Journal: Cell death & disease

Article Title: Cancer-derived exosomal HSPC111 promotes colorectal cancer liver metastasis by reprogramming lipid metabolism in cancer-associated fibroblasts.

doi: 10.1038/s41419-022-04506-4

Figure Lengend Snippet: Fig. 6 An increase in levels of acetyl-CoA and H3K27ac promotes CXCL5 secretion in CAFs. The effects of acetate treatment on acetyl-CoA levels in LX-2 cells incubated with ExoHCT116 (A) and ExoSW480 (B). Western blot showed the effects of acetate treatment on CXCL5 and H3K27ac levels in LX-2 cells incubated with ExoHCT116 (C) and ExoSW480 (D). E The effects of Trichostatin A (TSA) treatment on CXCL5 and H3K27ac levels in LX-2 cells incubated with ExoHCT116 KD and their relative control. F The effects of C646 treatment on CXCL5 and H3K27ac levels in LX-2 cells incubated with ExoSW480 OE and their relative control. G, H Chromatin immunoprecipitation (ChIP) assays using IgG as a control were performed with antibody against H3K23ac. Examination of H3K27 acetylation status in CXCL5 gene promoter region in LX-2 cells incubated with ExoHCT116 KD and ExoHCT116 KD Ctrl or in LX-2 cells incubated with ExoHCT116 KD and ExoHCT116 KD Ctrl and treated with TSA (G). Examination of H3K27 acetylation status in CXCL5 gene promoter region in LX-2 cells incubated with ExoSW480 OE and ExoSW480 OE Ctrl or in LX- 2 cells incubated with ExoSW480 OE and ExoSW480 OE Ctrl and treated with C646 (H). Each experiment was performed in triplicate. Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Cells were cultured in the presence of appropriate exosomes (20μg/mL) for 48 h before supernatants were collected, and the CXCL5 level was detected by CXCL5 ELISA kit (EK0728, BOSTER) according to the manufacturer’s protocols.

Techniques: Incubation, Western Blot, Control, Chromatin Immunoprecipitation

Journal: Cell death & disease

Article Title: Cancer-derived exosomal HSPC111 promotes colorectal cancer liver metastasis by reprogramming lipid metabolism in cancer-associated fibroblasts.

doi: 10.1038/s41419-022-04506-4

Figure Lengend Snippet: Fig. 8 A schematic diagram of mechanism of exosomal HSPC111 facilitates CRLM via reprogramming lipid metabolism in CAFs. Exosomal HSPC111 derived from CRC cells phosphorylates ACLY in CAFs, leading to the increased levels of acetyl-CoA and histone acetylation to secrete CXCL5, and resulting in CRC cells colonized in liver via the CXCL5-CXCR2 axis.

Article Snippet: Cells were cultured in the presence of appropriate exosomes (20μg/mL) for 48 h before supernatants were collected, and the CXCL5 level was detected by CXCL5 ELISA kit (EK0728, BOSTER) according to the manufacturer’s protocols.

Techniques: Derivative Assay

Journal: Molecular cell

Article Title: The MYCN oncoprotein is an RNA-binding accessory factor of the nuclear exosome targeting complex.

doi: 10.1016/j.molcel.2024.04.007

Figure Lengend Snippet: Figure 4. Exosome function controls the dynamics of the MYCN/MAX/MXD network (A) Immunoblot of IMR-32 cells stably expressing an shRNA targeting EXOSC10 treated for 72 h with Dox or EtOH as control. ACTB was loading control. Blot is representative of n = 3 independent replicates. (B) Read distribution of MYCN ChIP-Rx, MAX, MNT, and SIN3A cleavage under targets & release using nuclease (CUT&RUN) at the CDK5RAP3 and PHB genes. Cells treated as in (A) (n = 3 for MYCN ChIP-Rx; n = 2 for all C&Rs).

Article Snippet: 3 mg of each, MAX (Proteintech), MNT (Thermo Fisher Scientific), or SIN3A (Novus Biologicals) antibody were added per sample and incubated with shaking (800 rpm) at 4 C overnight.

