Journal: PLoS ONE

Article Title: JNK and Macroautophagy Activation by Bortezomib Has a Pro-Survival Effect in Primary Effusion Lymphoma Cells

doi: 10.1371/journal.pone.0075965

Figure Lengend Snippet: (A) Time course assay was performed on BC3 and BCBL1 cells treated with 20 nM of bortezomib (BZ) for 0, 4, 8, 16 hrs. Total cell lysates were prepared and immunoblotted with anti-BiP, anti-CHOP, anti IRE1α, anti-phospho-JNK (p-JNK 1/2) and t-JNK antibodies. Anti-tubulin was assayed as protein loading control. (B) Electron microscopy (EM) analysis was performed on BC3 and BCBL1 cells treated with Bortezomib (20 nM) for 16 hrs (BZ). Cells showed several signs of ER stress compared to untreated control (CT). More apoptotic features such as nuclear (N) condensation were observed in BCBL1. Er (Endoplasmic reticulum), G (Golgi Apparatus), N (Nucleus), Au (Autophagosomes), Ne (Nuclear Envelope), M (Mitochondria). Bars: 1 µm. Results are representative of three independent experiments.

Article Snippet: The BC3, BCBL1, JSC1 and BC1 PEL cell lines (ATCC) were cultured in RPMI 1640 10% Fetal Calf Serum (FBS) (Euroclone), glutamine and streptomycin (100 μg/ml) and penicillin (100U/ml) in 5% CO 2 at 37°C.

Techniques: Control, Electron Microscopy

Journal: PLoS ONE

Article Title: JNK and Macroautophagy Activation by Bortezomib Has a Pro-Survival Effect in Primary Effusion Lymphoma Cells

doi: 10.1371/journal.pone.0075965

Figure Lengend Snippet: (A) Electron microscopy (EM) on BC3 and BCBL1 cells treated with 20 nM of bortezomib after 16 hrs shows pronounced cell vacuolization (A a, a 4 ) with autophagosome membrane-bound compartment in different stage of maturation (A a 1, a 2 , a 4, a 5 ). Small intracellular structures consistent with aggresomes were evident in BCLB1 cells (A a 6 ). N (Nucleus), Au (Autophagosomes), M (Mitochondria), ag (aggresomes). Bars 1 µm. Results are representative of three independent experiments. (B) PEL cells were treated with bortezomib (20 nM), bafilomycin A (Baf) (10 nM) or combination of both. The western blot analysis was performed to detect the two isoforms of LC3 protein. The histograms indicate LC3-II/I and LC3-II/Actin ratio based on densitometric analysis (mean ± the standard deviation, n = 3 experiments). An antibody against β-actin was used as loading control.

Article Snippet: The BC3, BCBL1, JSC1 and BC1 PEL cell lines (ATCC) were cultured in RPMI 1640 10% Fetal Calf Serum (FBS) (Euroclone), glutamine and streptomycin (100 μg/ml) and penicillin (100U/ml) in 5% CO 2 at 37°C.

Techniques: Electron Microscopy, Membrane, Western Blot, Standard Deviation, Control

Journal: PLoS ONE

Article Title: JNK and Macroautophagy Activation by Bortezomib Has a Pro-Survival Effect in Primary Effusion Lymphoma Cells

