Journal: Biochemical and biophysical research communications

Article Title: Sortilin facilitates VLDL-B100 secretion by insulin sensitive McArdle RH7777 cells

doi: 10.1016/j.bbrc.2016.07.096

Figure Lengend Snippet: (A) VLDL-apo B and VLDL-B100 secretion were measured following 18 h incubation of McA cells in cDMEM (black bars) and in 1% BSA/DMEM (gray bars) by immuno slot blotting. (B) Representative standard curve generated from an immuno slot blot (duplicate standards) where VLDL protein is plotted against average chemiluminescence signal in arbitrary units (AU) generated using anti-rat B100 monoclonal antibody and HRP-anti-mouse IgG. (C) Immunoblots of McA cell lysates from four independent experiments comparing McA cells incubated in cDMEM with 1% BSA/DMEM. Anti-GAPDH blotting display equal protein loading. (D) Relative expression of Apob, Sort1 and Atf3 mRNA in McA cells incubated in 1% BSA/DMEM as a percentage of mRNA present in McA cells incubated in cDMEM. Results are averages from 4 independent experiments.* Indicates means are significantly different.

Article Snippet: McA cells were transfected using Fugene6 according to manufacturer’s protocol (Promega Corp., Madison WI) using three different pGIPZ based vectors expressing shRNAi targeting rat Sort1 mRNA (V2LMM_58553, V3LMM_450660, V3LMM_450662), and one scrambled, non-silencing control (GE Healthcare Dharmacon, Lafayette, CO).

Techniques: Incubation, Generated, Dot Blot, Western Blot, Expressing

Journal: Biochemical and biophysical research communications

Article Title: Sortilin facilitates VLDL-B100 secretion by insulin sensitive McArdle RH7777 cells

doi: 10.1016/j.bbrc.2016.07.096

Figure Lengend Snippet: (A) Following siRNA-mediated KD of sortilin, three clones of McA cells were selected representing high (H, McA60), medium (M, McA62) and low (L, McA53) sortilin expression evaluated by immunoblotting compared with wild type (WT) McA cells. The effect of increasing sortilin KD on VLDL-B100 secretion by McA cells incubated in cDMEM (B) or incubated in 1% BSA/DMEM (C). Results in (B) and (C) are averages of triplicate plates for each condition (n = 2 studies). * Indicates means are significantly different from the SCR McA cell line.

Article Snippet: McA cells were transfected using Fugene6 according to manufacturer’s protocol (Promega Corp., Madison WI) using three different pGIPZ based vectors expressing shRNAi targeting rat Sort1 mRNA (V2LMM_58553, V3LMM_450660, V3LMM_450662), and one scrambled, non-silencing control (GE Healthcare Dharmacon, Lafayette, CO).

Techniques: Clone Assay, Expressing, Western Blot, Incubation

Journal: Biochemical and biophysical research communications

Article Title: Sortilin facilitates VLDL-B100 secretion by insulin sensitive McArdle RH7777 cells

doi: 10.1016/j.bbrc.2016.07.096

Figure Lengend Snippet: (A) McA cell lines with variable sortilin KD were incubated with vehicle (black bars) or with 10 μM cpd984 (gray bars) for 18 h (3-100 mm plates per condition). Viability of McA cells was not compromised by incubations with cpd984 as there was no significant release of LDH into the medium compared with control incubations. VLDL was isolated from media of each plate and VLDL-B100 was quantified by immuno slot blotting. Results presented are averages of the 3 plates ± S.D. (B) McA cells were incubated for 18 h in 1% BSA/DMEM ± 10 μM cpd984. McA cells were then stimulated with a time course of 250 nM insulin (0, 5, 10 and 15 min). IRβ and AKT, pY-IRβ, and p-AKT (S473) were evaluated by immunoblotting using protein and phosphospecific antibodies.

Article Snippet: McA cells were transfected using Fugene6 according to manufacturer’s protocol (Promega Corp., Madison WI) using three different pGIPZ based vectors expressing shRNAi targeting rat Sort1 mRNA (V2LMM_58553, V3LMM_450660, V3LMM_450662), and one scrambled, non-silencing control (GE Healthcare Dharmacon, Lafayette, CO).