Techniques: Western Blot, Stable Transfection, Expressing, shRNA, Control

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 1. DDB1 interacts with and cleaved by NS3/4A. (A) DDB1 interacts with NS3/4A in overexpression system. The 293 cells were transfected with the indicated plasmids. Coimmunoprecipitation was performed with anti-Flag or control IgG. The immunoprecipitates were analyzed by immunoblot with anti-Flag anti-HA. The lysates were analyzed by immunoblots with anti-DDB1 or anti-HA. (B) Endogenous DDB1 interacts with NS3/4A in JFH-1 infected cells. Huh-7 cells (5 107) were mock-infected or infected with JFH-1 (Multiplicity of Infection, MOI: 0.3) for 3 days. Coimmunoprecipitation was performed with anti-DDB1 or control IgG. The immunoprecipitates were analyzed by immunoblot with anti-DDB1 and anti-NS3. The lysates were analyzed by immunoblots with anti-DDB1 or anti-NS3.

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Over Expression, Transfection, Control, Western Blot, Infection

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 2. NS3/4A cleaves DDB1 at C378. (A) Cleavage of DDB1 by NS3/4A is inhibited by the NS3/4A inhibitor VX-950. The 293 cells were transfected with N-terminal or C-terminal Flag-tagged DDB1 (N-Flag-DDB1 or DDB1-C-Flag respectively) and HA-NS3/4A. The transfected cells were treated with VX-950 (0.2 mM) or left untreated for 1 day before immunoblot analysis with anti-Flag or anti-HA. (B) Alignment of the junction sequences of NS proteins of HCV and the potential NS3/4A cleavage sites in TC-PTP, VISA, TRIF and DDB1. (C) NS3/4A cleaves DDB1 at C378. The 293 cells were transfected with the indicated plasmids and cells lysates were analyzed by immunoblots with anti-Flag or anti-HA. (D) DDB1 N-terminal cleavage product migrated similarly to overexpressed DDB1(1–378) mutant. The 293 cells were transfected with the indicated plasmids, treated with VX-950 or left untreated for 1 day before immunoblot analysis with with anti-Flag or anti-HA.

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Transfection, Western Blot, Mutagenesis

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 3. DDB1 plays a critical role in HCV replication. (A) Overexpression of DDB1 potentiates HCV RNA replication. Huh-7 cells (1 106) were transfected with the indicated amounts of Flag-DDB1 plasmid for 24 h and then cells were split and mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were determined by RT-qPCR and normalized to cellular GAPDH mRNA levels. The uninfected cell lysates were analyzed by immunoblots with anti-Flag or anti-b-actin. Graphs show mean7SD, n¼3. (B) Knockdown of DDB1 inhibits HCV RNA replication. Control or DDB1-RNAi knockdown Huh-7 cells were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The uninfected cells lysates were also analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show meanþSD, n¼3. (C) Knockdown of DDB1 inhibits HCV protein expression. Control or DDB1-RNAi knockdown Huh-7 cells were mock-infected or infected with JFH-1 for 3 days, and the cells were then analyzed by immunofluorescent staining with anti-E2 (red), and Hoechst (blue). (D) Knockdown of DDB1 inhibits production of infectious HCV particles. Control or DDB1-RNAi knockdown Huh-7 cells were mock-infected or infected with JFH-1 for 24 h. The cells were completely washed and fresh complete medium was added for 48 h. The JFH-1 infected medium was collected and diluted for infection of Huh-7.5.1 cells. Three days later, cells were analyzed by immunofluorescent staining with anti-E2 and HCV titers were calculated by counting positive stained cells foci. Graphs show mean7SD, n¼3. (E) Knockdown of DDB1 inhibits RNA replication of HCV subgenomic replicon. Control or DDB1-RNAi knockdown Huh-7 cells and Huh-7 Con1 subgenomic replicon cells were cultured for 3 days. The cells (2 106) were collected and intracellular HCV RNA levels were determined by RT-qPCR and normalized to cellular GAPDH mRNA levels. Cell lysates were analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show mean7SD, n¼3. (F) DDB1 has no effects on HCV entry. Control or DDB1-RNAi knockdown Huh-7 cells were infected with HCVpp for 3 days (NC: Negative Control, HCVpp pakaging without HCV E1E2). The lysates of infected cells were assayed by luciferase reporter assays and immunoblots with anti-DDB1 or anti-b-actin. Graphs show mean7SD, n¼3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Over Expression, Transfection, Plasmid Preparation, Infection, Quantitative RT-PCR, Western Blot, Knockdown, Control, Expressing, Staining, Cell Culture, Negative Control, Luciferase

