, 1:3000), SLC7A11 (26864-1-AP, 1:1500) (Proteintech, Wuhan, China); and p-AMPKα (Thr-172) (2531, 1:1000, CST, Boston, MA, USA).

Techniques: Inhibition, In Vitro, Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Western Blot, Knockdown

Journal: eLife

Article Title: Obesity-linked suppression of membrane-bound O -acyltransferase 7 (MBOAT7) drives non-alcoholic fatty liver disease

doi: 10.7554/eLife.49882

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-GPAT4 (Rabbit Polyclonal) , Novus BIologicals , Cat#: NB100-2390; RRID: AB_2273811 , 1:1000.

Techniques: Sequencing, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: The Journal of international medical research

Article Title: The therapeutic potential of irisin in alleviating acute lung injury via inflammation and ferroptosis modulation.

doi: 10.1177/03000605251340338

Figure Lengend Snippet: Figure 6. Western blotting analysis on the effects of irisin on the expression level of ferroptosis-associated proteins. (a) Representative western blotting images for the expression of ACSL4, COX-2, GPX4, p-AMPK, and t-AMPK in lung tissues. GAPDH was selected as the loading control protein. (b) Quantification analysis of the related bands of ACSL4, COX-2, GPX4, p-AMPK, and t-AMPK in lung tissues. Lung tissues were harvested on day 3 post-CLP. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. n ¼ 6 per group. Data are presented as means SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Subsequently, the membranes were incubated with rabbit anti-AMPK (MCE, Cat# HY-P80541), anti-p-AMPK (MCE, HYP80452), GPX4 (MCE, HY-P80450), ACSL4 (Santa Cruz Biotechnology, Dallas, Texas, USA; Cat# sc-365230), COX-2 (Abcam, Cambridge, United Kingdom; Cat# ab283574), and GAPDH (MCE, HY-P80137) antibodies at 4 C overnight.

Techniques: Western Blot, Expressing, Control