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