Journal: iScience

Article Title: Roseburia hominis enriched by baicalin reverses the non-response to metformin via upregulating linolenic acid metabolism

doi: 10.1016/j.isci.2025.113892

Figure Lengend Snippet: Baicalin combined with metformin improved the blood glucose levels in metformin non-responsive mice by activating the AMPK/ACC/CPT1 pathway (A–F) The serum concentrations of (A) TC, (B) LDL-C, (C) HDL-C, (D) IL-1β, (E) IL-6, and (F) IL-10. (G) Hematoxylin-eosin (H&E) staining of the liver, pancreas, epididymal adipose tissues, and oil red O staining of the liver (magnification, 30×; scale bars, 100 μm). (H) Western blot analysis of pAMPK (Thr172), ACC, and CPT1 proteins in the livers of mice after administration. Data are expressed as the mean ± SD (A‒F, n = 6–8; H, n = 3); ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, as determined by one-way ANOVA with Holm-Sidak’s post hoc test (A, B, C, E, F, and H) and Kruskal-Wallis test (D). NCD, normal chow diet; NR, non-response; Met, metformin; BA, baicalin; TC, total cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; IL-1β, interleukin-1β; IL-6, interleukin-6; IL-10, interleukin-10; AMPK, AMP-activated protein kinase; ACC, acetyl-CoA carboxylase; CPT1, carnitine palmitoyl transferase 1.

Article Snippet: These membranes were then incubated at 4°C overnight with primary antibodies against AMP-activated protein kinase (AMPK) (Proteintech, Cat# 10929-2-AP, RRID: AB_2169568 , 1:4,000), insulin receptor substrate 1 (IRS1) (Proteintech, Cat# 17509-1-AP, RRID: AB_10596914 , 1:1,000), phospho-AMPKα (Thr172) (40H9) (Cell Signaling Technology, Cat# 2535, RRID: AB_331250 , 1:1,000), phospho-IRS-1 (Ser636/639) (Cell Signaling Technology, Cat# 2388, RRID: AB_330339 , 1:1,000), phosphoenolpyruvate carboxykinase (PCK) (Proteintech, Cat# 16754-1-AP, RRID: AB_2160031 , 1:30,000), glucose-6-phosphatase (G6PC) (Proteintech, Cat# 66860-1-Ig, RRID: AB_2882199 , 1:5,000), mammalian target of rapamycin (mTOR) (Proteintech, Cat# 66888-1-Ig, RRID: AB_2882219 , 1:30,000), fatty acid desaturase 2 (FADS2) (Proteintech, Cat# 28034-1-AP, RRID: AB_2918142 , 1:4,000), phospholipase A2 group (PLA2G) (Proteintech, Cat# 18088-1-AP, RRID: AB_10859777 , 1:1,000), acetyl-CoA Carboxylase 1 (ACC1) (Proteintech, Cat# 21923-1-AP, RRID: AB_11042445 , 1:4,000), carnitine palmitoyl transferase 1 (CPT1) (Proteintech, Cat# 15184-1-AP, RRID: AB_2084676 , 1:50,000), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Proteintech, Cat# 10494-1-AP, RRID: AB_2263076 , 1:20,000), β-tubulin (Proteintech, Cat# 10094-1-AP, RRID: AB_2210695 , 1:10,000).

Techniques: Staining, Western Blot

Journal: iScience

Article Title: Roseburia hominis enriched by baicalin reverses the non-response to metformin via upregulating linolenic acid metabolism