Techniques: Incubation, Control, Isolation, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Doxorubicin cardiomyopathy is ameliorated by acacetin via Sirt1‐mediated activation of AMPK/Nrf2 signal molecules

doi: 10.1111/jcmm.15859

Figure Lengend Snippet: Effects of acacetin on related‐protein expression in ventricular tissues of mice treated with doxorubicin. A, Western blots of Nrf2, HO‐1, SOD1 and SOD2 in ventricular tissues of mice administered with vehicle (Control), doxorubicin (Dox) and acacetin prodrug plus doxorubicin. B, Summarized relative levels of Nrf2, HO‐1, SOD1 and SOD2 expression in ventricular tissues of mice from Western blots as shown in A. C, Western blots of Bcl‐2, Bax and cleaved caspase‐3 in ventricular tissues of mice with the same treatment as in A. C, Western blots and relative level of Sirt1 in ventricular tissues of mice with the same treatment as in A. D, Western blots and relative level of pAMPK and total AMPK (T‐AMPK) in ventricular tissues of mice with the same treatment as in A (n = 5 individual experiments, * P < 0.05, ** P < 0.01 vs control; # P < 0.05, ## P < 0.01 vs doxorubicin alone)

Article Snippet: Small interfering RNA (siRNA) molecules targeting rat Sirt1 mRNA (sc‐108043) was from Santa Cruz Biotechnology (Dallas, TX, USA), whereas siRNA molecules targeting rat AMPK and scrambled control siRNA were synthesized by RiboBio (Guangzhou, Guangdong, China).

Techniques: Expressing, Western Blot, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Doxorubicin cardiomyopathy is ameliorated by acacetin via Sirt1‐mediated activation of AMPK/Nrf2 signal molecules

doi: 10.1111/jcmm.15859

Figure Lengend Snippet: Effects of acacetin on Sirt1 and the dominant signal molecules. A, Western blots and relative level of Sirt1 in rat cardiomyoblasts in the absence and presence of 0.3, 1 or 3 μM acacetin. B, Western blots and mean relative levels of Sirt1 in rat cardiomyoblasts treated with 1 μM doxorubicin in the absence and presence of 0.3, 1 or 3 μM acacetin. C, Western blots and mean relative levels of pAMPK in rat cardiomyoblasts with the same treatment as in B. D, Western blots and mean relative levels of pLKB in rat cardiomyoblasts with the same treatment as in B. E, Western blots and relative level of pLKB and total LKB (t‐LKB) in rat cardiomyoblasts transfected with control siRNA or Sirt1 siRNA in the absence (V, vehicle) or presence of 3 μM acacetin. F, Western blots and relative level of pAMPK and total AMPK (t‐AMPK) in rat cardiomyoblasts transfected with control siRNA or Sirt1 siRNA and treated as in E. G, Western blots and relative level of Nrf2 in rat cardiomyoblasts with the same treatment as in E. H, Western blots and relative level of nuclei Nrf2 in rat cardiomyoblasts with the same treatment as in E. I, Relative Nrf2 mRNA level measured with real‐time PCR in rat cardiomyoblasts with the same treatment as in E. (n = 5 individual experiments, * P < 0.05, ** P < 0.01 vs control or vehicle of control siRNA; # P < 0.05, ## P < 0.01 vs control siRNA with acacetin)

Article Snippet: Small interfering RNA (siRNA) molecules targeting rat Sirt1 mRNA (sc‐108043) was from Santa Cruz Biotechnology (Dallas, TX, USA), whereas siRNA molecules targeting rat AMPK and scrambled control siRNA were synthesized by RiboBio (Guangzhou, Guangdong, China).

Techniques: Western Blot, Transfection, Control, Real-time Polymerase Chain Reaction

Journal: Frontiers in Pharmacology

Article Title: The Natural Flavone Acacetin Confers Cardiomyocyte Protection Against Hypoxia/Reoxygenation Injury via AMPK-Mediated Activation of Nrf2 Signaling Pathway

doi: 10.3389/fphar.2018.00497

Figure Lengend Snippet: Effects of acacetin on antioxidant-related proteins in cells with hypoxia/reoxygenation (H/R) exposure. Western blots and mean relative levels of Nrf2 (A) , HO-1 (B) , SOD1 (C) , SOD2 (D) in neonatal rat cardiomyocytes without (control) or with hypoxia/reoxygenation exposure in the absence (V, vehicle) or presence of 0.3, 1, or 3 μM acacetin. Western blots and mean relative levels of Nrf2 (E) , HO-1 (F) , SOD1 (G) , SOD2 (H) in H9C2 cardiomyoblasts without (control) or with hypoxia/reoxygenation exposure in the absence or presence of 0.3, 1, or 3 μM acacetin. Data were expressed as mean ± SEM and analyzed by one-way ANOVA followed by Bonferroni-test ( n = 5 individual experiments, ∗ P < 0.05, ∗∗ P < 0.01 vs. control; # P < 0.05 vs. hypoxia/reoxygenation alone).