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 4. DDB1 cleavage is required for HCV replication. (A) The indicated stable cell lines were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The uninfected cells lysates were also analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show meanþSD, n¼3. (B) The indicated stable cells were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. The cells were then analyzed by immunofluorescent staining with anti-E2 (red), and Hoechst (blue). (C) The indicated stable cell lines were mock-infected or infected with JFH-1 (MOI: 0.3) for 24 h. The cells were completely washed and fresh medium was added for 48 h. The JFH-1-containing medium was collected and diluted for infection of Huh-7.5.1 cells. Three days later, cells were analyzed by immunofluorescent staining with anti-E2 and HCV titers were calculated by counting positive stained cells foci. Graphs show meanþSD, n¼3. (D) Control or DDB1-RNAi knockdown Huh-7 cells were stably transduced with empty vector, DDB1, DDB1(C378R), off-target nonsense mutants of DDB1 or DDB1(C378R) respectively. Two days later, cells were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The cells lysates were also analyzed by immunoblots with anti-DDB1 or anti-actin. Graphs show meanþSD, n¼3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Stable Transfection, Infection, Quantitative RT-PCR, Western Blot, Staining, Control, Knockdown, Transduction, Plasmid Preparation

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 5. DDB1 cleavage products do not affect the HCV infection. (A) Huh-7 cells stably transduced with the indicated DDB1 truncation mutants were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The uninfected cell lysates were analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show meanþSD, n¼3. (B) Huh-7 cells stably transduced with the indicated DDB1 truncation mutants were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. The cells were then analyzed by immunofluorescent staining with anti-E2 (red), and Hoechst (blue). (C) Huh-7 cells stably transduced with the indicated DDB1 truncation mutants were mock-infected or infected with JFH-1 (MOI: 0.3) for 24 h. The cells were completely washed and fresh complete medium was added for 48 h. The JFH-1 infected medium was collected and diluted for infection of Huh-7.5.1 cells. Three days later, cells were analyzed by immunofluorescent staining with anti-E2 and HCV titers were calculated by counting positive stained cells foci. Graphs show meanþSD, n¼3. (D–F) Control or DDB1-RNAi-#1 (targeted sequence is within the cDNA fragment encoding aa379–1140) transduced Huh-7 cells were further transfected with empty vector, Flag-DDB1(1–378), Flag-DDB1(379–1140*) (*, a RNAi off-target mutant),or a combination of Flag-DDB1(1–378) and Flag-DDB1(379–1140*) by Lipofectamine 2000. One day post transfection, the cells were split and mock infected or infected with JFH-1(MOI: 0.3) for 3 (D, E) or 1 (F) day. Intracellular HCV RNA levels (D), intracellular viral particles (E), or viral titers in the medium (F) were then determined as described in (A–C). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Infection, Stable Transfection, Transduction, Quantitative RT-PCR, Western Blot, Staining, Control, Sequencing, Transfection, Plasmid Preparation, Mutagenesis