doi: 10.1016/j.isci.2025.113892

Figure Lengend Snippet: R. hominis treatment reversed the metformin NR phenotype in NR mice (A) Experimental protocol for administration of R. hominis in mice. (B) Oral glucose tolerance test (OGTT) curve and its area under the curve (AUC). (C) Insulin tolerance test (ITT) curve and its AUC. (D) Homeostasis model assessment of insulin resistance (HOMA-IR) after drug R. hominis . (E–J) The serum concentrations of (E) TC, (F) LDL-C, (G) HDL-C, (H) IL-1β, (I) IL-6, and (J) IL-10. (K) Hematoxylin-eosin (H&E) staining of the liver, pancreas, epididymal adipose tissues, and oil red O staining of the liver (magnification, 30×; scale bars, 100 μm). (L) Western blot analysis of AMPK/ACC/CPT1 proteins in the livers of mice after administration. Data are expressed as the mean ± SD ( n = 7–8); ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, as determined by one-way ANOVA with Holm-Sidak’s post hoc test. ANOVA, analysis of variance; ABX, antibiotic mixed; FMT, fecal microbial transplantation; NCD, normal chow diet; R.h, Roseburia hominis ; TC, total cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; IL-1β, interleukin-1β; IL-6, interleukin-6; IL-10, interleukin-10; AMPK, AMP-activated protein kinase; ACC, acetyl-CoA carboxylase; CPT1, carnitine palmitoyl transferase 1.

Article Snippet: These membranes were then incubated at 4°C overnight with primary antibodies against AMP-activated protein kinase (AMPK) (Proteintech, Cat# 10929-2-AP, RRID: AB_2169568 , 1:4,000), insulin receptor substrate 1 (IRS1) (Proteintech, Cat# 17509-1-AP, RRID: AB_10596914 , 1:1,000), phospho-AMPKα (Thr172) (40H9) (Cell Signaling Technology, Cat# 2535, RRID: AB_331250 , 1:1,000), phospho-IRS-1 (Ser636/639) (Cell Signaling Technology, Cat# 2388, RRID: AB_330339 , 1:1,000), phosphoenolpyruvate carboxykinase (PCK) (Proteintech, Cat# 16754-1-AP, RRID: AB_2160031 , 1:30,000), glucose-6-phosphatase (G6PC) (Proteintech, Cat# 66860-1-Ig, RRID: AB_2882199 , 1:5,000), mammalian target of rapamycin (mTOR) (Proteintech, Cat# 66888-1-Ig, RRID: AB_2882219 , 1:30,000), fatty acid desaturase 2 (FADS2) (Proteintech, Cat# 28034-1-AP, RRID: AB_2918142 , 1:4,000), phospholipase A2 group (PLA2G) (Proteintech, Cat# 18088-1-AP, RRID: AB_10859777 , 1:1,000), acetyl-CoA Carboxylase 1 (ACC1) (Proteintech, Cat# 21923-1-AP, RRID: AB_11042445 , 1:4,000), carnitine palmitoyl transferase 1 (CPT1) (Proteintech, Cat# 15184-1-AP, RRID: AB_2084676 , 1:50,000), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Proteintech, Cat# 10494-1-AP, RRID: AB_2263076 , 1:20,000), β-tubulin (Proteintech, Cat# 10094-1-AP, RRID: AB_2210695 , 1:10,000).

Techniques: Staining, Western Blot, Transplantation Assay

Journal: Cell death discovery

Article Title: Targeting oncogenic MAGEA6 sensitizes triple negative breast cancer to doxorubicin through its autophagy and ferroptosis by stabling AMPKα1.

doi: 10.1038/s41420-024-02196-9

Figure Lengend Snippet: Fig. 4 Inhibition of AMPK by MAGEA6 impacted the autophagy of TNBC. A, B In vitro ubiquitination assay displayed that AMPKα1 ubiquitination requires MAGEA6. C Co-immunoprecipitation assay stated the association between MAGEA6 and AMPKα1. D Western blotting showed that knockdown of MAGEA6 increased the levels of AMPKα1 and p-AMPKα (Thr-172). E Whether sh-MAGEA6 influenced autophagy via activating AMPK signaling was verified by Western blotting assay. *P < 0.05, **P < 0.01, ***P < 0.001; Data were obtained from three independent experiments. TNBC triple-negative breast cancer.