Article Snippet: Small interfering RNA (siRNA) molecules targeting rat Nrf2 mRNA (sc-156128) was from Santa Cruz Biotechnology (Dallas, TX, United States), while siRNA molecules targeting rat AMPK and scrambled control siRNA were synthesized by RiboBio, Co., Ltd. (Guangzhou, Guangdong, China).

Techniques: Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: The Natural Flavone Acacetin Confers Cardiomyocyte Protection Against Hypoxia/Reoxygenation Injury via AMPK-Mediated Activation of Nrf2 Signaling Pathway

doi: 10.3389/fphar.2018.00497

Figure Lengend Snippet: Silencing Nrf2 abolished cardiomyocytes protection of acacetin against hypoxia/reoxygenation insult. (A) Flow cytometry graphs showing cell viability, early apoptosis, late apoptosis, and dead cells in H9C2 cardiomyoblasts transfected with control siRNA or Nrf2 siRNA for 48 h, then subjected to hypoxia/reoxygenation in the absence (vehicle control) or presence of 3 μM acacetin. (B) Mean percent values of cell viability, early apoptosis, late apoptosis, and dead cells in H9C2 cardiomyoblasts transfected with control siRNA or Nrf2 siRNA for 48 h, then subjected to hypoxia/reoxygenation in the absence (vehicle control) or presence of 3 μM acacetin. (C) Flow cytometry graphs showing ROS production in H9C2 cardiomyoblasts with the treatment used in (A) . (D) Mean percent values of ROS production in H9C2 cardiomyoblasts. Data were expressed as mean ± SEM and analyzed by one-way ANOVA followed by Bonferroni-test ( n = 5 individual experiments, ∗ P < 0.05, ∗∗ P < 0.01 vs. control siRNA; # P < 0.05, ## P < 0.01 vs. control siRNA with acacetin).

Article Snippet: Small interfering RNA (siRNA) molecules targeting rat Nrf2 mRNA (sc-156128) was from Santa Cruz Biotechnology (Dallas, TX, United States), while siRNA molecules targeting rat AMPK and scrambled control siRNA were synthesized by RiboBio, Co., Ltd. (Guangzhou, Guangdong, China).

Techniques: Flow Cytometry, Transfection, Control

Journal: Frontiers in Pharmacology

Article Title: The Natural Flavone Acacetin Confers Cardiomyocyte Protection Against Hypoxia/Reoxygenation Injury via AMPK-Mediated Activation of Nrf2 Signaling Pathway

doi: 10.3389/fphar.2018.00497

Figure Lengend Snippet: Effects of silencing Nrf2 on antioxidant proteins in cells with hypoxia/reoxygenation insult. (A) Western blots and relative levels of Nrf2 in H9C2 cardiomyoblasts transfected with control siRNA or Nrf2 siRNA and subjected to hypoxia/reoxygenation insult in the absence (V, vehicle) or presence of 3 μM acacetin (Aca). (B) Western blots and relative levels of HO-1 in H9C2 cardiomyoblasts with the treatment used in (A) . (C) Western blots and relative levels of SOD1 in H9C2 cardiomyoblasts with the treatment used in (A) . (D) Western blots and relative levels of SOD2 in H9C2 cardiomyoblasts with the treatment used in (A) . Data were expressed as mean ± SEM and analyzed by one-way ANOVA followed by Bonferroni-test ( n = 5 individual experiments, ∗ P < 0.05, ∗∗ P < 0.01 vs. vehicle of control siRNA; ## P < 0.01 vs. control siRNA with acacetin).

Article Snippet: Small interfering RNA (siRNA) molecules targeting rat Nrf2 mRNA (sc-156128) was from Santa Cruz Biotechnology (Dallas, TX, United States), while siRNA molecules targeting rat AMPK and scrambled control siRNA were synthesized by RiboBio, Co., Ltd. (Guangzhou, Guangdong, China).