Journal: Cell Death and Differentiation

Article Title: Hepatocyte growth factor renders BRAF mutant human melanoma cell lines resistant to PLX4032 by downregulating the pro-apoptotic BH3-only proteins PUMA and BIM

doi: 10.1038/cdd.2016.96

Figure Lengend Snippet: Inhibition of BCL-XL but not inhibition of BCL-2 cooperates with PLX4032 in the killing of BRAFV600E mutant melanoma cell lines. (a) M14, UACC257, Malme3M and SKMEL5 BRAFV600E cell lines were treated with DMSO (vehicle; -) or PLX4032 (+ at 3 μM) in the presence of the pan-caspase inhibitor Q-VD-OPh for 24 h before western blot analysis to detect the indicated pro-survival BCL-2-like proteins. Probing with an antibody to HSP70 was used as a loading control. (b) BRAFV600E mutant melanoma cell lines from panel (a) were treated with DMSO, PLX4032 (3 μM) and/or (c) ABT-737 (1 μM; inhibitor of BCL-2, BCL-XL and BCL-W), ABT-199 (1 μM; inhibitor of BCL-2) or ABT-1155463 (1 μM; inhibitor of BCL-XL). Cell survival was assessed after 5 days of treatment by flow cytometric analysis. Living cells were identified as those negative for Annexin V and propidium iodide. The data are derived from three independent experimental replicates, each comprising the average of three technical replicates and are presented as mean±S.E.M.; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001

Article Snippet: Antibodies (clone) were purchased or obtained from the sources indicated: anti-BIM (Enzo Life Sciences, Farmingdale, NY, USA, cat# ADI-AAP-330-E), anti-PUMA (Abcam, Cambridge, UK, cat# 27669), anti-BMF (WEHI, clone 12E10), anti-NOXA (ProSci, Poway, CA, USA, cat# 2437), anti-MCL-1 (WEHI, clone 19C4-15), anti-BCL-2 (WEHI, clone Bcl-2-100), anti-BCL-XL (WEHI, clone 9C9), anti-pERK (Cell Signaling, Danvers, MA, USA, cat# 4370), anti-ERK (Cell Signaling, cat# 9102), anti-HSP70 (gift from Dr. R Anderson PMCC Melbourne, clone N6), anti-TP53 (Santa Cruz, Dallas, TX, USA, cat# 6243).

Techniques: Inhibition, Mutagenesis, Western Blot, Derivative Assay

Journal: Cell Death and Differentiation

Article Title: Hepatocyte growth factor renders BRAF mutant human melanoma cell lines resistant to PLX4032 by downregulating the pro-apoptotic BH3-only proteins PUMA and BIM

doi: 10.1038/cdd.2016.96

Figure Lengend Snippet: HGF inhibits PLX4032-induced upregulation of PUMA and BIM in cMET+ BRAF mutant melanoma cells and thereby protects them against PLX4032-induced killing. (a) cMET− (M14 and UACC257) and cMET+ (Malme3M and SKMEL5) BRAFV600E melanoma cell lines were treated with DMSO (vehicle; black bar) or PLX4032 (grey bars, at 3 μM) with the indicated concentrations of HGF (0, 3, 6, 12, 25, 50, 100, 200 ng/ml) for 5 days and cell survival was assessed by flow cytometry. Living cells were defined as those negative for Annexin V and propidium iodide. The concentration at which HGF causes significant protection from PLX4032-induced killing is indicated. Data are derived from three independent experimental replicates, each comprising the average of three technical replicates and are presented as mean±S.E.M.; *P<0.05, **P<0.01, ****P<0.0001. (b) cMET− (M14 and UACC257) and cMET+ (Malme3M and SKMEL5) BRAF mutant cells were treated with single agents or combinations of DMSO (vehicle), HGF (50 ng/ml) and PLX4032 (3 μM) for 24 h in the presence of Q-VD-OPh. The mRNA of cells was subjected to qRT-PCR analysis to measure the expression of pro-survival (BCL-2, BCL-X, BFL-1, MCL-1) and pro-apoptotic (BIM, PUMA) BCL-2 family members. The mRNA expression levels of the different genes were normalized to the levels of Gapdh and the data are presented as fold change relative to DMSO (vehicle)-treated control cells. The data are derived from three independent experimental replicates and are presented as mean±S.E.M. (c) Cells were treated as described in panel (b) and analyzed by western blotting for the expression of pro-survival (BCL-2, BCL-XL, MCL-1) and pro-apoptotic (BIM, PUMA) BCL-2 family members. Probing for HSP70 was used as a loading control