Article Snippet: The membranes were blocked with 5% skimmed milk, followed by incubating overnight with antibodies against MAGEA6 (PA575647, 1:500, Invitrogen, Carlsbad, CA, USA), LC3 (14600-1-AP, 1:3000), Cleaved caspase 3 (25128-1-AP, 1:1500), Cleaved PARP (13371-1-AP, 1:5000), AMPKα1 (66536-1-Ig, 1:3000), SLC7A11 (26864-1-AP, 1:1500) (Proteintech, Wuhan, China); and p-AMPKα (Thr-172) (2531, 1:1000, CST, Boston, MA, USA).

Techniques: Inhibition, In Vitro, Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Western Blot, Knockdown

Journal: Oncotarget

Article Title: Fyn phosphorylates AMPK to inhibit AMPK activity and AMP-dependent activation of autophagy

doi: 10.18632/oncotarget.11916

Figure Lengend Snippet: A. Whole cell lysates from gastrocnemius muscle of either FynKO mice, WT, HSA-FynB (FynB) or HSA-FynT (FynT) mice were homogenized and immnoblots were performed by AMPKα mAb and GAPDH B. Gastrocnemius muscle of either FynKO mice, WT, HSA-FynB (FynB) or HSA-FynT (FynT) mice were homogenized and immunoprecipitated with 4G10 mAb followed by immunoblotting with AMPK mAb. Blots are representative of 3 independent experiments. C. Signal quantification of tyrosine-phosphorylated AMPKα levels from (A) and (B). Statistical examination between each set was performed. *** P < 0.005.

Article Snippet: The pCMV6 Entry Myc-DDK tagged ORF clone of Homo sapiens AMPK α2 construct was obtained from Origene (Rockville, MD, USA) and used to generate the pCMV6-3Myc-2-AMPKα2-Myc DDK construct.

Techniques: Immunoprecipitation, Western Blot

Journal: Oncotarget

Article Title: Fyn phosphorylates AMPK to inhibit AMPK activity and AMP-dependent activation of autophagy

doi: 10.18632/oncotarget.11916

Figure Lengend Snippet: A., B. NIH3T3 cells were co-transfected with 3myc-AMPKα2-Flag and Fyn-CA-V5. 48 h later, cells extracts were prepared and immunoprecipitated with a Flag antibody. Immunoblots of lysates (A) and immunoprecipitation samples (B) were performed with the indicated antibodies. Blots are representative of 3 independent experiments. C., D. Forty-eight hours after co-transfection with 3myc-AMPKα2-Flag, Fyn-CA-V5 and pcDNA3-LKB1, HEK293T cells extracts were prepared and immunoprecipitation was performed using a Flag antibody. Immunoblots of lysates (C) and immunoprecipitation samples (D) were performed with the indicated antibodies. These are representative immunoblots independently performed 3 times. E. Signal quantification of tyrosine-phosphorylated AMPKα levels from (D). Statistical examination between each set was performed. F. Purified Human PRKAA2 (100 ng) and either mock or Flag-Fyn-CA (380 ng) were incubated with the kinase buffer and ATP at 30°C for 30 min. Samples were boiled and separated onto a 8% SDS-PAGE gel, followed by immunoblotting with the indicated antibodies. G., H. HeLa cells were co-transfected by either 3myc-AMPKα2 WT or Y436F (YF) with Fyn-CA-V5. Immunoprecipitation was performed using a Flag antibody. Immunoblots of lysates (G) and immunoprecipitation samples (H) were performed with the indicated antibodies. Images are representative of 4 independent experiments.

Article Snippet: The pCMV6 Entry Myc-DDK tagged ORF clone of Homo sapiens AMPK α2 construct was obtained from Origene (Rockville, MD, USA) and used to generate the pCMV6-3Myc-2-AMPKα2-Myc DDK construct.

