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: Journal of cachexia, sarcopenia and muscle

Article Title: Carnosol and its analogues attenuate muscle atrophy and fat lipolysis induced by cancer cachexia.

doi: 10.1002/jcsm.12710

Figure Lengend Snippet: Figure 2 Effects of CS, DCS, and DCSD on lipolysis of 3T3-L1 adipocyte in vitro. 3T3-L1 pre-adipocytes cells were differentiated into 3T3-L1 mature adipocyte for 6 days using differentiation medium. The lipolysis of 3T3-L1 mature adipocyte was induced by C26-tumour medium (1:1 dilution with fresh normal medium) and treated at the same time with different concentrations of CS, DCS, or DCSD, respectively for 48 h. (A) Effects of CS on the survival rate of 3T3-L1 mature adipocytes. (B) Oil Red O stained lipid droplets to detect the effect of CS on the lipolysis of 3T3-L1 mature adipocyte induced by C26-tumour medium. Scale bar, 50 μm. (C) Levels of free glycerol released in the 3T3-L1 mature adipocyte induced by C26-tumour medium with or without treatment of CS. (D) Concentrations of TG in the 3T3-L1 mature adipocyte induced by C26-tumour medium with or without treatment of CS. (E) Representative western blotting and quantification of phosphorylated HSL, total HSL, phosphorylated AMPK, total AMPK, phosphorylated p38 MAPK, total p38 MAPK, and UCP1 in the lipolysis of 3T3-L1 mature adipocyte induced by C26-tumour medium with or without treatment of CS. (F) Effects of DCS and DCSD on the survival rate of 3T3-L1 adipocytes. (G) Oil Red O stained lipid droplets to detect the effect of DCS or DCSD on the lipolysis of 3T3-L1 mature adipocyte induced by C26-tumour medium. Scale bar, 50 μm. (H) Levels of glycerol released in the 3T3-L1 mature adipocyte induced by C26-tumour medium with or without treatment of DCS or DCSD. (I) Concentration of TG in the 3T3-L1 mature adipocyte induced by C26-tumour medium with or without treatment of DCS or DCSD. (J, K) Representative western blotting and quantification of phosphorylated HSL, total HSL, phosphorylated AMPK, total AMPK, phosphorylated p38 MAPK, total p38 MAPK, and UCP1 in the lipolysis of 3T3-L1 mature adipocyte induced by C26-tumour medium with or without treatment of DCS or DCSD. Data presented are the mean ± SEM of three independent experiments. *vs. control group; #vs. C26-tumour medium. ***P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.01, ###P < 0.001.

Article Snippet: The primary antibodies used were as follows: mouse anti-MHCmonoclonal antibody (1:1000, DSHB, Iowa City, IA, United States), rabbit anti-MyoD polyclonal antibody (1:1000, Santa Cruz Biotechnology, Dallas, TX, United States), rabbit anti-AKT monoclonal antibody (1:1000, Cell Signaling Technology), rabbit anti-p-AKT monoclonal antibody (1:1000, Cell Signaling Technology), rabbit anti-NF-κB p65 monoclonal antibody (1:1000, Cell Signaling Technology), rabbit anti-p-NF-κB p65 monoclonal antibody (1:1000, Cell Signaling Technology), rabbit anti-MuRF-1 monoclonal antibody (1:1000, Abcam), rabbit anti-Atrogin-1 monoclonal antibody (1:1000, Abcam), rabbit anti-AMPKα monoclonal antibody (1:1000, Cell Signaling Technology), rabbit anti-p-AMPKα monoclonal antibody (1:1000, Cell Signaling Technology), rabbit anti-HSL polyclonal antibody (1:1000, Cell Signaling Technology), rabbit anti-p-HSL polyclonal antibody (1:1000, Cell Signaling Technology), rabbit anti-UCP1 monoclonal antibody (1:1000, Cell Signaling Technology), Actin-HRP (1: 5000, Santa Cruz Biotechnology), and GAPDH-HRP (1: 5000, Santa Cruz Biotechnology).

Techniques: In Vitro, Staining, Western Blot, Concentration Assay, Control

Journal: Journal of cachexia, sarcopenia and muscle

Article Title: Carnosol and its analogues attenuate muscle atrophy and fat lipolysis induced by cancer cachexia.

doi: 10.1002/jcsm.12710

Figure Lengend Snippet: Figure 8 Carnosol and its analogues attenuate cancer cachexia muscle atrophy and fat loss. CS, DCS, and DCSD inhibit the TNF-α production and inhibit the activation of NF-κB signalling in both muscle cells and adipocytes. In muscle cells, CS and its analogues inhibit ubiquitin-proteasome system (UPS) mediated by MURF-1, and Atrogin-1; activate the AKT signalling; promote the expression of MHC and MyoD; and thus ameliorate muscle atrophy. In adipocytes, CS and its analogues inhibit NF-κB signalling mediated inflammatory response and AMPK phosphorylation activation, decrease HSL phos- phorylation and expression of UCP1, and thus ameliorate fat loss.

