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  • 99
    Thermo Fisher bcl 2
    The mRNA ( A ) and protein ( B ) expressions of Bax, <t>Bcl-2</t> and Bcl-xL in human oral squamous carcinoma HSC-3 cells. * p
    Bcl 2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3409 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc bcl 2
    TNFα-activated caspase-9 apoptosis is regulated by mitochondrial fission. Notes: ( A and B ) Mitochondrial potential was observed via JC-1 staining. Red fluorescence of the JC-1 probe indicates the normal mitochondrial potential, whereas green fluorescence of the JC-1 probe means a defective mitochondrial potential. The red-to-green fluorescence intensity was recorded to quantify the mitochondrial potential. To perform the loss- and gain-of-function assays for mitochondrial fission, Mdivi-1, a pharmacological antagonist was used in TNFα-treated cells to inhibit mitochondrial fission activation. FCCP, an agonist for mitochondrial fission, was administered to the control group, which was set as the positive control group. ( C ) mPTP opening was measured in response to TNFα stress and/or mitochondrial fission inhibition. ( D and E ) Immunofluorescence assay for mitochondrial cyt-c translocation into nucleus. Nuclei were labeled by DAPI, and the colocalization of cyt-c and DAPI indicates the migration of mitochondrial cyt-c into nucleus. The relative expression of nuclear cyt-c was monitored. ( F ) Caspase-9 activity was determined via ELISA. TNFα-mediated caspase-9 activation could be abrogated by Mdivi-1. ( G – K ) Western blot was performed to analyze the alterations in mitochondrial apoptotic proteins. Bax and Bad are proapoptotic proteins, whereas <t>Bcl-2</t> and x-IAP are antiapoptotic proteins. TNFα regulated the balance of proapoptotic and antiapoptotic proteins via mitochondrial fission. * P
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    Santa Cruz Biotechnology bcl 2
    <t>bcl-2</t> interacts with HIF-1α in the nucleus. (A) Western blot analysis of bcl-2 and HIF-1α protein expression in nuclear (Nucl) and cytoplasmic (Cyto) protein extracts of M14 control (puro) and bcl-2 stably overexpressing (Bcl2/5) clones exposed to hypoxia or to normoxia for 24 h. LaminA/C (Lam A/C) and β-tubulin were used as markers for nuclear and cytoplasmic fraction, respectively. β-actin protein amounts are used to check equal loading and transfer of proteins. (B) Confocal laser scanning microscopy of immunofluorescence staining performed on Bcl2/5 stably overexpressing clone exposed to hypoxia or to normoxia for 24 h. Fixed cells were labelled with anti-bcl-2 ( green ) or anti-HIF-1α ( red ) antibodies. Nuclei were visualized using TO-PRO3® staining ( blue ). (C) Analysis of HIF-1α/bcl-2 interaction in Bcl2/5 stably overexpressing clone exposed to hypoxia for 24 h. Nuclear (Nucl) and cytoplasmic (Cyto) protein extracts were immunoprecipitated (IP) with anti-HIF-1α or anti-bcl-2, respectively, or control antibody (IgG) and then the Western blot analysis was performed using anti-bcl-2 or anti-HIF-1α antibodies. (A–C) Western blot and confocal analyses representative of two independent experiments with similar results are shown.
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    bcl 2  (Abcam)
    99
    Abcam bcl 2
    HNK inhibits proliferation and induces apoptosis of human OS cells. (A and B) Human Saos-2 and MG-63 OS cells were treated with or without 1–100 µ M HNK for 24 h, and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was conducted to assess viability. (C) Saos-2 and MG-63 cells were treated with or without 10 or 40 µ M HNK for 24 h, and apoptosis was measured using flow cytometry. (D) Proportion of apoptotic cells was quantified in three independent experiments. (E) Saos-2 and MG-63 cells were treated with or without 10 or 40 µ M of HNK for 24 h, and the protein expression levels of cleaved-caspase-3, cleaved-PARP, Bax and <t>Bcl-2</t> were analyzed by western blot analysis. Representative data from one of three individual experiments with similar results are presented. Data are presented as the mean ± standard deviation of three independent experiments. * P
    Bcl 2, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 6174 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti bcl 2
    <t>Bcl-2</t> interacts with human MLKL based on the presence of a BH3-like domain. a A predicted BH3-like domain in human MLKL is partially conserved across the indicated species. The consensus sequence for a BH3 domain is shown below. b Human MLKL interacts with Bcl-2. Various combinations of Flag-MLKL, Flag-MLKL L176A, Bcl-2, and Bcl-2 G145A were transfected into HEK-293T cells. Bcl-2 antibodies were used to immunoprecipitate the Bcl-2 proteins. The upper two blot panels show the co-immunoprecipitated Flag-MLKL and the immunoprecipitated Bcl-2 proteins. The lower three panels show the input proteins in cell lysates. c Phosphorylated MLKL interacts better with Bcl-2. Flag-MLKL, Flag-MLKL TS357-58ED (ED), and Flag-MLKL TS357-58AA (AA) were expressed in HEK-293T cells. The wild type and the AA mutant were also co-expressed with RIP3 The ED mutant is a phosphomimetic of RIP3 phosphorylated MLKL while the AA mutant cannot be phosphorylated at TS357-58. Anti-flag antibodies were used for co-immunoprecipitating Bcl-2. Western blots show all indicated proteins expressed in HEK-293T cells. d The endogenous Bcl-2 interacts the endogenous MLKL in THP-1 cells. The PMA treated THP-1 were used for immunoprecipitating Bcl-2 with or without treatment of TNFα 20 ng/ml (T), 100 nM Smac mimetic (S) and 20 µM z-VAD-FMK (Z) for 3 h. The lysates were used d for Bcl-2 immunoprecipitations. The pulled down of MLKL and the input proteins were blotted. ( e ) Bcl-2 recombinant protein binds with MLKL in vitro. The immunoprecipitated MLKL and its L176A mutant from HEK 293T cells were incubated with Bcl-2 protein for 1 h, and then heavily washed. Blots show the indicated proteins. f GST fusion BH3-like domain of MLKL binds Bcl-2 recombinant protein. The indicated GST fusion BH3 domains were expressed and purified from HEK-293T cells. Bcl-2 recombinant protein was added for in vitro binding with GST fusion BH3 domains.
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    Cell Signaling Technology Inc anti bcl 2
    Expression of Bax, <t>Bcl-2,</t> and caspase-3 in NCI-H292 cells. (a) Protein expression bands were detected by western blot. (b) The bar charts of Bax/ β -actin and Bcl-2/ β -actin. (c) The bar chart shows the ratio of Bcl-2/Bax. (d) Expression of caspase-3 normalized to β -actin. Compared with the control group, the expression of Bcl-2/Bax decreased and cleaved caspase-3 increased. # P
    Anti Bcl 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 3312 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc bcl2
    Expression of Bax, <t>Bcl-2,</t> and caspase-3 in NCI-H292 cells. (a) Protein expression bands were detected by western blot. (b) The bar charts of Bax/ β -actin and Bcl-2/ β -actin. (c) The bar chart shows the ratio of Bcl-2/Bax. (d) Expression of caspase-3 normalized to β -actin. Compared with the control group, the expression of Bcl-2/Bax decreased and cleaved caspase-3 increased. # P
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    Pharmingen bcl 2
    Three-week-old <t>Bcl-2</t> −/− mice have increased primary spongiosa due to both decreased number and dysfunctionality of osteoclasts. A , radiographic analysis of wild type ( WT ) and Bcl-2 −/− mice at 3 weeks of age. The arrows indicate the sclerotic regions at the metaphysis. Bar , 10 mm. B , histological section of tibiae of WT and Bcl-2 −/− mice (hematoxylin and eosin, von Kossa, and TRAP staining). The standardized regions of interest in the primary and secondary spongiosa are indicated by boxes . The insets of the lower panels indicate higher magnification views of the rectangular areas. Bars , 100 μm. C and D , histomorphometric analysis. Parameters of bone resorption ( C ) and bone formation ( D ) analyzed in the tibiae of WT and Bcl-2 −/− mice. Cancellous bone volume ( BV/TV ) is the percent of total marrow area (including trabeculae) occupied by cancellous bone. Trabecular thickness ( Tb.Th ) is the mean distance across individual trabeculae in micrometers. Trabecular number ( Tb.N ) per mm is calculated as (BV/TV)/Tb.Th. These variables can be used to evaluate trabecular connectivity. Eroded surface ( ES/BS ) is the percent of cancellous surface occupied by Howship's lacunae, with and without osteoclasts. Osteoblast surface ( Ob.S/BS ) and osteoclast surface ( Oc.S/BS ) identify the percent of cancellous surface occupied by osteoblasts and osteoclasts, respectively. Osteoid surface ( Osteoid S/BS ) is the percent of cancellous surface with unmineralized osteoid, with and without osteoblasts. Osteoid thickness ( Osteoid Th ) is the mean thickness, in micrometers, of the osteoid on cancellous surfaces. E , calcein double labeling of the mineralized matrix of proximal tibia at an interval of 2 days. WT mice ( n = 4), Bcl-2 −/− mice ( n = 4). Mineral appositional rate ( MAR ) is the rate (in μm/day) at which new bone is being added to cancellous surfaces. Bone formation rates ( BFR ) are estimates of cancellous bone volume that are being replaced annually. * indicates significantly different, p
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    Abcam anti bcl 2
    Three-week-old <t>Bcl-2</t> −/− mice have increased primary spongiosa due to both decreased number and dysfunctionality of osteoclasts. A , radiographic analysis of wild type ( WT ) and Bcl-2 −/− mice at 3 weeks of age. The arrows indicate the sclerotic regions at the metaphysis. Bar , 10 mm. B , histological section of tibiae of WT and Bcl-2 −/− mice (hematoxylin and eosin, von Kossa, and TRAP staining). The standardized regions of interest in the primary and secondary spongiosa are indicated by boxes . The insets of the lower panels indicate higher magnification views of the rectangular areas. Bars , 100 μm. C and D , histomorphometric analysis. Parameters of bone resorption ( C ) and bone formation ( D ) analyzed in the tibiae of WT and Bcl-2 −/− mice. Cancellous bone volume ( BV/TV ) is the percent of total marrow area (including trabeculae) occupied by cancellous bone. Trabecular thickness ( Tb.Th ) is the mean distance across individual trabeculae in micrometers. Trabecular number ( Tb.N ) per mm is calculated as (BV/TV)/Tb.Th. These variables can be used to evaluate trabecular connectivity. Eroded surface ( ES/BS ) is the percent of cancellous surface occupied by Howship's lacunae, with and without osteoclasts. Osteoblast surface ( Ob.S/BS ) and osteoclast surface ( Oc.S/BS ) identify the percent of cancellous surface occupied by osteoblasts and osteoclasts, respectively. Osteoid surface ( Osteoid S/BS ) is the percent of cancellous surface with unmineralized osteoid, with and without osteoblasts. Osteoid thickness ( Osteoid Th ) is the mean thickness, in micrometers, of the osteoid on cancellous surfaces. E , calcein double labeling of the mineralized matrix of proximal tibia at an interval of 2 days. WT mice ( n = 4), Bcl-2 −/− mice ( n = 4). Mineral appositional rate ( MAR ) is the rate (in μm/day) at which new bone is being added to cancellous surfaces. Bone formation rates ( BFR ) are estimates of cancellous bone volume that are being replaced annually. * indicates significantly different, p
    Anti Bcl 2, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1769 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Mimetics bcl 2
    Many anticancer agents mediate tumour cell killing though activation of the <t>BCL-2-regulated</t> apoptotic pathway. BH3-only proteins are activated transcriptionally and/or posttranscriptionally in a cytotoxic stimulus-specific manner by many anticancer agents,
    Bcl 2, supplied by Mimetics, used in various techniques. Bioz Stars score: 93/100, based on 956 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies bcl 2
    Melanoma-specific <t>bcl-2</t> establishes a suppressive microenvironmental condition that impairs T cell response. (A) Western blot analysis of bcl-2 protein expression in murine melanoma B16/F10 control (B16/F10 Control) or bcl-2 overexpressing (B16/F10 Bcl-2) cells. β-actin is shown as loading and transferring control. One representative western blot analysis out of two with similar results is reported. (B) Quantification of in vivo tumor growth after subcutaneous injection of B16/F10 control or bcl-2 overexpressing cells in C57/Bl6 mice. Quantification by cytofluorimetric analysis of (C) CD45 + cells among live cells, (D) cd11b + F4/80 + (TAM) among CD45 + cells, (E) CD206 + (left panel), and MHCII + (right panel) cells among TAM in B16/F10 control or bcl-2 overexpressing tumors. Quantification by cytofluorimetric analysis of (F) CD3 + among CD45 + cells (left panel), CD4 + and CD8 + among CD3 + cells (right panel), (G) IFNγ production and (H) CD44 + CD62L - among CD3 + infiltrating cells. (G) The expression level of IFNγ was analyzed after stimulation with brefeldin A, ionomycin, and PMA as reported in Methods section. (I) Quantification of in vivo tumor growth of C57/Bl6 mice subcutaneously injected with control (Control) or bcl-2 overexpressing (Bcl-2) B16/F10 cells and treated with vehicle or with clodronate liposomes. Tumor growth is expressed as % over Control+vehicle group. (C–H) The results were reported as % of positive cells and represent the average ±SD of two independent experiments. (B–H) P values were calculated between control and bcl-2 overexpressing tumors. *P
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    Becton Dickinson bcl 2
    IL-13 regulates <t>Bcl-2</t> expression through STAT-3 activation . (A) A time-dependent increase in IL-13 secretion post infection in vitro . Data are mean ± SEM ( n = 3), ** P
    Bcl 2, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 2136 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti bcl2
    Neuroprotective activity of 17-mer peptides. Markers of the cell death pathways were analyzed in rd1 mutant retinas after exposure to vehicle (MOCK) or 17-mer or 17-mer[H105A] (H105A) or 17-mer[R99A] (R99A). a Histogram representing the percentage of photoreceptors labeled by the fluorescent calpain activity assay ( N = 4; ± SD; *** P ≤ 0.001). b αII-spectrin was analyzed by immunoblot in total protein extracts from mouse retinas. The 145–150 kDa fragments resulting from calpain cleavage are marked by an asterisk. The immunoblot was normalized using anti-actin antibodies (lower panel) ( N = 3). c Immunoblotting of BAX protein in mitochondria enriched protein extracts. The immunoblot was normalized with anti-cytochrome c antibodies (lower panel) ( N = 3). d Total protein extracts were analyzed by immunoblot with an <t>anti-BCL2</t> antibody. The immunoblot was normalized using anti-actin antibodies (lower panel) ( N = 3). e Histogram representing the percentage of photoreceptors labeled with TUNEL (black) or by nuclear localization of AIF (white) ( N = 4; ± SD; *** P ≤ 0.001). f Immunoblotting of AIF protein in nuclear-enriched protein extracts. The immunoblot was normalized with anti-H3 histone antibodies (lower panel) ( N = 3)
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    bcl2  (Abcam)
    99
    Abcam bcl2
    Mir223 deficiency increased autophagy and resting microglia in the brains of EAE mice. ( A ) Flow cytometric analysis of microglia, demonstrating PTPRC and ITGAM cells isolated from the CNS of EAE mice (n = 6 mice per group), detected on the 15th day after the induction of EAE. The data are shown in a representative plot. ( B ) The absolute numbers of the cell subpopulations are shown; the black column is for mir223 −/- mice. Rest, ITGAM + PTPRC low ; mc, ITGAM + PTPRC hi and low . ( C ) Autophagy was measured in the brains of the mice. LC3 puncta were visualized by confocal imaging of microglia immunostained for LC3 and AIF1, followed by Alexa Fluor 488/555-conjugated secondary antibodies (green/red); nuclei were stained with DAPI. Representative images are shown. Scale bars: 20 µm. ( D ) and ( E ) <t>BCL2</t> and BECN1 expression were visualized by fluorescence imaging of microglia. Scale bars: 50 µm.
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    Abcam anti bcl 2 antibody e17
    Mir223 deficiency increased autophagy and resting microglia in the brains of EAE mice. ( A ) Flow cytometric analysis of microglia, demonstrating PTPRC and ITGAM cells isolated from the CNS of EAE mice (n = 6 mice per group), detected on the 15th day after the induction of EAE. The data are shown in a representative plot. ( B ) The absolute numbers of the cell subpopulations are shown; the black column is for mir223 −/- mice. Rest, ITGAM + PTPRC low ; mc, ITGAM + PTPRC hi and low . ( C ) Autophagy was measured in the brains of the mice. LC3 puncta were visualized by confocal imaging of microglia immunostained for LC3 and AIF1, followed by Alexa Fluor 488/555-conjugated secondary antibodies (green/red); nuclei were stained with DAPI. Representative images are shown. Scale bars: 20 µm. ( D ) and ( E ) <t>BCL2</t> and BECN1 expression were visualized by fluorescence imaging of microglia. Scale bars: 50 µm.
    Anti Bcl 2 Antibody E17, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 348 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Proteintech rabbit bcl2 polyclonal
    Mir223 deficiency increased autophagy and resting microglia in the brains of EAE mice. ( A ) Flow cytometric analysis of microglia, demonstrating PTPRC and ITGAM cells isolated from the CNS of EAE mice (n = 6 mice per group), detected on the 15th day after the induction of EAE. The data are shown in a representative plot. ( B ) The absolute numbers of the cell subpopulations are shown; the black column is for mir223 −/- mice. Rest, ITGAM + PTPRC low ; mc, ITGAM + PTPRC hi and low . ( C ) Autophagy was measured in the brains of the mice. LC3 puncta were visualized by confocal imaging of microglia immunostained for LC3 and AIF1, followed by Alexa Fluor 488/555-conjugated secondary antibodies (green/red); nuclei were stained with DAPI. Representative images are shown. Scale bars: 20 µm. ( D ) and ( E ) <t>BCL2</t> and BECN1 expression were visualized by fluorescence imaging of microglia. Scale bars: 50 µm.
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    Santa Cruz Biotechnology antibodies against bcl 2
    M14 xenografts carrying <t>bcl-2</t> protein lacking the BH4 domain show decreased proliferation and increased autophagy. (A) Representative images and (B) quantification of Ki67 immunostaining in tumor xenografts from M14 control (puro) and its derivatives
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    Becton Dickinson anti bcl 2
    Loss of Bak does not prevent thrombocytopaenia in <t>BCL-2</t> tg mice. ( a ) Blood platelet counts in WT (white), Bak − / − (light grey), BCL-2 tg (dark grey) and Bak − / − BCL-2 tg (black) mice at 6–8 weeks ( n =4–7 per genotype), 12 weeks ( n =9–14) and 24 weeks ( n =15–20). Bars represent mean±S.E.M. Statistical significance is shown only for 12-week-old mice; *** P
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    Thermo Fisher gene exp bcl2 hs00608023 m1
    Loss of Bak does not prevent thrombocytopaenia in <t>BCL-2</t> tg mice. ( a ) Blood platelet counts in WT (white), Bak − / − (light grey), BCL-2 tg (dark grey) and Bak − / − BCL-2 tg (black) mice at 6–8 weeks ( n =4–7 per genotype), 12 weeks ( n =9–14) and 24 weeks ( n =15–20). Bars represent mean±S.E.M. Statistical significance is shown only for 12-week-old mice; *** P
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    Image Search Results


    The mRNA ( A ) and protein ( B ) expressions of Bax, Bcl-2 and Bcl-xL in human oral squamous carcinoma HSC-3 cells. * p

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Lactobacillus casei Strain Shirota Enhances the In Vitro Antiproliferative Effect of Geniposide in Human Oral Squamous Carcinoma HSC-3 Cells

    doi: 10.3390/molecules23051069

    Figure Lengend Snippet: The mRNA ( A ) and protein ( B ) expressions of Bax, Bcl-2 and Bcl-xL in human oral squamous carcinoma HSC-3 cells. * p

    Article Snippet: Every group was isolated for 2 h using non-fat milk sealing liquid (5%), and thereafter, it was combined with primary antibody of caspase-3 (No. PA5-16332, 1:500 dilution, Thermo Fisher Scientific, Waltham, MA, USA), caspase-8 (No. MA5-11558, 1:2000 dilution, Thermo Fisher Scientific), caspase-9 (No. PA5-16355, 1:500 dilution, Thermo Fisher Scientific), Bax (No. MA5-14003, 1:100 dilution, Thermo Fisher Scientific), Bcl-2 (No. MA5-11757, 1:400 dilution, Thermo Fisher Scientific), Bcl-xL (No. MA5-15142, 1:1000 dilution, Thermo Fisher Scientific), Fas (No. ab82419, 1:1000 dilution, Abcam, Cambridge, MA, USA), FasL (No. ab15285, 1:1000 dilution, Abcam), p53 (No. MA5-12557, 1:200 dilution, Thermo Fisher Scientific), p21 (No. MA5-14949, 1:1000 dilution, Thermo Fisher Scientific), HIAP-1 (No. PA1-26473, 1:200 dilution, Thermo Fisher Scientific), HIAP-2 (No. PA5-47036, 1:400 dilution, Thermo Fisher Scientific), NF-κB (No. ab195854, 1:2000 dilution, Abcam), IκB-α (No. ab7217, 1:2000 dilution, Abcam), COX-2 (No. 35-8200, 1:500 dilution, Thermo Fisher Scientific), iNOS (No. PA3-030A, 1:2000 dilution, Thermo Fisher Scientific), MMP-2 (No. 436000, 1:500 dilution, Thermo Fisher Scientific), MMP-9 (No. MA5-15886, 1:1000 dilution, Thermo Fisher Scientific), TIMP-1 (No. MA1-773, 1:500 dilution, Thermo Fisher Scientific), TIMP-2 (No. MA5-12207, 1:200 dilution, Thermo Fisher Scientific) and β-actin (No. MA1-140, 1:500 dilution) at 4 °C for 12 h. Individually, each group was mixed with secondary antibody after being washed with TBST three times and incubated by shaking at 25 °C for 2 h, followed by rinsing with TBST three times.