Techniques: Transfection, Immunoprecipitation, Western Blot, Cotransfection, Purification, Incubation, SDS Page

Journal: Oncotarget

Article Title: Fyn phosphorylates AMPK to inhibit AMPK activity and AMP-dependent activation of autophagy

doi: 10.18632/oncotarget.11916

Figure Lengend Snippet: A. HEK293T cells were co-transfected with human siAMPK α1 and α2 subunits and either empty vector (pcDNA3), GST-rat AMPKα WT (WT) or Y430F mutant (equivalent to human Y436F (Y436F)) that are not affected by human AMPKa siRNA. 72 h after transfection immunoblots were performed with the indicated antibodies. B., C. S79 phosphorylation of ACC and total ACC levels in samples from (A) were assessed (B) and signal quantification of the expression levels of S79 phosphorylation of ACC normalized with total ACC were performed (C). Statistical examination between each sets were performed * p < 0.05, *** p < 0.005. D. , E . HEK293T cells were transfected by either 3myc-AMPKα2 WT or Y436F-Flag (Y436F). Co-immunoprecipitation was performed by either control IgG or AMPK β1/β2 polyclonal antibody. Immunoblots of lysates (D) and immunoprecipitation samples (E) were performed with the indicated antibodies. Images are representative of 3 independent experiments.

Article Snippet: The pCMV6 Entry Myc-DDK tagged ORF clone of Homo sapiens AMPK α2 construct was obtained from Origene (Rockville, MD, USA) and used to generate the pCMV6-3Myc-2-AMPKα2-Myc DDK construct.

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Phospho-proteomics, Expressing, Immunoprecipitation, Control

Journal: Oncotarget

Article Title: Fyn phosphorylates AMPK to inhibit AMPK activity and AMP-dependent activation of autophagy

doi: 10.18632/oncotarget.11916

Figure Lengend Snippet: A. HEK293T cells were incubated with TNFα (10 ng/ml) for 12, 24 or 36 h and then stimulated with 2 mM AICAR for 10 min. Immunoblots were performed with the indicated antibodies. These are representative images from 3 independent experiments. B., C. Signal quantifications of expression levels of phospho- T172 AMPKα corrected by total AMPKα (B) and levels of phospho- S79 of ACC to total ACC (C). The data are presented as mean ± s.e.m. Statistical examination between control and AICAR stimulated cells in each sets were performed. ** p < 0.01, *** p < 0.005.

Article Snippet: The pCMV6 Entry Myc-DDK tagged ORF clone of Homo sapiens AMPK α2 construct was obtained from Origene (Rockville, MD, USA) and used to generate the pCMV6-3Myc-2-AMPKα2-Myc DDK construct.

Techniques: Incubation, Western Blot, Expressing, Control

Journal: Oncotarget

Article Title: Fyn phosphorylates AMPK to inhibit AMPK activity and AMP-dependent activation of autophagy

doi: 10.18632/oncotarget.11916

Figure Lengend Snippet: A. Fyn kinase activity in HEK293T cells treated or not with TNFα (10ng/ml) for 36 h. These are representative images from 5 independent experiments *** p < 0.005. B. Fyn expression in HEK293T cells treated or not with TNFα for 36 h. C. After a 36-h incubation with 10ng/ml TNFα, HEK293T cells were treated or not with TNFα for 36 h and homogenized. Immunoprecipitation was performed with total AMPKα antibody followed by immunoblotting with the 4G10 and total AMPKα antibody. These are representative images from 4 independent experiments. D. Signal quantification (Y436 normalized to total AMPKα) from (C). (*** P < 0.005).

Article Snippet: The pCMV6 Entry Myc-DDK tagged ORF clone of Homo sapiens AMPK α2 construct was obtained from Origene (Rockville, MD, USA) and used to generate the pCMV6-3Myc-2-AMPKα2-Myc DDK construct.

Techniques: Activity Assay, Expressing, Incubation, Immunoprecipitation, Western Blot

Journal: Oncotarget

Article Title: Fyn phosphorylates AMPK to inhibit AMPK activity and AMP-dependent activation of autophagy

doi: 10.18632/oncotarget.11916

Figure Lengend Snippet: A. HEK293T cells were transfected with Fyn siRNAs. Cells were treated with TNFα (10 ng/ml) for 36 h with or without 2 mM AICAR for 10 min. Immunoblots were performed with the indicated antibodies. These are representative images from 3 independent experiments. B., C. Signal quantifications of expression levels of phospho- T172 AMPKα normalized to total AMPKα (B) and levels of S79 phosphorylation of ACC normalized to total ACC (C). The data are presented as mean ± SE. Statistical examination between control and AICAR stimulated cells in each sets were performed. * P < 0.05, ** P < 0.01.

