Journal: Oncotarget

Article Title: Aqueous Oldenlandia diffusa extracts inhibits colorectal cancer cells via activating AMP-activated protein kinase signalings

doi: 10.18632/oncotarget.9969

Figure Lengend Snippet: HCT-116 cells or patient-1-derived primary CRC cells were treated with or without applied ODE, cells were further cultured, expressions of listed proteins were tested by Western blots A , B , E and F . Stable HCT-116 cells expressing scramble control shRNA (“scr-shRNA”), AMPKα1-shRNA or dominant negative (dn)-AMPKα1 (“dnAMPKα1”) were treated with or without applied ODE, cells were further cultured for 6 h C. or 24 h D ., expressions of listed proteins were tested by Western blots. Kinase phosphorylations and Bcl-2/HIF-1α expressions were quantified. Data in this figure were repeated three times, and similar results were obtained.

Article Snippet: As described in our previous studies [ ], lentiviral particles containing the p53-shRNA-1 (Santa Cruz, sc-29435-V), p53 shRNA-2 (Genechem, Shanghai, China) or AMPKα1 shRNA (Santa Cruz, sc-29673-V) (10 μL/well) were added to cultured CRC cells for 24 h. Afterwards, cell culture medium was replaced with puromycin (0.5 μg/mL)-containing complete medium.

Techniques: Derivative Assay, Cell Culture, Western Blot, Expressing, Control, shRNA, Dominant Negative Mutation

Journal: Oncotarget

Article Title: Aqueous Oldenlandia diffusa extracts inhibits colorectal cancer cells via activating AMP-activated protein kinase signalings

doi: 10.18632/oncotarget.9969

Figure Lengend Snippet: Stable HCT-116 cells expressing scramble control shRNA (“scr-shRNA”), AMPKα1-shRNA or dominant negative (dn)-AMPKα1 (“dnAMPKα1”) as well as their parental cells were treated with or without applied ODE, cells were further cultured, cell viability ( A. , MTT assay), cell death ( B. , trypan blue staining assay) and cell apoptosis ( C. , Histone DNA ELISA assay) were tested. Primary CRC cells (patient-1-dervied), transfected with scramble control siRNA (“scr-siRNA”) or AMPKα1-siRNA (−1/−2), were treated with ODE for indicated time, expressions of listed proteins were shown D ., cell viability E . and apoptosis F . were tested similarly. Kinase phosphorylations were quantified (D). Data in this figure were repeated three times, and similar results were obtained. * p < 0.05 vs. “C” of “scr-shRNA”/“scr-siRNA” group. # p < 0.05 vs. “ODE” of “scr-shRNA”/“scr-siRNA” group.

Article Snippet: As described in our previous studies [ ], lentiviral particles containing the p53-shRNA-1 (Santa Cruz, sc-29435-V), p53 shRNA-2 (Genechem, Shanghai, China) or AMPKα1 shRNA (Santa Cruz, sc-29673-V) (10 μL/well) were added to cultured CRC cells for 24 h. Afterwards, cell culture medium was replaced with puromycin (0.5 μg/mL)-containing complete medium.

Techniques: Expressing, Control, shRNA, Dominant Negative Mutation, Cell Culture, MTT Assay, Staining, Enzyme-linked Immunosorbent Assay, Transfection

Journal: Oncotarget

Article Title: Aqueous Oldenlandia diffusa extracts inhibits colorectal cancer cells via activating AMP-activated protein kinase signalings

doi: 10.18632/oncotarget.9969

Figure Lengend Snippet: HCT-116 cells were treated with or without ODE at applied concentrations, cells were further cultured, expressions of listed proteins were tested by Western blots A and C ., the association between AMPKα1 (regular and p-) and p53 (regular and p-) was examined by co-immunoprecipitation (“Co-IP”) assay B ., IgG was also included as a Co-IP control (B). Stable HCT-116 cells expressing scramble-shRNA (“scr-shRNA”), AMPKα1-shRNA or dominant negative (dn)-AMPKα1 (“dnAMPKα1”) were treated with applied ODE, p53 (regular and p-) and Tubulin expressions were tested by Western blots D. Stable HCT-116 cells expressing scramble-shRNA (“scr-shRNA”) or p53-shRNA (“−1/−-2”) as well as their parental cells were treated with applied ODE, cell viability (MTT assay, E. ) and cell apoptosis (Histone DNA ELISA assay, F. ) were tested, expression of p53 in these cells was also shown (F, upper panel). p53 (regular and p-) and Tubulin expressions in ODE (50 μg/mL)-treated primary CRC cells (patient-1 derived) were shown G . p53 (regular and p-) and AMPKα1 expressions in ODE (50 μg/mL)-treated primary CRC cells with scramble control siRNA (“scr-siRNA”) or AMPKα1 siRNA (“−1/−2”) were shown H. Kinase phosphorylations and p53 expression were quantified. Data in this figure were repeated three times, and similar results were obtained. * p < 0.05 vs. “C” of “scr-shRNA” group. # p < 0.05 vs. “ODE” of “scr-shRNA” group.

