Journal: Redox Biology

Article Title: Adiponectin/AdiopR1 signaling prevents mitochondrial dysfunction and oxidative injury after traumatic brain injury in a SIRT3 dependent manner

doi: 10.1016/j.redox.2022.102390

Figure Lengend Snippet: Transcription and expressions of SIRT3 were regulated by AdipoR1/AMPK/PGC-1α signaling pathway in TBI models. (A–D) Western blot and statistical analysis of the phosphorylation of AMPK and expressions of SIRT3 and PGC-1α. (E) The transcription results of SIRT3 in AdipoR1 flox/flox and AdipoR1 CKO mice. **p < 0.01 and ***p < 0.001 vs sham group in each strain of mice, # p < 0.05 vs TBI group in each strain of mice, & p < 0.05, && p < 0.01, ns: no statistical significance. (F–I) Effects of AMPK phosphorylation inhibitor and AdipoRon treatment on AMPK phosphorylation and expressions of SIRT3 and PGC-1α after TBI. (J) Mechanism involved in protective effects of activated APN/AdipoR1 signaling after TBI. Data are presented as mean ± SD for n = 6. **p < 0.01 vs sham group, # p < 0.05 vs TBI group, $ p < 0.05 vs TBI + AdipoRon group.

Article Snippet: The membranes were immersed in 5% non-fat milk for 2 h and incubated at 4 °C overnight in the presence of primary rabbit polyclonal antibodies against Bcl-2 (12,789, Proteintech), Bax (50,599, Proteintech), AMPK (66,536, Proteintech), p-AMPK (2537, CST), PGC-1α (66,369, Proteintech), SIRT3 (10,099, Proteintech), PRDX3 (ab264354, Abcam), acetylated-Lysine (9441, CST), VDAC (55,259, Proteintech), and β-actin (AC026, ABclonal).

Techniques: Western Blot, Phospho-proteomics

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