doi: 10.1371/journal.pone.0075965

Figure Lengend Snippet: (A) BC3 and BCBL1 cells were treated with bortezomib (20 nM for 16 hrs) with or without pre-treatment with JNK inhibitor (SP600125) (20 µM). Total cell lysates were prepared and immunoblotted with the following antibodies: anti-LC3, anti-pJNK, anti-T-JNK and anti-p62. Anti-β-actin was used as loading control. The histograms indicate LC3-II/I and LC3-II/Actin ratio based on densitometric analysis (mean ± the standard deviation, n = 3 experiments). (B) BC3 cells were transfected with DN-JNK expression vector or with an control empty vector (CV) and after 16 hrs treated with bortezomib (20 nM) for an additional 16 hrs. Total cell lysates were prepared and immunoblotted with anti-LC3 and p62 antibodies. β-actin was used as internal control. The histograms indicate LC3-II/I and LC3-II/Actin ratio based on densitometric analysis (mean ± the standard deviation, n = 3 experiments). (C) BC3 and BCBL1 cells were treated with Bortezomib (20 nM) alone or in combination with JNK inhibitor (SP600125) (20 µM) or ERK inhibitor (PD98059) (10 µM) for 16 hrs. A western blotting was performed using the following antibodies: anti-pBcl2(S70) and anti-total Bcl2. β-actin was used as loading control.

Article Snippet: The BC3, BCBL1, JSC1 and BC1 PEL cell lines (ATCC) were cultured in RPMI 1640 10% Fetal Calf Serum (FBS) (Euroclone), glutamine and streptomycin (100 μg/ml) and penicillin (100U/ml) in 5% CO 2 at 37°C.

Techniques: Control, Standard Deviation, Transfection, Expressing, Plasmid Preparation, Western Blot

Journal: PLoS ONE

Article Title: JNK and Macroautophagy Activation by Bortezomib Has a Pro-Survival Effect in Primary Effusion Lymphoma Cells

doi: 10.1371/journal.pone.0075965

Figure Lengend Snippet: (A) Viability assay evaluated by trypan blue exclusion was performed in BC3 (left panel) and BCBL1 (right panel) cells treated with bortezomib (20 nM) or 3-MA (5 mM) alone or in combination for 16 hrs. Mean ± the standard deviation was also indicated (n = 3 experiments). * p-value = 0.05, ** p-value = 0.02. Western blotting analysis was performed on BC3 cells to evaluate the expression of cleaved (cl) PARP p85 fragment (middle panel). β-actin was used as loading control. (B) BC3 and BCBL1 cells were transfected with ATG5 siRNA or scramble siRNA (siRNASc), and than a western blot was performed with the anti-ATG5 antibody. β-actin was used as loading control. Viability assay evaluated by trypan blue exclusion was performed in BC3 and BCBL1 cells ATG5 or scramble-knocked down upon bortezomib treatment (20 nM) for 16 hrs. Mean ± the standard deviation was indicated (n = 3 experiments). ♦ p-value = 0.02, ♦♦ p-value = 0.03. Western blotting analysis was also performed on BC3 cells to evaluate the expression of PARP p85 fragment (cl PARP) and β-actin was used as internal control. (C) Cells viability assay on BC3 and BCBL1 cells treated with bortezomib (20 nM) and SP600125 (20 µM) alone or in combination for 16 hrs. The percentage of live cells was evaluated by trypan blue exclusion assay. Mean ± the standard deviation was indicated (n = 3 experiments). p-value = 0.01, p-value = 0.01. Western blotting analysis was performed on BC3 cells to evaluate the expression of PARP p85 fragment (cl PARP) and β-actin was used as loading control.

Article Snippet: The BC3, BCBL1, JSC1 and BC1 PEL cell lines (ATCC) were cultured in RPMI 1640 10% Fetal Calf Serum (FBS) (Euroclone), glutamine and streptomycin (100 μg/ml) and penicillin (100U/ml) in 5% CO 2 at 37°C.

Techniques: Viability Assay, Standard Deviation, Western Blot, Expressing, Control, Transfection, Trypan Blue Exclusion Assay