Article Snippet: The primary antibodies used were as follows: mouse anti-MHCmonoclonal antibody (1:1000, DSHB, Iowa City, IA, United States), rabbit anti-MyoD polyclonal antibody (1:1000, Santa Cruz Biotechnology, Dallas, TX, United States), rabbit anti-AKT monoclonal antibody (1:1000, Cell Signaling Technology), rabbit anti-p-AKT monoclonal antibody (1:1000, Cell Signaling Technology), rabbit anti-NF-κB p65 monoclonal antibody (1:1000, Cell Signaling Technology), rabbit anti-p-NF-κB p65 monoclonal antibody (1:1000, Cell Signaling Technology), rabbit anti-MuRF-1 monoclonal antibody (1:1000, Abcam), rabbit anti-Atrogin-1 monoclonal antibody (1:1000, Abcam), rabbit anti-AMPKα monoclonal antibody (1:1000, Cell Signaling Technology), rabbit anti-p-AMPKα monoclonal antibody (1:1000, Cell Signaling Technology), rabbit anti-HSL polyclonal antibody (1:1000, Cell Signaling Technology), rabbit anti-p-HSL polyclonal antibody (1:1000, Cell Signaling Technology), rabbit anti-UCP1 monoclonal antibody (1:1000, Cell Signaling Technology), Actin-HRP (1: 5000, Santa Cruz Biotechnology), and GAPDH-HRP (1: 5000, Santa Cruz Biotechnology).

Techniques: Analogues, Activation Assay, Ubiquitin Proteomics, Expressing, Phospho-proteomics

Journal: Scientific Reports

Article Title: Adiponectin is an endogenous anti-fibrotic mediator and therapeutic target

doi: 10.1038/s41598-017-04162-1

Figure Lengend Snippet: Attenuated adiponectin signaling in SSc skin biopsies. ( a ) Left, immunofluorescence using antibodies to phospho-AMP-activated protein kinase (Thr172) (p-AMPK; green) and α-smooth muscle actin (αSMA, red); nuclei stained with DAPI (blue). Skin biopsies from SSc patients (n = 20) and age-matched healthy controls (n = 4) were examined. Dotted lines indicate the border between the epidermis and dermis. Representative photomicrographs; scale bar = 50 µm. Inset illustrating interstitial myofibroblasts within lesional dermis that are p-AMPK negative, scale bar = 10 µm. Right, quantification of p-AMPK-positive cells within the dermis. Results are shown as pAMPK + cells/total nuclei, and p-AMPK/α-SMA double-positive cells/αSMA + cells. Values are means ± SD from three high-power fields in each biopsy. ( b ) Unsupervised cluster analysis. Heatmap demonstrating altered expression of genes (432) significantly correlated with adiponectin mRNA levels (r > 0.4, p < 0.005) measured in 70 SSc (left) and 20 control (right) skin biopsies. Red (green) color indicates higher (lower) levels of gene expression. Note that 37/70 subjects map to a subset with distinctly differentially regulated expression pattern. (c) Adiponectin signaling scores (described in Methods) were measured in skin biopsy transcriptome dataset (GSE49332). In addition to the significant difference between SSc and controls (red bars, mean ± SD, p = 0.04), note bimodal distribution in SSc, with 41 patients (blue) demonstrating normal adiponectin pathway scores and 29 (green) showing markedly decreased pathway scores. (d) Adiponectin pathway activation scores are significantly correlated with p-AMPK expression.

Article Snippet: Four μm thick paraffin-embedded sections of lesional skin or peritoneal membrane were incubated with rabbit anti-phospho-AMPK (Thr172) (1:100, Cell Signaling Technology, Beverly, MA), mouse anti-α-SMA (1:250, Dako, Carpinteria, CA), rabbit anti-α-SMA (1:200, Abcam, Cambridge, UK), rat anti-procollagen I (1:200, EMD Millipore, Billerica, MA), goat anti-type III collagen (1:200, Southern Biotech, Birmingham, AL), rabbit anti-Ki67 (1:500, Abcam) or rabbit anti-phospho FAK (Y397; 1:50, Abcam) primary antibodies, followed by species-appropriate secondary antibodies conjugated to Alexa Fluor 488, 594 or 647 (Invitrogen, Carlsbad, CA).

Techniques: Immunofluorescence, Staining, Expressing, Control, Gene Expression, Activation Assay

Journal: Scientific Reports

Article Title: Adiponectin is an endogenous anti-fibrotic mediator and therapeutic target

doi: 10.1038/s41598-017-04162-1

Figure Lengend Snippet: The anti-fibrotic effects of ADP355 are mediated by adiponectin receptor signaling and AMP protein kinase activation. Confluent cultures of human ( a,b ) or mouse ( c ) fibroblasts were incubated in media with ADP355 for 24 h ( a,c ) or 60 min ( b ). ( a ) Fibroblasts were transfected with AdipoR1/R2 siRNA (siR1/2), or scrambled control siRNA for 24 h prior to ADP355 and TGF-β. Results of qPCR shown as means ± SD of triplicate determinations. *p < 0.05, **p < 0.001. (b) Fibroblasts were immunostained with antibodies to phospho-AMP kinase (Thr172) (p-AMPK, green), or stained with DAPI (blue), and examined by confocal microscopy. Representative immunofluorescence photomicrographs. Original magnification x400. (c) Total RNA was examined by real-time qPCR. Results are the mean ± SD of triplicate determinations. *p < 0.05.

Article Snippet: Four μm thick paraffin-embedded sections of lesional skin or peritoneal membrane were incubated with rabbit anti-phospho-AMPK (Thr172) (1:100, Cell Signaling Technology, Beverly, MA), mouse anti-α-SMA (1:250, Dako, Carpinteria, CA), rabbit anti-α-SMA (1:200, Abcam, Cambridge, UK), rat anti-procollagen I (1:200, EMD Millipore, Billerica, MA), goat anti-type III collagen (1:200, Southern Biotech, Birmingham, AL), rabbit anti-Ki67 (1:500, Abcam) or rabbit anti-phospho FAK (Y397; 1:50, Abcam) primary antibodies, followed by species-appropriate secondary antibodies conjugated to Alexa Fluor 488, 594 or 647 (Invitrogen, Carlsbad, CA).

Techniques: Activation Assay, Incubation, Transfection, Control, Staining, Confocal Microscopy, Immunofluorescence