Journal: Journal of visualized experiments : JoVE

Article Title: Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids

doi: 10.3791/60303

Figure Lengend Snippet: LIST OF MATERIAL/EQUIPMENT

Article Snippet: Chromogranin A antibody (clone LK2H10) , Thermo Scientific , MA5-13096 , Antibodies for IHC.

Techniques: Staining, Software, Microscopy, RNA Binding Assay, Plasmid Preparation, Electron Microscopy, Immunohistochemistry

Journal: Cancer Biology & Medicine

Article Title: PGK1-coupled HSP90 stabilizes GSK3β expression to regulate the stemness of breast cancer stem cells

doi: 10.20892/j.issn.2095-3941.2020.0362

Figure Lengend Snippet: Glycogen synthase kinase-3 (GSK3β) is a client protein of heat shock protein 90 (Hsp90). (A) The interaction between GSK3β and Hsp90 in MCF-7ADR cells detected by a co-immunoprecipitation (IP) assay. GSK3β was immunoprecipitated from cell lysates using an anti-GSK3β antibody. Immunoblotting analyses were performed with the indicated antibodies. (B) The interaction of GSK3β with Hsp90 co-chaperones in MCF-7ADR cells using a co-IP assay. GSK3β was immunoprecipitated from cell lysates using an anti-GSK3β antibody. Immunoblotting analyses were performed with the indicated antibodies. (C) The effect of Hsp90 knockdown on GSK3β expression. MCF-7ADR cells were transfected with a control siRNA (NC) or Hsp90 siRNA for 48 h. Immunoblotting analyses were performed with the indicated antibodies. (D) The effect of 17-AAG on GSK3β expression in MCF-7ADR cells. The cells were treated with 17-AAG for 12 h, followed by co-treatment with MG132 for 12 h. Immunoblotting analyses were performed with the indicated antibodies. (E) The effect of 17-AAG on polyubiquitination of GSK3β. MCF-7ADR cells were treated with 17-AAG for 12 h, followed by co-treatment with MG132 for 12 h. GSK3β was immunoprecipitated from cell lysates using an anti-GSK3β antibody Immunoblotting analyses were performed with the indicated antibodies. All experiments were performed independently and repeated at least 3 times.

Article Snippet: Anti-Aha1 antibody and His-GSK3β protein were obtained from Sino Biological (Beijing, China).

Techniques: Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Expressing, Transfection

Journal: Cancer Biology & Medicine

Article Title: PGK1-coupled HSP90 stabilizes GSK3β expression to regulate the stemness of breast cancer stem cells

doi: 10.20892/j.issn.2095-3941.2020.0362

Figure Lengend Snippet: PGK1 acts as an Hsp90 co-chaperone specifically regulating glycogen synthase kinase-3 (GSK3β) expression. (A) The interaction of GSK3β with Hsp90 and PGK1 in MCF-7ADR cells. GSK3β was immunoprecipitated using an anti-GSK3β antibody. Immunoblotting analyses were performed with the indicated antibodies. (B) The interaction of PGK1 with chaperones, co-chaperones, and client proteins. MCF-7ADR cells were transfected with 2 μg PGK1-Flag plasmid for 48 h. PGK1-Flag was immunoprecipitated using an anti-flag antibody. Immunoblotting analyses were performed with the indicated antibodies. (C) The interaction of Hsp90 with GSK3β and PGK1 in MCF-7ADR cells. Hsp90 was immunoprecipitated from cell lysates using an anti-Hsp90 antibody. Immunoblotting analyses were performed with the indicated antibodies. (D) The interaction of Hsp90 with GSK3β and PGK1 in clinical breast cancer tissues of 3 patients. Hsp90 was immunoprecipitated from lysates using an anti-Hsp90 antibody. Immunoblotting analyses were performed with the indicated antibodies. (E, G) PGK1 depletion (E) or (G) overexpression on client protein expression of Hsp90. MCF-7ADR cells were transfected with PGK1 siRNA (E) or PGK1-Flag (G) for 48 h. Immunoblotting analyses were performed with the indicated antibodies. (F) The effect of PGK1 knockdown on GSK3β expression in the indicated cells. Immunoblotting analyses were performed with the indicated antibodies. (H) The effect of PGK1 knockdown on the interaction between Hsp90 and GSK3β. MCF-7ADR cells were transfected with PGK1 siRNA. After 36 h, cells were treated with 20 μM MG132 for 12 h, and then subjected to IP using an anti-Hsp90 antibody. Immunoblotting analyses were performed with the indicated antibodies. (I, J) The effect of HOP (I) or CDC37 (J) knockdown on GSK3β expression. MCF-7ADR cells were transfected with siRNA for 48 h. Immunoblotting analyses were performed with the indicated antibodies. All experiments were performed independently and repeated at least 3 times.