Techniques: Western Blot, Transfection, Control

Journal: Frontiers in Pharmacology

Article Title: The Natural Flavone Acacetin Confers Cardiomyocyte Protection Against Hypoxia/Reoxygenation Injury via AMPK-Mediated Activation of Nrf2 Signaling Pathway

doi: 10.3389/fphar.2018.00497

Figure Lengend Snippet: Effects of silencing Nrf2 on apoptosis- and inflammation-related proteins in cells with hypoxia/reoxygenation insult. Western blots and relative levels of Bcl-2 (A) , Bax (B) , and cleaved caspase-1 (C) in H9C2 cardiomyoblasts transfected with control siRNA or Nrf2 siRNA and subjected to hypoxia/reoxygenation insult in the absence (V, vehicle) or presence of 3 μM acacetin (Aca). Western blots and relative levels of IL-6 (D) , TRL-4 (E) , and IL-10 (F) in H9C2 cardiomyoblasts with the treatment used in (A–C) . Data were expressed as mean ± SEM and analyzed by one-way ANOVA followed by Bonferroni-test ( n = 5 individual experiments, ∗ P < 0.05, ∗∗ P < 0.01 vs. vehicle of control siRNA; ## P < 0.01 vs. control siRNA with acacetin).

Article Snippet: Small interfering RNA (siRNA) molecules targeting rat Nrf2 mRNA (sc-156128) was from Santa Cruz Biotechnology (Dallas, TX, United States), while siRNA molecules targeting rat AMPK and scrambled control siRNA were synthesized by RiboBio, Co., Ltd. (Guangzhou, Guangdong, China).

Techniques: Western Blot, Transfection, Control

Journal: Frontiers in Pharmacology

Article Title: The Natural Flavone Acacetin Confers Cardiomyocyte Protection Against Hypoxia/Reoxygenation Injury via AMPK-Mediated Activation of Nrf2 Signaling Pathway

doi: 10.3389/fphar.2018.00497

Figure Lengend Snippet: Nrf2 activation induced by acacetin is mediated by AMPK. (A) Western blots and relative level of pAMPK in H9C2 cardiomyoblasts treated by 0.3, 1, or 3 μM acacetin. (B) Western blots and relative level of Nrf2 in H9C2 cardiomyoblasts transfected with control siRNA or AMPK siRNA in the absence or presence of 3 μM acacetin. (C) Western blots and relative level of Nrf2 in left ventricular tissues of rats subjected to ischemia/reperfusion injury with treatment of acacetin or Compound C plus acacetin. Data were expressed as mean ± SEM and analyzed by one-way ANOVA followed by Bonferroni-test ( n = 5 individual experiments, ∗ P < 0.05 vs. sham, ∗∗ P < 0.01 vs. control or vehicle of control siRNA or sham; # P < 0.05 vs. acacetin; ## P < 0.01 vs. control siRNA with acacetin).

Article Snippet: Small interfering RNA (siRNA) molecules targeting rat Nrf2 mRNA (sc-156128) was from Santa Cruz Biotechnology (Dallas, TX, United States), while siRNA molecules targeting rat AMPK and scrambled control siRNA were synthesized by RiboBio, Co., Ltd. (Guangzhou, Guangdong, China).

Techniques: Activation Assay, Western Blot, Transfection, Control

Journal: Frontiers in Pharmacology

Article Title: The Natural Flavone Acacetin Confers Cardiomyocyte Protection Against Hypoxia/Reoxygenation Injury via AMPK-Mediated Activation of Nrf2 Signaling Pathway

doi: 10.3389/fphar.2018.00497

Figure Lengend Snippet: Schematic illustration showing the main cascade of molecules in cardiomyocytes subjected to ischemia/reperfusion insult and the mechanism of acacetin effects. Cardiomyocytes suffering from hypoxia/reoxygenation induces oxidative stress and triggers ROS production that induces inflammation and apoptosis via releasing the pro-inflammatory cytokines TLR-4 and IL-6 which increase the apoptotic proteins Bax and cleaved caspase-3, and decrease anti-inflammatory factor IL-10 and anti-apoptotic protein Bcl-2. Acacetin activates Nrf2 by stimulating AMPK, and increasing HO-1, SOD1, and SOD2, thereby conferring protection to cardiomyocytes against hypoxia/reoxygenation insult by reducing ROS production and inhibiting inflammation and apoptosis.

Article Snippet: Small interfering RNA (siRNA) molecules targeting rat Nrf2 mRNA (sc-156128) was from Santa Cruz Biotechnology (Dallas, TX, United States), while siRNA molecules targeting rat AMPK and scrambled control siRNA were synthesized by RiboBio, Co., Ltd. (Guangzhou, Guangdong, China).

Techniques: