Journal: Redox Biology

Article Title: Hepatic Regulator of G Protein Signaling 6 (RGS6) drives non-alcoholic fatty liver disease by promoting oxidative stress and ATM-dependent cell death

doi: 10.1016/j.redox.2021.102105

Figure Lengend Snippet: RGS6 KD in liver protects against hepatosteatosis and fibrosis in HFD-fed mice. Control and HFD (54.8% total fat content)-fed mice were administered scramble or RGS6-targeted shRNA via tail vein injection prior to initiation of the HFD (n = 6). Animals were sacrificed after 12 weeks of HFD feeding. (A) Immunoblotting for RGS6 in liver [L] and heart [H]. (B) Hepatic RGS6 expression measured via immunohistochemistry [scale bar = 100 μm]. (C) Immunblotting for RGS6, pATM and ATM with densitometric quantification. (D) Histological characterization of livers. Liver architecture (H & E), fibrosis (Masson Trichrome, Sirius Red), inflammation (F4/80), regeneration (PCNA) and lipid accumulation (Oil Red O) are depicted [scale bar = 100 μm]. (E) F4/80 and PCNA positive cells per microscope field (n = 10). (F) Hepatic collagen and hydroxyproline content (n = 6/group). (G) Liver enzyme (ALT & AST) and triglyceride levels. (H) Immunoblotting for markers of insulin sensitivity with corresponding densitometric quantification. (I) Liver MDA (n = 7). (J) Mitochondrial ROS (Mitosox) generation (n = 7). (K) Total and mitochondrial fatty acid oxidation (FAO) rate in liver (n = 7). (L) Liver metabolism as indicated by NADH/NAD + and Liver Lactate/Liver pyruvate (n = 7). β-Actin is used as a loading control for all immunoblots. Data were analyzed by two-way ANOVA with Sidak's post-hoc test. * P < 0.05, ** P < 0.01,*** P < 0.001, **** P < 0.0001. Data are presented as mean ± SEM. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Post isolation, cells were transfected with RGS6-targeted or scramble shRNA (Santa Cruz Biotechnology) and treated with palmitic acid (PA, 400 μM, 24 h) where indicated.

Techniques: Control, shRNA, Injection, Western Blot, Expressing, Immunohistochemistry, Microscopy

Journal: Redox Biology

Article Title: Hepatic Regulator of G Protein Signaling 6 (RGS6) drives non-alcoholic fatty liver disease by promoting oxidative stress and ATM-dependent cell death

doi: 10.1016/j.redox.2021.102105

Figure Lengend Snippet: RGS6, whose up-regulation is ROS driven, promotes mitochondrial dysfunction and cell death in hepatocytes. (A) Immunoblotting for RGS6 in human hepatocytes, murine hepatocytes, and HepaRG cells treated with PA (400 μM, 24 h). Densitometric quantification is provided (n = 3). (B) HepaRG cells were treated with PA (400 μM, 24 h) and/or H 2 O 2 (200 mM, 24 h) and immunoblotting performed for the detection of RGS6. Representative immunoblots and quantification are provided (n = 3). (C) HepaRG cells were treated with PA (400 μM, 24 h) and/or increasing concentrations of the H2O2 scavenger Peg-Cat (up to 200 U/mL, 1 h pre-treatment). Immunoblotting was performed for the detection of RGS6. Representative immunoblots and quantification are provided (n = 3). HepaRG cells were treated with increasing concentrations of PA (24 h) or a fixed drug dose (400 μM) 24 h following introduction of scramble or RGS6-targeted shRNA. The RGS6L isoform was reintroduced into a subset of RGS6 KD cells. (D) and (E) CM-H 2 DCFDA fluorescence (total ROS; n = 6–7). (F) Mitosox fluorescence (mitochondrial ROS; n = 7). (G) Mitochondrial membrane potential (n = 7) with and without the introduction of mitochondrial permeability transition pore blocker cyclosporin A (0.2 μM, 1 h pre-treatment) or mitochondrial calcium uniporter inhibitor Ru360 (50 μM, 1 h pre-treatment). (H) Mitochondrial calcium (Ca 2+ ) with and without Ru360 (50 μM, 1 h pre-treatment) (n = 7). (I) Cell death (TUNEL positive cells; n = 9). Data were analyzed by one- or two-way ANOVA with Sidak's post-hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns = not significant. Data are presented as mean ± SEM.

