Journal: Carcinogenesis

Article Title: Fisetin induces autophagic cell death through suppression of mTOR signaling pathway in prostate cancer cells

doi: 10.1093/carcin/bgq115

Figure Lengend Snippet: Effect of fisetin on mTOR target proteins. (A) Fisetin-mediated changes in mTOR target protein S6K70 and its substrates rpS6 and (B) eIF4B. (C) Western blot results of 4EBP1 and eIF4E are shown. Three different forms of 4EBP1 can be resolved by electrophoresis, the hyperphosphorylated γ form, the intermediately phosphorylated β form and the most mobile and non-phosphorylated α form. In western blots, anti-β-actin antibody was used to verify equal loading. Effect of fisetin on the formation of translation initiation complex by 7-methyl-GTP-Sepharose chromatography. (D) Cells were treated with 40, 80 and 120 μM of fisetin followed by affinity precipitation (AP) of the lysate with 7-methyl-GTP-Sepharose. The resulting affinity complex was washed and subjected to western blot using antibodies against 4EBP1, eIF4G and eIF4E. Effect of fisetin on Cap-dependent protein translation. (E) The structure of the bicistronic luciferase reporter construct is presented. The cassette is transcriptionally regulated by cytomegalovirus (CMV) promoter. The expression of Renilla luciferase (RLuc) is directed by Cap-dependent translation, whereas the firefly luciferase (FLuc) expression is driven by hepatitis C virus (HCV) internal ribosomal entry site (IRES)-dependent translation, i.e. Cap-independent translation. A stem-loop structure is inserted into the bicistronic structure to prevent the leaky scanning of the ribosome. (F) Cells grown in six-well plates were transfected with 1 μg of bicistronic plasmid per well and treated with fisetin 24 h after transfection. After 24 h of treatment, the luciferase activities of Renilla and firefly were determined using Dual Luciferase Reporter Assay System. The result is presented as relative Cap-dependent translation (%) compared with control that is calculated by (Renilla luciferase activity/Firefly luciferase activity) × 100. The data shown are representative results of three independent experiments and are presented as mean ± SEM; *P < 0.05 versus control.

Article Snippet: The non-targeting- and Beclin1-specific small interfering RNAs (siRNAs) were purchased from Dharmacon (Lafayette, CO) and cells were transfected using the nucleofection kit V specific for PC-3 transfection from Amaxa Biosystems (Gaithersburg, MD).

Techniques: Western Blot, Electrophoresis, Chromatography, Affinity Precipitation, Luciferase, Construct, Expressing, Transfection, Plasmid Preparation, Reporter Assay, Activity Assay

Journal: Carcinogenesis

Article Title: Fisetin induces autophagic cell death through suppression of mTOR signaling pathway in prostate cancer cells

doi: 10.1093/carcin/bgq115

Figure Lengend Snippet: Effect of fisetin on induction of autophagy. (A) Detection of autophagy by MDC. Cells were treated with either 120 μM of fisetin or VP16 for 48 h followed by detection of autophagy and apoptosis by MDC and Hoechst 33258, respectively. Autophagosome formation is indicated by arrows and fragmented chromatin is indicated by arrow heads; ×600. (B) Detection of autophagy by GFP-LC3. PC3 cells were transfected with GFP-LC3 and treated with or without 120 μM of fisetin for 24 h. Fluorescent microscope was used to detect the punctuate pattern (indicated by arrows) of GFP-LC3 (top panel). Also the number of cells with bright GFP-LC3 dots of total number of cells from five different fields was counted and compared with that of control (bottom panel). The data are presented as mean ± SEM; *P < 0.05 versus control, ×600. Effect of (C) Z-VAD and (D) CQ on fisetin and VP16-treated cells. Cells were treated with combination of fisetin (120 μM), VP16 (120 μM), Z-VAD (100 μM) and CQ (10 μM) as indicated. After 48 h of treatment, cells were subjected to viability assay. The data shown represent three independent experiments and are presented as mean ± SEM; *P < 0.05 versus paired control. (E) Effect of Beclin1-knockdown in PC3 cells. Western blot of Beclin1 is shown. Cells were transiently transfected with scrambled siRNA (sc) or indicated doses of Beclin1 siRNA (siBeclin1). Anti-β-actin antibody was used as loading control. (F) Viability assay of Beclin1-knockdown PC3 cells treated with fisetin or VP16. Equal number of cells was seeded in six-well plates and transfected with 100 pmol of scrambled siRNA (scrambled) or Beclin1 siRNA (siBeclin1). After 24 h of transfection, cells were treated with indicated doses of fisetin or VP16 for another 24 h followed by MTT assay. The average viability of scrambled siRNA-transfected cells was set to 100%. The data represent average of six independent experiments and are shown as mean ± SEM; *P < 0.05 versus control. (G) Effect of fisetin treatment on LC3-II protein expression in Beclin1-knockdown PC3 cells. Cells were transfected with 100 pmol of scrambled siRNA (scrambled) or Beclin1 siRNA (siBeclin1). After 24 h of transfection, cells were treated with indicated doses of fisetin or VP16 for another 24 h followed by western blot analysis. Western blot of β-actin was used to verify equal loading.