Article Snippet: Anti-Aha1 antibody and His-GSK3β protein were obtained from Sino Biological (Beijing, China).

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

Journal: Cancer Biology & Medicine

Article Title: PGK1-coupled HSP90 stabilizes GSK3β expression to regulate the stemness of breast cancer stem cells

doi: 10.20892/j.issn.2095-3941.2020.0362

Figure Lengend Snippet: PGK1 binds to the C-terminus of Hsp90 in the “closed” state to promote the interaction between glycogen synthase kinase-3 GSK3β and Hsp90. (A) Constructed 293T cells stably expressing flag-tagged Hsp90α full-length (WT), N-, M-, or C- domains. Empty vector was used as a control. (B) The interaction of different Hsp90α domains with PGK1 and GSK3β. Different domains of Flag-Hsp90α were immunoprecipitated using an anti-Flag antibody. Immunoblotting analyses were performed with the indicated antibodies. (C, D) The interaction of PGK1 with the C-terminus (C) or M-domain (D) of Hsp90α in vitro . A total of 5 μg GST-C/M-Hsp90α and 2.5 μg His-PGK1 were incubated at 30 °C for 1 h. Immunoprecipitation (IP) was performed with an anti-GST antibody. Immunoblotting analyses were performed with the indicated antibodies. (E) The interaction of PGK1 with GSK3β in vitro . A total of 2.5 μg His-GSK3β and 2.5 μg His-PGK1 were incubated at 30 °C for 1 h. IP was performed with an anti-GSK3β antibody. Immunoblotting analyses were performed with the indicated antibodies. (F) PGK1 regulates the interaction between C-terminal Hsp90α and GSK3β in vitro . A total of 5 μg GST-C-Hsp90α and 2.5 μg His-GSK3β were incubated with or without 2.5 μg His-PGK1. IP was performed with an anti-GST antibody. Immunoblotting analyses were performed with the indicated antibodies. (G) The interaction of PGK1 with different conformations of Hsp90α. Flag-tagged Hsp90α wild type, D93A, or E47A mutant was stably expressed in 293T cells. Flag-tagged Hsp90 were immunoprecipitated. Immunoblotting analyses were performed with the indicated antibodies. (H) The binding mode of PGK1 with the “closed” conformation of Hsp90β. The N-terminal and C-terminal of Hsp90β are colored in blue and magenta, respectively, while the PGK1 is shown in green. All experiments were performed independently and repeated at least 3 times.

Article Snippet: Anti-Aha1 antibody and His-GSK3β protein were obtained from Sino Biological (Beijing, China).

Techniques: Construct, Stable Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, In Vitro, Incubation, Mutagenesis, Binding Assay

Journal: Cancer Biology & Medicine

Article Title: PGK1-coupled HSP90 stabilizes GSK3β expression to regulate the stemness of breast cancer stem cells

doi: 10.20892/j.issn.2095-3941.2020.0362

Figure Lengend Snippet: Hsp90 inhibitors targeting different domains of Hsp90 exhibit distinct inhibitory effects on BCSCs. (A) Flow cytometry (FCM) sorting of CD44 + CD24 −/low cells (BCSCs) and CD44 + CD24 + cells (non-BCSCs) from MCF-7ADR cells. (B) Immunofluorescence analysis of BCSCs and non-BCSCs. CD44 + CD24 −/low cells and CD44 + CD24 + cells were stained with an anti-GSK3β pS9 antibody and 4′,6-diamidino-2-phenylindole. (C) Immunoblotting analysis of BCSCs and non-BCSCs were performed with the indicated antibodies. (D) The effect of glycogen synthase kinase-3 knockdown on ALDH1A1 expression. MCF-7ADR cells were transfected with siRNA for 48 h. Immunoblot analyses were performed with the indicated antibodies. (E) The effect of PGK1 knockdown on ALDH1A1 expression. MCF-7ADR cells were transfected with siRNA for 48 h. Immunoblot analyses were performed with the indicated antibodies. (F, G) The effects of HDN-1 (F) and 17-AAG (G) on the proliferation of MCF-7ADR cells. The cells were treated with HDN-1 or 17-AAG for 48 h. Cell proliferation was determined using the MTT assay. (H-K) The effects of HDN-1 (H, I) and 17-AAG (J, K) on ALDH1A1 expression in MCF-7ADR cells. Cells were treated with HDN-1 or 17-AAG for 48 h. Immunoblot analyses of BCSCs and non-BCSCs were performed with the indicated antibodies. (L-O) The effects of HDN-1 (L, M) and 17-AAG (N, O) on mammosphere formation of MCF-7ADR cells. A total of 2,000 MCF-7ADR cells per well were cultured in serum-free medium and then treated with different concentrations of HDN-1 or 17-AAG. After 7 days, images were captured using a light microscope with a camera, and the number of mammospheres was quantified. The scale bar is 10.0 μm. * P < 0.05; ** P < 0.01 vs . dimethyl sulfoxide. All experiments were performed independently and repeated at least 3 times.