Article Snippet: The pCMV6 Entry Myc-DDK tagged ORF clone of Homo sapiens AMPK α2 construct was obtained from Origene (Rockville, MD, USA) and used to generate the pCMV6-3Myc-2-AMPKα2-Myc DDK construct.

Techniques: Transfection, Western Blot, Expressing, Phospho-proteomics, Control

Journal: Oncotarget

Article Title: Fyn phosphorylates AMPK to inhibit AMPK activity and AMP-dependent activation of autophagy

doi: 10.18632/oncotarget.11916

Figure Lengend Snippet: A. HEK293T cells were co-transfected with siRNAs of human siAMPK α1 and α2 subunits and either empty vector (pcDNA3), GST-rat AMPKα WT (WT) or Y430F mutant (equivalent to human Y436F (Y436F)) to be replaced. Immunoblots were performed 72 h after transfection. B. HEK293T cells were transfected with Fyn siRNAs. Cells were treated with 10ng/ml TNFα for 36 h with or without 2 mM AICAR for 10 min. Immunoblots were performed with the indicated antibodies. These are representative images from 3 independent experiments. C. Signal quantifications of the expression levels of phospho-S555 ULK1 to total ULK1. The data are presented as mean ± SE. Statistical examination between control and AICAR stimulated cells in each sets were performed * P < 0.05, ** P < 0.01.

Article Snippet: The pCMV6 Entry Myc-DDK tagged ORF clone of Homo sapiens AMPK α2 construct was obtained from Origene (Rockville, MD, USA) and used to generate the pCMV6-3Myc-2-AMPKα2-Myc DDK construct.

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Expressing, Control

Journal: Nature Communications

Article Title: Redefining the role of AMPK in autophagy and the energy stress response

doi: 10.1038/s41467-023-38401-z

Figure Lengend Snippet: a The phosphorylation of ULK1 at Ser758 by mTORC1 stabilizes the AMPK-ULK1 interaction. HEK293T cells lacking ULK1 were transiently transfected with either myc-tagged WT or mutant ULK1, with or without Rheb. Anti-myc immunoprecipitates were analyzed for the presence of endogenous AMPKα. b AMPK activation stabilizes the AMPK-ULK1 interaction. HEK293T cells lacking ULK1 were transiently transfected with myc-tagged ULK1. The cells were incubated in either full or amino acid-deprived medium, or treatment with Torin1 in the presence or absence of A769662 for 2 h. Anti-myc immunoprecipitates were analyzed for endogenous AMPKα. c Quantitative analysis of the AMPK-ULK1 interaction and ULK1 activity from b . d AMPK kinase activity is required to stabilize the AMPK-ULK1 interaction. HEK293T cells lacking AMPKα were transiently transfected with either WT or KD AMPKα1 and myc-tagged ULK1. The cells were treated as described in b . e , f AMPK activation stabilizes the AMPK-ULK1 interaction. ULK1 KO HEK293T cells that were transduced with myc-ULK1 were treated with the indicated conditions for 1 h. g Schematics summarizing how mTORC1 and AMPK regulate the AMPK-ULK1 interaction and ULK1 activity. h AMPK-dependent events, beyond the phosphorylation of ULK1 Ser758 by mTORC1, stabilize the AMPK-ULK1 interaction. ULK1 KO HEK293T cells were transiently transfected with myc-tagged WT or the S758A ULK1 mutant. The cells were treated as described in b . i Summary of AMPK- and mTORC1-mediated phosphorylation sites on ULK1 and the impact of their alanine substitution on Ser758 phosphorylation and the stability of the AMPK-ULK1 interaction. The residue numbers are based on the human ULK1 sequence. j , k The phosphorylation of ULK1 at either Ser556 or Thr660 is necessary for AMPK to stabilize the AMPK-ULK1 interaction. l Quantitative analysis of the AMPK-ULK1 interaction from k . The statistical analysis in this figure was performed as described in Fig. . The concentrations of the chemicals used for the experiments in this figure are the same as described in Figs. 1, 2 and Supplementary Fig. 2. Source data are provided as a Source Data file.

