Journal: American Journal of Cancer Research

Article Title: High expression of peroxisomal D-bifunctional protein in cytosol regulates apoptosis and energy metabolism of hepatocellular carcinoma cells via PI3K/AKT pathway

doi:

Figure Lengend Snippet: Cytosolic DBP promoted HepG2 cell proliferation via PI3K/AKT/FOXO3a/Bim pathway. A, B. Representative WB images of DBP, p-AKT, AKT, p-FOXO3a, FOXO3a, p-JNK, JNK, and Bim. β-actin served as a loading control. The quantitation of WB data was presented. C. Representative WB images of FOXO3a in the nuclear fraction. LAMN and GAPDH were nuclear and cytosolic markers, respectively. The quantitation of WB data was presented. D. The IF staining of FOXO3a (red), DAPI (blue), and their merge (purple). Quantitation of PCC data was presented. Scale bars = 10 μm. E. qRT-PCR to detect the expression of Bim. F. Apoptosis was assessed by flow cytometry with propidium iodide (PI)/Annexin V staining and the corresponding apoptosis ratio (%). Q2 region indicated late apoptotic cells with necrosis cells and mechanically damaged cells. Q4 indicated early apoptotic cells. G. Colony formation assay. H. CCK-8 assay. HepG2 cells were infected with control adenovirus (Empty), DBP-SKL, or DBP-deSKL. HepG2 cells were also treated with non-specific siRNA (siNC), DBP specific siRNA (siDBP), or AKT specific siRNA (siAKT). MK: MK2206, AKT inhibitor. LY: LY294002, PI3K inhibitor. Data were presented as mean ± SD. *P<0.05, **P<0.01 and ***P<0.001 vs. Empty group; #P<0.05, ##P<0.01 and ###P<0.001 vs. DBP-SKL group; •P<0.05, ••P<0.01 and •••P<0.001 vs. DBP-deSKL group.

Article Snippet: Phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (GC15485; 20 mmol/L) and AKT inhibitor MK2206 (GC16304; 10 mmol/L) were purchased from GlpBio, New York, USA.

Techniques: Control, Quantitation Assay, Staining, Quantitative RT-PCR, Expressing, Flow Cytometry, Colony Assay, CCK-8 Assay, Infection

Journal: American Journal of Cancer Research

Article Title: High expression of peroxisomal D-bifunctional protein in cytosol regulates apoptosis and energy metabolism of hepatocellular carcinoma cells via PI3K/AKT pathway

doi:

Figure Lengend Snippet: Mitochondrial localization of DBP was p-AKT-dependent. (A-C) The IF staining of DBP (red) in MK2206-treated DBP-SKL- and DBP-deSKL-expressing HepG2 cells. Cells were also co-stained with peroxisomal marker PMP70 (green) (A), mitochondrial marker COXIV (green) (B), and GAPDH (green) (C). Scale bars = 10 μm. PCC data were quantified. (D) WB of p-DBP in MK2206-treated mitochondria of DBP-SKL- or DBP-deSKL-overexpressing HepG2 cells. (E) CO-IP of DBP and p-Akt in DBP-SKL-expressing cells. (F) The IF staining of DBP (red) with p-AKT (green) in MK2206-treated DBP-SKL-expressing HepG2 cells. PCC data were quantified. Scale bar = 10 μm. (G) WB of p-DBP in tumor tissues and the adjacent liver tissues. (H) WB of p-DBP in DBP-SKL-overexpressing cells. HepG2 cells were infected with control adenovirus (Empty), DBP-SKL, or DBP-deSKL. MK: MK2206, AKT inhibitor. Data were presented as mean ± SD. ***P<0.001 vs. Empty group; ###P<0.001 vs. DBP-SKL group; •••P<0.001 vs. DBP-deSKL group.

Article Snippet: Phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (GC15485; 20 mmol/L) and AKT inhibitor MK2206 (GC16304; 10 mmol/L) were purchased from GlpBio, New York, USA.

Techniques: Staining, Expressing, Marker, Co-Immunoprecipitation Assay, Infection, Control

Journal: American Journal of Cancer Research

Article Title: High expression of peroxisomal D-bifunctional protein in cytosol regulates apoptosis and energy metabolism of hepatocellular carcinoma cells via PI3K/AKT pathway

doi:

Figure Lengend Snippet: Cytosolic DBP regulated the production of glycogen and ATP in HepG2 cells via PI3K/AKT/GSK3β signaling pathway. A, B. WB of p-GSK3β and GSK3β. β-actin: loading control. Data were quantified. C. Glycogen levels in the indicated cells. D. Glucose uptake as indicated by 2-NBDG fluorescence intensity in the treated cells. Scale bar (left) = 100 μm, scale bar (right) = 30 μm. E. Glycogen staining by PAS in tumors and the adjacent liver tissues of HCC patients (n = 4). PAS-positive staining (magenta) was marked by arrows. Scale bars = 100 μm. F. WB of p-GSK3β in the mitochondrial fraction of cells. COXIV and β-actin: mitochondrial and cytosolic markers, respectively. Data were quantified. G. The IF staining of p-GSK3β (green), mitochondrial COXIV (red), and their merge (yellow). PCC data were quantified. Scale bars = 10 μm. H. The enzymatic activity of mitochondrial respiratory chain complex III in the treated cells. I. ATP levels in the treated cells. HepG2 cells were infected with control adenovirus (Empty), DBP-SKL, or DBP-deSKL. HepG2 cells were also treated with non-specific siRNA (siNC), DBP specific siRNA (siDBP), AKT specific siRNA (siAKT). MK: MK2206, AKT inhibitor. Data were presented as mean ± SD. *P<0.05, **P<0.01 and ***P<0.001 vs. Empty group; #P<0.05, ##P<0.01 vs. DBP-SKL group; •P<0.05, ••P<0.01 and •••P<0.001 vs. DBP-deSKL group.