Article Snippet: Anti-Aha1 antibody and His-GSK3β protein were obtained from Sino Biological (Beijing, China).

Techniques: Flow Cytometry, Immunofluorescence, Staining, Western Blot, Expressing, Transfection, MTT Assay, Cell Culture, Light Microscopy

Journal: Cancer Biology & Medicine

Article Title: PGK1-coupled HSP90 stabilizes GSK3β expression to regulate the stemness of breast cancer stem cells

doi: 10.20892/j.issn.2095-3941.2020.0362

Figure Lengend Snippet: 17-AAG and HDN-1 have distinct effects on Hsp90-PGK1 interaction and subsequent expressions of GSK3β and β-catenin. (A, C) The effect of HDN-1 in a dose-dependent (A) or time-course (C) treatment on the expressions of proteins related to Akt-GSK3β-β-catenin signaling in MCF-7ADR cells for 24 h. (B) The effect of HDN-1 on the phosphorylation level of β-catenin at Ser 45 in MCF-7ADR cells. (D) The effect of HDN-1 on polyubiquitination of glycogen synthase kinase-3 (GSK3β). MCF-7ADR cells were treated with HDN-1 for 12 h, followed by co-treatment with MG132 for 12 h. GSK3β was immunoprecipitated from cell lysates using an anti-GSK3β antibody. (E, F) The effect of 17-AAG in a dose-dependent (E) or time-course (F) treatment on the expression of Akt, GSK3β, p-β-catenin at Ser45, and β-catenin in MCF-7ADR cells. (G, H) The effects of HDN-1 (G) and 17-AAG (H) on the expressions of Akt, GSK3β, and β-catenin. The 293T cells were treated with HDN-1 or 17-AAG for 24 h. (I, J) The effects of HDN-1 (I) and 17-AAG (J) on the binding of Hsp90 to Akt and GSK3β. MCF-7ADR cells were treated with HDN-1 or 17-AAG. Hsp90 was immunoprecipitated from cell lysates using an anti-Hsp90 antibody. (K, L) The effects of HDN-1 (K) and 17-AAG (L) on PGK1 expression. (M) The effects of HDN-1 and 17-AAG on the formation of the Hsp90α-PGK1-GSK3β complex in vitro . Hsp90α, His-PGK1, and His-GSK3β were incubated, followed by treatment with 1 μM HDN-1 or 17-AAG at 4 °C for 1 h. Immunoprecipitation was performed using an anti-Hsp90 antibody. (N) The effects of HDN-1 and 17-AAG on the interaction between PGK1 and the “closed” conformational Hsp90α. The 293T cells expressing Flag-tagged Hsp90-E47A were treated with HDN-1 or 17-AAG for 24 h. Flag-Hsp90-E47A was then immunoprecipitated from cell lysates. Immunoblotting analyses were performed with the indicated antibodies. All experiments were performed independently and repeated at least 3 times.

Article Snippet: Anti-Aha1 antibody and His-GSK3β protein were obtained from Sino Biological (Beijing, China).

Techniques: Immunoprecipitation, Expressing, Binding Assay, In Vitro, Incubation, Western Blot

Journal: Cancer Biology & Medicine

Article Title: PGK1-coupled HSP90 stabilizes GSK3β expression to regulate the stemness of breast cancer stem cells

doi: 10.20892/j.issn.2095-3941.2020.0362

Figure Lengend Snippet: Schematic of the distinct effects of HND-1 and 17-AAG on stemness of breast cancer by affecting Hsp90 regulation of glycogen synthase kinase-3 (GSK3β). The 17-AAG and HDN-1 had distinct effects on Hsp90-PGK1 interaction, resulting in different changes and the instability of GSK3β. They all inhibited AKT expression and its phosphorylation. HDN-1 significantly inhibited the Wnt-β-catenin cascade, yet 17-AAG did not affect the signaling pathway, leading to different effects on the stemness of breast cancer.

Article Snippet: Anti-Aha1 antibody and His-GSK3β protein were obtained from Sino Biological (Beijing, China).

Techniques: Expressing