Article Snippet: As described in our previous studies [ ], lentiviral particles containing the p53-shRNA-1 (Santa Cruz, sc-29435-V), p53 shRNA-2 (Genechem, Shanghai, China) or AMPKα1 shRNA (Santa Cruz, sc-29673-V) (10 μL/well) were added to cultured CRC cells for 24 h. Afterwards, cell culture medium was replaced with puromycin (0.5 μg/mL)-containing complete medium.

Techniques: Cell Culture, Western Blot, Co-Immunoprecipitation Assay, Control, Expressing, shRNA, Dominant Negative Mutation, MTT Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Mediators of Inflammation

Article Title: Loss of AMP-Activated Protein Kinase Induces Mitochondrial Dysfunction and Proinflammatory Response in Unstimulated Abcd1-Knockout Mice Mixed Glial Cells

doi: 10.1155/2015/176983

Figure Lengend Snippet: Adenosine monophosphate activated protein kinase (AMPK α 1) levels are similar in vivo in wild type (WT) and Abcd1-knockout (KO) central nervous systems and in vitro in mixed glial cells. (a) Age-matched (3-month-old) WT and Abcd1-KO mice were sacrificed and the brains and spinal cords harvested for AMPK α 1 protein (i) and mRNA (ii) levels. (b) In primary mixed glial cells from WT and Abcd1-KO mice, AMPK α 1 protein (i) and mRNA (ii) levels were similar. (b) Abcd1-KO mixed glial cells were silenced for scrambled control (Scr) or AMPK α 1 as described in . Lentiviral vector silencing with AMPK α 1-shRNA significantly decreased the AMPK α 1 protein (i) and mRNA (ii) levels in Abcd1-KO mice primary mixed glial cells. Results represent the mean ± SD of triplicates from three different experiments. *** P < 0.001 AMPK α 1 silenced compared with scrambled (Scr) silenced.

Article Snippet: Transduction-ready mouse shRNA lentiviral particles (10 6 TU/mL) for AMPK α 1 (consisting of a pool of 3–5 constructs and puromycin selection gene; sc-29674-V) and control shRNA lentiviral particles (Scr) (10 6 TU/mL, sc-108080) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: In Vivo, Knock-Out, In Vitro, Control, Plasmid Preparation, shRNA

Journal: Mediators of Inflammation

Article Title: Loss of AMP-Activated Protein Kinase Induces Mitochondrial Dysfunction and Proinflammatory Response in Unstimulated Abcd1-Knockout Mice Mixed Glial Cells

doi: 10.1155/2015/176983

Figure Lengend Snippet: Mitochondrial complex subunit expression and levels in wild type, Abcd1-knockout (KO), and adenosine monophosphate activated protein kinase- (AMPK α 1-) deleted Abcd1-KO primary mixed glial cells. Wild type (WT) and Abcd1-KO primary mixed glial cells were cultured as described in . Abcd1-KO mixed glial cells were silenced for scrambled control (Scr) or AMPK α 1 as described in . mRNA (a) and protein (b) levels of complex subunits were significantly reduced in Abcd1-KO mixed glial cells deleted for AMPK α 1. (c) Densitometric ratio of mitochondrial subunit levels versus actin western blots. Results represent the mean ± SD of triplicates from two different experiments. * P < 0.05; ** P < 0.01; *** P < 0.001. COM: complex, NS: nonsignificant.