Article Snippet: Post isolation, cells were transfected with RGS6-targeted or scramble shRNA (Santa Cruz Biotechnology) and treated with palmitic acid (PA, 400 μM, 24 h) where indicated.

Techniques: Western Blot, shRNA, Fluorescence, Membrane, Permeability, TUNEL Assay

Journal: Redox Biology

Article Title: Hepatic Regulator of G Protein Signaling 6 (RGS6) drives non-alcoholic fatty liver disease by promoting oxidative stress and ATM-dependent cell death

doi: 10.1016/j.redox.2021.102105

Figure Lengend Snippet: RGS6, ATM and ROS function in the same pathway to promote hyperlipidemia-dependent hepatotoxicity. Scramble or RGS6-targeted shRNA was introduced into human hepatocytes (A, B) or HepaRG cells (A, C–F) 24 h prior to treatment with PA (400 μM, 24 h), NAC (5 mM, 1 h pre-treatment) and/or the ATM inhibitor KU55933 (ATMi; 5 μM, 1 h pre-treatment) where indicated. In a subset of experiments RGS6 deletion constructs were introduced via transfection prior to drug treatments. (A) Immunoblotting for pATM and ATM. Representative westerns and a densitometric quantification are provided (n = 3). β-Actin is used as a loading control for all immunoblots. (B) Cell death (cytoplasmic histone-associated DNA fragments; n = 5) ± RGS6 KD, ATMi, and/or NAC in human hepatocytes. (C) CM-H 2 DCFDA fluorescence (n = 6) and (D) cell death (cytoplasmic histone-associated DNA fragments; n = 6) ± RGS6 KD and/or ATMi. (E) CM-H 2 DCFDA fluorescence (n = 5); (F) cell death (cytoplasmic histone-associated DNA fragments; n = 6); and albumin production (n = 5) ± RGS6 KD and/or RGS6 deletion constructs. Data were analyzed by one- or two-way ANOVA with Sidak's post-hoc test. * P < 0.05, ** P < 0.01, **** P < 0.0001. ns = not significant. Data are presented as mean ± SEM.

Article Snippet: Post isolation, cells were transfected with RGS6-targeted or scramble shRNA (Santa Cruz Biotechnology) and treated with palmitic acid (PA, 400 μM, 24 h) where indicated.

Techniques: shRNA, Construct, Transfection, Western Blot, Control, Fluorescence

Journal: Redox Biology

Article Title: Hepatic Regulator of G Protein Signaling 6 (RGS6) drives non-alcoholic fatty liver disease by promoting oxidative stress and ATM-dependent cell death

doi: 10.1016/j.redox.2021.102105

Figure Lengend Snippet: RGS6 and ATM form a co-precipitable complex in hepatocytes. (A) Reciprocal ATM and RGS6 co-immunoprecipitation in the human hepatocyte cell line HepaRG. (B) Immunoprecipitation of ATM with RGS6 deletion constructs in HepaRG cells. Densitometric quantification was performed, and the relative quantity of ATM protein pulled down with each RGS6 construct is indicated. (C) In silico modeling of the putative ATM-RGS6 complex revealed key RGS6 residues predicted to support a direct interaction between RGS6 and ATM (D348, D371, K374, D379, and R419 in green). Red = ATM Protein; Blue = RGS6; Surface is shown for ATM-interacting amino acids (AA) of RGS6. (D) Co-immunoprecipitation of ATM with RGS6 point mutants in HepaRG cells. Densitometric quantification was performed, and the relative quantity of ATM protein pulled down with each RGS6 construct is indicated. Data are representative of at least 3 independent experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: RGS6 gene silencing via small hairpin RNA (shRNA) delivery in vivo – shRNA against RGS6 was purchased from Santacruz Biotechnology (Paso Robles, CA, USA).