    Techniques:

    TNFα-activated caspase-9 apoptosis is regulated by mitochondrial fission. Notes: ( A and B ) Mitochondrial potential was observed via JC-1 staining. Red fluorescence of the JC-1 probe indicates the normal mitochondrial potential, whereas green fluorescence of the JC-1 probe means a defective mitochondrial potential. The red-to-green fluorescence intensity was recorded to quantify the mitochondrial potential. To perform the loss- and gain-of-function assays for mitochondrial fission, Mdivi-1, a pharmacological antagonist was used in TNFα-treated cells to inhibit mitochondrial fission activation. FCCP, an agonist for mitochondrial fission, was administered to the control group, which was set as the positive control group. ( C ) mPTP opening was measured in response to TNFα stress and/or mitochondrial fission inhibition. ( D and E ) Immunofluorescence assay for mitochondrial cyt-c translocation into nucleus. Nuclei were labeled by DAPI, and the colocalization of cyt-c and DAPI indicates the migration of mitochondrial cyt-c into nucleus. The relative expression of nuclear cyt-c was monitored. ( F ) Caspase-9 activity was determined via ELISA. TNFα-mediated caspase-9 activation could be abrogated by Mdivi-1. ( G – K ) Western blot was performed to analyze the alterations in mitochondrial apoptotic proteins. Bax and Bad are proapoptotic proteins, whereas Bcl-2 and x-IAP are antiapoptotic proteins. TNFα regulated the balance of proapoptotic and antiapoptotic proteins via mitochondrial fission. * P

    Journal: OncoTargets and therapy

    Article Title: TNFα promotes glioblastoma A172 cell mitochondrial apoptosis via augmenting mitochondrial fission and repression of MAPK–ERK–YAP signaling pathways

    doi: 10.2147/OTT.S184337

    Figure Lengend Snippet: TNFα-activated caspase-9 apoptosis is regulated by mitochondrial fission. Notes: ( A and B ) Mitochondrial potential was observed via JC-1 staining. Red fluorescence of the JC-1 probe indicates the normal mitochondrial potential, whereas green fluorescence of the JC-1 probe means a defective mitochondrial potential. The red-to-green fluorescence intensity was recorded to quantify the mitochondrial potential. To perform the loss- and gain-of-function assays for mitochondrial fission, Mdivi-1, a pharmacological antagonist was used in TNFα-treated cells to inhibit mitochondrial fission activation. FCCP, an agonist for mitochondrial fission, was administered to the control group, which was set as the positive control group. ( C ) mPTP opening was measured in response to TNFα stress and/or mitochondrial fission inhibition. ( D and E ) Immunofluorescence assay for mitochondrial cyt-c translocation into nucleus. Nuclei were labeled by DAPI, and the colocalization of cyt-c and DAPI indicates the migration of mitochondrial cyt-c into nucleus. The relative expression of nuclear cyt-c was monitored. ( F ) Caspase-9 activity was determined via ELISA. TNFα-mediated caspase-9 activation could be abrogated by Mdivi-1. ( G – K ) Western blot was performed to analyze the alterations in mitochondrial apoptotic proteins. Bax and Bad are proapoptotic proteins, whereas Bcl-2 and x-IAP are antiapoptotic proteins. TNFα regulated the balance of proapoptotic and antiapoptotic proteins via mitochondrial fission. * P

    Article Snippet: The primary antibodies used in the present study are described as follows: p-ERK (1:1,000, #ab176660; Abcam, Cambridge, MA, USA), t-ERK (1:1,000, #ab54230; Abcam), Yap (1:1,000; #14074; Cell Signaling Technology, Danvers, MA, USA), complex III subunit core (CIII-core2, 1:1,000, #459220; Invitrogen, Merck KGaA, Darmstadt, Germany), complex II (CII-30, 1:1,000, #ab110410; Abcam), complex IV subunit II (CIV-II, 1:1,000, #ab110268; Abcam), Drp1 (1:1,000, #ab56788; Abcam), Fis1 (1:1,000, #ab71498; Abcam), Opa1 (1:1,000, #ab42364; Abcam), Mfn2 (1:1,000, #ab57602; Abcam), Mff (1:1,000, #86668; Cell Signaling Technology), Tom20 (1:1,000, #ab186735; Abcam), Bcl-2 (1:1,000, #3498; Cell Signaling Technology), Bax (1:1,000, #2772; Cell Signaling Technology), Bcl-2 (1:1,000, #3498; Cell Signaling Technology), Bad (1:1,000, #ab90435; Abcam), and x-IAP (1:1,000, #ab28151; Abcam).

    Techniques: Staining, Fluorescence, Activation Assay, Positive Control, Inhibition, Immunofluorescence, Translocation Assay, Labeling, Migration, Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot

    bcl-2 interacts with HIF-1α in the nucleus. (A) Western blot analysis of bcl-2 and HIF-1α protein expression in nuclear (Nucl) and cytoplasmic (Cyto) protein extracts of M14 control (puro) and bcl-2 stably overexpressing (Bcl2/5) clones exposed to hypoxia or to normoxia for 24 h. LaminA/C (Lam A/C) and β-tubulin were used as markers for nuclear and cytoplasmic fraction, respectively. β-actin protein amounts are used to check equal loading and transfer of proteins. (B) Confocal laser scanning microscopy of immunofluorescence staining performed on Bcl2/5 stably overexpressing clone exposed to hypoxia or to normoxia for 24 h. Fixed cells were labelled with anti-bcl-2 ( green ) or anti-HIF-1α ( red ) antibodies. Nuclei were visualized using TO-PRO3® staining ( blue ). (C) Analysis of HIF-1α/bcl-2 interaction in Bcl2/5 stably overexpressing clone exposed to hypoxia for 24 h. Nuclear (Nucl) and cytoplasmic (Cyto) protein extracts were immunoprecipitated (IP) with anti-HIF-1α or anti-bcl-2, respectively, or control antibody (IgG) and then the Western blot analysis was performed using anti-bcl-2 or anti-HIF-1α antibodies. (A–C) Western blot and confocal analyses representative of two independent experiments with similar results are shown.

    Journal: PLoS ONE

    Article Title: Bcl-2 Regulates HIF-1? Protein Stabilization in Hypoxic Melanoma Cells via the Molecular Chaperone HSP90

    doi: 10.1371/journal.pone.0011772

    Figure Lengend Snippet: bcl-2 interacts with HIF-1α in the nucleus. (A) Western blot analysis of bcl-2 and HIF-1α protein expression in nuclear (Nucl) and cytoplasmic (Cyto) protein extracts of M14 control (puro) and bcl-2 stably overexpressing (Bcl2/5) clones exposed to hypoxia or to normoxia for 24 h. LaminA/C (Lam A/C) and β-tubulin were used as markers for nuclear and cytoplasmic fraction, respectively. β-actin protein amounts are used to check equal loading and transfer of proteins. (B) Confocal laser scanning microscopy of immunofluorescence staining performed on Bcl2/5 stably overexpressing clone exposed to hypoxia or to normoxia for 24 h. Fixed cells were labelled with anti-bcl-2 ( green ) or anti-HIF-1α ( red ) antibodies. Nuclei were visualized using TO-PRO3® staining ( blue ). (C) Analysis of HIF-1α/bcl-2 interaction in Bcl2/5 stably overexpressing clone exposed to hypoxia for 24 h. Nuclear (Nucl) and cytoplasmic (Cyto) protein extracts were immunoprecipitated (IP) with anti-HIF-1α or anti-bcl-2, respectively, or control antibody (IgG) and then the Western blot analysis was performed using anti-bcl-2 or anti-HIF-1α antibodies. (A–C) Western blot and confocal analyses representative of two independent experiments with similar results are shown.

    Article Snippet: Immunoprecipitation were also performed using multiple antibodies recognizing different epitopes on the bcl-2 (Santa Cruz Biotecnology) and HIF-1α (Santa Cruz Biotecnology; Novus Biologicals, Littleton, CO) protein.

    Techniques: Western Blot, Expressing, Stable Transfection, Clone Assay, Laser Capture Microdissection, Confocal Laser Scanning Microscopy, Immunofluorescence, Staining, Immunoprecipitation

    bcl-2 interacts with HIF-1α. (A) Analysis of HIF-1α/bcl-2 protein interaction in M14 control (puro) and stably bcl-2 overexpressing (Bcl2/5, Bcl2/37) clones exposed to hypoxia for 24 h. Whole cell lysates were immunoprecipitated (IP) with anti-bcl-2 or control (IgG) antibodies and then the Western blot analysis was performed using anti-HIF-1α and anti-bcl-2 antibodies. Analysis of HIF-1α/bcl-2 protein interaction in (B) M14 control (puro) and stably bcl-2 overexpressing (Bcl2/5, Bcl2/37) clones or (C,D) in PLF2 and JR8 control cells (PLF2/puro, JR8/puro) and stably bcl-2 overexpressing (PLF2/Bcl-2, JR8/Bcl-2) cells, exposed to MG132 (10 µM, 6 h). Whole cell lysates were immunoprecipitated with anti-HIF-1α or control (IgG) antibodies and then the Western blot analysis was performed using anti-HIF-1α and anti-bcl-2 antibodies. (A–D) β-actin protein amounts are used to check equal loading and transfer of proteins. Western blot analyses representative of two independent experiments with similar results are shown.

    Journal: PLoS ONE

    Article Title: Bcl-2 Regulates HIF-1? Protein Stabilization in Hypoxic Melanoma Cells via the Molecular Chaperone HSP90

    doi: 10.1371/journal.pone.0011772

    Figure Lengend Snippet: bcl-2 interacts with HIF-1α. (A) Analysis of HIF-1α/bcl-2 protein interaction in M14 control (puro) and stably bcl-2 overexpressing (Bcl2/5, Bcl2/37) clones exposed to hypoxia for 24 h. Whole cell lysates were immunoprecipitated (IP) with anti-bcl-2 or control (IgG) antibodies and then the Western blot analysis was performed using anti-HIF-1α and anti-bcl-2 antibodies. Analysis of HIF-1α/bcl-2 protein interaction in (B) M14 control (puro) and stably bcl-2 overexpressing (Bcl2/5, Bcl2/37) clones or (C,D) in PLF2 and JR8 control cells (PLF2/puro, JR8/puro) and stably bcl-2 overexpressing (PLF2/Bcl-2, JR8/Bcl-2) cells, exposed to MG132 (10 µM, 6 h). Whole cell lysates were immunoprecipitated with anti-HIF-1α or control (IgG) antibodies and then the Western blot analysis was performed using anti-HIF-1α and anti-bcl-2 antibodies. (A–D) β-actin protein amounts are used to check equal loading and transfer of proteins. Western blot analyses representative of two independent experiments with similar results are shown.

    Article Snippet: Immunoprecipitation were also performed using multiple antibodies recognizing different epitopes on the bcl-2 (Santa Cruz Biotecnology) and HIF-1α (Santa Cruz Biotecnology; Novus Biologicals, Littleton, CO) protein.

    Techniques: Stable Transfection, Clone Assay, Immunoprecipitation, Western Blot

    bcl-2 promotes HIF-1α protein stability preventing its ubiquitin-mediated degradation. (A) Western blot analysis (left panel) and quantification (right panel) of HIF-1α protein expression in M14 control (puro) and bcl-2 stably overexpressing (Bcl2/5, Bcl2/37) clones exposed to hypoxia for the indicated time. (B) Pulse analysis of HIF-1α protein synthesis rate in cells exposed to [ 35 S]–labeled methionine and cysteine for the indicated time. (C) Western blot analysis (left panel) and quantification (right panel) of HIF-1α protein expression in cells exposed to hypoxia for 24 h and then treated with Cyclohexamide (CHX, 50 µg/ml) for the indicated time. (D) Pulse-chase analysis of HIF-1α protein (left panel) and quantification (right panel) in cells plated under dense conditions, pulsed for 45 min with [ 35 S]–labeled methionine and cysteine and chased for the indicated time. (B,D) Whole cell lysates were immunoprecipitated (IP) with anti-HIF-1α antibody and subjected to SDS-PAGE. (E) Western blot analysis of HIF-1α ubiquitination in the cells exposed to MG132 (10 µM, 6 h) or to hypoxia for 24 h. Whole cell lysates were immunoprecipitated (IP) with anti-HIF-1α antibody and then the Western blot analysis was performed using anti-Ubiquitin antibody. (A,C) β-actin protein amounts are used to check equal loading and transfer of proteins and to quantify relative HIF-1α protein levels. (A–E) Western blot, pulse and pulse-chase analyses representative of two independent experiments with similar results are shown. (A,C,D) Densitometric analysis (right panel) of the relative Western blot or Pulse-chase analysis (left panel) was performed using Molecular Analyst Software and normalized with relative controls.

    Journal: PLoS ONE

    Article Title: Bcl-2 Regulates HIF-1? Protein Stabilization in Hypoxic Melanoma Cells via the Molecular Chaperone HSP90

    doi: 10.1371/journal.pone.0011772

    Figure Lengend Snippet: bcl-2 promotes HIF-1α protein stability preventing its ubiquitin-mediated degradation. (A) Western blot analysis (left panel) and quantification (right panel) of HIF-1α protein expression in M14 control (puro) and bcl-2 stably overexpressing (Bcl2/5, Bcl2/37) clones exposed to hypoxia for the indicated time. (B) Pulse analysis of HIF-1α protein synthesis rate in cells exposed to [ 35 S]–labeled methionine and cysteine for the indicated time. (C) Western blot analysis (left panel) and quantification (right panel) of HIF-1α protein expression in cells exposed to hypoxia for 24 h and then treated with Cyclohexamide (CHX, 50 µg/ml) for the indicated time. (D) Pulse-chase analysis of HIF-1α protein (left panel) and quantification (right panel) in cells plated under dense conditions, pulsed for 45 min with [ 35 S]–labeled methionine and cysteine and chased for the indicated time. (B,D) Whole cell lysates were immunoprecipitated (IP) with anti-HIF-1α antibody and subjected to SDS-PAGE. (E) Western blot analysis of HIF-1α ubiquitination in the cells exposed to MG132 (10 µM, 6 h) or to hypoxia for 24 h. Whole cell lysates were immunoprecipitated (IP) with anti-HIF-1α antibody and then the Western blot analysis was performed using anti-Ubiquitin antibody. (A,C) β-actin protein amounts are used to check equal loading and transfer of proteins and to quantify relative HIF-1α protein levels. (A–E) Western blot, pulse and pulse-chase analyses representative of two independent experiments with similar results are shown. (A,C,D) Densitometric analysis (right panel) of the relative Western blot or Pulse-chase analysis (left panel) was performed using Molecular Analyst Software and normalized with relative controls.

    Article Snippet: Immunoprecipitation were also performed using multiple antibodies recognizing different epitopes on the bcl-2 (Santa Cruz Biotecnology) and HIF-1α (Santa Cruz Biotecnology; Novus Biologicals, Littleton, CO) protein.

    Techniques: Western Blot, Expressing, Stable Transfection, Clone Assay, Labeling, Pulse Chase, Immunoprecipitation, SDS Page, Software

    bcl-2 forms a complex with HSP90 and HIF-1α proteins. (A) Western blot analysis of HIF-1α protein expression in M14 control cells (puro) and bcl-2 stably overexpressing (Bcl2/5, Bcl2/37) clones treated with 17-AAG under hypoxia or exposed to normoxia for 24 h. (B) HRE-dependent transcriptional activity in the cells treated with 17-AAG from 0.05 to 2 µM under hypoxia or exposed to normoxia for 24 h. Relative luciferase activity of each sample was normalized to untreated cells exposed to normoxic conditions. Results represent the average ± SD of 3 independent experiments performed in triplicate. p values were calculated relative to untreated cells exposed to hypoxic conditions, *p≤0.01. (C) Western blot analysis of HSP90 protein expression in parental M14 cells, control (puro) and bcl-2 stably overexpressing (Bcl2/5, Bcl2/37) clones. (D) Analysis of HIF-1α/HSP90 interaction in the cells exposed to hypoxia for 24 h. Whole cell lysates were immunoprecipitated (IP) with anti-HIF-1α or control (IgG) antibodies and then the Western blot analysis was performed using anti-HSP90 and anti-HIF-1α antibodies. (E) Analysis of HSP90/HIF-1α and HSP90/bcl-2 interactions in the cells exposed to hypoxia for 24 h. Cell lysates were immunoprecipitated (IP) with anti-HSP90 or control (IgG) antibodies and then the Western blot analysis was performed using anti-HIF-1α, anti-bcl-2 and anti-HSP90 antibodies. (F) Analysis of HIF-1α/HSP90 and HIF-1α/bcl-2 interactions in the cells treated with 0.5 µM 17-AAG for 24 h under hypoxia. Whole cell lysates were immunoprecipitated (IP) with anti-HIF-1α antibody and then the Western blot analysis was performed using specific anti-HSP90 and bcl-2 antibodies. (G) Analysis of HSP90/HIF-1α/bcl-2 protein complex in the cells exposed to hypoxia for 24 h. Whole cell lysates were sequentially immunoprecipitated with anti-HIF-1α (IP1) and anti-bcl-2 antibodies (IP2) and then the Western blot analysis was performed using anti-HSP90 antibody. (A,C) β-actin protein amounts are used to check equal loading and transfer of proteins.

    Journal: PLoS ONE

    Article Title: Bcl-2 Regulates HIF-1? Protein Stabilization in Hypoxic Melanoma Cells via the Molecular Chaperone HSP90

    doi: 10.1371/journal.pone.0011772

    Figure Lengend Snippet: bcl-2 forms a complex with HSP90 and HIF-1α proteins. (A) Western blot analysis of HIF-1α protein expression in M14 control cells (puro) and bcl-2 stably overexpressing (Bcl2/5, Bcl2/37) clones treated with 17-AAG under hypoxia or exposed to normoxia for 24 h. (B) HRE-dependent transcriptional activity in the cells treated with 17-AAG from 0.05 to 2 µM under hypoxia or exposed to normoxia for 24 h. Relative luciferase activity of each sample was normalized to untreated cells exposed to normoxic conditions. Results represent the average ± SD of 3 independent experiments performed in triplicate. p values were calculated relative to untreated cells exposed to hypoxic conditions, *p≤0.01. (C) Western blot analysis of HSP90 protein expression in parental M14 cells, control (puro) and bcl-2 stably overexpressing (Bcl2/5, Bcl2/37) clones. (D) Analysis of HIF-1α/HSP90 interaction in the cells exposed to hypoxia for 24 h. Whole cell lysates were immunoprecipitated (IP) with anti-HIF-1α or control (IgG) antibodies and then the Western blot analysis was performed using anti-HSP90 and anti-HIF-1α antibodies. (E) Analysis of HSP90/HIF-1α and HSP90/bcl-2 interactions in the cells exposed to hypoxia for 24 h. Cell lysates were immunoprecipitated (IP) with anti-HSP90 or control (IgG) antibodies and then the Western blot analysis was performed using anti-HIF-1α, anti-bcl-2 and anti-HSP90 antibodies. (F) Analysis of HIF-1α/HSP90 and HIF-1α/bcl-2 interactions in the cells treated with 0.5 µM 17-AAG for 24 h under hypoxia. Whole cell lysates were immunoprecipitated (IP) with anti-HIF-1α antibody and then the Western blot analysis was performed using specific anti-HSP90 and bcl-2 antibodies. (G) Analysis of HSP90/HIF-1α/bcl-2 protein complex in the cells exposed to hypoxia for 24 h. Whole cell lysates were sequentially immunoprecipitated with anti-HIF-1α (IP1) and anti-bcl-2 antibodies (IP2) and then the Western blot analysis was performed using anti-HSP90 antibody. (A,C) β-actin protein amounts are used to check equal loading and transfer of proteins.

    Article Snippet: Immunoprecipitation were also performed using multiple antibodies recognizing different epitopes on the bcl-2 (Santa Cruz Biotecnology) and HIF-1α (Santa Cruz Biotecnology; Novus Biologicals, Littleton, CO) protein.

    Techniques: Western Blot, Expressing, Stable Transfection, Clone Assay, Activity Assay, Luciferase, Immunoprecipitation

    bcl-2 modulation regulates HIF-1α protein expression in conditions strictly dependent on oxygen avaibility. (A) Western blot analysis of HIF-1α and bcl-2 protein expression in total extracts of M14 cells transfected with siRNA targeting bcl-2 mRNA (si-bcl-2) or with a control scrambled si-RNA (si-contr) and then exposed to normoxia or hypoxia for 24 h. (B) Western blot analysis of HIF-1α and HIF-1β protein expression in total extracts of M14 control (puro) and bcl-2 stably overexpressing (Bcl2/5, Bcl2/37) cells plated under low (sparse) or high (dense) cell density conditions, or cultured under normoxia for 4 days or under hypoxia for 24 h. Western blot analysis of HIF-1α and HIF-1β protein expression in total extracts of the cells plated under high cell density conditions and (C) exposed to 24 h shaking or (D) cultured with different volumes of medium. (E) Western blot analysis of HIF-1α and HIF-1β protein expression in total extracts of cells exposed to Insulin (100 nM) or Epidermal Growth Factor (EGF, 20 ng/ml) for 24 h. (A–E) β-actin protein amounts are used to check equal loading and transfer of proteins. Western blot analyses representative of two independent experiments with similar results are shown.

    Journal: PLoS ONE

    Article Title: Bcl-2 Regulates HIF-1? Protein Stabilization in Hypoxic Melanoma Cells via the Molecular Chaperone HSP90

    doi: 10.1371/journal.pone.0011772

    Figure Lengend Snippet: bcl-2 modulation regulates HIF-1α protein expression in conditions strictly dependent on oxygen avaibility. (A) Western blot analysis of HIF-1α and bcl-2 protein expression in total extracts of M14 cells transfected with siRNA targeting bcl-2 mRNA (si-bcl-2) or with a control scrambled si-RNA (si-contr) and then exposed to normoxia or hypoxia for 24 h. (B) Western blot analysis of HIF-1α and HIF-1β protein expression in total extracts of M14 control (puro) and bcl-2 stably overexpressing (Bcl2/5, Bcl2/37) cells plated under low (sparse) or high (dense) cell density conditions, or cultured under normoxia for 4 days or under hypoxia for 24 h. Western blot analysis of HIF-1α and HIF-1β protein expression in total extracts of the cells plated under high cell density conditions and (C) exposed to 24 h shaking or (D) cultured with different volumes of medium. (E) Western blot analysis of HIF-1α and HIF-1β protein expression in total extracts of cells exposed to Insulin (100 nM) or Epidermal Growth Factor (EGF, 20 ng/ml) for 24 h. (A–E) β-actin protein amounts are used to check equal loading and transfer of proteins. Western blot analyses representative of two independent experiments with similar results are shown.

    Article Snippet: Immunoprecipitation were also performed using multiple antibodies recognizing different epitopes on the bcl-2 (Santa Cruz Biotecnology) and HIF-1α (Santa Cruz Biotecnology; Novus Biologicals, Littleton, CO) protein.

    Techniques: Expressing, Western Blot, Transfection, Stable Transfection, Cell Culture

    HIF-1α prolyl hydroxylation is not required for bcl-2-induced increase of HIF-1α expression and HIF-1 activity in hypoxia. (A) Western blot analysis of HIF-1α, bcl-2 and PHD2 protein expression and (B) HRE-dependent transcriptional activity in M14 cells stably expressing HA-HIF-1α wild-type (HIF1α wt ) or mutated (HIF1α PP/AA ), after transiently transfection with control vector (empty), bcl-2 or PHD2 expressing vectors, and then exposure to hypoxia for 24 h. (A) β-actin protein amounts are used to check equal loading and transfer of proteins. Western blot analyses representative of two independent experiments with similar results are shown. (B) Relative luciferase activity of each sample were normalized to the control vector transfected cells. Results represent the mean ± SD of 3 independent experiments performed in triplicate, * p≤0.01.

    Journal: PLoS ONE

    Article Title: Bcl-2 Regulates HIF-1? Protein Stabilization in Hypoxic Melanoma Cells via the Molecular Chaperone HSP90

    doi: 10.1371/journal.pone.0011772

    Figure Lengend Snippet: HIF-1α prolyl hydroxylation is not required for bcl-2-induced increase of HIF-1α expression and HIF-1 activity in hypoxia. (A) Western blot analysis of HIF-1α, bcl-2 and PHD2 protein expression and (B) HRE-dependent transcriptional activity in M14 cells stably expressing HA-HIF-1α wild-type (HIF1α wt ) or mutated (HIF1α PP/AA ), after transiently transfection with control vector (empty), bcl-2 or PHD2 expressing vectors, and then exposure to hypoxia for 24 h. (A) β-actin protein amounts are used to check equal loading and transfer of proteins. Western blot analyses representative of two independent experiments with similar results are shown. (B) Relative luciferase activity of each sample were normalized to the control vector transfected cells. Results represent the mean ± SD of 3 independent experiments performed in triplicate, * p≤0.01.

    Article Snippet: Immunoprecipitation were also performed using multiple antibodies recognizing different epitopes on the bcl-2 (Santa Cruz Biotecnology) and HIF-1α (Santa Cruz Biotecnology; Novus Biologicals, Littleton, CO) protein.

    Techniques: Expressing, Activity Assay, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Luciferase

    HSP90β is the mediator of HIF-1α induction by bcl-2 under hypoxic conditions. (A) Western blot analysis of HSP90α and HSP90β protein expression in M14 control (puro) and bcl-2 stably overexpressing (Bcl2/5, Bcl2/37) clones exposed to hypoxia or to normoxia for 24 h. (B) Analysis of HSP90α/HIF-1α and HSP90β/HIF-1α interactions in the cells exposed to hypoxia for 24 h. Protein extracts were immunoprecipitated (IP) with anti-HIF-1α and then Western blot analysis was performed using anti-HSP90α and anti-HSP90β antibodies. (C,D) Western blot analysis of HIF-1α, HSP90α and HSP90β protein expression in bcl-2 stably overexpressing cells transiently transfected with short hairpin construct targeting HSP90β (shHSP90β), HSP90α (shHSP90α) or with control vector (shNC) and exposed to hypoxia or to normoxia for 24 h. (A,C,D) β-actin protein amounts are used to check equal loading and transfer of proteins. (A–D) Western blot analyses representative of two independent experiments with similar results are shown.

    Journal: PLoS ONE

    Article Title: Bcl-2 Regulates HIF-1? Protein Stabilization in Hypoxic Melanoma Cells via the Molecular Chaperone HSP90

    doi: 10.1371/journal.pone.0011772

    Figure Lengend Snippet: HSP90β is the mediator of HIF-1α induction by bcl-2 under hypoxic conditions. (A) Western blot analysis of HSP90α and HSP90β protein expression in M14 control (puro) and bcl-2 stably overexpressing (Bcl2/5, Bcl2/37) clones exposed to hypoxia or to normoxia for 24 h. (B) Analysis of HSP90α/HIF-1α and HSP90β/HIF-1α interactions in the cells exposed to hypoxia for 24 h. Protein extracts were immunoprecipitated (IP) with anti-HIF-1α and then Western blot analysis was performed using anti-HSP90α and anti-HSP90β antibodies. (C,D) Western blot analysis of HIF-1α, HSP90α and HSP90β protein expression in bcl-2 stably overexpressing cells transiently transfected with short hairpin construct targeting HSP90β (shHSP90β), HSP90α (shHSP90α) or with control vector (shNC) and exposed to hypoxia or to normoxia for 24 h. (A,C,D) β-actin protein amounts are used to check equal loading and transfer of proteins. (A–D) Western blot analyses representative of two independent experiments with similar results are shown.

    Article Snippet: Immunoprecipitation were also performed using multiple antibodies recognizing different epitopes on the bcl-2 (Santa Cruz Biotecnology) and HIF-1α (Santa Cruz Biotecnology; Novus Biologicals, Littleton, CO) protein.

    Techniques: Western Blot, Expressing, Stable Transfection, Clone Assay, Immunoprecipitation, Transfection, Construct, Plasmid Preparation

    Regulation of Bcl2 by activated JNK. (A) Activated JNK causes Bcl2 phosphorylation in vivo. CHO cells were transfected without and with a plasmid expression vector for human Bcl2 (20 ng). The cells were cotransfected with the empty expression vector or expression vectors for JNK1 and MKK7-JNK1 fusion proteins. The cells were harvested at 40 h posttransfection and the expression of Bcl2 was examined by immunoblot analysis. (B) Bcl2 phosphorylation is suppressed by increased expression of Bcl2. CHO cells were cotransfected with an expression vector for the Flag-MKK7-JNK1 fusion protein together with increasing amounts of an expression vector for human Bcl2 (0, 10, 20, 40, 60, and 80 ng). The cells were harvested at 40 h posttransfection and the expression of Bcl2 and Flag-MKK7-JNK1 was examined by immunoblot analysis. (C) CHO cells were transfected with an empty vector (control) or an expression vector for the MKK7-JNK1 fusion protein. The effect of expression of Bcl2 was examined (20 ng of plasmid). Cell viability at 40 h posttransfection was examined using a luciferase reporter plasmid. The data presented represent the means ± SD of data obtained for three independent experiments.

    Journal: Molecular and Cellular Biology

    Article Title: The Bax Subfamily of Bcl2-Related Proteins Is Essential for Apoptotic Signal Transduction by c-Jun NH2-Terminal Kinase

    doi: 10.1128/MCB.22.13.4929-4942.2002

    Figure Lengend Snippet: Regulation of Bcl2 by activated JNK. (A) Activated JNK causes Bcl2 phosphorylation in vivo. CHO cells were transfected without and with a plasmid expression vector for human Bcl2 (20 ng). The cells were cotransfected with the empty expression vector or expression vectors for JNK1 and MKK7-JNK1 fusion proteins. The cells were harvested at 40 h posttransfection and the expression of Bcl2 was examined by immunoblot analysis. (B) Bcl2 phosphorylation is suppressed by increased expression of Bcl2. CHO cells were cotransfected with an expression vector for the Flag-MKK7-JNK1 fusion protein together with increasing amounts of an expression vector for human Bcl2 (0, 10, 20, 40, 60, and 80 ng). The cells were harvested at 40 h posttransfection and the expression of Bcl2 and Flag-MKK7-JNK1 was examined by immunoblot analysis. (C) CHO cells were transfected with an empty vector (control) or an expression vector for the MKK7-JNK1 fusion protein. The effect of expression of Bcl2 was examined (20 ng of plasmid). Cell viability at 40 h posttransfection was examined using a luciferase reporter plasmid. The data presented represent the means ± SD of data obtained for three independent experiments.

    Article Snippet: This phosphorylation has been reported to inhibit the prosurvival function of Bcl2 and Bcl-XL ( , , , ), although this conclusion is controversial since some studies indicate that phosphorylation may enhance the antiapoptotic actions of Bcl2 ( , , ) and may stabilize Bcl2 (and thus increase prosurvival signaling) by preventing ubiquitin-mediated degradation of Bcl2 ( , ).

    Techniques: In Vivo, Transfection, Plasmid Preparation, Expressing, Luciferase

    The proapoptotic Bcl2 family protein Bid is not required for JNK-dependent apoptosis. (A) Bid is not required for JNK-dependent apoptosis. Wild-type and Bid −/− mouse fibroblasts were transfected with empty vector (control) or expression vectors for MKK7-JNK fusion proteins. Cell viability at 40 h posttransfection was examined using a luciferase reporter plasmid. The data presented represent the means ± SD of data obtained for three independent experiments. (B) Endogenous JNK is not required for apoptosis caused by ectopic expression of activated JNK. Wild-type and Jnk1 −/− Jnk2 −/− mouse fibroblasts were transfected with empty vector (control) or expression vectors for MKK7-JNK fusion proteins. Cell viability at 40 h posttransfection was examined using a luciferase reporter plasmid. The data presented represent the means ± SD of data obtained for three independent experiments.

    Journal: Molecular and Cellular Biology

    Article Title: The Bax Subfamily of Bcl2-Related Proteins Is Essential for Apoptotic Signal Transduction by c-Jun NH2-Terminal Kinase

    doi: 10.1128/MCB.22.13.4929-4942.2002

    Figure Lengend Snippet: The proapoptotic Bcl2 family protein Bid is not required for JNK-dependent apoptosis. (A) Bid is not required for JNK-dependent apoptosis. Wild-type and Bid −/− mouse fibroblasts were transfected with empty vector (control) or expression vectors for MKK7-JNK fusion proteins. Cell viability at 40 h posttransfection was examined using a luciferase reporter plasmid. The data presented represent the means ± SD of data obtained for three independent experiments. (B) Endogenous JNK is not required for apoptosis caused by ectopic expression of activated JNK. Wild-type and Jnk1 −/− Jnk2 −/− mouse fibroblasts were transfected with empty vector (control) or expression vectors for MKK7-JNK fusion proteins. Cell viability at 40 h posttransfection was examined using a luciferase reporter plasmid. The data presented represent the means ± SD of data obtained for three independent experiments.

    Article Snippet: This phosphorylation has been reported to inhibit the prosurvival function of Bcl2 and Bcl-XL ( , , , ), although this conclusion is controversial since some studies indicate that phosphorylation may enhance the antiapoptotic actions of Bcl2 ( , , ) and may stabilize Bcl2 (and thus increase prosurvival signaling) by preventing ubiquitin-mediated degradation of Bcl2 ( , ).

    Techniques: Transfection, Plasmid Preparation, Expressing, Luciferase

    The proapoptotic Bcl2 family proteins Bax and Bak are required for JNK-dependent apoptosis. (A) Bax and Bak are required for JNK-dependent apoptosis. Wild-type and Bax −/− Bak −/− mouse fibroblasts were transfected with empty vector (control) or expression vectors for JNK1 or MKK7-JNK fusion proteins. Cell viability at 40 h posttransfection was examined using a luciferase reporter plasmid. The data presented represent the means ± SD of data obtained for three independent experiments. (B) Endogenous JNK is not required for apoptosis caused by Bax and Bak. Wild-type and Jnk1 −/− Jnk2 −/− mouse fibroblasts were transfected with empty vector (control) or expression vectors for Bax, Bak, or tBid. Cell viability at 40 h posttransfection was examined using a luciferase reporter plasmid. The data presented represent the means ± SD of data obtained for three independent experiments. (C) Bax is not activated by UV radiation in JNK-deficient murine fibroblasts. Wild-type and Jnk1 −/− Jnk2 −/− fibroblasts were exposed to UV radiation (80 J/m 2 ) and incubated (24 h). The cells were fixed and stained with a conformation-specific antibody to Bax and Texas Red-conjugated goat anti-mouse antibody. The Bax antibody stains activated and membrane-targeted Bax but not cytoplasmic Bax. DNA was stained with DAPI (blue).

    Journal: Molecular and Cellular Biology

    Article Title: The Bax Subfamily of Bcl2-Related Proteins Is Essential for Apoptotic Signal Transduction by c-Jun NH2-Terminal Kinase

    doi: 10.1128/MCB.22.13.4929-4942.2002

    Figure Lengend Snippet: The proapoptotic Bcl2 family proteins Bax and Bak are required for JNK-dependent apoptosis. (A) Bax and Bak are required for JNK-dependent apoptosis. Wild-type and Bax −/− Bak −/− mouse fibroblasts were transfected with empty vector (control) or expression vectors for JNK1 or MKK7-JNK fusion proteins. Cell viability at 40 h posttransfection was examined using a luciferase reporter plasmid. The data presented represent the means ± SD of data obtained for three independent experiments. (B) Endogenous JNK is not required for apoptosis caused by Bax and Bak. Wild-type and Jnk1 −/− Jnk2 −/− mouse fibroblasts were transfected with empty vector (control) or expression vectors for Bax, Bak, or tBid. Cell viability at 40 h posttransfection was examined using a luciferase reporter plasmid. The data presented represent the means ± SD of data obtained for three independent experiments. (C) Bax is not activated by UV radiation in JNK-deficient murine fibroblasts. Wild-type and Jnk1 −/− Jnk2 −/− fibroblasts were exposed to UV radiation (80 J/m 2 ) and incubated (24 h). The cells were fixed and stained with a conformation-specific antibody to Bax and Texas Red-conjugated goat anti-mouse antibody. The Bax antibody stains activated and membrane-targeted Bax but not cytoplasmic Bax. DNA was stained with DAPI (blue).

    Article Snippet: This phosphorylation has been reported to inhibit the prosurvival function of Bcl2 and Bcl-XL ( , , , ), although this conclusion is controversial since some studies indicate that phosphorylation may enhance the antiapoptotic actions of Bcl2 ( , , ) and may stabilize Bcl2 (and thus increase prosurvival signaling) by preventing ubiquitin-mediated degradation of Bcl2 ( , ).

    Techniques: Transfection, Plasmid Preparation, Expressing, Luciferase, Incubation, Staining

    HNK inhibits proliferation and induces apoptosis of human OS cells. (A and B) Human Saos-2 and MG-63 OS cells were treated with or without 1–100 µ M HNK for 24 h, and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was conducted to assess viability. (C) Saos-2 and MG-63 cells were treated with or without 10 or 40 µ M HNK for 24 h, and apoptosis was measured using flow cytometry. (D) Proportion of apoptotic cells was quantified in three independent experiments. (E) Saos-2 and MG-63 cells were treated with or without 10 or 40 µ M of HNK for 24 h, and the protein expression levels of cleaved-caspase-3, cleaved-PARP, Bax and Bcl-2 were analyzed by western blot analysis. Representative data from one of three individual experiments with similar results are presented. Data are presented as the mean ± standard deviation of three independent experiments. * P

    Journal: International Journal of Molecular Medicine

    Article Title: Honokiol suppresses proliferation and induces apoptosis via regulation of the miR-21/PTEN/PI3K/AKT signaling pathway in human osteosarcoma cells

    doi: 10.3892/ijmm.2018.3433

    Figure Lengend Snippet: HNK inhibits proliferation and induces apoptosis of human OS cells. (A and B) Human Saos-2 and MG-63 OS cells were treated with or without 1–100 µ M HNK for 24 h, and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was conducted to assess viability. (C) Saos-2 and MG-63 cells were treated with or without 10 or 40 µ M HNK for 24 h, and apoptosis was measured using flow cytometry. (D) Proportion of apoptotic cells was quantified in three independent experiments. (E) Saos-2 and MG-63 cells were treated with or without 10 or 40 µ M of HNK for 24 h, and the protein expression levels of cleaved-caspase-3, cleaved-PARP, Bax and Bcl-2 were analyzed by western blot analysis. Representative data from one of three individual experiments with similar results are presented. Data are presented as the mean ± standard deviation of three independent experiments. * P

    Article Snippet: Antibodies of apoptosis-associated proteins were as follows: cleaved PARP (ab32064), Bax (ab25901), cleaved caspase-3 (ab13847) and Bcl-2 (ab32503), purchased from Abcam.

    Techniques: Flow Cytometry, Cytometry, Expressing, Western Blot, Standard Deviation

    Bcl-2 interacts with human MLKL based on the presence of a BH3-like domain. a A predicted BH3-like domain in human MLKL is partially conserved across the indicated species. The consensus sequence for a BH3 domain is shown below. b Human MLKL interacts with Bcl-2. Various combinations of Flag-MLKL, Flag-MLKL L176A, Bcl-2, and Bcl-2 G145A were transfected into HEK-293T cells. Bcl-2 antibodies were used to immunoprecipitate the Bcl-2 proteins. The upper two blot panels show the co-immunoprecipitated Flag-MLKL and the immunoprecipitated Bcl-2 proteins. The lower three panels show the input proteins in cell lysates. c Phosphorylated MLKL interacts better with Bcl-2. Flag-MLKL, Flag-MLKL TS357-58ED (ED), and Flag-MLKL TS357-58AA (AA) were expressed in HEK-293T cells. The wild type and the AA mutant were also co-expressed with RIP3 The ED mutant is a phosphomimetic of RIP3 phosphorylated MLKL while the AA mutant cannot be phosphorylated at TS357-58. Anti-flag antibodies were used for co-immunoprecipitating Bcl-2. Western blots show all indicated proteins expressed in HEK-293T cells. d The endogenous Bcl-2 interacts the endogenous MLKL in THP-1 cells. The PMA treated THP-1 were used for immunoprecipitating Bcl-2 with or without treatment of TNFα 20 ng/ml (T), 100 nM Smac mimetic (S) and 20 µM z-VAD-FMK (Z) for 3 h. The lysates were used d for Bcl-2 immunoprecipitations. The pulled down of MLKL and the input proteins were blotted. ( e ) Bcl-2 recombinant protein binds with MLKL in vitro. The immunoprecipitated MLKL and its L176A mutant from HEK 293T cells were incubated with Bcl-2 protein for 1 h, and then heavily washed. Blots show the indicated proteins. f GST fusion BH3-like domain of MLKL binds Bcl-2 recombinant protein. The indicated GST fusion BH3 domains were expressed and purified from HEK-293T cells. Bcl-2 recombinant protein was added for in vitro binding with GST fusion BH3 domains.

    Journal: Cell Death Discovery

    Article Title: Bcl-2 regulates pyroptosis and necroptosis by targeting BH3-like domains in GSDMD and MLKL

    doi: 10.1038/s41420-019-0230-2

    Figure Lengend Snippet: Bcl-2 interacts with human MLKL based on the presence of a BH3-like domain. a A predicted BH3-like domain in human MLKL is partially conserved across the indicated species. The consensus sequence for a BH3 domain is shown below. b Human MLKL interacts with Bcl-2. Various combinations of Flag-MLKL, Flag-MLKL L176A, Bcl-2, and Bcl-2 G145A were transfected into HEK-293T cells. Bcl-2 antibodies were used to immunoprecipitate the Bcl-2 proteins. The upper two blot panels show the co-immunoprecipitated Flag-MLKL and the immunoprecipitated Bcl-2 proteins. The lower three panels show the input proteins in cell lysates. c Phosphorylated MLKL interacts better with Bcl-2. Flag-MLKL, Flag-MLKL TS357-58ED (ED), and Flag-MLKL TS357-58AA (AA) were expressed in HEK-293T cells. The wild type and the AA mutant were also co-expressed with RIP3 The ED mutant is a phosphomimetic of RIP3 phosphorylated MLKL while the AA mutant cannot be phosphorylated at TS357-58. Anti-flag antibodies were used for co-immunoprecipitating Bcl-2. Western blots show all indicated proteins expressed in HEK-293T cells. d The endogenous Bcl-2 interacts the endogenous MLKL in THP-1 cells. The PMA treated THP-1 were used for immunoprecipitating Bcl-2 with or without treatment of TNFα 20 ng/ml (T), 100 nM Smac mimetic (S) and 20 µM z-VAD-FMK (Z) for 3 h. The lysates were used d for Bcl-2 immunoprecipitations. The pulled down of MLKL and the input proteins were blotted. ( e ) Bcl-2 recombinant protein binds with MLKL in vitro. The immunoprecipitated MLKL and its L176A mutant from HEK 293T cells were incubated with Bcl-2 protein for 1 h, and then heavily washed. Blots show the indicated proteins. f GST fusion BH3-like domain of MLKL binds Bcl-2 recombinant protein. The indicated GST fusion BH3 domains were expressed and purified from HEK-293T cells. Bcl-2 recombinant protein was added for in vitro binding with GST fusion BH3 domains.

    Article Snippet: Reagents and antibodies Antibodies used for immunoblotting analysis were the following: anti-IL-1β (#12703), anti-caspase-1 (#3866), anti-caspase-4 (#4450), anti-caspase-5 (#46680), anti-MLKL (#14993), anti-phospho-MLKL (#91689), anti-RIP-3 (#13526), anti-phospho-RIP3 (#93654) and anti-Myc-tag (#2278) (Cell Signaling Technology); and anti-Flag (Sigma-Aldrich, F1804), anti-Bcl-2 (Santa Cruz Biotechnology, sc-783), anti-actin conjugated to horseradish peroxidase (Sigma-Aldrich, A3854), anti-GAPDH conjugated to horseradish peroxidase (Proteintech, HRP-60004), goat anti-rabbit HRP-linked antibody (#7074), and horse anti-mouse HRP-linked antibody (#7076) (Cell Signaling Technology).

    Techniques: Sequencing, Transfection, Immunoprecipitation, Mutagenesis, Western Blot, Recombinant, In Vitro, Incubation, Purification, Binding Assay

    Transient expression of Bcl-2, but Bcl-2 G145A mutant impairs RIP3 induced MLKL phosphorylation and oligomerization. a Cell lysates from HEK-293T cells transfected with the indicated constructs were separated into cytosol and membrane enriched fractions using a detergent-based phase separation method. The indicated proteins in the two fractions were immunoblotted. The membranes were first blotted with anti-phospho-MLKL antibody (upper panel), and then reblotted with anti-Flag antibody (large middle panel). Bcl-2 and RIP3 immunoblots are shown in the lower panels. Actin and the TOM20 immunoblots verify equal loading of the cytosol and membrane fractions. b In vitro kinase assay. The immunoprecipitated and purified Flag-MLKL served a substrate of RIP3 kinase, which was expressed in HEK-293T cells. Phospho-MLKL (S358) antibodies were used to detect the amounts of phosphorylated MLKL. Bcl-2 recombinant protein was added to the assay. Immunoblots show all indicated proteins. c GST fusion BH3-like domain of MLKL reduces Bcl-2’s inhibition of RIP3 mediated MLKL phosphorylation. The indicated proteins were expressed in HEK-293T cells, the lysates were used for immunoblots. The immunoblotted proteins are shown. d The MLKL L176A mutation interferes with Bcl2’s inhibition of RIP3 mediated MLKL phosphorylation. The indicated proteins were transiently expressed in HEK-293T cells and identified by immunoblotting.

    Journal: Cell Death Discovery

    Article Title: Bcl-2 regulates pyroptosis and necroptosis by targeting BH3-like domains in GSDMD and MLKL

    doi: 10.1038/s41420-019-0230-2

    Figure Lengend Snippet: Transient expression of Bcl-2, but Bcl-2 G145A mutant impairs RIP3 induced MLKL phosphorylation and oligomerization. a Cell lysates from HEK-293T cells transfected with the indicated constructs were separated into cytosol and membrane enriched fractions using a detergent-based phase separation method. The indicated proteins in the two fractions were immunoblotted. The membranes were first blotted with anti-phospho-MLKL antibody (upper panel), and then reblotted with anti-Flag antibody (large middle panel). Bcl-2 and RIP3 immunoblots are shown in the lower panels. Actin and the TOM20 immunoblots verify equal loading of the cytosol and membrane fractions. b In vitro kinase assay. The immunoprecipitated and purified Flag-MLKL served a substrate of RIP3 kinase, which was expressed in HEK-293T cells. Phospho-MLKL (S358) antibodies were used to detect the amounts of phosphorylated MLKL. Bcl-2 recombinant protein was added to the assay. Immunoblots show all indicated proteins. c GST fusion BH3-like domain of MLKL reduces Bcl-2’s inhibition of RIP3 mediated MLKL phosphorylation. The indicated proteins were expressed in HEK-293T cells, the lysates were used for immunoblots. The immunoblotted proteins are shown. d The MLKL L176A mutation interferes with Bcl2’s inhibition of RIP3 mediated MLKL phosphorylation. The indicated proteins were transiently expressed in HEK-293T cells and identified by immunoblotting.

    Article Snippet: Reagents and antibodies Antibodies used for immunoblotting analysis were the following: anti-IL-1β (#12703), anti-caspase-1 (#3866), anti-caspase-4 (#4450), anti-caspase-5 (#46680), anti-MLKL (#14993), anti-phospho-MLKL (#91689), anti-RIP-3 (#13526), anti-phospho-RIP3 (#93654) and anti-Myc-tag (#2278) (Cell Signaling Technology); and anti-Flag (Sigma-Aldrich, F1804), anti-Bcl-2 (Santa Cruz Biotechnology, sc-783), anti-actin conjugated to horseradish peroxidase (Sigma-Aldrich, A3854), anti-GAPDH conjugated to horseradish peroxidase (Proteintech, HRP-60004), goat anti-rabbit HRP-linked antibody (#7074), and horse anti-mouse HRP-linked antibody (#7076) (Cell Signaling Technology).

    Techniques: Expressing, Mutagenesis, Transfection, Construct, Western Blot, In Vitro, Kinase Assay, Immunoprecipitation, Purification, Recombinant, Inhibition

    Bcl-2 regulates TSZ induced-necroptosis in HT-29 cells. a Stable expression of Bcl-2, but Bcl-2 G145A decreases TSZ induced-MLKL phosphorylation and oligomerization. The HT-29 cells stably expressing a vector control, Bcl-2, or Bcl-2 G145A were treated with TNF-α 20 ng/ml (T), 100 nM Smac mimetic (S) and 20 µM z-VAD-FMK (Z) for 5 h. DMSO served as a vehicle control treatment. The stimulated cells were fractionated into the cytosol and membrane fractions by detergent phase separation and subjected to immunoblotting. pMLKL (top panel), total MLKL (2nd panel). Bcl-2, Actin, TOM20 levels are shown in the lower blots. b Bcl-2 reduces TSZ induced cell death. Stably transfected HT-29 cells were stimulated with TSZ 12 h, and then the cells were stained with SYTOX green to assess cell death. Data are means and ± SD of four samples. ** p

    Journal: Cell Death Discovery

    Article Title: Bcl-2 regulates pyroptosis and necroptosis by targeting BH3-like domains in GSDMD and MLKL

    doi: 10.1038/s41420-019-0230-2

    Figure Lengend Snippet: Bcl-2 regulates TSZ induced-necroptosis in HT-29 cells. a Stable expression of Bcl-2, but Bcl-2 G145A decreases TSZ induced-MLKL phosphorylation and oligomerization. The HT-29 cells stably expressing a vector control, Bcl-2, or Bcl-2 G145A were treated with TNF-α 20 ng/ml (T), 100 nM Smac mimetic (S) and 20 µM z-VAD-FMK (Z) for 5 h. DMSO served as a vehicle control treatment. The stimulated cells were fractionated into the cytosol and membrane fractions by detergent phase separation and subjected to immunoblotting. pMLKL (top panel), total MLKL (2nd panel). Bcl-2, Actin, TOM20 levels are shown in the lower blots. b Bcl-2 reduces TSZ induced cell death. Stably transfected HT-29 cells were stimulated with TSZ 12 h, and then the cells were stained with SYTOX green to assess cell death. Data are means and ± SD of four samples. ** p

    Article Snippet: Reagents and antibodies Antibodies used for immunoblotting analysis were the following: anti-IL-1β (#12703), anti-caspase-1 (#3866), anti-caspase-4 (#4450), anti-caspase-5 (#46680), anti-MLKL (#14993), anti-phospho-MLKL (#91689), anti-RIP-3 (#13526), anti-phospho-RIP3 (#93654) and anti-Myc-tag (#2278) (Cell Signaling Technology); and anti-Flag (Sigma-Aldrich, F1804), anti-Bcl-2 (Santa Cruz Biotechnology, sc-783), anti-actin conjugated to horseradish peroxidase (Sigma-Aldrich, A3854), anti-GAPDH conjugated to horseradish peroxidase (Proteintech, HRP-60004), goat anti-rabbit HRP-linked antibody (#7074), and horse anti-mouse HRP-linked antibody (#7076) (Cell Signaling Technology).

    Techniques: Expressing, Stable Transfection, Plasmid Preparation, Transfection, Staining

    Bcl-2 interacts with GSDMD. a A predicted BH3-like domain in GSDMD is conserved across the indicated species. The consensus BH3 domain sequence is shown. b Endogenous Bcl-2 interacts with the endogenous GSDMD. Lysates from THP-1 cells treated with the indicated inflammasome activators (LPS 50 ng/ml 5 h, nigericin 5 µM 30 min) were used for Bcl-2 immunoprecipitations. The blots of Bcl-2 that co-immunoprecipitated with GSDMD are showed on the upper panel. The lower panels show the input proteins in cell lysates. c GSDMD-CT does not interact with Bcl-2. Lysates from HEK-293T cells transfected with the indicated constructs were immunoprecipitated using anti-Flag antibodies conjugated agarose beads to pull down Flag-GSDMD. Matched non-specific antibodies were used for the mock immunoprecipitation. The left panel shows the input proteins, and the right panel shows the interacting proteins. d GSDMD-NT and its BH3-like domain are required for the interaction between GSDMD and Bcl-2. The lysates from HEK-293T cells transfected with the indicated constructs were immunoprecipitated using anti-flag agarose beads. The upper panel shows the interacted proteins, and the lower panel shows the input proteins. e BH3-like domain of GSDMD is required for the interaction with Bcl-2. The lysates from HEK-293T cells transiently expressing the indicated constructs were used to immunoprecipitated Flag tagged GSDMD. The upper panel shows the co-immunoprecipitated proteins, and the lower panel shows the input proteins. f Bcl-2 recombinant protein binds GSDMD in vitro. Immunoprecipitated GSDMD or its L150Q mutant from HEK-293T cells were incubated with Bcl-2 recombinant protein, or the same amount of BSA as a negative control for one hour, then heavily washed. The upper panel shows Bcl-2 bound with GSDMD. The lower panel shows Bcl-2 bound to GSDMD wild type and L150Q mutant. g GST fusion BH3-like domain of GSDMD binds with Bcl-2 recombinant protein. The indicated GST fusion BH3 domains were expressed and purified from HEK-293T cells. Bcl-2 recombinant protein was added for in vitro binding with indicated GST fusion proteins.

    Journal: Cell Death Discovery

    Article Title: Bcl-2 regulates pyroptosis and necroptosis by targeting BH3-like domains in GSDMD and MLKL

    doi: 10.1038/s41420-019-0230-2

    Figure Lengend Snippet: Bcl-2 interacts with GSDMD. a A predicted BH3-like domain in GSDMD is conserved across the indicated species. The consensus BH3 domain sequence is shown. b Endogenous Bcl-2 interacts with the endogenous GSDMD. Lysates from THP-1 cells treated with the indicated inflammasome activators (LPS 50 ng/ml 5 h, nigericin 5 µM 30 min) were used for Bcl-2 immunoprecipitations. The blots of Bcl-2 that co-immunoprecipitated with GSDMD are showed on the upper panel. The lower panels show the input proteins in cell lysates. c GSDMD-CT does not interact with Bcl-2. Lysates from HEK-293T cells transfected with the indicated constructs were immunoprecipitated using anti-Flag antibodies conjugated agarose beads to pull down Flag-GSDMD. Matched non-specific antibodies were used for the mock immunoprecipitation. The left panel shows the input proteins, and the right panel shows the interacting proteins. d GSDMD-NT and its BH3-like domain are required for the interaction between GSDMD and Bcl-2. The lysates from HEK-293T cells transfected with the indicated constructs were immunoprecipitated using anti-flag agarose beads. The upper panel shows the interacted proteins, and the lower panel shows the input proteins. e BH3-like domain of GSDMD is required for the interaction with Bcl-2. The lysates from HEK-293T cells transiently expressing the indicated constructs were used to immunoprecipitated Flag tagged GSDMD. The upper panel shows the co-immunoprecipitated proteins, and the lower panel shows the input proteins. f Bcl-2 recombinant protein binds GSDMD in vitro. Immunoprecipitated GSDMD or its L150Q mutant from HEK-293T cells were incubated with Bcl-2 recombinant protein, or the same amount of BSA as a negative control for one hour, then heavily washed. The upper panel shows Bcl-2 bound with GSDMD. The lower panel shows Bcl-2 bound to GSDMD wild type and L150Q mutant. g GST fusion BH3-like domain of GSDMD binds with Bcl-2 recombinant protein. The indicated GST fusion BH3 domains were expressed and purified from HEK-293T cells. Bcl-2 recombinant protein was added for in vitro binding with indicated GST fusion proteins.

    Article Snippet: Reagents and antibodies Antibodies used for immunoblotting analysis were the following: anti-IL-1β (#12703), anti-caspase-1 (#3866), anti-caspase-4 (#4450), anti-caspase-5 (#46680), anti-MLKL (#14993), anti-phospho-MLKL (#91689), anti-RIP-3 (#13526), anti-phospho-RIP3 (#93654) and anti-Myc-tag (#2278) (Cell Signaling Technology); and anti-Flag (Sigma-Aldrich, F1804), anti-Bcl-2 (Santa Cruz Biotechnology, sc-783), anti-actin conjugated to horseradish peroxidase (Sigma-Aldrich, A3854), anti-GAPDH conjugated to horseradish peroxidase (Proteintech, HRP-60004), goat anti-rabbit HRP-linked antibody (#7074), and horse anti-mouse HRP-linked antibody (#7076) (Cell Signaling Technology).

    Techniques: Sequencing, Immunoprecipitation, Transfection, Construct, Expressing, Recombinant, In Vitro, Mutagenesis, Incubation, Negative Control, Purification, Binding Assay

    Bcl-2 reduces the caspase-1/-4/-5 generated P30.5 GSDMD fragment but enhances the P10 N-terminal fragment. a Cell lysates from HEK-293T cells transiently expressing the indicated constructs were used for the immunoblots. The Flag-GSDMD-full length, Flag-N-terminal (NT) fragment P30.5, and the Flag-NT fragment P10 proteins were detected with the anti-Flag antibody (upper panels), and the caspase proteins blotted with anti-Myc antibody (middle panels). The expressed Bcl-2 was immunoblotted with anti-Bcl-2 antibody. The GAPDH blot serves as a loading control. b , c BH3-like domain of GSDMD is required for Bcl-2 to regulate GSDMD cleaved by caspase-1, -4 or -5. Immunoblots of cell lysates from HEK-293T cells transiently transfected with Flag-GSDMD or Flag-GSDMD L150Q and the indicated constructs. The full length, P30.5, and P10 N-terminal fragments were detected with anti-Flag antibody (upper panels). Caspase-1/-4/-5 were detected with anti-Myc antibody. Bcl-2 was immunoblotted with anti-Bcl-2 antibody. d Trypan blue exclusion was used to quantitate cell death. The HEK-293T cells were transfected with the indicated constructs for 24 h. Data are means and ± SD of four samples. **p

    Journal: Cell Death Discovery

    Article Title: Bcl-2 regulates pyroptosis and necroptosis by targeting BH3-like domains in GSDMD and MLKL

    doi: 10.1038/s41420-019-0230-2

    Figure Lengend Snippet: Bcl-2 reduces the caspase-1/-4/-5 generated P30.5 GSDMD fragment but enhances the P10 N-terminal fragment. a Cell lysates from HEK-293T cells transiently expressing the indicated constructs were used for the immunoblots. The Flag-GSDMD-full length, Flag-N-terminal (NT) fragment P30.5, and the Flag-NT fragment P10 proteins were detected with the anti-Flag antibody (upper panels), and the caspase proteins blotted with anti-Myc antibody (middle panels). The expressed Bcl-2 was immunoblotted with anti-Bcl-2 antibody. The GAPDH blot serves as a loading control. b , c BH3-like domain of GSDMD is required for Bcl-2 to regulate GSDMD cleaved by caspase-1, -4 or -5. Immunoblots of cell lysates from HEK-293T cells transiently transfected with Flag-GSDMD or Flag-GSDMD L150Q and the indicated constructs. The full length, P30.5, and P10 N-terminal fragments were detected with anti-Flag antibody (upper panels). Caspase-1/-4/-5 were detected with anti-Myc antibody. Bcl-2 was immunoblotted with anti-Bcl-2 antibody. d Trypan blue exclusion was used to quantitate cell death. The HEK-293T cells were transfected with the indicated constructs for 24 h. Data are means and ± SD of four samples. **p

    Article Snippet: Reagents and antibodies Antibodies used for immunoblotting analysis were the following: anti-IL-1β (#12703), anti-caspase-1 (#3866), anti-caspase-4 (#4450), anti-caspase-5 (#46680), anti-MLKL (#14993), anti-phospho-MLKL (#91689), anti-RIP-3 (#13526), anti-phospho-RIP3 (#93654) and anti-Myc-tag (#2278) (Cell Signaling Technology); and anti-Flag (Sigma-Aldrich, F1804), anti-Bcl-2 (Santa Cruz Biotechnology, sc-783), anti-actin conjugated to horseradish peroxidase (Sigma-Aldrich, A3854), anti-GAPDH conjugated to horseradish peroxidase (Proteintech, HRP-60004), goat anti-rabbit HRP-linked antibody (#7074), and horse anti-mouse HRP-linked antibody (#7076) (Cell Signaling Technology).

    Techniques: Generated, Expressing, Construct, Western Blot, Transfection

    Bcl-2 expression levels in THP-1 cells affect NLRP3 inflammasome activation. a Expression of Bcl-2 but not Bcl-2 G145A in THP-1 cells impairs LPS and nigericin (Nig.) triggered inflammasome activation. Precipitated proteins from cell supernatants (Sup.) and from cell lysates (Ly.) were immunoblotted for the indicated proteins. b A siRNA directed against Bcl-2 or a scramble control siRNA were transfected into THP-1 cells. The cells were treated with LPS and nigericin and precipitated proteins from cell supernatants and the cell lysates were used for immunoblotting the indicated proteins. c NLRP3 inflammasome was reconstituted in HEK-293T cells by exogenous expression of all needed elements. Nigericin addition induced inflammasome activation, which was monitored by the presence of cleaved caspase-1. d , e Bcl-2 expression does not impact cytosol LPS triggered caspase-4 or -5 activation. Cell lysates prepared from HEK-293T cells transfected with the indicated constructs in the presence or absence of LPS transfection. The indicated proteins were detected by immunoblot analysis. The presence of intracellular LPS triggers caspase-4 and caspase-5 cleavage.

    Journal: Cell Death Discovery

    Article Title: Bcl-2 regulates pyroptosis and necroptosis by targeting BH3-like domains in GSDMD and MLKL

    doi: 10.1038/s41420-019-0230-2

    Figure Lengend Snippet: Bcl-2 expression levels in THP-1 cells affect NLRP3 inflammasome activation. a Expression of Bcl-2 but not Bcl-2 G145A in THP-1 cells impairs LPS and nigericin (Nig.) triggered inflammasome activation. Precipitated proteins from cell supernatants (Sup.) and from cell lysates (Ly.) were immunoblotted for the indicated proteins. b A siRNA directed against Bcl-2 or a scramble control siRNA were transfected into THP-1 cells. The cells were treated with LPS and nigericin and precipitated proteins from cell supernatants and the cell lysates were used for immunoblotting the indicated proteins. c NLRP3 inflammasome was reconstituted in HEK-293T cells by exogenous expression of all needed elements. Nigericin addition induced inflammasome activation, which was monitored by the presence of cleaved caspase-1. d , e Bcl-2 expression does not impact cytosol LPS triggered caspase-4 or -5 activation. Cell lysates prepared from HEK-293T cells transfected with the indicated constructs in the presence or absence of LPS transfection. The indicated proteins were detected by immunoblot analysis. The presence of intracellular LPS triggers caspase-4 and caspase-5 cleavage.

    Article Snippet: Reagents and antibodies Antibodies used for immunoblotting analysis were the following: anti-IL-1β (#12703), anti-caspase-1 (#3866), anti-caspase-4 (#4450), anti-caspase-5 (#46680), anti-MLKL (#14993), anti-phospho-MLKL (#91689), anti-RIP-3 (#13526), anti-phospho-RIP3 (#93654) and anti-Myc-tag (#2278) (Cell Signaling Technology); and anti-Flag (Sigma-Aldrich, F1804), anti-Bcl-2 (Santa Cruz Biotechnology, sc-783), anti-actin conjugated to horseradish peroxidase (Sigma-Aldrich, A3854), anti-GAPDH conjugated to horseradish peroxidase (Proteintech, HRP-60004), goat anti-rabbit HRP-linked antibody (#7074), and horse anti-mouse HRP-linked antibody (#7076) (Cell Signaling Technology).

    Techniques: Expressing, Activation Assay, Transfection, Construct

    Bcl-2 induces caspase-1/-4/-5 to cleave GSDMD at D87. a – c Cell lysates from HEK-293T cells transfected with the Flag-GSDMD, Flag-GSDMD D87A, and Flag-GSDMD D275A along with Myc-Casp-1 ( a ), Myc-Casp-4 ( b ), or Myc-Casp-5 ( c ) in the presence or absence of Bcl-2 were used for immunoblotting. Antibodies raised against amino acids 154–252 of GSDMD were used to detect the C-terminal fragment P42.8 of Flag-GSDMD. The membranes were re-blotted with anti-Flag antibodies to detect the N-terminal fragments of Flag-GSDMD cleaved at D87 or D275. Myc-tagged caspase-1, -4 and -5 were detected with tag specific antibodies. Bcl-2 and GAPDH were detected with specific antibodies. d Bcl-2 recombinant protein reduces the cleavage of GSDMD driven by active caspase-3 in vitro. The immunoprecipitated Flag-GSDMD served as a caspase-3 substrate and was incubated with active caspase-3 with or without Bcl-2 protein in caspase assay buffer. The upper panel shows GSDMD full length, merged IgG heavy chain, IgG light chain and cleaved GSDMD N-terminal P10 at longer exposure (LE). The middle panel shows the shorter exposure (SE). The bottom panel shows the added Bcl-2 recombinant protein.