Article Snippet: The non-targeting- and Beclin1-specific small interfering RNAs (siRNAs) were purchased from Dharmacon (Lafayette, CO) and cells were transfected using the nucleofection kit V specific for PC-3 transfection from Amaxa Biosystems (Gaithersburg, MD).

Techniques: Transfection, Microscopy, Viability Assay, Western Blot, MTT Assay, Expressing

Journal: Oncotarget

Article Title: Identification of a structurally novel BTK mutation that drives ibrutinib resistance in CLL

doi: 10.18632/oncotarget.11932

Figure Lengend Snippet: A. Patient's absolute lymphocytosis (ALC) is plotted over disease and treatment course. The four sample collection time points, S1-S4, are shown. Blue section indicates time before CLL histologic progression; Red, time after progression; Green, time after ibr treatment, and Purple, time after RT relapse. The transient lymphocytosis following ibr treatment (green peak) is shown. BM, bone marrow; LN, lymph node; PB, peripheral blood; PE, pleural effusion. B. Morphologic progression of CLL to RT of large B-cell lymphoma. S1 (100x) , bone marrow biopsy at the time of diagnosis; proliferation centers (black stars) can be seen as vaguely pale nodular areas at low magnification. S1 (400x) , at high magnification occasional scattered prolymphocytes (white arrows) are seen. S2 (100x) , lymph node core biopsy taken at the time of CLL histologic progression. The lymph node architecture is effaced by a diffuse proliferation of small CLL cells. S2 (400x) , high magnification shows an increase in large atypical lymphoid cells and mitotic figures (white arrowheads). S4 (400x) , cell block from pleural effusion collected shortly before patient expired. Sheets of large lymphoid cells are present. S4 (1000x) , higher magnification shows highly atypical cells with frequent mitotic figures. C. Integrative Genomics Viewer (IGV) profile of BTK T316A mutation of the 3 samples. Data presented are results sequenced using the CLL panel (See Methods).

Article Snippet: For cell transfection with BTK WT, BTK C481S and BTK T316A mutant constructs, Amaxa Nucleofection technology was applied according to the manufacturer's protocols (Amaxa, Cologne, Germany; kit V, Program U-13).

Techniques: Biomarker Discovery, Blocking Assay, Mutagenesis

Journal: Oncotarget

Article Title: Identification of a structurally novel BTK mutation that drives ibrutinib resistance in CLL

doi: 10.18632/oncotarget.11932

Figure Lengend Snippet: A. Growth of TMD8 cells transfected with BTK T316A, C481S, and WT BTK constructs. The transfected cells were cultured with either 100 nM ibr or DMSO. The live cell numbers were counted daily to 7 days. The results represent four independent experiments. B. Cell proliferation evaluated with the BrdU incorporation assay. Cells transfected with WT BTK and T316A were treated with 100 nM ibr for 3 days and were labeled with 10 μM BrdU for 2 hrs.

Article Snippet: For cell transfection with BTK WT, BTK C481S and BTK T316A mutant constructs, Amaxa Nucleofection technology was applied according to the manufacturer's protocols (Amaxa, Cologne, Germany; kit V, Program U-13).

Techniques: Transfection, Construct, Cell Culture, BrdU Incorporation Assay, Labeling