Journal: Nutrients

Article Title: Hydroxycitric Acid Inhibits Chronic Myelogenous Leukemia Growth through Activation of AMPK and mTOR Pathway

doi: 10.3390/nu14132669

Figure Lengend Snippet: Hydroxycitric acid promotes AMPK phosphorylation in CML cells. CML cell lines were treated for 24 h with different concentrations of HCA. An equal amount of protein from each condition was separated on SDS PAGE (12% gel) and an immunoblot was performed using specific antibodies against pT172 AMPK and total AMPK. Vinculin was used as an internal control. ( A ) K562 ( B ) MEG-01 ( C ) KYO-1 ( D ) SHK-1. For the K562, MEG-01, KYO-1, and SHK-1, samples were loaded in duplicate. One was probed with total AMPK and the another one was probed with pT172 AMPK. Upper vinculin is referred to as pAMPK, and lower vinculin is referred to as the total AMPK. The bar graph beside each figure panel reflects the band intensity evaluated as optical density and represented as fold change for treated vs. untreated cells normalized for vinculin. ** p < 0.01, * p < 0.05 treated vs. untreated cells.

Article Snippet: Endogenous AMPK was immunoprecipitated using AMPKα antibody (Cell-Signaling, Danvers, MA, USA; #2523S).

Techniques: Phospho-proteomics, SDS Page, Western Blot, Control

Journal: Nutrients

Article Title: Hydroxycitric Acid Inhibits Chronic Myelogenous Leukemia Growth through Activation of AMPK and mTOR Pathway

doi: 10.3390/nu14132669

Figure Lengend Snippet: AMPK interacts with ACLY. ( A ) Co-immunoprecipitation of AMPK and ACLY in K562 cells. Endogenous AMPK was immunoprecipitated with antibody against total AMPK followed by Western blotting with anti-AMPK and anti-ACLY (upper panel). Bar graph showing the ratio of optical density of immunoprecipitated (IP) band of ACLY and AMPK after normalization with the respective input (lower panel). ( B ) Co-immunoprecipitation of AMPK and ACLY in K562 cells upon treatment with HCA (upper panel). Bar graph of optical density of immunoprecipitated ACLY and AMPK in each condition normalized to respective input band (left lower panel). Ratio of optical density of immunoprecipitated (IP) band of ACLY and AMPK after normalization with the control (right lower panel).

Article Snippet: Endogenous AMPK was immunoprecipitated using AMPKα antibody (Cell-Signaling, Danvers, MA, USA; #2523S).

Techniques: Immunoprecipitation, Western Blot, Control

Journal: Nutrients

Article Title: Hydroxycitric Acid Inhibits Chronic Myelogenous Leukemia Growth through Activation of AMPK and mTOR Pathway

doi: 10.3390/nu14132669

Figure Lengend Snippet: HCA stimulates unfolded protein response pathway. Cell lysate was prepared from K562 cells treated with indicated concentration of HCA. Proteins were separated and immunoblotted with antibody against desired protein. ( A ) HCA induces concomitant activation of AMPK and mTORC1 pathways in K562 cells. Samples were loaded in duplicate. One of them was used to immunoblot with phosphor form and the other was used to immunoblot respective total protein. Upper vinculin is referred to as p-AMPK, p-p70S6K, and pS6 ribosomal protein; lower vinculin is referred to as total AMPK, p70S6K, and S6 ribosomal protein. ( B ) HCA-treated K562 show upregulation of unfolded protein response markers ATF4 and p-elF2α. Upper vinculin is referred to as peIF2α and ATF4; lower vinculin is referred to as eIF2α. The bar graph beside each figure panel reflects the band intensity evaluated as optical density and represented as fold change for treated vs. untreated cells normalized for vinculin. ** p < 0.01, * p < 0.05 treated vs. untreated cells.

Article Snippet: Endogenous AMPK was immunoprecipitated using AMPKα antibody (Cell-Signaling, Danvers, MA, USA; #2523S).

Techniques: Concentration Assay, Activation Assay, Western Blot