Techniques: Immunoprecipitation, Construct, In Silico

Journal: Redox Biology

Article Title: Hepatic Regulator of G Protein Signaling 6 (RGS6) drives non-alcoholic fatty liver disease by promoting oxidative stress and ATM-dependent cell death

doi: 10.1016/j.redox.2021.102105

Figure Lengend Snippet: ATM and RGS6 are up-regulated in NAFLD. (A) Histological characterization of NAFLD human liver samples. Detectable disruption of liver architecture (H & E), fibrosis (Masson Trichrome), inflammation (F4/80), regeneration (PCNA) and cell death (TUNEL) [scale bar = 100 μm] was observed in livers from NAFLD patients. F4/80, PCNA, and TUNEL positive cells are quantified (n = 10). (B) Immunohistochemical staining for RGS6 and ATM in liver of control and NAFLD patient liver samples (n = 10). Representative images and quantification across samples are shown. (C) NAFLD samples were stratified based on RGS6 expression (low, n = 7; medium, n-10; high, n = 9) and immunoblotting performed for RGS6, ATM, and γH2AX. (D) Densitometric quantification was performed, and protein content expressed relative to RGS6 low samples. β-Actin is used as a loading control for all immunoblots.(E) Correlation between ATM and RGS6 expression across 48 liver samples detected via immunoblotting. A simple linear regression was used to determine the goodness of fit (r 2 ) and degree to which the slope of the line deviates from zero ( P ). Data were analyzed by student's t-test or one-way ANOVA with Sidak's post-hoc test. * P < 0.05, *** P < 0.001, **** P < 0.0001. ns = not significant. Data are presented as mean ± SEM.

Article Snippet: RGS6 gene silencing via small hairpin RNA (shRNA) delivery in vivo – shRNA against RGS6 was purchased from Santacruz Biotechnology (Paso Robles, CA, USA).

Techniques: Disruption, TUNEL Assay, Immunohistochemical staining, Staining, Control, Expressing, Western Blot

Journal: Redox Biology

Article Title: Hepatic Regulator of G Protein Signaling 6 (RGS6) drives non-alcoholic fatty liver disease by promoting oxidative stress and ATM-dependent cell death

doi: 10.1016/j.redox.2021.102105

Figure Lengend Snippet: RGS6 levels are high in the livers of NAFLD patients with co-morbid DM, marked insulin resistance, and a pronounced inflammatory burden. (A) Expression of RGS6, pATM/ATM, and γH2AX in livers from control, NAFLD and NAFLD with co-morbid DM patients (n = 4–10). Representative immunoblots and densitometric quantification are included. (B) Immunoblotting (representative image and quantification) for RGS6, ATM, and γH2AX in NAFLD patient samples stratified based on HOMA-IR score (n = 13). (C) Representative images of RGS6 and CD68 staining in livers [scale bar = 100 μm] from NAFLD samples at each steatosis grade with corresponding correlation (n = 32). A simple linear regression was used to determine the goodness of fit (r 2 ) and degree to which the slope of the line deviates from zero ( P ). β-Actin is used as a loading control for all immunoblots. Data were analyzed by student's t-test or one-way ANOVA with Sidak's post-hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns = not significant. Data are presented as mean ± SEM.

Article Snippet: RGS6 gene silencing via small hairpin RNA (shRNA) delivery in vivo – shRNA against RGS6 was purchased from Santacruz Biotechnology (Paso Robles, CA, USA).

Techniques: Expressing, Control, Western Blot, Staining

Journal: Redox Biology

Article Title: Hepatic Regulator of G Protein Signaling 6 (RGS6) drives non-alcoholic fatty liver disease by promoting oxidative stress and ATM-dependent cell death

doi: 10.1016/j.redox.2021.102105

Figure Lengend Snippet: RGS6 KD in liver protects against hepatosteatosis and fibrosis in HFD-fed mice. Control and HFD (54.8% total fat content)-fed mice were administered scramble or RGS6-targeted shRNA via tail vein injection prior to initiation of the HFD (n = 6). Animals were sacrificed after 12 weeks of HFD feeding. (A) Immunoblotting for RGS6 in liver [L] and heart [H]. (B) Hepatic RGS6 expression measured via immunohistochemistry [scale bar = 100 μm]. (C) Immunblotting for RGS6, pATM and ATM with densitometric quantification. (D) Histological characterization of livers. Liver architecture (H & E), fibrosis (Masson Trichrome, Sirius Red), inflammation (F4/80), regeneration (PCNA) and lipid accumulation (Oil Red O) are depicted [scale bar = 100 μm]. (E) F4/80 and PCNA positive cells per microscope field (n = 10). (F) Hepatic collagen and hydroxyproline content (n = 6/group). (G) Liver enzyme (ALT & AST) and triglyceride levels. (H) Immunoblotting for markers of insulin sensitivity with corresponding densitometric quantification. (I) Liver MDA (n = 7). (J) Mitochondrial ROS (Mitosox) generation (n = 7). (K) Total and mitochondrial fatty acid oxidation (FAO) rate in liver (n = 7). (L) Liver metabolism as indicated by NADH/NAD + and Liver Lactate/Liver pyruvate (n = 7). β-Actin is used as a loading control for all immunoblots. Data were analyzed by two-way ANOVA with Sidak's post-hoc test. * P < 0.05, ** P < 0.01,*** P < 0.001, **** P < 0.0001. Data are presented as mean ± SEM. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: RGS6 gene silencing via small hairpin RNA (shRNA) delivery in vivo – shRNA against RGS6 was purchased from Santacruz Biotechnology (Paso Robles, CA, USA).

Techniques: Control, shRNA, Injection, Western Blot, Expressing, Immunohistochemistry, Microscopy

Journal: Redox Biology

Article Title: Hepatic Regulator of G Protein Signaling 6 (RGS6) drives non-alcoholic fatty liver disease by promoting oxidative stress and ATM-dependent cell death

doi: 10.1016/j.redox.2021.102105

Figure Lengend Snippet: RGS6, whose up-regulation is ROS driven, promotes mitochondrial dysfunction and cell death in hepatocytes. (A) Immunoblotting for RGS6 in human hepatocytes, murine hepatocytes, and HepaRG cells treated with PA (400 μM, 24 h). Densitometric quantification is provided (n = 3). (B) HepaRG cells were treated with PA (400 μM, 24 h) and/or H 2 O 2 (200 mM, 24 h) and immunoblotting performed for the detection of RGS6. Representative immunoblots and quantification are provided (n = 3). (C) HepaRG cells were treated with PA (400 μM, 24 h) and/or increasing concentrations of the H2O2 scavenger Peg-Cat (up to 200 U/mL, 1 h pre-treatment). Immunoblotting was performed for the detection of RGS6. Representative immunoblots and quantification are provided (n = 3). HepaRG cells were treated with increasing concentrations of PA (24 h) or a fixed drug dose (400 μM) 24 h following introduction of scramble or RGS6-targeted shRNA. The RGS6L isoform was reintroduced into a subset of RGS6 KD cells. (D) and (E) CM-H 2 DCFDA fluorescence (total ROS; n = 6–7). (F) Mitosox fluorescence (mitochondrial ROS; n = 7). (G) Mitochondrial membrane potential (n = 7) with and without the introduction of mitochondrial permeability transition pore blocker cyclosporin A (0.2 μM, 1 h pre-treatment) or mitochondrial calcium uniporter inhibitor Ru360 (50 μM, 1 h pre-treatment). (H) Mitochondrial calcium (Ca 2+ ) with and without Ru360 (50 μM, 1 h pre-treatment) (n = 7). (I) Cell death (TUNEL positive cells; n = 9). Data were analyzed by one- or two-way ANOVA with Sidak's post-hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns = not significant. Data are presented as mean ± SEM.

Article Snippet: RGS6 gene silencing via small hairpin RNA (shRNA) delivery in vivo – shRNA against RGS6 was purchased from Santacruz Biotechnology (Paso Robles, CA, USA).

Techniques: Western Blot, shRNA, Fluorescence, Membrane, Permeability, TUNEL Assay

Journal: Redox Biology

Article Title: Hepatic Regulator of G Protein Signaling 6 (RGS6) drives non-alcoholic fatty liver disease by promoting oxidative stress and ATM-dependent cell death

doi: 10.1016/j.redox.2021.102105

Figure Lengend Snippet: RGS6 expression is sufficient to drive hepatic ATM phosphorylation in vivo and oxidative stress and cell death in hepatocytes. (A) Control and HFD-fed mice were administered vehicle or a viral construct encoding RGS6L via tail vein injection prior to initiation of the HFD (n = 3). Animals were sacrificed after 12 weeks of HFD feeding. Immunoblotting was performed to detect RGS6, pATM/ATM, and γH2AX and densitometric quantification provided. β-Actin is used as a loading control for all immunoblots. (B–D) HepaRG cells were treated with PA (400 μM, 24 h) 24 h following transfection with RGS6L. (B) CM-H 2 DCFDA fluorescence (n = 7) and apoptosis as measured via (C) cytoplasmic histone-associated DNA fragments (n = 7) or (D) TUNEL positive cells (n = 7). Data were analyzed by two-way ANOVA with Sidak's post-hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns = not significant. Data are presented as mean ± SEM.