Journal: Cell Proliferation

Article Title: Apigenin induces apoptosis via downregulation of S‐phase kinase‐associated protein 2‐mediated induction of p27Kip1 in primary effusion lymphoma cells

doi: 10.1111/j.1365-2184.2009.00662.x

Figure Lengend Snippet: (a) Apigenin suppresses growth of PEL cells. BC1, BC3, BCBL1 and HBL‐6 cells were incubated in 0–100 μm apigenin for 24 h. Cell death assays were performed using trypan blue exclusion dye as described in the Materials and methods section. The graph displays mean ± SD (standard deviation) of three independent experiments with replicates of six wells for all doses, and vehicle control for each experiment. *P < 0.05, statistically significant (Student’s t‐test). (b) Apigenin treatment increases G2/M population at 25 μm and sub‐G1 (apoptotic) populations at 50 μm. BC1, BC3, BCBL1 and HBL‐6 cells were treated with 25 and 50 μm apigenin for 24 h. Thereafter, the cells were washed, fixed and stained with propidium iodide, and analysed for DNA content by flow cytometry as described in the Materials and methods section. At least three independent experiments were performed for all cell lines. Results from one representative experiment shown. (c) Apigenin‐induced apoptosis detected by Annexin V/PI dual staining. Cells were treated with 25 and 50 μm apigenin (as indicated) for 24 h and were subsequently stained with flourescein‐conjugated annexin‐V and propidium iodide (PI) and analysed by flow cytometry. (d) BC1 and BC3 cells were treated with 25 and 50 μm apigenin for 24 h and DNA was extracted and separated by electrophoresis on 1.5% agarose gel.

Article Snippet: Human PEL (BC1, BC3, BCBL1 and HBL‐6) cell lines were obtained from American Type Culture Collection (ATCC), (Rockville, MD, USA) and cultured in RPMI 1640 medium supplemented with 10% (v/v) foetal bovine serum (FBS), 100 U/ml penicillin and 100 U/ml streptomycin at 37 °C in a humidified atmosphere containing 5% CO 2 .

Techniques: Incubation, Standard Deviation, Control, Staining, Flow Cytometry, Electrophoresis, Agarose Gel Electrophoresis

Journal: Cell Proliferation

Article Title: Apigenin induces apoptosis via downregulation of S‐phase kinase‐associated protein 2‐mediated induction of p27Kip1 in primary effusion lymphoma cells

doi: 10.1111/j.1365-2184.2009.00662.x

Figure Lengend Snippet: Apigenin inhibits constitutive active AKT, Cyclin D1, Rb, SKP2 and p27 in PEL cells. (a) BC1, BC3 and BCBL1 cells were treated with various doses of apigenin as indicated and cytoplasmic extracts were prepared. Twenty micrograms protein from each sample was separated on SDS–PAGE and immunoblotted with phospho‐Akt‐Ser473, phospho‐Akt‐Thr308, total AKT, cyclin D1, phospho‐Rb, SKP2 and p27 antibodies. Blots were probed with antibody against beta‐actin for equal loading (bottom panel). (b) AKT siRNA expression inactivates AKT, down‐regulates SKP2 and accumulates p27Kip1. BC1 cells were transfected with Scrambled siRNA (100 nm) and AKT siRNA (50 and 100 nm) with Lipofectamine as described in the Materials and methods section. After 48 h transfection, cells were lysed and equal amounts of proteins were separated by SDS–PAGE, transferred to Immobilon membrane, and immunoblotted with antibodies against p‐AKT, SKP2, p27‐Kip1 and beta actin, as indicated. Representative of three separate independent experiments depicted in this figure. (c) SKP2 siRNA expression down‐regulates SKP2 and accumulates p27Kip1. BC1 cells were transfected with Scrambled siRNA (100 nm) and SKP2 siRNA (50 and 100 nm) with Lipofectamine as described in the Materials and methods section. After 48 h transfection, cells were lysed and equal amounts of protein were separated by SDS–PAGE, transferred to Immobilon membrane and immunoblotted with antibodies against SKP2, P27Kip1 and beta actin as indicated. Representative of three separate independent experiments depicted. (d) Apigenin treatment causes inactivation of downstream targets of AKT: BC1, BC3 and BCBL1 cells were treated with 25 and 50 μm apigenin and cytoplasmic extracts were prepared. Twenty micrograms protein from each sample was separated on SDS–PAGE and immunoblotted with phospho‐FOXO1, FOXO‐1, phospho‐Bad, Bad and actin for equal loading. (e) 5 × 106 cells were treated with and without apigenin for 24 h, immunoprecipitated using Bad antibody and proteins were separated on SDS–PAGE. Blots were probed with 14:3:3 Sigma antibody. Lysates were separated on SDS–PAGE, and immunoblotted with Bad antibody to insure equal loading.