Article Snippet: Human WT AMPKα1 (Addgene #79010) and KD AMPKα1 (Addgene #79011) DNAs and rat WT AMPKα2 (Addgene #15991) and K45R AMPKα2 (kinase inactive mutant) (Addgene #15992) DNAs were subcloned into pLV-EF1a-IRES-Puro or pLV-EF1a-IRES-blast.

Techniques: Phospho-proteomics, Transfection, Mutagenesis, Activation Assay, Incubation, Activity Assay, Transduction, Residue, Sequencing

Journal: Nature Communications

Article Title: Redefining the role of AMPK in autophagy and the energy stress response

doi: 10.1038/s41467-023-38401-z

Figure Lengend Snippet: a The phosphorylation of ULK1 at Ser758 by mTORC1 stabilizes the AMPK-ULK1 interaction. HEK293T cells lacking ULK1 were transiently transfected with either myc-tagged WT or mutant ULK1, with or without Rheb. Anti-myc immunoprecipitates were analyzed for the presence of endogenous AMPKα. b AMPK activation stabilizes the AMPK-ULK1 interaction. HEK293T cells lacking ULK1 were transiently transfected with myc-tagged ULK1. The cells were incubated in either full or amino acid-deprived medium, or treatment with Torin1 in the presence or absence of A769662 for 2 h. Anti-myc immunoprecipitates were analyzed for endogenous AMPKα. c Quantitative analysis of the AMPK-ULK1 interaction and ULK1 activity from b . d AMPK kinase activity is required to stabilize the AMPK-ULK1 interaction. HEK293T cells lacking AMPKα were transiently transfected with either WT or KD AMPKα1 and myc-tagged ULK1. The cells were treated as described in b . e , f AMPK activation stabilizes the AMPK-ULK1 interaction. ULK1 KO HEK293T cells that were transduced with myc-ULK1 were treated with the indicated conditions for 1 h. g Schematics summarizing how mTORC1 and AMPK regulate the AMPK-ULK1 interaction and ULK1 activity. h AMPK-dependent events, beyond the phosphorylation of ULK1 Ser758 by mTORC1, stabilize the AMPK-ULK1 interaction. ULK1 KO HEK293T cells were transiently transfected with myc-tagged WT or the S758A ULK1 mutant. The cells were treated as described in b . i Summary of AMPK- and mTORC1-mediated phosphorylation sites on ULK1 and the impact of their alanine substitution on Ser758 phosphorylation and the stability of the AMPK-ULK1 interaction. The residue numbers are based on the human ULK1 sequence. j , k The phosphorylation of ULK1 at either Ser556 or Thr660 is necessary for AMPK to stabilize the AMPK-ULK1 interaction. l Quantitative analysis of the AMPK-ULK1 interaction from k . The statistical analysis in this figure was performed as described in Fig. . The concentrations of the chemicals used for the experiments in this figure are the same as described in Figs. 1, 2 and Supplementary Fig. 2. Source data are provided as a Source Data file.

Article Snippet: Human WT AMPKα1 (Addgene #79010) and KD AMPKα1 (Addgene #79011) DNAs and rat WT AMPKα2 (Addgene #15991) and K45R AMPKα2 (kinase inactive mutant) (Addgene #15992) DNAs were subcloned into pLV-EF1a-IRES-Puro or pLV-EF1a-IRES-blast.

