Journal: Cell metabolism

Article Title: Systematic dissection of the metabolic-apoptotic interface in AML reveals heme biosynthesis to be a regulator of drug sensitivity

doi: 10.1016/j.cmet.2019.01.011

Figure Lengend Snippet:

Article Snippet: Cat# ab14745, RRID:AB_2213640; Rabbit polyclonal anti-Bax (N-terminus) EMD Millipore Cat# 06–499, RRID:AB_310143 Rabbit monoclonal anti-Bcl-2 (clone D55G8) antibody Cell Signaling Technology Cat# 4223, RRID:AB_1903909 Rabbit monoclonal anti-Bcl-xL (clone 54H6) antibody Cell Signaling Technology Cat# 2764, RRID:AB_2228008 Rabbit monoclonal anti-Mcl-1 (clone S-19) antibody Santa Cruz Biotechnology Cat# sc-819, RRID:AB_2144105 Rabbit monoclonal anti-Bim (clone C34C5) antibody Cell Signaling Technology Cat# 2933, RRID:AB_1030947 Rabbit polyclonal anti-Bid antibody Cell Signaling Technology Cat# 2002, RRID:AB_10692485 Rabbit monoclonal anti-total Bad (clone D24A9) antibody Cell Signaling Technology Cat# 9239, RRID:AB_2062127 Rabbit monoclonal anti-phospho-Bad (S112) (clone 40A9) antibody Cell Signaling Technology Cat# 5284, RRID:AB_560884 Rabbit monoclonal anti-phospho-Bad (S136) (clone D25H8) antibody Cell Signaling Technology Cat# 4366, RRID:AB_10547878 Rabbit monoclonal anti-PUMA (clone D30C10) antibody Cell Signaling Technology Cat# 12450 Rabbit monoclonal anti-NOXA (clone D8L7U) antibody Cell Signaling Technology Cat# 14766 Bacterial and Virus Strains lentiCRISPR v2 Sanjana et al. Nat.

Techniques: Virus, Expressing, Plasmid Preparation, Recombinant, Cell Fractionation, Membrane, Cell Culture, Cell Viability Assay, Activity Assay, Isolation, shRNA, Metabolic Labelling, Software

Journal: Journal of Virology

Article Title: Functional genomics screens reveal a role for TBC1D24 and SV2B in antibody-dependent enhancement of dengue virus infection

doi: 10.1128/jvi.01582-24

Figure Lengend Snippet: TBC1D24 and SV2B are required for efficient ADE but not direct infection. ( A–B ) (Left) Representative dose-response ADE curves for K562 ( A ) TBC1D24 KO clone, ( B ) SV2B KO clone, and genetically trans -complemented K562 KO cells infected with DENV2-GFP in the presence of serially diluted human anti-DENV IgG monoclonal antibody J9 . In each experiment, a K562 WT cell pool and an FcgRIIa KO clone were included as a control. Data points and error bars represent the mean and range of infection in duplicate wells, respectively. The graphs shown are representative of at least three independent experiments. (Right) Quantification of the area under the curve normalized to unmutagenized (WT) K562 cells from three independent dose-response ADE experiments, each represented as a data point. Horizontal lines and error bars indicate mean and standard deviation, respectively. P -values shown are from multiple paired student’s t -tests adjusted using the Benjamini-Hochberg method. ( C ) Efficiency of DENV2-GFP infection of the indicated K562-DCSIGN cells in the absence of antibodies. Shown are percentage infections for individual KO clones (data points) normalized to unmutagenized (WT) K562-DCSIGN cell pool, median (horizontal line within box), 25th to 75th percentile (box), and minimum and maximum (whiskers). Data are representative of two independent experiments, each performed in duplicate wells. Comparisons of direct infection efficiency of each KO cell line to WT were not statistically significant ( P > 0.05) as determined by multiple unpaired student’s t -tests adjusted using the Benjamini-Hochberg method.

Article Snippet: DENV1 UIS 998 (isolated in 2007, Cat# NR-49713), DENV2 US/BID-V594/2006 (isolated in 2006, Cat# NR-43280), DENV3/US/BID-V1043/2006 (isolated in 2006, Cat# NR-43282), and DENV4 strain UIS497 (isolated in 2004, Cat# NR-49724) were obtained from BEI Resources (Manassas, VA) and propagated on C6/36 cells.

Techniques: Infection, Control, Standard Deviation, Clone Assay

Journal: Journal of Virology

Article Title: Functional genomics screens reveal a role for TBC1D24 and SV2B in antibody-dependent enhancement of dengue virus infection

doi: 10.1128/jvi.01582-24

Figure Lengend Snippet: The role of TBC1D24 and SV2B in ADE is not limited to a single DENV serotype, antibody, or cell line. ( A–B ) Dose-response ADE assays performed on K562 cells with ( A ) DENV2-GFP in the presence of convalescent sera from three independent DENV-immune donors or ( B ) fully infectious DENV1-4 in the presence of anti-DENV monoclonal IgG antibody ( J9 ). Data points represent the mean and the error bars represent the range of infection in duplicate wells, respectively. The graphs shown are representative of two independent experiments. ( C–D ) Dose-response ADE assays performed on clonal U937 cells with a KO in ( C ) SV2B or ( D ) TBC1D24 using DENV2-GFP in the presence of J9 monoclonal antibody. Data points represent the mean and the error bars represent the range of infection in duplicate wells. The data shown is representative of four independent experiments, each performed in duplicate wells. In each experiment, a WT U937 cell pool and a U937 FcgRIIa KO clone were included as controls.

Article Snippet: DENV1 UIS 998 (isolated in 2007, Cat# NR-49713), DENV2 US/BID-V594/2006 (isolated in 2006, Cat# NR-43280), DENV3/US/BID-V1043/2006 (isolated in 2006, Cat# NR-43282), and DENV4 strain UIS497 (isolated in 2004, Cat# NR-49724) were obtained from BEI Resources (Manassas, VA) and propagated on C6/36 cells.

Techniques: Infection

Journal: Journal of Virology

Article Title: Functional genomics screens reveal a role for TBC1D24 and SV2B in antibody-dependent enhancement of dengue virus infection

doi: 10.1128/jvi.01582-24

Figure Lengend Snippet: TBC1D24 and SV2B mediate efficient binding of DENV-IgG complexes to cells. ( A ) Quantitative RT-PCR of DENV RNA harvested from cell-surface and internalized virions in K562 cells at the indicated time points (x-axis) (see Materials and Methods for details). Bars represent the mean normalized to WT at each time point from at least four independent experiments (data points) performed in duplicate wells and error bars show the standard deviation. ( B ) Relative luminescence of the indicated K562 cells electroporated with Renilla luciferase-expressing DENV2 replicon and lysed at indicated time points. Values for each cell line were normalized to the corresponding 0 h time point to account for differences in electroporation efficiencies. Data points show the mean of four independent experiments, and error bars show the standard deviation. ( C ) Median fluorescence intensity of FcgRIIa expression in permeabilized (total expression) or non-permeabilized (surface expression) K562 cells. Bars represent the mean from at least four independent experiments (data points) and error bars show the standard deviation. ( D ) Histograms of FcgRIIa expression from a representative experiment of the data shown in ( C ). For ( A ) and ( C ), the P -values shown are from multiple unpaired student’s t -tests adjusted using the Benjamini-Hochberg method.

Article Snippet: DENV1 UIS 998 (isolated in 2007, Cat# NR-49713), DENV2 US/BID-V594/2006 (isolated in 2006, Cat# NR-43280), DENV3/US/BID-V1043/2006 (isolated in 2006, Cat# NR-43282), and DENV4 strain UIS497 (isolated in 2004, Cat# NR-49724) were obtained from BEI Resources (Manassas, VA) and propagated on C6/36 cells.

Techniques: Binding Assay, Quantitative RT-PCR, Standard Deviation, Luciferase, Expressing, Electroporation, Fluorescence

Journal: bioRxiv

Article Title: Differential regulation of BAX and BAK apoptotic activity revealed by a novel small molecule

doi: 10.1101/2024.08.04.605933

Figure Lengend Snippet: (A) Experimental workflow, and selection criteria employed for the discovery of inhibitors of human BAX-driven apoptosis. (B) Schematics of assays measuring different steps in the apoptosis pathway. (C) Dose-response curves for 17 hit compounds with mitochondria from BAK -/- KMS-12-PE and BAK -/- HeLa in JC-1 fluorescence assay. Data are from 1 independent experiment. (D) Chemical structures of Compound 1. (E) Dose-response curve for Compound 1 tested with BAK -/- KMS-12-PE in the primary screen measured by CellTiter-Glo ® . Data are mean ± S.E.M. of 2 independent experiments. (F) Chemical structures of WEHI-3773. (G) Dose-response curve for WEHI-3773 tested with BAK -/- KMS-12-PE in the primary screen measured by CellTiter-Glo ® . Data are mean ± S.E.M. of 2 independent experiments. (H) Dose response viability curves of BAK -/- KMS-12-PE in the presence of EC 70 concentration of ABT-737, measured by PI uptake and flow cytometry. Data are mean ± S.E.M. of 3 independent experiments. (I) Impact of WEHI-3773 on mitochondrial membrane potential measured by JC-1 staining and flow cytometry. Plasma-membrane-permeabilized BAK -/- KMS-12-PE were co-treated with EC 50 concentrations of human BIM-BH3 peptides. Data are mean ± S.E.M. of 3 independent experiments.

Article Snippet: BH3 mimetics venetoclax (cat# S8048, Selleckchem/Jomar Life Research), ABT-737 (Cat#A-1002, Active Biochem), A-1331852 (WEHI Chemical Biology Division) and S63845 (Cat#A-6044, Active Biochem) were used.

Techniques: Selection, Fluorescence, Concentration Assay, Flow Cytometry, Membrane, Staining

Journal: bioRxiv

Article Title: Differential regulation of BAX and BAK apoptotic activity revealed by a novel small molecule

doi: 10.1101/2024.08.04.605933

Figure Lengend Snippet: (A) Dose response viability curves of BAK -/- HeLa in the presence of EC 60 concentrations of ABT-737 and S63845, measured by PI uptake and flow cytometry. Data are mean ± S.E.M. of 3 independent experiments. (B) Impact of WEHI-3773 on mitochondrial membrane potential measured by JC-1 staining and flow cytometry. Plasma-membrane-permeabilized BAK -/- HeLa were co-treated with EC 60 concentrations of human BIM-BH3 peptides. Data are mean ± S.E.M. of 3 independent experiments. (C) Representative images of colony formation assay using BAK -/- HeLa cells after 14 days of culture in the presence of ABT-737 and S63845 together with WEHI-3773, Q-VD.oph or DMSO. (D) Quantification of colony number. Data are as mean values and S.E.M of 3 independent experiments. (E) Intra-cellular staining, and flow cytometry to measure conformationally activated BAX and cytochrome c within BAK -/- HeLa cells. (F) Western blot analysis of BAK -/- HeLa cells treated with WEHI-3773, ABT-737 and S63845 at indicated concentrations for 2 hours. Representative blots from at least n = 3 independent experiments are shown.

Article Snippet: BH3 mimetics venetoclax (cat# S8048, Selleckchem/Jomar Life Research), ABT-737 (Cat#A-1002, Active Biochem), A-1331852 (WEHI Chemical Biology Division) and S63845 (Cat#A-6044, Active Biochem) were used.

Techniques: Flow Cytometry, Membrane, Staining, Colony Assay, Western Blot

Journal: bioRxiv

Article Title: Differential regulation of BAX and BAK apoptotic activity revealed by a novel small molecule

doi: 10.1101/2024.08.04.605933

Figure Lengend Snippet: (A) Western blot analysis of Vdac2 -/- or Vdac2 +/+ MEF treated with WEHI-3773 alone at indicated concentrations for 2 hours. Representative blots of at least 3 independent experiments are shown. (B) Dose response viability curves of Bak -/- MEFs measured by PI uptake and flow cytometry. Cells were pre-treated with WEHI-3773 for 2 h first, then EC 70 concentrations of ABT-737 plus S63845 were added for another 22 h before measurement. Data are mean ± S.E.M. of 3 independent experiments. (C) Western blot analysis of BAK -/- mitochondria treated with WEHI-3773 or human BIM-BH3 peptide at indicated concentrations for 30 min, followed by blue native PAGE. Representative blots of at least 3 independent experiments are shown. (D) Proposed working model shows that WEHI-3773 acts on inhibiting BAX:VDAC2 interaction on mitochondria (gray) to limit MOMP and release of mitochondrial factors (brown dots).

Article Snippet: BH3 mimetics venetoclax (cat# S8048, Selleckchem/Jomar Life Research), ABT-737 (Cat#A-1002, Active Biochem), A-1331852 (WEHI Chemical Biology Division) and S63845 (Cat#A-6044, Active Biochem) were used.

Techniques: Western Blot, Flow Cytometry, Blue Native PAGE

Journal: bioRxiv

Article Title: Differential regulation of BAX and BAK apoptotic activity revealed by a novel small molecule

doi: 10.1101/2024.08.04.605933

Figure Lengend Snippet: (A) Dose response viability curves of KMS-12-PE in the presence of EC 50 concentration of ABT-737, measured by PI uptake and flow cytometry. Data are mean ± S.E.M. of 3 independent experiments. (B) Dose response viability curves of HeLa in the presence of EC 50 concentrations of ABT-737 plus S63845, measured by PI uptake and flow cytometry. Data are mean ± S.E.M. of 3 independent experiments. (C) Impact of WEHI-3773 on mitochondrial membrane potential measured by JC-1 staining and flow cytometry. Plasma-membrane-permeabilized BAX -/- or WT KMS-12-PE were co-treated with EC 50 concentrations of human BIM-BH3 peptides. Data are mean ± S.E.M. of 3 independent experiments. (D) Dose response viability curves of Bax -/- MEF measured by PI uptake and flow cytometry. Cells were pre-treated with WEHI-3773 for 2 h, then EC 30 concentrations of ABT-737 plus S63845 were added for another 22 h before measurement. Data are mean ± S.E.M. of 3 independent experiments. (E) Western blot analysis of BAX -/- HeLa treated with WEHI-3773, ABT-737 and S63845 at indicated concentrations for 2 h, followed by blue native PAGE. Representative blots of at least 3 independent experiments are shown. (F) Intra-cellular staining, and flow cytometry to measure conformationally activated BAK and preserved cytochrome c level within BAX -/- HeLa cells. (G) Schematic model for WEHI-3773 inhibiting the interaction of VDAC2 with BAK / BAX S184L on mitochondria (gray) to potentiate MOMP and release of mitochondrial factors (brown dots) into cytosol. (H) Dose response viability curves of Bak -/- Bax -/- MEFs re-expressing indicated BAX variants, measured by PI uptake and flow cytometry. Cells were pre-treated with WEHI-3773 for 2 h first, then EC 30 or EC 70 concentrations of ABT-737 plus S63845 were added for another 22 h before measurement. Data is mean ± S.E.M. of 3 independent experiments. (I) Intra-cellular staining and flow cytometry to measure conformationally activated BAX in BAK -/- or WT HeLa. (J) Working model of WEHI-3773 in WT cells.

Article Snippet: BH3 mimetics venetoclax (cat# S8048, Selleckchem/Jomar Life Research), ABT-737 (Cat#A-1002, Active Biochem), A-1331852 (WEHI Chemical Biology Division) and S63845 (Cat#A-6044, Active Biochem) were used.

Techniques: Concentration Assay, Flow Cytometry, Membrane, Staining, Western Blot, Blue Native PAGE, Expressing

Journal: bioRxiv

Article Title: Differential regulation of BAX and BAK apoptotic activity revealed by a novel small molecule

doi: 10.1101/2024.08.04.605933

Figure Lengend Snippet: (A) Western blot of lysates from MV4;11 cells transduced with empty vector or BAX gRNA. (B) BAX -/- MV4;11 cells were treated with the indicated concentration of BH3 mimetics with or without WEHI-3773 (200 nM) prior to assessment of cell viability after 48 h. (C-E) BAX -/- MV4;11 cells were treated with the indicated concentration of venetoclax (C), S63845 (D) or combination of venetoclax and S63845 (E), with or without WEHI-3773 prior to assessment of cell viability after 48 h. Data is mean ± S.E.M. of 3 independent experiments. Two-way ANOVA with Tukey test; **p < 0.01, ***p < 0.001. (F-H) OCI-AML3 cells rendered resistant to venetoclax ( BCL-2i R ) were treated with the indicated concentration of venetoclax (F), S63845 (G) or combination of venetoclax and S63845 (H), with or without WEHI-3773 prior to assessment of cell viability after 48 h. Data is mean ± S.E.M. of 3 independent experiments. Two-way ANOVA with Tukey test; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: BH3 mimetics venetoclax (cat# S8048, Selleckchem/Jomar Life Research), ABT-737 (Cat#A-1002, Active Biochem), A-1331852 (WEHI Chemical Biology Division) and S63845 (Cat#A-6044, Active Biochem) were used.

Techniques: Western Blot, Transduction, Plasmid Preparation, Concentration Assay

Journal: Translational Cancer Research

Article Title: Neochlorogenic acid inhibits gastric cancer cell growth through apoptosis and cell cycle arrest

doi: 10.21037/tcr-2025-696

Figure Lengend Snippet: Western blot analysis reveals NCA-mediated modulation of apoptosis-associated proteins in HGC-27 and NUGC-3 cells. Following a 48-hour treatment with control or NCA, the expression of BAX, CASP3, BID, CYCS, and BCL2 was examined by Western blotting. β-actin was used as a loading control. Protein levels were normalized to β-actin and expressed as fold changes relative to the untreated control group. Bar graphs depict the quantification of relative protein levels. Data represent mean ± standard deviation from three independent experiments (n=3). Statistical significance was determined by Student’s t -test. *, P<0.05; **, P<0.01; ***, P<0.001. BAX, BCL2-associated X protein; BCL2, B-cell lymphoma 2; BID, BH3 interacting domain death agonist; CASP3, cysteinyl aspartate specific proteinase 3; CYCS, cytochrome c, somatic; NCA, neochlorogenic acid.

Article Snippet: The membranes were then incubated overnight at 4 °C with primary antibodies for BCL2 (B-cell lymphoma 2) (Cat. No. bs-0032R; Bioss, Beijing, China), BID (BH3 interacting domain death agonist) (Cat. No. 10988-1-AP), CYCS (cytochrome c, somatic) (Cat. No. 10993-1-AP) from Proteintech (Wuhan, China), as well as β-actin (Cat. No. 81115-1-RR, Proteintech, Wuhan, China) and BAX (BCL2-associated X protein) (Cat. No. 50599-2-Ig, Proteintech, Wuhan, China).

Techniques: Western Blot, Control, Expressing, Standard Deviation

Journal: Oncotarget

Article Title: Induction of apoptosis and suppression of tumor growth by Nur77-derived Bcl-2 converting peptide in chemoresistant lung cancer cells