Article Snippet: Phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (GC15485; 20 mmol/L) and AKT inhibitor MK2206 (GC16304; 10 mmol/L) were purchased from GlpBio, New York, USA.

Techniques: Control, Fluorescence, Staining, Activity Assay, Infection

Journal: Science signaling

Article Title: The transcriptional repressor REST drives lineage-stage-specific chromatin compaction at Ptch1 and AKT hyperactivation in medulloblastoma

doi: 10.1126/scisignal.aan8680

Figure Lengend Snippet: (A to C) IHC was performed with p-AKTSer473 or PTEN specific antibodies in (A) Ptch+/− and Ptch+/−/RESTTG tumors (n=3) (B) DAOY and DAOY-REST xenografts (n=3) and (C) human SHH- subgroup patient derived xenografts (n=3). (D) PTEN mRNA expression profile was measured by microarray. Hierarchical clustering based on expression levels of neuronal differentiation markers divided the SHH MB patient samples into six distinct clusters. Each dot corresponds to one individual patient. (E) Pten mRNA expression was measured in WT and\RESTTG CGNPs after culturing with proliferation or differentiation media. Graph represents fold change compared to WT proliferating controls. WT data represents the mean ± S.D. from triplicate samples, RESTTG data represents two individual pups. (F) Western blot analysis was used to measure p-AktSer473 and total Akt protein expression in WT (n=2) and RESTTG (n=2) CGNPs after culturing with proliferation or differentiation media. Representative Western images of p-AktSer473, Akt, and input control histone H3 are shown. (G) p-AktSer473 and total Akt protein expression were measured after 5 hours of treatment of proliferating and differentiating WT and RESTTG CGNPs with MK2206 (1 or 5μM) (n=2). Representative Western images of p-AktSer473, Akt and histone H3 (control) are shown. For (A, B, and C), scale bars = 20 μm (40X). For (E), p values for qRT-PCR were calculated by paired two-tailed t test of ΔCp values: significance is indicated as not significant (ns), p<0.05 (*), p<0.01 (**), p<0.001 (***), or p<0.0001 (****).

Article Snippet: DAOY, UW228, DAOY-HR vs DAOY-LR and UW426-HR vs UW426-LR cells were cultured in 96-well microplates and treated with various concentration of the AKT inhibitor MK2206 (US Biological Life Science), HDAC inhibitor MS275 (Cayman Chemicals, catalog # 13284) and G9a inhibitor UNC0638 (Cayman Chemicals, catalog # 10734), either alone or in combination for the time period indicated in the figures.

Techniques: Derivative Assay, Expressing, Microarray, Western Blot, Control, Quantitative RT-PCR, Two Tailed Test

Journal: Science signaling

Article Title: The transcriptional repressor REST drives lineage-stage-specific chromatin compaction at Ptch1 and AKT hyperactivation in medulloblastoma

doi: 10.1126/scisignal.aan8680

Figure Lengend Snippet: (A) Western blotting for basal protein abundance of REST, p-AKTSer473, total AKT, and histone H3 (control) in DAOY, UW426 and UW228 cells. Representative blots are shown; long/short indicate exposure times. (B and C) Western blotting for total and phosphorylated (p-AKTSer473) protein abundance after either (B) shRNA-mediated REST knockdown in UW228 and DAOY cells or (C) REST overexpression in DAOY cells. Blots are representative images of 3 experiments. (D) MTT assay-derived proliferation of UW228 and DAOY cells treated with various doses of MK2206 for 24, 48 or 72 hours. Data are means ± S.D. of 3 independent assays. (E) Western blotting for abundance of p-AKTSer473, total AKT, cleaved Caspase-3, cleaved PARP and histone H3 (control) to assess induction of apoptosis following treatment of UW228 cells with MK2206 (5 μM) for 12 or 24 hours. Representative blots are shown (n=3).

Article Snippet: DAOY, UW228, DAOY-HR vs DAOY-LR and UW426-HR vs UW426-LR cells were cultured in 96-well microplates and treated with various concentration of the AKT inhibitor MK2206 (US Biological Life Science), HDAC inhibitor MS275 (Cayman Chemicals, catalog # 13284) and G9a inhibitor UNC0638 (Cayman Chemicals, catalog # 10734), either alone or in combination for the time period indicated in the figures.

Techniques: Western Blot, Quantitative Proteomics, Control, shRNA, Knockdown, Over Expression, MTT Assay, Derivative Assay