Techniques: Phospho-proteomics, Transfection, Mutagenesis, Activation Assay, Incubation, Activity Assay, Transduction, Residue, Sequencing

Journal: bioRxiv

Article Title: Stromal mediated DNA damage promotes high grade serous ovarian cancer initiation

doi: 10.1101/2024.05.23.595550

Figure Lengend Snippet: A) Western blot and quantification of AMPKα1 and pAMPKα1 from nMSC (N=4) and hrMSCs (N=6) patient lines. B) Western blot and quantification of AMPKα1 and pAMPKα1 following BC1618 treatment for 24 hours. C) BC1618 treated hrMSCs were analyzed by flow cytometry for 4-HNE-PE and MDA-APC fluorescence intensity. D) We hypothesize that restoration of hrMSC AMPKα1 expression will ameliorate DNA damage in FTE cells. E) Western blot and quantification of MSCs transduced with lentiviral particles containing either GFP-tagged AMPKα1 (hrMSC OE) or GFP-empty vector constructs (hrMSC EV). Endogenous and exogenous AMPKα1 and pAMPKα1 were combined for quantification. F) Quantification of 4-HNE-PE and MDA-APC fluorescence intensity by flow cytometry in transduced cell lines (n=2). G) Representative 20X images of FTE that were co-cultured at a 1:1 ratio with transduced hrMSCs. 53BP1 foci were quantified by fluorescence microscopy. >3 fields per condition were imaged and analyzed. P-values were determined by ordinary one-way ANOVA with Tukey’s multiple comparison analysis.

Article Snippet: Lentiviral particles endcoding PRKAA1 -mGFP (RC218572L2V) or empty vector mGFP control constructs (PS100071V) were purchased from OriGene.

Techniques: Western Blot, Flow Cytometry, Fluorescence, Expressing, Transduction, Plasmid Preparation, Construct, Cell Culture, Microscopy, Comparison

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Hypoxia induces arginase II expression and increases viable human pulmonary artery smooth muscle cell numbers via AMPKα 1 signaling

doi: 10.1152/ajplung.00117.2016

Figure Lengend Snippet: AMPK overexpression induces arginase II expression and increases viable hPASMC numbers. Human PASMCs were seeded on six-well plates and transfected with an empty pCMV6-Entry vector with in-frame MYC/DDK tags or pCMV6-Entry vector with in-frame MYC/DDK tags containing AMPKα1, AMPKα2, or AMPKα1/2. The transfected hPASMCs were placed in 21% O2 (normoxia) or 1% O2 (hypoxia) for 48 h then protein was analyzed by Western blot analysis. Protein was assayed for myc and arginase II expression by Western blot analysis and was quantified by densitometry. Representative Western blots are shown for myc (A) and arginase II and β-actin (B). Data are shown as fold-change ± SE relative to empty vector control (vControl) (n = 4–6) (C). Human PASMCs were seeded on six-well plates and transfected with an empty pCMV6-Entry vector with in-frame MYC/DDK tags or pCMV6-Entry vector with in-frame MYC/DDK tags containing AMPKα1, AMPKα2, or AMPKα(1+2). The transfected hPASMCs were seeded onto six-well plates at 104 cells per well and incubated for 120 h in normoxia or hypoxia, and viable cell numbers were determined using trypan blue exclusion. D: data are shown as viable cell numbers as a percentage change from the number of seeded cells (n = 6). *Significant difference compared with vControl, P < 0.01. †Significant difference compared with hypoxia, vControl, P < 0.05.

Article Snippet: The cells were transfected with empty vector (vControl, cat. no. {"type":"entrez-nucleotide","attrs":{"text":"CW103174","term_id":"54795715","term_text":"CW103174"}} CW103174 , lot no. 120457, OriGene; final concentration: 1 μg/ml), vector-AMPKα1 (cat. no. RC518572, lot no. 130457, OriGene), vector-AMPKα2 (cat. no. RC510226, lot no. SR013124, OriGene), or vector-AMPKα 1+2 in transfection medium [Opti-MEM (200 μl, cat. no. 22600, lot no.460294, Thermo Fisher Scientific, Pittsburgh, PA)+DNA Turbofect (2 μl/μg, cat. no. R0531, lot no. 00197193; Thermo Fisher), serum/antibiotic-free media (800 μl)] for 6 h. Nontransfected hPASMCs were used as a control.