    Journal: Cell Death Discovery

    Article Title: Bcl-2 regulates pyroptosis and necroptosis by targeting BH3-like domains in GSDMD and MLKL

    doi: 10.1038/s41420-019-0230-2

    Figure Lengend Snippet: Bcl-2 induces caspase-1/-4/-5 to cleave GSDMD at D87. a – c Cell lysates from HEK-293T cells transfected with the Flag-GSDMD, Flag-GSDMD D87A, and Flag-GSDMD D275A along with Myc-Casp-1 ( a ), Myc-Casp-4 ( b ), or Myc-Casp-5 ( c ) in the presence or absence of Bcl-2 were used for immunoblotting. Antibodies raised against amino acids 154–252 of GSDMD were used to detect the C-terminal fragment P42.8 of Flag-GSDMD. The membranes were re-blotted with anti-Flag antibodies to detect the N-terminal fragments of Flag-GSDMD cleaved at D87 or D275. Myc-tagged caspase-1, -4 and -5 were detected with tag specific antibodies. Bcl-2 and GAPDH were detected with specific antibodies. d Bcl-2 recombinant protein reduces the cleavage of GSDMD driven by active caspase-3 in vitro. The immunoprecipitated Flag-GSDMD served as a caspase-3 substrate and was incubated with active caspase-3 with or without Bcl-2 protein in caspase assay buffer. The upper panel shows GSDMD full length, merged IgG heavy chain, IgG light chain and cleaved GSDMD N-terminal P10 at longer exposure (LE). The middle panel shows the shorter exposure (SE). The bottom panel shows the added Bcl-2 recombinant protein.

    Article Snippet: Reagents and antibodies Antibodies used for immunoblotting analysis were the following: anti-IL-1β (#12703), anti-caspase-1 (#3866), anti-caspase-4 (#4450), anti-caspase-5 (#46680), anti-MLKL (#14993), anti-phospho-MLKL (#91689), anti-RIP-3 (#13526), anti-phospho-RIP3 (#93654) and anti-Myc-tag (#2278) (Cell Signaling Technology); and anti-Flag (Sigma-Aldrich, F1804), anti-Bcl-2 (Santa Cruz Biotechnology, sc-783), anti-actin conjugated to horseradish peroxidase (Sigma-Aldrich, A3854), anti-GAPDH conjugated to horseradish peroxidase (Proteintech, HRP-60004), goat anti-rabbit HRP-linked antibody (#7074), and horse anti-mouse HRP-linked antibody (#7076) (Cell Signaling Technology).

    Techniques: Transfection, Recombinant, In Vitro, Immunoprecipitation, Incubation, Caspase Assay

    LARP1 expression in ovarian tumors correlates with BIK and BCL2 expression. ( A ) Correlation of mRNA expression (Log 2 RPKM) in ovarian tumors ( n = 412) between LARP1 and BCL2 or BIK (Pearson R). Data from TCGA Ovarian RNAseq cohort (tcga-data.nci.nih.gov). ( B ) Comparison of upper and lower quartiles of LARP1 expression (Log 2 RPKM, n = 103 in each), by BCL2 or BIK expression (Wilcoxon test). Data as before. ( C ) A summary of the role of LARP1 in the ovarian cancer cell. As a component of mRNP complexes containing transcripts of survival-associated genes, LARP1 acts to simultaneously stabilize anti-apoptotic mRNAs, whilst destabilizing pro-apoptotic transcripts. The net effect is to promote apoptosis evasion, enhancing tumorigenicity, chemoresistance and cancer stem cell (CSC)-like traits.

    Journal: Nucleic Acids Research

    Article Title: The RNA-binding protein LARP1 is a post-transcriptional regulator of survival and tumorigenesis in ovarian cancer

    doi: 10.1093/nar/gkv1515

    Figure Lengend Snippet: LARP1 expression in ovarian tumors correlates with BIK and BCL2 expression. ( A ) Correlation of mRNA expression (Log 2 RPKM) in ovarian tumors ( n = 412) between LARP1 and BCL2 or BIK (Pearson R). Data from TCGA Ovarian RNAseq cohort (tcga-data.nci.nih.gov). ( B ) Comparison of upper and lower quartiles of LARP1 expression (Log 2 RPKM, n = 103 in each), by BCL2 or BIK expression (Wilcoxon test). Data as before. ( C ) A summary of the role of LARP1 in the ovarian cancer cell. As a component of mRNP complexes containing transcripts of survival-associated genes, LARP1 acts to simultaneously stabilize anti-apoptotic mRNAs, whilst destabilizing pro-apoptotic transcripts. The net effect is to promote apoptosis evasion, enhancing tumorigenicity, chemoresistance and cancer stem cell (CSC)-like traits.

    Article Snippet: Blocking and antibody incubation was performed according to antibody manufacturers’ instructions with anti-LARP1 (rabbit, SDIX-Novus Biologicals), anti-BCL2 (mouse, Santa Cruz), anti-BIK (goat, Santa Cruz) and anti-HSP60 (rabbit, Abcam) antibodies.

    Techniques: Expressing

    LARP1 regulates stability at the level of the 3′ UTR and binds the BCL2 3′ UTR via its DM15 domain. ( A ) Schematics outlining construction of 3′-untranslated region (3′ UTR) reporter constructs for BIK and BCL2. Both were compared relative to a control vector with no additional 3′ UTR sequence. ( B ) OVCAR8 and SKOV3 cells were co-transfected with Renilla luciferase 3′ UTR constructs and a control Firefly luciferase control vector. Renilla luciferase activity following LARP1 knockdown was determined for each 3′ UTR construct cells. Data was normalized to Firefly luciferase activity. ( C ) OVCAR8 cells were co-transfected with Renilla luciferase 3′ UTR constructs and a control Firefly luciferase control vector and Renilla luciferase mRNA abundance following LARP1 knockdown was determined using RT-qPCR. ( D ) Electrophoretic mobility shift assay (EMSA) of LARP1 with a fragment of the BCL2 3′ UTR (construct 3). The affinity of this interaction is indicated. ( E ) Competition experiment analysed by EMSA of LARP1 pre-bound to radiolabelled RNA representing the 5′ TOP of RPS6 and competed with cold competitors, as indicated. ( F ) Confocal immunofluorescence microscopy of SKOV3 cells treated with sodium arsenite to trigger aggregation of mRNP bodies. Cells were stained for LARP1 protein (green) and either the P-body marker DCP1a or stress granule marker PABP (both red). Scale bar 10 μm (top) and 25 μm (bottom).

    Journal: Nucleic Acids Research

    Article Title: The RNA-binding protein LARP1 is a post-transcriptional regulator of survival and tumorigenesis in ovarian cancer

    doi: 10.1093/nar/gkv1515

    Figure Lengend Snippet: LARP1 regulates stability at the level of the 3′ UTR and binds the BCL2 3′ UTR via its DM15 domain. ( A ) Schematics outlining construction of 3′-untranslated region (3′ UTR) reporter constructs for BIK and BCL2. Both were compared relative to a control vector with no additional 3′ UTR sequence. ( B ) OVCAR8 and SKOV3 cells were co-transfected with Renilla luciferase 3′ UTR constructs and a control Firefly luciferase control vector. Renilla luciferase activity following LARP1 knockdown was determined for each 3′ UTR construct cells. Data was normalized to Firefly luciferase activity. ( C ) OVCAR8 cells were co-transfected with Renilla luciferase 3′ UTR constructs and a control Firefly luciferase control vector and Renilla luciferase mRNA abundance following LARP1 knockdown was determined using RT-qPCR. ( D ) Electrophoretic mobility shift assay (EMSA) of LARP1 with a fragment of the BCL2 3′ UTR (construct 3). The affinity of this interaction is indicated. ( E ) Competition experiment analysed by EMSA of LARP1 pre-bound to radiolabelled RNA representing the 5′ TOP of RPS6 and competed with cold competitors, as indicated. ( F ) Confocal immunofluorescence microscopy of SKOV3 cells treated with sodium arsenite to trigger aggregation of mRNP bodies. Cells were stained for LARP1 protein (green) and either the P-body marker DCP1a or stress granule marker PABP (both red). Scale bar 10 μm (top) and 25 μm (bottom).

    Article Snippet: Blocking and antibody incubation was performed according to antibody manufacturers’ instructions with anti-LARP1 (rabbit, SDIX-Novus Biologicals), anti-BCL2 (mouse, Santa Cruz), anti-BIK (goat, Santa Cruz) and anti-HSP60 (rabbit, Abcam) antibodies.

    Techniques: Construct, Plasmid Preparation, Sequencing, Transfection, Luciferase, Activity Assay, Quantitative RT-PCR, Electrophoretic Mobility Shift Assay, Immunofluorescence, Microscopy, Staining, Marker

    LARP1 is a component of BCL2-containing mRNP complexes and promotes transcript stability. ( A ) Schematic of LARP1 RNA-immunoprecipitation (RIP) with representative western blot of LARP1 protein following LARP1-immunoprecipiation in OVCAR8 and SKOV3 cells. ( B ) RT-qPCR analysis of BCL2 and BIK mRNA obtained from RIP with anti-LARP1 and IgG control antibodies in OVCAR8 cells. OZA1 and 28S were included as negative controls. ( C ) Representative cell images following LARP1 knockdown and 8 h exposure to actinomycin D (Scale bar 200 μm). ( D ) Following transient knockdown of LARP1, OVCAR8 cells were treated with actinomycin D to halt transcription and apoptosis (as determined by cleaved Caspase 3/7) was monitored using the CaspaseGlo assay (data normalized to T = 0, at the time of actinomycin-D administration). ** P

    Journal: Nucleic Acids Research

    Article Title: The RNA-binding protein LARP1 is a post-transcriptional regulator of survival and tumorigenesis in ovarian cancer

    doi: 10.1093/nar/gkv1515

    Figure Lengend Snippet: LARP1 is a component of BCL2-containing mRNP complexes and promotes transcript stability. ( A ) Schematic of LARP1 RNA-immunoprecipitation (RIP) with representative western blot of LARP1 protein following LARP1-immunoprecipiation in OVCAR8 and SKOV3 cells. ( B ) RT-qPCR analysis of BCL2 and BIK mRNA obtained from RIP with anti-LARP1 and IgG control antibodies in OVCAR8 cells. OZA1 and 28S were included as negative controls. ( C ) Representative cell images following LARP1 knockdown and 8 h exposure to actinomycin D (Scale bar 200 μm). ( D ) Following transient knockdown of LARP1, OVCAR8 cells were treated with actinomycin D to halt transcription and apoptosis (as determined by cleaved Caspase 3/7) was monitored using the CaspaseGlo assay (data normalized to T = 0, at the time of actinomycin-D administration). ** P

    Article Snippet: Blocking and antibody incubation was performed according to antibody manufacturers’ instructions with anti-LARP1 (rabbit, SDIX-Novus Biologicals), anti-BCL2 (mouse, Santa Cruz), anti-BIK (goat, Santa Cruz) and anti-HSP60 (rabbit, Abcam) antibodies.

    Techniques: Immunoprecipitation, Western Blot, Quantitative RT-PCR, Caspase-Glo Assay

    BCL2 can rescue the LARP1 apoptotic phenotype. ( A ) BCL2 promoter activity following LARP1 knockdown ( firefly luciferase mRNA normalized to renilla luciferase mRNA control). ( B ) Western blot analysis of BCL2 protein levels following LARP1 knockdown. ( C ) Western blot analysis of BCL2 protein levels following BCL2 knockdown. ( D ) Percentage of Annexin V-positive OVCAR8 cells, determined by flow cytometry, following transient BCL2 knockdown, with and without co-treatment with cisplatin (25 μM). ( E ) Percentage of CD133-posititve OVCAR3 cells, determined by flow cytometry, following transient knockdown of BCL2. ( F ) Percentage of Annexin V-positive OVCAR8 cells, determined by flow cytometry, following LARP1 knockdown or transfection with control siRNA and treatment with cisplatin (25 μM), with co-transfection of a Flag-tagged control (F-empty) or BCL2-overexpression (F-BCL2) construct. Minimum of three experimental repeats. Student t -test. Error bars indicate SEM.

    Journal: Nucleic Acids Research

    Article Title: The RNA-binding protein LARP1 is a post-transcriptional regulator of survival and tumorigenesis in ovarian cancer

    doi: 10.1093/nar/gkv1515

    Figure Lengend Snippet: BCL2 can rescue the LARP1 apoptotic phenotype. ( A ) BCL2 promoter activity following LARP1 knockdown ( firefly luciferase mRNA normalized to renilla luciferase mRNA control). ( B ) Western blot analysis of BCL2 protein levels following LARP1 knockdown. ( C ) Western blot analysis of BCL2 protein levels following BCL2 knockdown. ( D ) Percentage of Annexin V-positive OVCAR8 cells, determined by flow cytometry, following transient BCL2 knockdown, with and without co-treatment with cisplatin (25 μM). ( E ) Percentage of CD133-posititve OVCAR3 cells, determined by flow cytometry, following transient knockdown of BCL2. ( F ) Percentage of Annexin V-positive OVCAR8 cells, determined by flow cytometry, following LARP1 knockdown or transfection with control siRNA and treatment with cisplatin (25 μM), with co-transfection of a Flag-tagged control (F-empty) or BCL2-overexpression (F-BCL2) construct. Minimum of three experimental repeats. Student t -test. Error bars indicate SEM.

    Article Snippet: Blocking and antibody incubation was performed according to antibody manufacturers’ instructions with anti-LARP1 (rabbit, SDIX-Novus Biologicals), anti-BCL2 (mouse, Santa Cruz), anti-BIK (goat, Santa Cruz) and anti-HSP60 (rabbit, Abcam) antibodies.

    Techniques: Activity Assay, Luciferase, Western Blot, Flow Cytometry, Cytometry, Transfection, Cotransfection, Over Expression, Construct

    Expression of Bax, Bcl-2, and caspase-3 in NCI-H292 cells. (a) Protein expression bands were detected by western blot. (b) The bar charts of Bax/ β -actin and Bcl-2/ β -actin. (c) The bar chart shows the ratio of Bcl-2/Bax. (d) Expression of caspase-3 normalized to β -actin. Compared with the control group, the expression of Bcl-2/Bax decreased and cleaved caspase-3 increased. # P

    Journal: Journal of Immunology Research

    Article Title: Poly-L-Arginine Induces Apoptosis of NCI-H292 Cells via ERK1/2 Signaling Pathway

    doi: 10.1155/2018/3651743

    Figure Lengend Snippet: Expression of Bax, Bcl-2, and caspase-3 in NCI-H292 cells. (a) Protein expression bands were detected by western blot. (b) The bar charts of Bax/ β -actin and Bcl-2/ β -actin. (c) The bar chart shows the ratio of Bcl-2/Bax. (d) Expression of caspase-3 normalized to β -actin. Compared with the control group, the expression of Bcl-2/Bax decreased and cleaved caspase-3 increased. # P

    Article Snippet: Anti-Bax, anti-Bcl-2, and anti-caspase-3 were from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Expressing, Western Blot

    Expression of Bax, Bcl-2, caspase-3, and p-ERK/ERK in NCI-H292 cells treated with PLA and PD98059. (a) Protein expression bands were detected by Western blot. (b) The bar chart of Bcl-2/Bax. (c) The bar chart of caspase-3/ β -actin. (d) The bar chart of p-ERK/ERK. Compared with PLA group, PD98059 significantly inhibited the activation of ERK1/2 and also blocked the regulatory effect of PLA on Bcl-2/Bax and cleaved caspase-3. ### P

    Journal: Journal of Immunology Research

    Article Title: Poly-L-Arginine Induces Apoptosis of NCI-H292 Cells via ERK1/2 Signaling Pathway

    doi: 10.1155/2018/3651743

    Figure Lengend Snippet: Expression of Bax, Bcl-2, caspase-3, and p-ERK/ERK in NCI-H292 cells treated with PLA and PD98059. (a) Protein expression bands were detected by Western blot. (b) The bar chart of Bcl-2/Bax. (c) The bar chart of caspase-3/ β -actin. (d) The bar chart of p-ERK/ERK. Compared with PLA group, PD98059 significantly inhibited the activation of ERK1/2 and also blocked the regulatory effect of PLA on Bcl-2/Bax and cleaved caspase-3. ### P

    Article Snippet: Anti-Bax, anti-Bcl-2, and anti-caspase-3 were from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Expressing, Proximity Ligation Assay, Western Blot, Activation Assay

    Effect of TiO 2 NP exposure on the apoptosis of placenta. Notes: ( A ) Representative immunoblotting images of Casp-3, Bax and Bcl-2 proteins in the TiO 2 NP-treated placentas. ( B – D ) Densitometric values from Western blot analyses of Bcl-2 ( B ), Bax ( C ) and cleaved-Casp-3 ( D ) proteins. The data are normalized to β-actin expression (but cleaved-Casp-3 is normalized to Casp-3) and shown as mean ± SEM of 6 animals. * P

    Journal: International Journal of Nanomedicine

    Article Title: Gestational exposure to titanium dioxide nanoparticles impairs the placentation through dysregulation of vascularization, proliferation and apoptosis in mice

    doi: 10.2147/IJN.S152400

    Figure Lengend Snippet: Effect of TiO 2 NP exposure on the apoptosis of placenta. Notes: ( A ) Representative immunoblotting images of Casp-3, Bax and Bcl-2 proteins in the TiO 2 NP-treated placentas. ( B – D ) Densitometric values from Western blot analyses of Bcl-2 ( B ), Bax ( C ) and cleaved-Casp-3 ( D ) proteins. The data are normalized to β-actin expression (but cleaved-Casp-3 is normalized to Casp-3) and shown as mean ± SEM of 6 animals. * P

    Article Snippet: Nonspecific binding was blocked with 5% BSA for 1 h at room temperature and blotting membranes were incubated with the following primary antibodies at 4°C overnight: rabbit anti-Bcl-2, Bax, caspase-3 (Casp-3) and mouse anti-β-actin (Cell Signaling).

    Techniques: Western Blot, Expressing

    Effects of KHG26792 on MPP + -induced imbalance in the expression of Bax and Bcl-2, and caspase-3 activity. (A) Total cell extracts were subjected to Western blotting analysis with antibodies against Bcl-2 and Bax. β-actin was used as a loading control. (B, C) Densitometric analysis is shown for the relative expression level of each protein. (D) Caspase-3 activity. Data from three independent experiments are presented as the means ± SD (*P

    Journal: BMB Reports

    Article Title: 3-(Naphthalen-2-yl(propoxy)methyl)azetidine hydrochloride attenuates MPP+-induced cytotoxicity by regulating oxidative stress and mitochondrial dysfunction in SH-SY5Y cells

    doi: 10.5483/BMBRep.2018.51.11.123

    Figure Lengend Snippet: Effects of KHG26792 on MPP + -induced imbalance in the expression of Bax and Bcl-2, and caspase-3 activity. (A) Total cell extracts were subjected to Western blotting analysis with antibodies against Bcl-2 and Bax. β-actin was used as a loading control. (B, C) Densitometric analysis is shown for the relative expression level of each protein. (D) Caspase-3 activity. Data from three independent experiments are presented as the means ± SD (*P

    Article Snippet: Antibodies against Bcl-2, Bax, and β-actin were purchased from Cell Signaling Technology (Beverly, MA).