Article Snippet: Antibodies (clone) were purchased or obtained from the sources indicated: anti-BIM (Enzo Life Sciences, Farmingdale, NY, USA, cat# ADI-AAP-330-E), anti-PUMA (Abcam, Cambridge, UK, cat# 27669), anti-BMF (WEHI, clone 12E10), anti-NOXA (ProSci, Poway, CA, USA, cat# 2437), anti-MCL-1 (WEHI, clone 19C4-15), anti-BCL-2 (WEHI, clone Bcl-2-100), anti-BCL-XL (WEHI, clone 9C9), anti-pERK (Cell Signaling, Danvers, MA, USA, cat# 4370), anti-ERK (Cell Signaling, cat# 9102), anti-HSP70 (gift from Dr. R Anderson PMCC Melbourne, clone N6), anti-TP53 (Santa Cruz, Dallas, TX, USA, cat# 6243).

Techniques: Mutagenesis, Flow Cytometry, Concentration Assay, Derivative Assay, Quantitative RT-PCR, Expressing, Western Blot

Journal: Cell Death and Differentiation

Article Title: Hepatocyte growth factor renders BRAF mutant human melanoma cell lines resistant to PLX4032 by downregulating the pro-apoptotic BH3-only proteins PUMA and BIM

doi: 10.1038/cdd.2016.96

Figure Lengend Snippet: The combination of PLX4032 and BCL-XL inhibition can partially overcome HGF-mediated resistance of cMET+ BRAF mutant melanoma cells. (a) Malme3M and SKMEL5 cells were treated with DMSO (vehicle), PLX4032 (3 μM) alone or in combination with ABT-737 (1 μM) (blue bars), ABT-199 (1 μM) (green bars), A-1155463 (1 μM) (red bars), with or without addition of HGF (50 ng/ml) for 5 days and cell survival was assessed by flow cytometry. Living cells were identified as those negative for Annexin V and PI. The data are derived from three independent experimental replicates, each comprising the average of three technical replicates. The data were analyzed using Student's t-test and are presented as mean±S.E.M.; *P<0.05, **P<0.01. (b) Under steady-state conditions, low levels of pro-apoptotic BH3-only proteins are present in the BRAF mutant melanoma cells. Hence, pro-survival proteins are able to neutralize BAX and BAK to cause survival of the cell. In the presence of PLX4032, PUMA and BIM are getting induced, which leads to sequestration of pro-survival proteins, allowing BAX and BAK to induce apoptosis. In the presence of HGF, PLX4032 mediated upregulation of PUMA and BIM is substantially diminished, and this is the underlying molecular mechanism that causes melanoma cells to develop resistance to PLX4032-induced killing. When combining PLX4032 and the BCL-XL-specific inhibitor, A-1155463, HGF-mediated resistance can partially be overcome

Article Snippet: Antibodies (clone) were purchased or obtained from the sources indicated: anti-BIM (Enzo Life Sciences, Farmingdale, NY, USA, cat# ADI-AAP-330-E), anti-PUMA (Abcam, Cambridge, UK, cat# 27669), anti-BMF (WEHI, clone 12E10), anti-NOXA (ProSci, Poway, CA, USA, cat# 2437), anti-MCL-1 (WEHI, clone 19C4-15), anti-BCL-2 (WEHI, clone Bcl-2-100), anti-BCL-XL (WEHI, clone 9C9), anti-pERK (Cell Signaling, Danvers, MA, USA, cat# 4370), anti-ERK (Cell Signaling, cat# 9102), anti-HSP70 (gift from Dr. R Anderson PMCC Melbourne, clone N6), anti-TP53 (Santa Cruz, Dallas, TX, USA, cat# 6243).

Techniques: Inhibition, Mutagenesis, Flow Cytometry, Derivative Assay