Techniques: Over Expression, Expressing, Transfection, Plasmid Preparation, Western Blot, Incubation

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Hypoxia induces arginase II expression and increases viable human pulmonary artery smooth muscle cell numbers via AMPKα 1 signaling

doi: 10.1152/ajplung.00117.2016

Figure Lengend Snippet: AMPKα1 knockdown prevents hypoxia-induced arginase II protein expression. Human PASMCs were transfected with AMPKα isoform-specific siRNA or scramble siRNA for 24 h, and then exposed to 21% O2 (normoxia) or 1% O2 (hypoxia) for 48 h. Protein was assayed for AMPKα1, AMPKα2, or arginase II protein expression by Western blot and was quantified by densitometry. Representative Western blots are shown for AMPKα1 (A), AMPKα2 (B), and arginase II and β-actin (C). Data are shown as means ± SE relative to normoxia, scramble siRNA-treated controls (n = 3–10). *Significant difference compared with normoxia, scramble siRNA-treated, P < 0.05. †Significant difference, arginase II vs. hypoxia, scramble siRNA-treated, P < 0.0001.

Article Snippet: The cells were transfected with empty vector (vControl, cat. no. {"type":"entrez-nucleotide","attrs":{"text":"CW103174","term_id":"54795715","term_text":"CW103174"}} CW103174 , lot no. 120457, OriGene; final concentration: 1 μg/ml), vector-AMPKα1 (cat. no. RC518572, lot no. 130457, OriGene), vector-AMPKα2 (cat. no. RC510226, lot no. SR013124, OriGene), or vector-AMPKα 1+2 in transfection medium [Opti-MEM (200 μl, cat. no. 22600, lot no.460294, Thermo Fisher Scientific, Pittsburgh, PA)+DNA Turbofect (2 μl/μg, cat. no. R0531, lot no. 00197193; Thermo Fisher), serum/antibiotic-free media (800 μl)] for 6 h. Nontransfected hPASMCs were used as a control.

Techniques: Expressing, Transfection, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Hypoxia induces arginase II expression and increases viable human pulmonary artery smooth muscle cell numbers via AMPKα 1 signaling

doi: 10.1152/ajplung.00117.2016

Figure Lengend Snippet: Knockdown of AMPKα1, but not AMPKα2, attenuates hypoxic activation of AMPK. Human PASMCs were treated with AMPKα isoform-specific siRNA or scramble siRNA for 24 h, and then exposed to 1% O2 (hypoxia) for 48 h. Protein was assayed for p-AMPK, total AMPK, p-ACC, or total ACC protein expression by Western blot and was quantified by densitometry. Representative Western blots are shown for p-AMPK, total AMPK (A), p-ACC, total ACC, and β-actin (B). Data are shown as fold-change ± SE relative to scramble siRNA-treated controls (n = 3–7). *Significant difference, compared with scramble siRNA-treated, P < 0.01. †Significant difference, compared with siAMPKα1-treated, P < 0.05.

Article Snippet: The cells were transfected with empty vector (vControl, cat. no. {"type":"entrez-nucleotide","attrs":{"text":"CW103174","term_id":"54795715","term_text":"CW103174"}} CW103174 , lot no. 120457, OriGene; final concentration: 1 μg/ml), vector-AMPKα1 (cat. no. RC518572, lot no. 130457, OriGene), vector-AMPKα2 (cat. no. RC510226, lot no. SR013124, OriGene), or vector-AMPKα 1+2 in transfection medium [Opti-MEM (200 μl, cat. no. 22600, lot no.460294, Thermo Fisher Scientific, Pittsburgh, PA)+DNA Turbofect (2 μl/μg, cat. no. R0531, lot no. 00197193; Thermo Fisher), serum/antibiotic-free media (800 μl)] for 6 h. Nontransfected hPASMCs were used as a control.

Techniques: Activation Assay, Expressing, Western Blot