    Techniques: Expressing, Activity Assay, Western Blot

    Three-week-old Bcl-2 −/− mice have increased primary spongiosa due to both decreased number and dysfunctionality of osteoclasts. A , radiographic analysis of wild type ( WT ) and Bcl-2 −/− mice at 3 weeks of age. The arrows indicate the sclerotic regions at the metaphysis. Bar , 10 mm. B , histological section of tibiae of WT and Bcl-2 −/− mice (hematoxylin and eosin, von Kossa, and TRAP staining). The standardized regions of interest in the primary and secondary spongiosa are indicated by boxes . The insets of the lower panels indicate higher magnification views of the rectangular areas. Bars , 100 μm. C and D , histomorphometric analysis. Parameters of bone resorption ( C ) and bone formation ( D ) analyzed in the tibiae of WT and Bcl-2 −/− mice. Cancellous bone volume ( BV/TV ) is the percent of total marrow area (including trabeculae) occupied by cancellous bone. Trabecular thickness ( Tb.Th ) is the mean distance across individual trabeculae in micrometers. Trabecular number ( Tb.N ) per mm is calculated as (BV/TV)/Tb.Th. These variables can be used to evaluate trabecular connectivity. Eroded surface ( ES/BS ) is the percent of cancellous surface occupied by Howship's lacunae, with and without osteoclasts. Osteoblast surface ( Ob.S/BS ) and osteoclast surface ( Oc.S/BS ) identify the percent of cancellous surface occupied by osteoblasts and osteoclasts, respectively. Osteoid surface ( Osteoid S/BS ) is the percent of cancellous surface with unmineralized osteoid, with and without osteoblasts. Osteoid thickness ( Osteoid Th ) is the mean thickness, in micrometers, of the osteoid on cancellous surfaces. E , calcein double labeling of the mineralized matrix of proximal tibia at an interval of 2 days. WT mice ( n = 4), Bcl-2 −/− mice ( n = 4). Mineral appositional rate ( MAR ) is the rate (in μm/day) at which new bone is being added to cancellous surfaces. Bone formation rates ( BFR ) are estimates of cancellous bone volume that are being replaced annually. * indicates significantly different, p

    Journal: The Journal of Biological Chemistry

    Article Title: Anti-apoptotic Molecule Bcl-2 Regulates the Differentiation, Activation, and Survival of Both Osteoblasts and Osteoclasts *

    doi: 10.1074/jbc.M109.016915

    Figure Lengend Snippet: Three-week-old Bcl-2 −/− mice have increased primary spongiosa due to both decreased number and dysfunctionality of osteoclasts. A , radiographic analysis of wild type ( WT ) and Bcl-2 −/− mice at 3 weeks of age. The arrows indicate the sclerotic regions at the metaphysis. Bar , 10 mm. B , histological section of tibiae of WT and Bcl-2 −/− mice (hematoxylin and eosin, von Kossa, and TRAP staining). The standardized regions of interest in the primary and secondary spongiosa are indicated by boxes . The insets of the lower panels indicate higher magnification views of the rectangular areas. Bars , 100 μm. C and D , histomorphometric analysis. Parameters of bone resorption ( C ) and bone formation ( D ) analyzed in the tibiae of WT and Bcl-2 −/− mice. Cancellous bone volume ( BV/TV ) is the percent of total marrow area (including trabeculae) occupied by cancellous bone. Trabecular thickness ( Tb.Th ) is the mean distance across individual trabeculae in micrometers. Trabecular number ( Tb.N ) per mm is calculated as (BV/TV)/Tb.Th. These variables can be used to evaluate trabecular connectivity. Eroded surface ( ES/BS ) is the percent of cancellous surface occupied by Howship's lacunae, with and without osteoclasts. Osteoblast surface ( Ob.S/BS ) and osteoclast surface ( Oc.S/BS ) identify the percent of cancellous surface occupied by osteoblasts and osteoclasts, respectively. Osteoid surface ( Osteoid S/BS ) is the percent of cancellous surface with unmineralized osteoid, with and without osteoblasts. Osteoid thickness ( Osteoid Th ) is the mean thickness, in micrometers, of the osteoid on cancellous surfaces. E , calcein double labeling of the mineralized matrix of proximal tibia at an interval of 2 days. WT mice ( n = 4), Bcl-2 −/− mice ( n = 4). Mineral appositional rate ( MAR ) is the rate (in μm/day) at which new bone is being added to cancellous surfaces. Bone formation rates ( BFR ) are estimates of cancellous bone volume that are being replaced annually. * indicates significantly different, p

    Article Snippet: Interestingly, although the expression of runx2 and osterix was equivalent between these two cell preparations on day 3 of culture, the increase in their expression observed in Bcl-2 +/− Bim +/− cells was not apparent in Bcl-2 −/− Bim +/− cells, and the levels of runx2 and osterix expression were significantly lower in Bcl-2 −/− Bim +/− cells than Bcl-2 +/− Bim +/− cells after 3 weeks of culture ( G ).

    Techniques: Mouse Assay, Staining, Labeling

    Bcl-2 is required for survival of osteoclasts. A , TRAP staining of WT and Bcl-2 −/− osteoclasts in 12 h after cytokine withdrawal. B , survival rate of WT and Bcl-2 −/− osteoclasts after cytokine withdrawal. C , Western blotting analysis of cleaved caspase-3 in total cell lysates from WT and Bcl-2 −/− osteoclasts after cytokine withdrawal. D , TRAP staining of WT (empty vector) and Bcl-2 −/− osteoclasts 12 h after cytokine withdrawal. Osteoclasts were retrovirally transfected with empty vector or pMx Bcl-2-puro. E , survival of WT (empty vector) and Bcl-2 −/− osteoclasts retrovirally transfected with empty vector or pMx Bcl-2-puro. The osteoclast survival rate was represented as the percentage of morphologically intact TRAP-positive cells. **, significantly different p

    Journal: The Journal of Biological Chemistry

    Article Title: Anti-apoptotic Molecule Bcl-2 Regulates the Differentiation, Activation, and Survival of Both Osteoblasts and Osteoclasts *

    doi: 10.1074/jbc.M109.016915

    Figure Lengend Snippet: Bcl-2 is required for survival of osteoclasts. A , TRAP staining of WT and Bcl-2 −/− osteoclasts in 12 h after cytokine withdrawal. B , survival rate of WT and Bcl-2 −/− osteoclasts after cytokine withdrawal. C , Western blotting analysis of cleaved caspase-3 in total cell lysates from WT and Bcl-2 −/− osteoclasts after cytokine withdrawal. D , TRAP staining of WT (empty vector) and Bcl-2 −/− osteoclasts 12 h after cytokine withdrawal. Osteoclasts were retrovirally transfected with empty vector or pMx Bcl-2-puro. E , survival of WT (empty vector) and Bcl-2 −/− osteoclasts retrovirally transfected with empty vector or pMx Bcl-2-puro. The osteoclast survival rate was represented as the percentage of morphologically intact TRAP-positive cells. **, significantly different p

    Article Snippet: Interestingly, although the expression of runx2 and osterix was equivalent between these two cell preparations on day 3 of culture, the increase in their expression observed in Bcl-2 +/− Bim +/− cells was not apparent in Bcl-2 −/− Bim +/− cells, and the levels of runx2 and osterix expression were significantly lower in Bcl-2 −/− Bim +/− cells than Bcl-2 +/− Bim +/− cells after 3 weeks of culture ( G ).

    Techniques: Staining, Western Blot, Plasmid Preparation, Transfection

    Bcl-2 is essential for the later stage differentiation, mineralization, and survival of osteoblasts. A , proliferation of WT and Bcl-2 −/− osteoblasts was assessed by MTT assay. Each collection of osteoblasts was cultured in α-MEM, 10% FBS. B , alkaline phosphatase ( ALP ) and alizarin red S staining of WT and Bcl-2 −/− osteoblasts obtained from calvaria. C , mRNA expression of osteoblastic markers as determined by real time PCR. D , TUNEL staining of WT and Bcl-2 −/− osteoblasts incubated with osteogenic medium for 3 weeks. E , proportion of TUNEL-positive osteoblasts incubated with osteogenic medium for 3 weeks. ** significantly different, p

    Journal: The Journal of Biological Chemistry

    Article Title: Anti-apoptotic Molecule Bcl-2 Regulates the Differentiation, Activation, and Survival of Both Osteoblasts and Osteoclasts *

    doi: 10.1074/jbc.M109.016915

    Figure Lengend Snippet: Bcl-2 is essential for the later stage differentiation, mineralization, and survival of osteoblasts. A , proliferation of WT and Bcl-2 −/− osteoblasts was assessed by MTT assay. Each collection of osteoblasts was cultured in α-MEM, 10% FBS. B , alkaline phosphatase ( ALP ) and alizarin red S staining of WT and Bcl-2 −/− osteoblasts obtained from calvaria. C , mRNA expression of osteoblastic markers as determined by real time PCR. D , TUNEL staining of WT and Bcl-2 −/− osteoblasts incubated with osteogenic medium for 3 weeks. E , proportion of TUNEL-positive osteoblasts incubated with osteogenic medium for 3 weeks. ** significantly different, p

    Article Snippet: Interestingly, although the expression of runx2 and osterix was equivalent between these two cell preparations on day 3 of culture, the increase in their expression observed in Bcl-2 +/− Bim +/− cells was not apparent in Bcl-2 −/− Bim +/− cells, and the levels of runx2 and osterix expression were significantly lower in Bcl-2 −/− Bim +/− cells than Bcl-2 +/− Bim +/− cells after 3 weeks of culture ( G ).

    Techniques: MTT Assay, Cell Culture, ALP Assay, Staining, Expressing, Real-time Polymerase Chain Reaction, TUNEL Assay, Incubation

    Bcl-2 promotes osteoclast differentiation. A , flow cytometry of bone marrow cells obtained from WT and Bcl-2 −/− mice. Expression of c-Kit and Mac-1 (CD11b) on c-Fms + bone marrow cells was analyzed by FACS. B , proliferation of M-CSF-dependent osteoclast precursors assessed by MTT assay. C , differentiation of osteoclasts was assessed by TRAP solution assay after osteoclastogenesis was induced by RANKL stimulation. ** indicate significantly different, p

    Journal: The Journal of Biological Chemistry

    Article Title: Anti-apoptotic Molecule Bcl-2 Regulates the Differentiation, Activation, and Survival of Both Osteoblasts and Osteoclasts *

    doi: 10.1074/jbc.M109.016915

    Figure Lengend Snippet: Bcl-2 promotes osteoclast differentiation. A , flow cytometry of bone marrow cells obtained from WT and Bcl-2 −/− mice. Expression of c-Kit and Mac-1 (CD11b) on c-Fms + bone marrow cells was analyzed by FACS. B , proliferation of M-CSF-dependent osteoclast precursors assessed by MTT assay. C , differentiation of osteoclasts was assessed by TRAP solution assay after osteoclastogenesis was induced by RANKL stimulation. ** indicate significantly different, p

    Article Snippet: Interestingly, although the expression of runx2 and osterix was equivalent between these two cell preparations on day 3 of culture, the increase in their expression observed in Bcl-2 +/− Bim +/− cells was not apparent in Bcl-2 −/− Bim +/− cells, and the levels of runx2 and osterix expression were significantly lower in Bcl-2 −/− Bim +/− cells than Bcl-2 +/− Bim +/− cells after 3 weeks of culture ( G ).

    Techniques: Flow Cytometry, Cytometry, Mouse Assay, Expressing, FACS, MTT Assay

    Loss of a single Bim allele restored abnormal bone resorption but not bone formation in Bcl-2 −/− mice. A, left , radiographic analysis of WT and Bcl-2 −/− mice at 3 weeks of age, and Bcl-2 +/− Bim +/− and Bcl-2 −/− Bim +/− mice at 16 weeks of age. A, right , micro-computed tomography analysis of femurs in Bcl-2 +/− Bim +/− and Bcl-2 −/− Bim +/− mice at 16 weeks of age. Bar , 10 mm. B , quantification of micro-computed tomography data in Bcl-2 +/− Bim +/− and Bcl-2 −/− Bim +/− mice at 16 weeks of age. *, significantly different, p

    Journal: The Journal of Biological Chemistry

    Article Title: Anti-apoptotic Molecule Bcl-2 Regulates the Differentiation, Activation, and Survival of Both Osteoblasts and Osteoclasts *

    doi: 10.1074/jbc.M109.016915

    Figure Lengend Snippet: Loss of a single Bim allele restored abnormal bone resorption but not bone formation in Bcl-2 −/− mice. A, left , radiographic analysis of WT and Bcl-2 −/− mice at 3 weeks of age, and Bcl-2 +/− Bim +/− and Bcl-2 −/− Bim +/− mice at 16 weeks of age. A, right , micro-computed tomography analysis of femurs in Bcl-2 +/− Bim +/− and Bcl-2 −/− Bim +/− mice at 16 weeks of age. Bar , 10 mm. B , quantification of micro-computed tomography data in Bcl-2 +/− Bim +/− and Bcl-2 −/− Bim +/− mice at 16 weeks of age. *, significantly different, p

    Article Snippet: Interestingly, although the expression of runx2 and osterix was equivalent between these two cell preparations on day 3 of culture, the increase in their expression observed in Bcl-2 +/− Bim +/− cells was not apparent in Bcl-2 −/− Bim +/− cells, and the levels of runx2 and osterix expression were significantly lower in Bcl-2 −/− Bim +/− cells than Bcl-2 +/− Bim +/− cells after 3 weeks of culture ( G ).

    Techniques: Mouse Assay, Micro-CT

    Bcl-2 is necessary for bone resorbing activity of osteoclasts. A , resorption pits generated by WT and Bcl-2 −/− osteoclasts. After osteoclasts were generated on collagen gel-coated dishes, they were replated on dentine slices and cultured for an additional 24 h. The resorption areas were visualized by staining the dentine slices with 1% toluidine blue and measured using an image analysis system (Microanalyzer). **, significantly different, p

    Journal: The Journal of Biological Chemistry

    Article Title: Anti-apoptotic Molecule Bcl-2 Regulates the Differentiation, Activation, and Survival of Both Osteoblasts and Osteoclasts *

    doi: 10.1074/jbc.M109.016915

    Figure Lengend Snippet: Bcl-2 is necessary for bone resorbing activity of osteoclasts. A , resorption pits generated by WT and Bcl-2 −/− osteoclasts. After osteoclasts were generated on collagen gel-coated dishes, they were replated on dentine slices and cultured for an additional 24 h. The resorption areas were visualized by staining the dentine slices with 1% toluidine blue and measured using an image analysis system (Microanalyzer). **, significantly different, p

    Article Snippet: Interestingly, although the expression of runx2 and osterix was equivalent between these two cell preparations on day 3 of culture, the increase in their expression observed in Bcl-2 +/− Bim +/− cells was not apparent in Bcl-2 −/− Bim +/− cells, and the levels of runx2 and osterix expression were significantly lower in Bcl-2 −/− Bim +/− cells than Bcl-2 +/− Bim +/− cells after 3 weeks of culture ( G ).

    Techniques: Activity Assay, Generated, Cell Culture, Staining

    Many anticancer agents mediate tumour cell killing though activation of the BCL-2-regulated apoptotic pathway. BH3-only proteins are activated transcriptionally and/or posttranscriptionally in a cytotoxic stimulus-specific manner by many anticancer agents,

    Journal: Cell Death and Differentiation

    Article Title: The BCL-2 protein family, BH3-mimetics and cancer therapy

    doi: 10.1038/cdd.2015.50

    Figure Lengend Snippet: Many anticancer agents mediate tumour cell killing though activation of the BCL-2-regulated apoptotic pathway. BH3-only proteins are activated transcriptionally and/or posttranscriptionally in a cytotoxic stimulus-specific manner by many anticancer agents,

    Article Snippet: Some cancers (such as CLL) with very high expression of BCL-2 (and possibly also cancers expressing high levels of BCL-XL and/or BCL-W) respond robustly to the existing BH3-mimetics when used as single agents.

    Techniques: Activation Assay

    Role of the BCL-2-Regulated Apoptotic Pathway in Mouse Models of Tumorigenesis

    Journal: Cell Death and Differentiation

    Article Title: The BCL-2 protein family, BH3-mimetics and cancer therapy

    doi: 10.1038/cdd.2015.50

    Figure Lengend Snippet: Role of the BCL-2-Regulated Apoptotic Pathway in Mouse Models of Tumorigenesis

    Article Snippet: Some cancers (such as CLL) with very high expression of BCL-2 (and possibly also cancers expressing high levels of BCL-XL and/or BCL-W) respond robustly to the existing BH3-mimetics when used as single agents.

    Techniques:

    Role of the BCL-2-Regulated Apoptotic Pathway in Human Cancer

    Journal: Cell Death and Differentiation

    Article Title: The BCL-2 protein family, BH3-mimetics and cancer therapy

    doi: 10.1038/cdd.2015.50

    Figure Lengend Snippet: Role of the BCL-2-Regulated Apoptotic Pathway in Human Cancer

    Article Snippet: Some cancers (such as CLL) with very high expression of BCL-2 (and possibly also cancers expressing high levels of BCL-XL and/or BCL-W) respond robustly to the existing BH3-mimetics when used as single agents.

    Techniques:

    The BCL-2 family members interact to regulate initiation of apoptosis. In healthy cells, the BCL-2-like pro-survival proteins safeguard mitochondrial outer membrane integrity and cell survival by preventing the activation of BAX and BAK. Under conditions

    Journal: Cell Death and Differentiation

    Article Title: The BCL-2 protein family, BH3-mimetics and cancer therapy

    doi: 10.1038/cdd.2015.50

    Figure Lengend Snippet: The BCL-2 family members interact to regulate initiation of apoptosis. In healthy cells, the BCL-2-like pro-survival proteins safeguard mitochondrial outer membrane integrity and cell survival by preventing the activation of BAX and BAK. Under conditions

    Article Snippet: Some cancers (such as CLL) with very high expression of BCL-2 (and possibly also cancers expressing high levels of BCL-XL and/or BCL-W) respond robustly to the existing BH3-mimetics when used as single agents.

    Techniques: Activation Assay

    Melanoma-specific bcl-2 establishes a suppressive microenvironmental condition that impairs T cell response. (A) Western blot analysis of bcl-2 protein expression in murine melanoma B16/F10 control (B16/F10 Control) or bcl-2 overexpressing (B16/F10 Bcl-2) cells. β-actin is shown as loading and transferring control. One representative western blot analysis out of two with similar results is reported. (B) Quantification of in vivo tumor growth after subcutaneous injection of B16/F10 control or bcl-2 overexpressing cells in C57/Bl6 mice. Quantification by cytofluorimetric analysis of (C) CD45 + cells among live cells, (D) cd11b + F4/80 + (TAM) among CD45 + cells, (E) CD206 + (left panel), and MHCII + (right panel) cells among TAM in B16/F10 control or bcl-2 overexpressing tumors. Quantification by cytofluorimetric analysis of (F) CD3 + among CD45 + cells (left panel), CD4 + and CD8 + among CD3 + cells (right panel), (G) IFNγ production and (H) CD44 + CD62L - among CD3 + infiltrating cells. (G) The expression level of IFNγ was analyzed after stimulation with brefeldin A, ionomycin, and PMA as reported in Methods section. (I) Quantification of in vivo tumor growth of C57/Bl6 mice subcutaneously injected with control (Control) or bcl-2 overexpressing (Bcl-2) B16/F10 cells and treated with vehicle or with clodronate liposomes. Tumor growth is expressed as % over Control+vehicle group. (C–H) The results were reported as % of positive cells and represent the average ±SD of two independent experiments. (B–H) P values were calculated between control and bcl-2 overexpressing tumors. *P

    Journal: Journal for Immunotherapy of Cancer

    Article Title: Melanoma-specific bcl-2 promotes a protumoral M2-like phenotype by tumor-associated macrophages

    doi: 10.1136/jitc-2019-000489

    Figure Lengend Snippet: Melanoma-specific bcl-2 establishes a suppressive microenvironmental condition that impairs T cell response. (A) Western blot analysis of bcl-2 protein expression in murine melanoma B16/F10 control (B16/F10 Control) or bcl-2 overexpressing (B16/F10 Bcl-2) cells. β-actin is shown as loading and transferring control. One representative western blot analysis out of two with similar results is reported. (B) Quantification of in vivo tumor growth after subcutaneous injection of B16/F10 control or bcl-2 overexpressing cells in C57/Bl6 mice. Quantification by cytofluorimetric analysis of (C) CD45 + cells among live cells, (D) cd11b + F4/80 + (TAM) among CD45 + cells, (E) CD206 + (left panel), and MHCII + (right panel) cells among TAM in B16/F10 control or bcl-2 overexpressing tumors. Quantification by cytofluorimetric analysis of (F) CD3 + among CD45 + cells (left panel), CD4 + and CD8 + among CD3 + cells (right panel), (G) IFNγ production and (H) CD44 + CD62L - among CD3 + infiltrating cells. (G) The expression level of IFNγ was analyzed after stimulation with brefeldin A, ionomycin, and PMA as reported in Methods section. (I) Quantification of in vivo tumor growth of C57/Bl6 mice subcutaneously injected with control (Control) or bcl-2 overexpressing (Bcl-2) B16/F10 cells and treated with vehicle or with clodronate liposomes. Tumor growth is expressed as % over Control+vehicle group. (C–H) The results were reported as % of positive cells and represent the average ±SD of two independent experiments. (B–H) P values were calculated between control and bcl-2 overexpressing tumors. *P

    Article Snippet: Immunoreactions were revealed by Bond Polymer Refine Detection in an automated autostainer (BondTM Max, Leica Biosystems, Milan, Italy) using anti-F4/80 (SP115, ThermoFisher), -CD206 (Abcam, Cambridge, UK) or -bcl-2 (124, Dako, Milan, Italy) antibodies.

    Techniques: Western Blot, Expressing, Transferring, In Vivo, Injection, Mouse Assay

    Bcl-2 drives expression of COX-2/PGE2 axis and IL-1β, IL-8, IL-17, CCL2 in melanoma cells. Analysis of COX-2 expression by (A) Western blot and (B) qRT-PCR analyses in M14 human melanoma control (M14 Control) and bcl-2 overexpressing (M14 Bcl-2/6) cells. (A) β-actin is shown as loading and transferring control. One representative western blot analysis out of two with similar results is reported. The numbers indicate densitometric analysis relative to control. (C) ELISA of PGE2 levels in CM derived from M14 control and bcl-2 overexpressing cells. PGE2 levels were normalized to the number of adherent cells. (D) qRT-PCR and (E) ELISA analyses of IL-1β, IL-8 and IL-17 expression in M14 control and bcl-2 overexpressing cells. (E) Protein levels were normalized to the number of adherent cells. (F) qRT-PCR analysis of IL-1β (IL-1R1), IL-8 (CXCR1), IL-17 (IL-17RA) receptors in M14 control and bcl-2 overexpressing cells. (G) qRT-PCR analysis of CCL2, CSF-1 and SDF-1 mRNA levels in M14 control and bcl-2 overexpressing cells. (B–G) Fold induction relative to control is reported. The results represent the average±SEM (B, D, F, G) or ±SD (C, E) of three independent experiments. *P

    Journal: Journal for Immunotherapy of Cancer

    Article Title: Melanoma-specific bcl-2 promotes a protumoral M2-like phenotype by tumor-associated macrophages

    doi: 10.1136/jitc-2019-000489

    Figure Lengend Snippet: Bcl-2 drives expression of COX-2/PGE2 axis and IL-1β, IL-8, IL-17, CCL2 in melanoma cells. Analysis of COX-2 expression by (A) Western blot and (B) qRT-PCR analyses in M14 human melanoma control (M14 Control) and bcl-2 overexpressing (M14 Bcl-2/6) cells. (A) β-actin is shown as loading and transferring control. One representative western blot analysis out of two with similar results is reported. The numbers indicate densitometric analysis relative to control. (C) ELISA of PGE2 levels in CM derived from M14 control and bcl-2 overexpressing cells. PGE2 levels were normalized to the number of adherent cells. (D) qRT-PCR and (E) ELISA analyses of IL-1β, IL-8 and IL-17 expression in M14 control and bcl-2 overexpressing cells. (E) Protein levels were normalized to the number of adherent cells. (F) qRT-PCR analysis of IL-1β (IL-1R1), IL-8 (CXCR1), IL-17 (IL-17RA) receptors in M14 control and bcl-2 overexpressing cells. (G) qRT-PCR analysis of CCL2, CSF-1 and SDF-1 mRNA levels in M14 control and bcl-2 overexpressing cells. (B–G) Fold induction relative to control is reported. The results represent the average±SEM (B, D, F, G) or ±SD (C, E) of three independent experiments. *P

    Article Snippet: Immunoreactions were revealed by Bond Polymer Refine Detection in an automated autostainer (BondTM Max, Leica Biosystems, Milan, Italy) using anti-F4/80 (SP115, ThermoFisher), -CD206 (Abcam, Cambridge, UK) or -bcl-2 (124, Dako, Milan, Italy) antibodies.