Article Snippet: Human PEL (BC1, BC3, BCBL1 and HBL‐6) cell lines were obtained from American Type Culture Collection (ATCC), (Rockville, MD, USA) and cultured in RPMI 1640 medium supplemented with 10% (v/v) foetal bovine serum (FBS), 100 U/ml penicillin and 100 U/ml streptomycin at 37 °C in a humidified atmosphere containing 5% CO 2 .

Techniques: SDS Page, Expressing, Transfection, Membrane, Immunoprecipitation

Journal: Cell Proliferation

Article Title: Apigenin induces apoptosis via downregulation of S‐phase kinase‐associated protein 2‐mediated induction of p27Kip1 in primary effusion lymphoma cells

doi: 10.1111/j.1365-2184.2009.00662.x

Figure Lengend Snippet: Apigenin‐induced mitochondrial signalling pathways in primary effusion lymphoma cells. (a) Apigenin treatment causes alteration in Bcl‐2 expression. BC1 cells were treated with various doses of apigenin. Cells were lysed and equal amounts of proteins were separated by SDS–PAGE, transferred to Immobilon membrane, and immunoblotted with antibodies against Bax, Bcl‐2 and beta actin as indicated (upper panel). Data obtained from immunoblot analyses of Bax and Bcl‐2 were used to evaluate effects of apigenin on Bax/Bcl‐2 ratio. Densitometric analysis of Bax and Bcl‐2 bands was performed using AlphaImager Software (San Leandro, CA, USA), and data (relative density normalized to β‐actin) were plotted as Bax/Bcl‐2 ratio. (b) Apigenin‐induced Bax activation. After treating with 50 μm apigenin for indicated time periods, BC1 and BC3 cells were lysed in 1% Chaps lysis buffer and subjected to immunoprecipitation with either anti‐Bax 6A7 antibody or non‐specific IgG, for detection of conformationally changed Bax protein. In addition, total cell lysates were applied directly to SDS–PAGE, transferred to Immobilon membrane and immunoblotted with specific anti‐Bax polyclonal antibody. (c) Loss of mitochondrial potential by apigenin treatment. Cells were treated with and without 25 and 50 μm apigenin for 24 h. Live cells with intact mitochondrial membrane potential (red bars) and dead cells with lost mitochondrial membrane potential (green bars) were measured by JC‐1 staining and analysed by flow cytometry as described in the Materials and methods section. Average of three independent experiments is depicted. (d) Apigenin‐induced release of cytochrome c. BC1 and BC3 cells were treated with and without 25 and 50 μm apigenin for 24 h. Mitochondrial‐free cytoplasmic fractions as well as mitochondrial extracts were isolated as described in the Materials and Methods sections. Cell extracts were separated on SDS–PAGE, transferred to PVDF membrane, and immunoblotted with an antibody against cytochrome c. Beta‐actin was used for equal loading.

Article Snippet: Human PEL (BC1, BC3, BCBL1 and HBL‐6) cell lines were obtained from American Type Culture Collection (ATCC), (Rockville, MD, USA) and cultured in RPMI 1640 medium supplemented with 10% (v/v) foetal bovine serum (FBS), 100 U/ml penicillin and 100 U/ml streptomycin at 37 °C in a humidified atmosphere containing 5% CO 2 .

Techniques: Expressing, SDS Page, Membrane, Western Blot, Software, Activation Assay, Lysis, Immunoprecipitation, Staining, Flow Cytometry, Isolation