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