Article Snippet: Transduction-ready mouse shRNA lentiviral particles (10 6 TU/mL) for AMPK α 1 (consisting of a pool of 3–5 constructs and puromycin selection gene; sc-29674-V) and control shRNA lentiviral particles (Scr) (10 6 TU/mL, sc-108080) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Expressing, Knock-Out, Cell Culture, Control, Western Blot

Journal: Mediators of Inflammation

Article Title: Loss of AMP-Activated Protein Kinase Induces Mitochondrial Dysfunction and Proinflammatory Response in Unstimulated Abcd1-Knockout Mice Mixed Glial Cells

doi: 10.1155/2015/176983

Figure Lengend Snippet: Mitochondrial bioenergetics in adenosine monophosphate activated protein kinase- (AMPK α 1-) deleted Abcd1-knockout (KO) primary mixed glial cells. (a) Proliferator-activated receptor gamma coactivator 1-alpha (PGC-1 α ) protein and mRNA levels were significantly reduced in Abcd1-KO cells when deleted for AMPK α 1. (b) Abcd1-KO mice mixed glial cells (1.5 × 10 4 cells/well) were plated in XF 96 V3-PS cell culture plates (Seahorse Bioscience) and deleted for AMPK α 1 as described in . Mitochondrial oxygen consumption rate (OCR) was measured by sequential addition of oligomycin, FCCP, and antimycin/rotenone to measure basal and FCCP-uncoupled OCR. (c) Basal and FCCP-linked OCR was similar between wild type (WT) and Abcd1-KO mixed glial cells but decreased in Abcd1-KO mixed glial cells deleted for AMPK α 1. (d) A parallel 96-well plate was used for adenosine triphosphate (ATP) measurement. Results represent the mean ± SD of triplicates from three different experiments. *** P < 0.001. NS: nonsignificant.

Article Snippet: Transduction-ready mouse shRNA lentiviral particles (10 6 TU/mL) for AMPK α 1 (consisting of a pool of 3–5 constructs and puromycin selection gene; sc-29674-V) and control shRNA lentiviral particles (Scr) (10 6 TU/mL, sc-108080) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Knock-Out, Cell Culture

Journal: Mediators of Inflammation

Article Title: Loss of AMP-Activated Protein Kinase Induces Mitochondrial Dysfunction and Proinflammatory Response in Unstimulated Abcd1-Knockout Mice Mixed Glial Cells

doi: 10.1155/2015/176983

Figure Lengend Snippet: Adenosine monophosphate activated protein kinase- (AMPK α 1-) deletion induces a spontaneous inflammatory response in Abcd1-KO mixed glial cells. Abcd1-KO mice primary mixed glial cells were plated in 6-well plates and deleted for AMPK α 1 using lentiviral vector. Inducible nitric oxide synthase (iNOS) protein (a) and mRNA levels (b) were induced in Abcd1-KO mixed glial cells when deleted for AMPK α 1 using lentiviral particles. Results represent the mean ± SD of triplicates from three different experiments. *** P < 0.001.

Article Snippet: Transduction-ready mouse shRNA lentiviral particles (10 6 TU/mL) for AMPK α 1 (consisting of a pool of 3–5 constructs and puromycin selection gene; sc-29674-V) and control shRNA lentiviral particles (Scr) (10 6 TU/mL, sc-108080) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Plasmid Preparation

Journal: Biochimica et biophysica acta

Article Title: Autophagy-dependent and -independent involvement of AMP-activated protein kinase in 6-hydroxydopamine toxicity to SH-SY5Y neuroblastoma cells.

doi: 10.1016/j.bbadis.2012.08.006

Figure Lengend Snippet: Fig. 3. 6-OHDA induced autophagy is associated with modulation of AMPK/mTOR signaling. (A) SH-SY5Y cells were incubated with 6-OHDA (50 μM) and the activation of AMPK/ mTOR signaling molecules was assessed by immunoblotting at the indicated time points. The blots from a representative of three experiments are presented with the densitometry data above the relevant bands. (B) Control shRNA- or AMPK shRNA-transfected cells were treated with 6-OHDA for 16 h and the intracellular acidification was analyzed by flow cytometry after acridine orange (AO) staining (the insert shows immunoblot confirmation of AMPK knockdown). The data are mean±SD values from three independent experiments (#pb0.05 vs. untreated control; *pb0.05 vs. 6-OHDA-treated control). (C) Control shRNA- or AMPK shRNA-transfected cells were treated with 6-OHDA (50 μM) for 8 h and the activation of AMPK/mTOR signaling molecules, LC3 conversion and p62 levels were assessed by immunoblotting. The blots from one of at least two experiments with similar results are presented with the densitometry data above the relevant bands.