doi: 10.18632/oncotarget.25437

Figure Lengend Snippet: (A) Increased Bcl-2 expression in Paclitaxel resistant H460 cells. Western blot analysis of H460 parental and derived paclitaxel resistant cells. (B) High Bcl-2 expression in multidrug resistant H69AR and paclitaxel resistant H460 cells compared to parental H460 cells. Western blot analysis of multidrug resistant H69AR and H460 parental and paclitaxel resistant cells probed with indicated antibodies. (C) Increased phosphorylation of Bcl-2 at serine 70 in H460 paclitaxel resistant cells. Western blot analysis of H460 parental and paclitaxel resistant cells blotted with indicated antibodies. (D) Correlation of high Bcl-2 and increased resistance to paclitaxel. Western blot analysis of H460 parental and paclitaxel resistant cells treated with paclitaxel for 48 hours and probed with indicated antibodies. (E) Analysis of Bcl-2, Mcl-1 and Bcl-xL mRNA levels in parental and resistant H460 cells using quantitative real-time PCR. (F) Bcl-2 protein stability is not altered in parental and paclitaxel resistant cells. The level of Bcl-2 was detected by Western blot and GAPDH was used as a loading control. (G) Bcl-2 expression alone confers resistance to paclitaxel. Flow cytometry-based analysis of viability in H460 parental cells expressing Bcl-2 or control vector, after treatment with 100 nM paclitaxel for 48 hours; right panel: Western blot analysis of transfected cells show increased Bcl-2 expression. Results are the mean±s.d. Two-way ANOVA, with Sidaks multiple comparison post-test, **** = P<0.0001.

Article Snippet: Primary antibody Bcl-2 BH3 domain specific (cat# AP1303a) (Abgent, San Diego, CA) was added at a 1:30 dilution overnight at 4°C and then washed three times using cold PBS.

Techniques: Expressing, Western Blot, Derivative Assay, Real-time Polymerase Chain Reaction, Flow Cytometry, Plasmid Preparation, Transfection

Journal: Oncotarget

Article Title: Induction of apoptosis and suppression of tumor growth by Nur77-derived Bcl-2 converting peptide in chemoresistant lung cancer cells

doi: 10.18632/oncotarget.25437

Figure Lengend Snippet: (A) Annexin V staining of H460 parental and paclitaxel resistant cells after expression of control GFP or GFP-Nur77 peptide for 72 hours. Histogram gate indicates difference in annexin V positive cells between H460 parental (P) (blue) and paclitaxel resistant (R) (orange) cells. Data is representative of 3 independent experiments and quantification of apoptosis is shown in the right panel. (B) Annexin V staining of H460 parental (P) (blue) and resistant (R) (orange) cells after expression of control GFP or GFP-Nur77 peptide for after 96 hours. Histogram gate indicates difference in annexin V positive cells between H460 parental and paclitaxel resistant cells. Data is representative of 3 independent experiments and quantification of apoptosis is shown in the right panel. (C) Annexin V staining of H460 parental and resistant cells after expression of GFP-Nur77 peptide with or without co-treatment with paclitaxel (10 nM). Histogram gates indicate difference in annexin V positive cells between vehicle and paclitaxel 10 nM (Upper panels), Nur77 peptide expression alone and Nur77 expression combined with paclitaxel 10 nM (Lower panels). Data is representative of 3 independent experiments and quantification of apoptosis is shown in the right panel. (D) Annexin V staining of H69AR multidrug resistant lung cancer cells after expression of control GFP (black) or GFP-Nur77 peptide (blue), histogram gate indicates difference in annexin V positive cells between GFP and GFP-Nur77 peptide expression. The right panel is the quantification of percentage of apoptotic cells from the histograms. (E) Expression of Nur77 peptide induces cell death in a Bcl-2-dependent manner in multidrug resistant H69AR lung cancer cells. Flow cytometry-based analysis of cell death in Bcl-2 knockout H69AR CRISPR lines after expression of GFP-Nur77 peptide for 36 hours relative to control GFP expression. Suppression of Bcl-2 expression is confirmed by Western blot analysis of H69AR CRISPR lines expressing control CRISPR plasmid (Bcl-2 WT) and Bcl-2 targeted gRNA CRISPR plasmid (Bcl-2 KO). Results are the of mean±s.d. unpaired two-tailed t-test **** =P<0.0001.

Article Snippet: Primary antibody Bcl-2 BH3 domain specific (cat# AP1303a) (Abgent, San Diego, CA) was added at a 1:30 dilution overnight at 4°C and then washed three times using cold PBS.