    Techniques: Expressing, Western Blot, Quantitative RT-PCR, Transferring, Enzyme-linked Immunosorbent Assay, Derivative Assay

    IL-1β secretion plays an autocrine role in controlling the expression of bcl-2-induced factors. qRT-PCR analysis of the indicated molecules in human melanoma M14 control and bcl-2 overexpressing cells untreated (M14 Bcl-2/6) or treated for 24 hours with (A) anti-IL-1β, (B) anti-IL-17, and (C) anti-IL-8 blocking antibodies (M14 Bcl-2/6 AbIL). (A–C) P values were calculated between bcl-2 overexpressing cells untreated and treated with antibody. Fold induction relative to control is reported. *P

    Journal: Journal for Immunotherapy of Cancer

    Article Title: Melanoma-specific bcl-2 promotes a protumoral M2-like phenotype by tumor-associated macrophages

    doi: 10.1136/jitc-2019-000489

    Figure Lengend Snippet: IL-1β secretion plays an autocrine role in controlling the expression of bcl-2-induced factors. qRT-PCR analysis of the indicated molecules in human melanoma M14 control and bcl-2 overexpressing cells untreated (M14 Bcl-2/6) or treated for 24 hours with (A) anti-IL-1β, (B) anti-IL-17, and (C) anti-IL-8 blocking antibodies (M14 Bcl-2/6 AbIL). (A–C) P values were calculated between bcl-2 overexpressing cells untreated and treated with antibody. Fold induction relative to control is reported. *P

    Article Snippet: Immunoreactions were revealed by Bond Polymer Refine Detection in an automated autostainer (BondTM Max, Leica Biosystems, Milan, Italy) using anti-F4/80 (SP115, ThermoFisher), -CD206 (Abcam, Cambridge, UK) or -bcl-2 (124, Dako, Milan, Italy) antibodies.

    Techniques: Expressing, Quantitative RT-PCR, Blocking Assay

    Bcl-2 drives transcriptional activation of IL-1β, IL-17, RORa, RORc and CCL2 in a NF-κB-dependent manner. (A) Western blot analysis of NF-κB subunit p65 in nuclear and cytosol extracts in M14 human melanoma control (M14 Control) and bcl-2 overexpressing clone (M14 Bcl-2/6). HSP90 and histone H3 are shown as loading, transferring and cytoplasmic/nuclear purification control. One representative western blot analysis out of two with similar results is reported. The numbers indicate densitometric analysis relative to control. ChIP analysis of (B) NF-κB subunit p65 recruitment, (C) histone H3 acetylation and (D) Pol II recruitment at IL-1β, IL-8, COX-2 and CCL2 promoters in M14 control and two bcl-2 overexpressing clones (M14 Bcl-2/6, M14 Bcl-2/37). The results are reported as % Input – NoAb. (E) qRT-PCR analysis of IL-1β, IL-8, IL-17, RORa, RORc, COX-2, CCL2 expression in M14 control, Bcl-2/6 and Bcl-2/6 IKBSR cells. (F) qRT-PCR analysis of CD206, IL-10, CCL1, CCL22, IL-12 and COX-2 expression in M-DM after exposure to CM from M14 control, Bcl-2/6 or Bcl-2/6 IKBSR cells. (B–F) Fold induction relative to control cells and the average±SEM of three experiments is reported. P values were calculated between (B–D) control and bcl-2 overexpressing cells or between (E, F) Bcl-2/6 cells and Bcl-2/6 overexpressing IKBSR cells, *P

    Journal: Journal for Immunotherapy of Cancer

    Article Title: Melanoma-specific bcl-2 promotes a protumoral M2-like phenotype by tumor-associated macrophages

    doi: 10.1136/jitc-2019-000489

    Figure Lengend Snippet: Bcl-2 drives transcriptional activation of IL-1β, IL-17, RORa, RORc and CCL2 in a NF-κB-dependent manner. (A) Western blot analysis of NF-κB subunit p65 in nuclear and cytosol extracts in M14 human melanoma control (M14 Control) and bcl-2 overexpressing clone (M14 Bcl-2/6). HSP90 and histone H3 are shown as loading, transferring and cytoplasmic/nuclear purification control. One representative western blot analysis out of two with similar results is reported. The numbers indicate densitometric analysis relative to control. ChIP analysis of (B) NF-κB subunit p65 recruitment, (C) histone H3 acetylation and (D) Pol II recruitment at IL-1β, IL-8, COX-2 and CCL2 promoters in M14 control and two bcl-2 overexpressing clones (M14 Bcl-2/6, M14 Bcl-2/37). The results are reported as % Input – NoAb. (E) qRT-PCR analysis of IL-1β, IL-8, IL-17, RORa, RORc, COX-2, CCL2 expression in M14 control, Bcl-2/6 and Bcl-2/6 IKBSR cells. (F) qRT-PCR analysis of CD206, IL-10, CCL1, CCL22, IL-12 and COX-2 expression in M-DM after exposure to CM from M14 control, Bcl-2/6 or Bcl-2/6 IKBSR cells. (B–F) Fold induction relative to control cells and the average±SEM of three experiments is reported. P values were calculated between (B–D) control and bcl-2 overexpressing cells or between (E, F) Bcl-2/6 cells and Bcl-2/6 overexpressing IKBSR cells, *P

    Article Snippet: Immunoreactions were revealed by Bond Polymer Refine Detection in an automated autostainer (BondTM Max, Leica Biosystems, Milan, Italy) using anti-F4/80 (SP115, ThermoFisher), -CD206 (Abcam, Cambridge, UK) or -bcl-2 (124, Dako, Milan, Italy) antibodies.

    Techniques: Activation Assay, Western Blot, Transferring, Purification, Chromatin Immunoprecipitation, Clone Assay, Quantitative RT-PCR, Expressing

    IL-1β plays a central role in the bcl-2-mediated effect on TAM and T cell functions. (A) Quantification of tumor volume in C57/Bl6 mice subcutaneously injected with control (Control) or bcl-2 overexpressing (Bcl-2) B16/F10 cells and treated with vehicle or with kineret (1 mg/kg, daily) from day 3 to day 12. Quantification by cytofluorimetric analysis of (B) CD45 + cells among live cells, (C) cd11b + F4/80 + (TAM) among CD45 + cells, (D) CD3 + among CD45 + cells (left panel), CD4 + (middle panel) and CD8 + (right panel) among CD3 + cells, (E) CD206 + (left panel), and MHCII + cells (right panel) among TAM in B16/F10 control or bcl-2 overexpressing tumors treated with vehicle or with kineret as reported in (A). Quantification by cytofluorimetric analysis of (F) IFNγ production and (G) CD44 + CD62L - among CD3 + infiltrating cells in B16/F10 control or bcl-2 overexpressing tumors treated with vehicle or with kineret as reported in (A). The results were reported as % of positive cells. (H) Quantification of bcl-2 and CD163 association in metastatic melanoma specimens, p=0.036. (I) Representative images of IHC analysis of bcl-2 and CD163 expression in patient #8 (bcl-2 low; CD163 low) and patient #13 (bcl-2 high; CD163 high). (A–G) The results represent the average ±SD of two independent experiments. P values were calculated between control and bcl-2 overexpressing tumors (#) and between tumors treated with vehicle or kineret (*), *p

    Journal: Journal for Immunotherapy of Cancer

    Article Title: Melanoma-specific bcl-2 promotes a protumoral M2-like phenotype by tumor-associated macrophages

    doi: 10.1136/jitc-2019-000489

    Figure Lengend Snippet: IL-1β plays a central role in the bcl-2-mediated effect on TAM and T cell functions. (A) Quantification of tumor volume in C57/Bl6 mice subcutaneously injected with control (Control) or bcl-2 overexpressing (Bcl-2) B16/F10 cells and treated with vehicle or with kineret (1 mg/kg, daily) from day 3 to day 12. Quantification by cytofluorimetric analysis of (B) CD45 + cells among live cells, (C) cd11b + F4/80 + (TAM) among CD45 + cells, (D) CD3 + among CD45 + cells (left panel), CD4 + (middle panel) and CD8 + (right panel) among CD3 + cells, (E) CD206 + (left panel), and MHCII + cells (right panel) among TAM in B16/F10 control or bcl-2 overexpressing tumors treated with vehicle or with kineret as reported in (A). Quantification by cytofluorimetric analysis of (F) IFNγ production and (G) CD44 + CD62L - among CD3 + infiltrating cells in B16/F10 control or bcl-2 overexpressing tumors treated with vehicle or with kineret as reported in (A). The results were reported as % of positive cells. (H) Quantification of bcl-2 and CD163 association in metastatic melanoma specimens, p=0.036. (I) Representative images of IHC analysis of bcl-2 and CD163 expression in patient #8 (bcl-2 low; CD163 low) and patient #13 (bcl-2 high; CD163 high). (A–G) The results represent the average ±SD of two independent experiments. P values were calculated between control and bcl-2 overexpressing tumors (#) and between tumors treated with vehicle or kineret (*), *p

    Article Snippet: Immunoreactions were revealed by Bond Polymer Refine Detection in an automated autostainer (BondTM Max, Leica Biosystems, Milan, Italy) using anti-F4/80 (SP115, ThermoFisher), -CD206 (Abcam, Cambridge, UK) or -bcl-2 (124, Dako, Milan, Italy) antibodies.

    Techniques: Mouse Assay, Injection, Immunohistochemistry, Expressing

    Melanoma cells expressing bcl-2 promote migration and polarization of macrophages to a M2-type phenotype. qRT-PCR analysis of CD206, IL-10, CCL1, CCL22, IL-12, COX-2 mRNA levels in (A) THP-1 cells and (B) human M-DM after 24 hours exposure to serum free medium (M0 macrophages) or to CM from M14 human melanoma control or bcl-2 overexpressing cells. (C) ELISA of IL-10 and IL-12 protein secretion in M-DM stimulated as reported in (B). (D) qRT-PCR analysis of CD206, IL-10, CCL1, CCL22, IL-12, COX-2 mRNA levels in M-DM stimulated with CM derived from M14 melanoma control or bcl-2 silenced cells. (E) qRT-PCR analysis of IL-1β, IL-8 and VEGF mRNA levels in M-DM stimulated as reported in (B). (F) Representative images (left panels) and relative quantification (right panel) of THP-1 cell migration in response to CM from M14 control (CM M14 Control) or bcl-2 overexpressing (CM M14 Bcl-2/6) melanoma cells. The values are reported as number of migrated cells/field. The quantification was performed by counting the number of migrated cells in at least 10 fields for each condition. The results are reported as fold induction relative to (A, B) M0 macrophages or to (C) melanoma control cells. The results are reported as % of mRNA variation in macrophages exposed to CM derived from (D) bcl-2 silenced cells versus control ones and from (E) bcl-2 overexpressing cells versus control ones. The average±SEM (A, B, D, E) or ±SD (C, F) of three independent experiments is reported. *P

    Journal: Journal for Immunotherapy of Cancer

    Article Title: Melanoma-specific bcl-2 promotes a protumoral M2-like phenotype by tumor-associated macrophages

    doi: 10.1136/jitc-2019-000489

    Figure Lengend Snippet: Melanoma cells expressing bcl-2 promote migration and polarization of macrophages to a M2-type phenotype. qRT-PCR analysis of CD206, IL-10, CCL1, CCL22, IL-12, COX-2 mRNA levels in (A) THP-1 cells and (B) human M-DM after 24 hours exposure to serum free medium (M0 macrophages) or to CM from M14 human melanoma control or bcl-2 overexpressing cells. (C) ELISA of IL-10 and IL-12 protein secretion in M-DM stimulated as reported in (B). (D) qRT-PCR analysis of CD206, IL-10, CCL1, CCL22, IL-12, COX-2 mRNA levels in M-DM stimulated with CM derived from M14 melanoma control or bcl-2 silenced cells. (E) qRT-PCR analysis of IL-1β, IL-8 and VEGF mRNA levels in M-DM stimulated as reported in (B). (F) Representative images (left panels) and relative quantification (right panel) of THP-1 cell migration in response to CM from M14 control (CM M14 Control) or bcl-2 overexpressing (CM M14 Bcl-2/6) melanoma cells. The values are reported as number of migrated cells/field. The quantification was performed by counting the number of migrated cells in at least 10 fields for each condition. The results are reported as fold induction relative to (A, B) M0 macrophages or to (C) melanoma control cells. The results are reported as % of mRNA variation in macrophages exposed to CM derived from (D) bcl-2 silenced cells versus control ones and from (E) bcl-2 overexpressing cells versus control ones. The average±SEM (A, B, D, E) or ±SD (C, F) of three independent experiments is reported. *P

    Article Snippet: Immunoreactions were revealed by Bond Polymer Refine Detection in an automated autostainer (BondTM Max, Leica Biosystems, Milan, Italy) using anti-F4/80 (SP115, ThermoFisher), -CD206 (Abcam, Cambridge, UK) or -bcl-2 (124, Dako, Milan, Italy) antibodies.

    Techniques: Expressing, Migration, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Derivative Assay

    Prognostic impact of MYC/BCL2 coexpression in DLBCL is independent of MYC/BCL2 corearrangement and TP53 mutation status. (A-B) OS (A) and PFS (B) of patients with MYC / BCL2 double-hit DLBCL. (C-D) OS (C) and PFS (D) of patients with MYC + BCL2 + DLBCL in

    Journal: Blood

    Article Title: MYC/BCL2 protein coexpression contributes to the inferior survival of activated B-cell subtype of diffuse large B-cell lymphoma and demonstrates high-risk gene expression signatures: a report from The International DLBCL Rituximab-CHOP Consortium Program

    doi: 10.1182/blood-2012-10-460063

    Figure Lengend Snippet: Prognostic impact of MYC/BCL2 coexpression in DLBCL is independent of MYC/BCL2 corearrangement and TP53 mutation status. (A-B) OS (A) and PFS (B) of patients with MYC / BCL2 double-hit DLBCL. (C-D) OS (C) and PFS (D) of patients with MYC + BCL2 + DLBCL in

    Article Snippet: MYC (clone Y69; Epitomics) expression showed a distinct nuclear pattern and BCL2 (clone 124; DAKO) expression exhibited a cytoplasmic pattern.

    Techniques: Mutagenesis

    MYC/BCL2 protein coexpression predicts poor prognosis in DLBCL

    Journal: Blood

    Article Title: MYC/BCL2 protein coexpression contributes to the inferior survival of activated B-cell subtype of diffuse large B-cell lymphoma and demonstrates high-risk gene expression signatures: a report from The International DLBCL Rituximab-CHOP Consortium Program

    doi: 10.1182/blood-2012-10-460063

    Figure Lengend Snippet: MYC/BCL2 protein coexpression predicts poor prognosis in DLBCL

    Article Snippet: MYC (clone Y69; Epitomics) expression showed a distinct nuclear pattern and BCL2 (clone 124; DAKO) expression exhibited a cytoplasmic pattern.

    Techniques:

    Gene expression signature of DLBCL with MYC/BCL2 coexpression

    Journal: Blood

    Article Title: MYC/BCL2 protein coexpression contributes to the inferior survival of activated B-cell subtype of diffuse large B-cell lymphoma and demonstrates high-risk gene expression signatures: a report from The International DLBCL Rituximab-CHOP Consortium Program

    doi: 10.1182/blood-2012-10-460063

    Figure Lengend Snippet: Gene expression signature of DLBCL with MYC/BCL2 coexpression

    Article Snippet: MYC (clone Y69; Epitomics) expression showed a distinct nuclear pattern and BCL2 (clone 124; DAKO) expression exhibited a cytoplasmic pattern.

    Techniques: Expressing

    Prognostic impact of MYC/BCL2 coexpression in DLBCL risk-stratified according to clinicopathologic parameters. (A-B) OS (A) and PFS (B) of patients with MYC + BCL2 + DLBCL of the GCB subtype in the training set. (C-D) OS (C) and PFS (D) of patients with

    Journal: Blood

    Article Title: MYC/BCL2 protein coexpression contributes to the inferior survival of activated B-cell subtype of diffuse large B-cell lymphoma and demonstrates high-risk gene expression signatures: a report from The International DLBCL Rituximab-CHOP Consortium Program

    doi: 10.1182/blood-2012-10-460063

    Figure Lengend Snippet: Prognostic impact of MYC/BCL2 coexpression in DLBCL risk-stratified according to clinicopathologic parameters. (A-B) OS (A) and PFS (B) of patients with MYC + BCL2 + DLBCL of the GCB subtype in the training set. (C-D) OS (C) and PFS (D) of patients with

    Article Snippet: MYC (clone Y69; Epitomics) expression showed a distinct nuclear pattern and BCL2 (clone 124; DAKO) expression exhibited a cytoplasmic pattern.

    Techniques:

    Gene expression signature of DLBCL with MYC/BCL2 coexpression. Comparison of GEPs of DLBCL with MYC/BCL2 coexpression (149 cases) vs DLBCL negative for MYC and BCL2 expression (88 cases). A total of 153 genes corresponding to 219 probe sets were differentially

    Journal: Blood

    Article Title: MYC/BCL2 protein coexpression contributes to the inferior survival of activated B-cell subtype of diffuse large B-cell lymphoma and demonstrates high-risk gene expression signatures: a report from The International DLBCL Rituximab-CHOP Consortium Program

    doi: 10.1182/blood-2012-10-460063

    Figure Lengend Snippet: Gene expression signature of DLBCL with MYC/BCL2 coexpression. Comparison of GEPs of DLBCL with MYC/BCL2 coexpression (149 cases) vs DLBCL negative for MYC and BCL2 expression (88 cases). A total of 153 genes corresponding to 219 probe sets were differentially

    Article Snippet: MYC (clone Y69; Epitomics) expression showed a distinct nuclear pattern and BCL2 (clone 124; DAKO) expression exhibited a cytoplasmic pattern.

    Techniques: Expressing

    Frequency of BCL2 and MYC expression in COO subtypes of DLBCL. (A) Relative frequency of the ABC vs GCB subtype in DLBCL positive for BCL2 expression, MYC expression, or MYC/BCL2 coexpression in the training set. (B) Frequency of BCL2 expression, MYC

    Journal: Blood

    Article Title: MYC/BCL2 protein coexpression contributes to the inferior survival of activated B-cell subtype of diffuse large B-cell lymphoma and demonstrates high-risk gene expression signatures: a report from The International DLBCL Rituximab-CHOP Consortium Program

    doi: 10.1182/blood-2012-10-460063

    Figure Lengend Snippet: Frequency of BCL2 and MYC expression in COO subtypes of DLBCL. (A) Relative frequency of the ABC vs GCB subtype in DLBCL positive for BCL2 expression, MYC expression, or MYC/BCL2 coexpression in the training set. (B) Frequency of BCL2 expression, MYC

    Article Snippet: MYC (clone Y69; Epitomics) expression showed a distinct nuclear pattern and BCL2 (clone 124; DAKO) expression exhibited a cytoplasmic pattern.

    Techniques: Expressing

    MYC/BCL2 coexpression contributes to the inferior prognosis of ABC-DLBCL. (A-B) OS (A) and PFS (B) of the ABC vs GCB subtype of DLBCL in the entire training set. COO classification of 411 cases was based on GEP results and 55 cases based on IHC results.

    Journal: Blood

    Article Title: MYC/BCL2 protein coexpression contributes to the inferior survival of activated B-cell subtype of diffuse large B-cell lymphoma and demonstrates high-risk gene expression signatures: a report from The International DLBCL Rituximab-CHOP Consortium Program

    doi: 10.1182/blood-2012-10-460063

    Figure Lengend Snippet: MYC/BCL2 coexpression contributes to the inferior prognosis of ABC-DLBCL. (A-B) OS (A) and PFS (B) of the ABC vs GCB subtype of DLBCL in the entire training set. COO classification of 411 cases was based on GEP results and 55 cases based on IHC results.

    Article Snippet: MYC (clone Y69; Epitomics) expression showed a distinct nuclear pattern and BCL2 (clone 124; DAKO) expression exhibited a cytoplasmic pattern.