Article Snippet: RGS6 gene silencing via small hairpin RNA (shRNA) delivery in vivo – shRNA against RGS6 was purchased from Santacruz Biotechnology (Paso Robles, CA, USA).

Techniques: Expressing, Phospho-proteomics, In Vivo, Control, Construct, Injection, Western Blot, Transfection, Fluorescence, TUNEL Assay

Journal: Redox Biology

Article Title: Hepatic Regulator of G Protein Signaling 6 (RGS6) drives non-alcoholic fatty liver disease by promoting oxidative stress and ATM-dependent cell death

doi: 10.1016/j.redox.2021.102105

Figure Lengend Snippet: RGS6, ATM and ROS function in the same pathway to promote hyperlipidemia-dependent hepatotoxicity. Scramble or RGS6-targeted shRNA was introduced into human hepatocytes (A, B) or HepaRG cells (A, C–F) 24 h prior to treatment with PA (400 μM, 24 h), NAC (5 mM, 1 h pre-treatment) and/or the ATM inhibitor KU55933 (ATMi; 5 μM, 1 h pre-treatment) where indicated. In a subset of experiments RGS6 deletion constructs were introduced via transfection prior to drug treatments. (A) Immunoblotting for pATM and ATM. Representative westerns and a densitometric quantification are provided (n = 3). β-Actin is used as a loading control for all immunoblots. (B) Cell death (cytoplasmic histone-associated DNA fragments; n = 5) ± RGS6 KD, ATMi, and/or NAC in human hepatocytes. (C) CM-H 2 DCFDA fluorescence (n = 6) and (D) cell death (cytoplasmic histone-associated DNA fragments; n = 6) ± RGS6 KD and/or ATMi. (E) CM-H 2 DCFDA fluorescence (n = 5); (F) cell death (cytoplasmic histone-associated DNA fragments; n = 6); and albumin production (n = 5) ± RGS6 KD and/or RGS6 deletion constructs. Data were analyzed by one- or two-way ANOVA with Sidak's post-hoc test. * P < 0.05, ** P < 0.01, **** P < 0.0001. ns = not significant. Data are presented as mean ± SEM.

Article Snippet: RGS6 gene silencing via small hairpin RNA (shRNA) delivery in vivo – shRNA against RGS6 was purchased from Santacruz Biotechnology (Paso Robles, CA, USA).

Techniques: shRNA, Construct, Transfection, Western Blot, Control, Fluorescence

Journal: Redox Biology

Article Title: Hepatic Regulator of G Protein Signaling 6 (RGS6) drives non-alcoholic fatty liver disease by promoting oxidative stress and ATM-dependent cell death

doi: 10.1016/j.redox.2021.102105

Figure Lengend Snippet: Schematic depicting the role of RGS6 in hyperlipidemia-driven hepatotoxicity. Oxidative stress drives RGS6 up-regulation, which represents an amplification node facilitating further pathogenic ROS generation. By interacting with ATM, RGS6 promotes mitochondrial dysfunction, which compromised fatty acid oxidation leading to hepatic steatosis and, in the face of overwhelming cellular stress, can trigger cell death. RGS6 is also expressed in hepatic stellate cells (HSCs) where RGS6 up-regulation leads to oxidative stress, actions likely also contributing to hepatic inflammation and fibrosis. Finally, RGS6 also suppresses markers of insulin sensitivity, of particular relevance to patients with co-morbid diabetes mellitus.

Article Snippet: RGS6 gene silencing via small hairpin RNA (shRNA) delivery in vivo – shRNA against RGS6 was purchased from Santacruz Biotechnology (Paso Robles, CA, USA).

Techniques: Amplification