Techniques: Staining, Expressing, Flow Cytometry, Knock-Out, CRISPR, Western Blot, Plasmid Preparation, Two Tailed Test

Journal: Oncotarget

Article Title: Induction of apoptosis and suppression of tumor growth by Nur77-derived Bcl-2 converting peptide in chemoresistant lung cancer cells

doi: 10.18632/oncotarget.25437

Figure Lengend Snippet: (A) Effect of NuBCP-9, NuBCP-9/AA (inactive form) on H460 parental and paclitaxel resistant lung cancer cells after 72 hours of treatment in 1% serum. Two-way ANOVA with Dunnett's multi comparisons post-test, **** =P<0.0001. (B) Annexin V staining of H460 parental and paclitaxel resistant cells treated with vehicle or 10 μM NuBCP-9 for 24 hours in 1% serum. Results are the mean±s.d. of three technical replicates. Two-way ANOVA with Dunnett's multi comparisons post-test, *** =P<0.001, **** =P<0.0001. (C) NuBCP-9 induces conformational change in Bcl-2 and exposes its BH3 domain. Parental and Resistant H460 cells were treated for 24 hours with vehicle or NuBCP-9 (10 μM) and immunostained with Bcl-2 BH3 specific antibody (upper panel), or Bcl-2 conformation independent antibody (lower panel) and analyzed by flow cytometry. Shift of peak to the right indicates BH3 domain exposure in upper panel. There is no such shift in the lower panels. (D) Effect of Paclitaxel, Doxorubicin (Doxo), NuBCP-9 and NuBCP-9/AA (inactive form) on multidrug resistant H69AR lung cancer cells after 72 hours of treatment in 1% serum. Two-way ANOVA with Dunnett's multi comparisons post-test, *** =P<0.001, **** =P<0.0001. (E) Annexin V staining of H69AR multidrug resistant cancer cells treated with vehicle, doxorubicin (100 nM) or NuBCP-9 (10 μM) for 36 hours. Results are the mean±s.d. of three technical replicates. Unpaired student t-test, ** =P<0.01. (F) NuBCP-9 reduces growth of multidrug resistant lung cancer cell 3D spheroids. H69AR spheroid cultures were grown for 48 hours and were then treated for 72 hours with vehicle, NuBCP-9 (6 and 10 μM). Percentage viability is calculated relative to vehicle treatment. Unpaired student t-test, **** =P<0.0001. (G) Representative images of zebrafish xenograft 1-day post injection (dpi) and 4 dpi. Red indicates dyed H460 resistant cells. (H) NuBCP-9 suppresses growth of paclitaxel resistant H460 cells in a zebrafish xenograft model. Growth of H460 paclitaxel resistant cells in xenograft zebrafish model, pre-treated with vehicle or NuBCP-9 (10 μM) for 6 hours prior to injection into zebrafish. Paclitaxel-resistant H460 cells were pre-treated with NuBCP-9 to ensure delivery of the peptide and avoid absorption issues in zebrafish embryos. n= 34 for vehicle and n = 27 for NuBCP-9. Results are the mean±SEM of two independent experiments. Students t-test, * P<0.05.

Article Snippet: Primary antibody Bcl-2 BH3 domain specific (cat# AP1303a) (Abgent, San Diego, CA) was added at a 1:30 dilution overnight at 4°C and then washed three times using cold PBS.

Techniques: Staining, Flow Cytometry, Injection

Journal: Oncotarget

Article Title: Induction of apoptosis and suppression of tumor growth by Nur77-derived Bcl-2 converting peptide in chemoresistant lung cancer cells

doi: 10.18632/oncotarget.25437

Figure Lengend Snippet: Lung cancer cells that have acquired resistance to paclitaxel express increased levels of Bcl-2 compared to the paclitaxel sensitive cells. High Bcl-2 in the chemoresistant cells confers increased sensitivity to Nur77 derived Bcl-2 converting peptide (NuBCP-9). NuBCP-9 converts Bcl-2 from a pro-survival protein into a killer protein through a conformational change exposing its BH3 domain and induces apoptosis in the paclitaxel resistant cells.

Article Snippet: Primary antibody Bcl-2 BH3 domain specific (cat# AP1303a) (Abgent, San Diego, CA) was added at a 1:30 dilution overnight at 4°C and then washed three times using cold PBS.

Techniques: Derivative Assay