Article Snippet: The short hairpin RNA (shRNA) targeting human LC3β or AMPKα 1/2 genes, as well as scrambled control shRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Incubation, Activation Assay, Western Blot, Control, shRNA, Transfection, Cytometry, Staining, Knockdown

Journal: Biochimica et biophysica acta

Article Title: Autophagy-dependent and -independent involvement of AMP-activated protein kinase in 6-hydroxydopamine toxicity to SH-SY5Y neuroblastoma cells.

doi: 10.1016/j.bbadis.2012.08.006

Figure Lengend Snippet: Fig. 4. Autophagy is involved in neurotoxicity of 6-OHDA. (A, B) SH-SY5Y cells were incubated with 6-OHDA (50 μM) in the presence or absence of the autophagy inhibitors 3-methyl adenine (3-MA; 4 mM), wortmannin (Wort; 100 nM), bafilomycin A1 (Baf; 2 nM), NH4Cl (10 mM) and chloroquine (Cq; 20 μM). After 24 h, cell viability was assessed by MTT (A) and LDH release test (B). (C–I) SH-SY5Y cells transfected with control or LC3β shRNA were treated with 6-OHDA (the inset in C shows immunoblot verification of LC3β knockdown). Intracellular acidification (C), phosphatidylserine externalization (F), caspase activation (G), ROS production (H) or superoxide levels (I) were examined by flow cytometry after 16 h. Cell viability was assessed by crystal violet (D) and LDH release test (E) after 24 h. The data are mean±SD values of triplicate measurements from a representative of three experiments (A, B, D, E) or mean±SD values from three independent experiments (C, F–I) (p#b0.05 vs. untreated control; *pb0.05 vs. 6-OHDA-treated control).

Article Snippet: The short hairpin RNA (shRNA) targeting human LC3β or AMPKα 1/2 genes, as well as scrambled control shRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Incubation, Transfection, Control, shRNA, Western Blot, Knockdown, Activation Assay, Cytometry

Journal: Biochimica et biophysica acta

Article Title: Autophagy-dependent and -independent involvement of AMP-activated protein kinase in 6-hydroxydopamine toxicity to SH-SY5Y neuroblastoma cells.

doi: 10.1016/j.bbadis.2012.08.006

Figure Lengend Snippet: Fig. 5. AMPK/mTOR signaling is involved in neurotoxicity of 6-OHDA. (A–E) Control shRNA- or AMPK shRNA-transfected SH-SY5Y cells were treated with 6-OHDA and the cell viability was assessed by crystal violet (A) or LDH test (B) after 24 h. Phosphatidylserine externalization (C), caspase activation (D) and ROS or superoxide production (E) were examined by flow cytometry after 16 h. (F) SH-SY5Y cells were incubated with 6-OHDA (50 μM) in the presence or absence of rapamycin (RAP) and the cell viability was determined by MTT test after 24 h. The data are mean±SD values of triplicate measurements from a representative of three experiments (A, B, F) or mean±SD values from three independent experiments (C–E) (p#b0.05 vs. untreated control; *pb0.05 vs. 6-OHDA-treated control).

Article Snippet: The short hairpin RNA (shRNA) targeting human LC3β or AMPKα 1/2 genes, as well as scrambled control shRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Control, shRNA, Transfection, Activation Assay, Cytometry, Incubation

Journal: Biochimica et biophysica acta

Article Title: Autophagy-dependent and -independent involvement of AMP-activated protein kinase in 6-hydroxydopamine toxicity to SH-SY5Y neuroblastoma cells.

doi: 10.1016/j.bbadis.2012.08.006

Figure Lengend Snippet: Fig. 6. AMPK/p38 signaling is involved in neurotoxicity of 6-OHDA. (A) SH-SY5Y cells were incubated with 6-OHDA (50 μM) and the phosphorylation of p38 was assessed by immunoblotting at the indicated time points. (B, C) SH-SY5Y cells transfected with control shRNA (B, C), LC3 shRNA (B) or AMPK shRNA (C) were treated with 6-OHDA (50 μM) for 8 h and p38 activation was determined by immunoblotting. (D–F) SH-SY5Y cells were incubated with 6-OHDA (50 μM) in the presence or absence of p38 inhibitor SB 203580 (10 μM). Phosphorylation of p38 and AMPK, as well as LC3 conversion were assessed by immunoblot after 8 h (D), while the cell viability was determined by crystal violet staining (E) and LDH test (F) after 24 h of incubation. The representative blots are presented with the densitometry data above the relevant bands, while the data in (E, F) are mean±SD values of triplicate measurements from a representative of three experiments (p#b0.05 vs. untreated control; *pb0.05 vs. 6-OHDA-treated control).

Article Snippet: The short hairpin RNA (shRNA) targeting human LC3β or AMPKα 1/2 genes, as well as scrambled control shRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Incubation, Phospho-proteomics, Western Blot, Transfection, Control, shRNA, Activation Assay, Staining