    Techniques: Immunohistochemistry

    MYC/BCL2 coexpression contributes to the different gene expression profiles between GCB and ABC subtypes of DLBCL. (A) GEP comparison between the ABC vs GCB subtype of DLBCL with MYC/BCL2 coexpression. Of 157 cases of MYC + BCL2 + DLBCL, GEP was successfully

    Journal: Blood

    Article Title: MYC/BCL2 protein coexpression contributes to the inferior survival of activated B-cell subtype of diffuse large B-cell lymphoma and demonstrates high-risk gene expression signatures: a report from The International DLBCL Rituximab-CHOP Consortium Program

    doi: 10.1182/blood-2012-10-460063

    Figure Lengend Snippet: MYC/BCL2 coexpression contributes to the different gene expression profiles between GCB and ABC subtypes of DLBCL. (A) GEP comparison between the ABC vs GCB subtype of DLBCL with MYC/BCL2 coexpression. Of 157 cases of MYC + BCL2 + DLBCL, GEP was successfully

    Article Snippet: MYC (clone Y69; Epitomics) expression showed a distinct nuclear pattern and BCL2 (clone 124; DAKO) expression exhibited a cytoplasmic pattern.

    Techniques: Expressing

    Prognostic impact of MYC/BCL2 coexpression in DLBCL. (A-B) OS (A) and PFS (B) of patients with DLBCL with MYC/BCL2 coexpression (MYC + BCL2 + ) in the training set. (C-D) OS of patients with MYC + DLBCL in the presence (C) or absence (D) of BCL2 coexpression

    Journal: Blood

    Article Title: MYC/BCL2 protein coexpression contributes to the inferior survival of activated B-cell subtype of diffuse large B-cell lymphoma and demonstrates high-risk gene expression signatures: a report from The International DLBCL Rituximab-CHOP Consortium Program

    doi: 10.1182/blood-2012-10-460063

    Figure Lengend Snippet: Prognostic impact of MYC/BCL2 coexpression in DLBCL. (A-B) OS (A) and PFS (B) of patients with DLBCL with MYC/BCL2 coexpression (MYC + BCL2 + ) in the training set. (C-D) OS of patients with MYC + DLBCL in the presence (C) or absence (D) of BCL2 coexpression

    Article Snippet: MYC (clone Y69; Epitomics) expression showed a distinct nuclear pattern and BCL2 (clone 124; DAKO) expression exhibited a cytoplasmic pattern.

    Techniques:

    IL-13 regulates Bcl-2 expression through STAT-3 activation . (A) A time-dependent increase in IL-13 secretion post infection in vitro . Data are mean ± SEM ( n = 3), ** P

    Journal: Frontiers in Immunology

    Article Title: Leishmania donovani-Induced Increase in Macrophage Bcl-2 Favors Parasite Survival

    doi: 10.3389/fimmu.2016.00456

    Figure Lengend Snippet: IL-13 regulates Bcl-2 expression through STAT-3 activation . (A) A time-dependent increase in IL-13 secretion post infection in vitro . Data are mean ± SEM ( n = 3), ** P

    Article Snippet: Membrane blocking was performed in 5% fat free milk (Santa Cruz Biotechnology, Cat. No. sc-2325) followed by incubation with any of the primary antibodies like, Bcl-2 (1:1000) (BD Biosciences, Cat. No. 51-6511GR), Bcl-xL (1:1000) (Cell Signaling Technologies, Cat. No. 54H6), Mcl-1 (1:1000) (BD Biosciences, Cat. No. 559027), Bad (1:1000) (Santa Cruz Biotechnology, Cat. No. sc-492), p-Bad (1:1000) (Santa Cruz Biotechnology, Cat. No. sc-271963), TLR-2 (1:1000) (Novus Biologicals, Cat. No. NBP1-77843), pERK-1/2 (1:1000) (Cell Signaling Technologies, Cat. No. 9101), p-STAT-3 (1:1000) (Cell Signaling Technologies, Cat. No. D3A7), p-PI3K (1:1000) (Cell Signaling Technologies, Cat. No. 4428), p-Akt (1:1000) (Cell Signaling Technologies, Cat. No. 9271), iNOS (1:1000) (Santa Cruz Biotechnology, Cat. No. sc-8310), and β-tubulin (1:4000) (Thermo, Cat. No. RB-9249-P1).

    Techniques: Expressing, Activation Assay, Infection, In Vitro

    Bcl-2 and iNOS protein levels in the human VL subjects . (A) The western blot shows Bcl-2 expression in the PBMCs obtained from the blood of human VL patients. The adjacent bar graph shows densitometric average of the patient (P1-P9) Bcl-2 levels as compared to healthy controls. * P ≤ 0.05; Mann–Whitney test. (B) Blots show iNOS protein levels in the PBMCs of VL patients (P1–P5), pre and post treatment. The adjacent plot shows densitometric comparison of iNOS levels pre and post treatment. The next bar graph shows nitrite measurements in the serum of the same patients, pre and post treatment. * P ≤ 0.05; Wilcoxon signed-rank test. (C) Schematic showing the involvement of signaling pathways during Leishmania infection.

    Journal: Frontiers in Immunology

    Article Title: Leishmania donovani-Induced Increase in Macrophage Bcl-2 Favors Parasite Survival

    doi: 10.3389/fimmu.2016.00456

    Figure Lengend Snippet: Bcl-2 and iNOS protein levels in the human VL subjects . (A) The western blot shows Bcl-2 expression in the PBMCs obtained from the blood of human VL patients. The adjacent bar graph shows densitometric average of the patient (P1-P9) Bcl-2 levels as compared to healthy controls. * P ≤ 0.05; Mann–Whitney test. (B) Blots show iNOS protein levels in the PBMCs of VL patients (P1–P5), pre and post treatment. The adjacent plot shows densitometric comparison of iNOS levels pre and post treatment. The next bar graph shows nitrite measurements in the serum of the same patients, pre and post treatment. * P ≤ 0.05; Wilcoxon signed-rank test. (C) Schematic showing the involvement of signaling pathways during Leishmania infection.

    Article Snippet: Membrane blocking was performed in 5% fat free milk (Santa Cruz Biotechnology, Cat. No. sc-2325) followed by incubation with any of the primary antibodies like, Bcl-2 (1:1000) (BD Biosciences, Cat. No. 51-6511GR), Bcl-xL (1:1000) (Cell Signaling Technologies, Cat. No. 54H6), Mcl-1 (1:1000) (BD Biosciences, Cat. No. 559027), Bad (1:1000) (Santa Cruz Biotechnology, Cat. No. sc-492), p-Bad (1:1000) (Santa Cruz Biotechnology, Cat. No. sc-271963), TLR-2 (1:1000) (Novus Biologicals, Cat. No. NBP1-77843), pERK-1/2 (1:1000) (Cell Signaling Technologies, Cat. No. 9101), p-STAT-3 (1:1000) (Cell Signaling Technologies, Cat. No. D3A7), p-PI3K (1:1000) (Cell Signaling Technologies, Cat. No. 4428), p-Akt (1:1000) (Cell Signaling Technologies, Cat. No. 9271), iNOS (1:1000) (Santa Cruz Biotechnology, Cat. No. sc-8310), and β-tubulin (1:4000) (Thermo, Cat. No. RB-9249-P1).

    Techniques: Western Blot, Expressing, MANN-WHITNEY, Infection

    Bcl-2 increase contributes to iNOS suppression . (A) Western blot showing iNOS levels during control treatments and upon infection for 24 h. (B) iNOS levels at 12 and 24 h post infection. LPS has been taken as a positive control. (C) Blot showing higher iNOS expression at 12 h post-infection during Bcl-2 downregulation by siRNA to Bcl-2. Note no significant change after inhibition with siRNA to Mcl-1. Ld, Leishmania donovani ; KD, knockdown; Scr, scrambled siRNA. (D) Western blot of THP1-MDM extracts at 12 h post-infection, showing higher levels of iNOS when Bcl-2 protein was inhibited by ABT-199/ABT-263. The bar graphs below the western blots in (A–D) show nitrite levels in soups obtained from corresponding experiments. Note the correspondence between iNOS expression and the nitrite levels in all the above experiments. (E) Nitrite concentrations at 12 h after treatment with Bcl-2 inhibitor ABT-199 and LNMA, iNOS inhibitor. Data are mean ± SEM ( n = 3). * P ≤ 0.05; Mann–Whitney test. (F) Parasite burden 12 h postinfection in THP-1 MDM in the presence of Bcl-2 inhibitor ABT-199 and iNOS inhibitor, LNMA. Data are mean ± SEM ( n = 3). *** P ≤ 0.001; Mann–Whitney test. (G) Photomicrograph of infected THP-1 MDM treated with LNMA and ABT-199. Note the loss of ABT-199-conferred reduction in parasite burden upon inhibition of iNOS activity by LNMA. The yellow arrows indicate intracellular parasites. (H) Representative Western blots from the lysates of hMDMs showing increased iNOS expression at 12 h upon Bcl-2 KD and in the presence of Bcl-2 inhibitors ABT-199/ABT-263. The bar graphs below represent corresponding nitrite levels. Ld, Leishmania donovani ; KD, knockdown; Scr, scrambled siRNA. Data are mean ± SEM ( n = 3). * P ≤ 0.05; Mann–Whitney test. (I) First three bar graphs show nitrite concentrations at 12 h after parasite infection of mPMs upon Bcl-2, Mcl-1 KD or treatments with Bcl-2 inhibitors, ABT-199/ABT-263 and upon inhibition of iNOS activity using LNMA treatment. The fourth bar graph shows increased parasite burden in LNMA-treated mPMs even in the presence of ABT-199. Ld, Leishmania donovani ; KD, knockdown; Scr, scrambled siRNA; mPM, mouse peritoneal macrophage. Data are mean ± SEM ( n = 3). * P ≤ 0.05, ** P ≤ 0.01; Mann–Whitney test.

    Journal: Frontiers in Immunology

    Article Title: Leishmania donovani-Induced Increase in Macrophage Bcl-2 Favors Parasite Survival

    doi: 10.3389/fimmu.2016.00456

    Figure Lengend Snippet: Bcl-2 increase contributes to iNOS suppression . (A) Western blot showing iNOS levels during control treatments and upon infection for 24 h. (B) iNOS levels at 12 and 24 h post infection. LPS has been taken as a positive control. (C) Blot showing higher iNOS expression at 12 h post-infection during Bcl-2 downregulation by siRNA to Bcl-2. Note no significant change after inhibition with siRNA to Mcl-1. Ld, Leishmania donovani ; KD, knockdown; Scr, scrambled siRNA. (D) Western blot of THP1-MDM extracts at 12 h post-infection, showing higher levels of iNOS when Bcl-2 protein was inhibited by ABT-199/ABT-263. The bar graphs below the western blots in (A–D) show nitrite levels in soups obtained from corresponding experiments. Note the correspondence between iNOS expression and the nitrite levels in all the above experiments. (E) Nitrite concentrations at 12 h after treatment with Bcl-2 inhibitor ABT-199 and LNMA, iNOS inhibitor. Data are mean ± SEM ( n = 3). * P ≤ 0.05; Mann–Whitney test. (F) Parasite burden 12 h postinfection in THP-1 MDM in the presence of Bcl-2 inhibitor ABT-199 and iNOS inhibitor, LNMA. Data are mean ± SEM ( n = 3). *** P ≤ 0.001; Mann–Whitney test. (G) Photomicrograph of infected THP-1 MDM treated with LNMA and ABT-199. Note the loss of ABT-199-conferred reduction in parasite burden upon inhibition of iNOS activity by LNMA. The yellow arrows indicate intracellular parasites. (H) Representative Western blots from the lysates of hMDMs showing increased iNOS expression at 12 h upon Bcl-2 KD and in the presence of Bcl-2 inhibitors ABT-199/ABT-263. The bar graphs below represent corresponding nitrite levels. Ld, Leishmania donovani ; KD, knockdown; Scr, scrambled siRNA. Data are mean ± SEM ( n = 3). * P ≤ 0.05; Mann–Whitney test. (I) First three bar graphs show nitrite concentrations at 12 h after parasite infection of mPMs upon Bcl-2, Mcl-1 KD or treatments with Bcl-2 inhibitors, ABT-199/ABT-263 and upon inhibition of iNOS activity using LNMA treatment. The fourth bar graph shows increased parasite burden in LNMA-treated mPMs even in the presence of ABT-199. Ld, Leishmania donovani ; KD, knockdown; Scr, scrambled siRNA; mPM, mouse peritoneal macrophage. Data are mean ± SEM ( n = 3). * P ≤ 0.05, ** P ≤ 0.01; Mann–Whitney test.

    Article Snippet: Membrane blocking was performed in 5% fat free milk (Santa Cruz Biotechnology, Cat. No. sc-2325) followed by incubation with any of the primary antibodies like, Bcl-2 (1:1000) (BD Biosciences, Cat. No. 51-6511GR), Bcl-xL (1:1000) (Cell Signaling Technologies, Cat. No. 54H6), Mcl-1 (1:1000) (BD Biosciences, Cat. No. 559027), Bad (1:1000) (Santa Cruz Biotechnology, Cat. No. sc-492), p-Bad (1:1000) (Santa Cruz Biotechnology, Cat. No. sc-271963), TLR-2 (1:1000) (Novus Biologicals, Cat. No. NBP1-77843), pERK-1/2 (1:1000) (Cell Signaling Technologies, Cat. No. 9101), p-STAT-3 (1:1000) (Cell Signaling Technologies, Cat. No. D3A7), p-PI3K (1:1000) (Cell Signaling Technologies, Cat. No. 4428), p-Akt (1:1000) (Cell Signaling Technologies, Cat. No. 9271), iNOS (1:1000) (Santa Cruz Biotechnology, Cat. No. sc-8310), and β-tubulin (1:4000) (Thermo, Cat. No. RB-9249-P1).

    Techniques: Western Blot, Infection, Positive Control, Expressing, Inhibition, MANN-WHITNEY, Activity Assay

    TLR-2 expression increases upon infection . (A) Western blot shows a time-dependent increase in TLR-2 expression by THP-1 MDMs after infection with L. donovani parasite. The blots below show siRNA-mediated KD of TLR-2 and reduced induction of Bcl-2 protein in TLR-2 KD macrophages. Blots are representative of three independent experiments. The bar graph alongside shows slightly reduced parasite uptake by TLR-2 KD THP-1 MDMs. Next placed are the immunofluorescence photomicrographs showing enhanced expression of macrophage TLR-2 receptors at 12 and 24 h post infection. (B) Blot shows increased TLR-2 expression by hMDMs upon L. donovani infection. The blot below shows infection-induced Bcl-2 expression was diminished in TLR-2 KD hMDMs. Ld, Leishmania donovani ; KD, knockdown; Scr, scrambled siRNA. Blots are representative of three independent experiments. The adjacent bar graph shows a significant reduction in the parasite uptake by TLR-2 KD hMDMs. A minimum of 200 cells were counted. Data are mean ± SEM ( n = 3). * P ≤ 0.05; Mann–Whitney test. (C) Blot shows a time-dependent increase in TLR-2 protein in mPM post infection. Unlike peritoneal macrophages from WT animals, no significant increase in the expression of Bcl-2 protein was observed in TLR-2 KO mPMs in response to infection. Blots in this figure are representative of at least 3–4 experiments. The adjacent bar graph shows that mPMs obtained from TLR-2 KO mice show significantly lesser uptake of parasites as compared to macrophages obtained from WT animals. The parasite uptake was measured by counting average number of internalized parasites per macrophage 3 h postinfection. WT, wild type; KO, knockout. Data are mean ± SEM ( n = 3); * P ≤ 0.05; Mann–Whitney test.

    Journal: Frontiers in Immunology

    Article Title: Leishmania donovani-Induced Increase in Macrophage Bcl-2 Favors Parasite Survival

    doi: 10.3389/fimmu.2016.00456

    Figure Lengend Snippet: TLR-2 expression increases upon infection . (A) Western blot shows a time-dependent increase in TLR-2 expression by THP-1 MDMs after infection with L. donovani parasite. The blots below show siRNA-mediated KD of TLR-2 and reduced induction of Bcl-2 protein in TLR-2 KD macrophages. Blots are representative of three independent experiments. The bar graph alongside shows slightly reduced parasite uptake by TLR-2 KD THP-1 MDMs. Next placed are the immunofluorescence photomicrographs showing enhanced expression of macrophage TLR-2 receptors at 12 and 24 h post infection. (B) Blot shows increased TLR-2 expression by hMDMs upon L. donovani infection. The blot below shows infection-induced Bcl-2 expression was diminished in TLR-2 KD hMDMs. Ld, Leishmania donovani ; KD, knockdown; Scr, scrambled siRNA. Blots are representative of three independent experiments. The adjacent bar graph shows a significant reduction in the parasite uptake by TLR-2 KD hMDMs. A minimum of 200 cells were counted. Data are mean ± SEM ( n = 3). * P ≤ 0.05; Mann–Whitney test. (C) Blot shows a time-dependent increase in TLR-2 protein in mPM post infection. Unlike peritoneal macrophages from WT animals, no significant increase in the expression of Bcl-2 protein was observed in TLR-2 KO mPMs in response to infection. Blots in this figure are representative of at least 3–4 experiments. The adjacent bar graph shows that mPMs obtained from TLR-2 KO mice show significantly lesser uptake of parasites as compared to macrophages obtained from WT animals. The parasite uptake was measured by counting average number of internalized parasites per macrophage 3 h postinfection. WT, wild type; KO, knockout. Data are mean ± SEM ( n = 3); * P ≤ 0.05; Mann–Whitney test.

    Article Snippet: Membrane blocking was performed in 5% fat free milk (Santa Cruz Biotechnology, Cat. No. sc-2325) followed by incubation with any of the primary antibodies like, Bcl-2 (1:1000) (BD Biosciences, Cat. No. 51-6511GR), Bcl-xL (1:1000) (Cell Signaling Technologies, Cat. No. 54H6), Mcl-1 (1:1000) (BD Biosciences, Cat. No. 559027), Bad (1:1000) (Santa Cruz Biotechnology, Cat. No. sc-492), p-Bad (1:1000) (Santa Cruz Biotechnology, Cat. No. sc-271963), TLR-2 (1:1000) (Novus Biologicals, Cat. No. NBP1-77843), pERK-1/2 (1:1000) (Cell Signaling Technologies, Cat. No. 9101), p-STAT-3 (1:1000) (Cell Signaling Technologies, Cat. No. D3A7), p-PI3K (1:1000) (Cell Signaling Technologies, Cat. No. 4428), p-Akt (1:1000) (Cell Signaling Technologies, Cat. No. 9271), iNOS (1:1000) (Santa Cruz Biotechnology, Cat. No. sc-8310), and β-tubulin (1:4000) (Thermo, Cat. No. RB-9249-P1).

    Techniques: Expressing, Infection, Western Blot, Immunofluorescence, MANN-WHITNEY, Mouse Assay, Knock-Out

    MEK-MAPK/ERK but not PI3K-Akt activation regulates Bcl-2 increase in THP-1 MDM . (A) Representative Western blots for pPI3K, pAkt, and pERK upon infection with L. donovani . PI3K is phosphorylated during early infection within 30–60 min. Note pretreatment with wortmannin, which is an inhibitor of PI3K phosphorylation, completely abrogates Akt phosphorylation. mpi, minutes post infection. (B) Blots showing effect of PI3K and ERK inhibitors on Bcl-2 induction during infection. The first blot shows significant decrease of infection-induced Bcl-2 on inhibition of ERK phosphorylation with U0126 and PD98059 (MEK1/2 inhibitors). The second blot in the figure shows no significant difference in the expression of Bcl-2 protein in the presence of PI3K inhibitors, wortmannin and LY294002 at 12 h postinfection. The third blot shows stabilization of the Bad protein in the presence of PI3K inhibitors showing PI3K mediated degradation of Bad protein during infection. Ld, Leishmania donovani ; Veh, vehicle (DMSO). The blots in this figure are representative of three independent experiments. (C) Nitrite concentration measurements 12 h post infection in the presence of ERK or PI3K inhibitors. (D) Parasite burden in the presence of ERK or PI3K inhibitors. Note reduced parasite burden in the presence of ERK inhibitors. (E,F) show nitrite measurements and parasite burden respectively for hMDMs in the presence of ERK/PI3K inhibitors. A minimum of 200 cells was counted for estimating parasite burden. Data are mean ± SEM ( n = 3); * P ≤ 0.05; Mann–Whitney test.

    Journal: Frontiers in Immunology

    Article Title: Leishmania donovani-Induced Increase in Macrophage Bcl-2 Favors Parasite Survival

    doi: 10.3389/fimmu.2016.00456

    Figure Lengend Snippet: MEK-MAPK/ERK but not PI3K-Akt activation regulates Bcl-2 increase in THP-1 MDM . (A) Representative Western blots for pPI3K, pAkt, and pERK upon infection with L. donovani . PI3K is phosphorylated during early infection within 30–60 min. Note pretreatment with wortmannin, which is an inhibitor of PI3K phosphorylation, completely abrogates Akt phosphorylation. mpi, minutes post infection. (B) Blots showing effect of PI3K and ERK inhibitors on Bcl-2 induction during infection. The first blot shows significant decrease of infection-induced Bcl-2 on inhibition of ERK phosphorylation with U0126 and PD98059 (MEK1/2 inhibitors). The second blot in the figure shows no significant difference in the expression of Bcl-2 protein in the presence of PI3K inhibitors, wortmannin and LY294002 at 12 h postinfection. The third blot shows stabilization of the Bad protein in the presence of PI3K inhibitors showing PI3K mediated degradation of Bad protein during infection. Ld, Leishmania donovani ; Veh, vehicle (DMSO). The blots in this figure are representative of three independent experiments. (C) Nitrite concentration measurements 12 h post infection in the presence of ERK or PI3K inhibitors. (D) Parasite burden in the presence of ERK or PI3K inhibitors. Note reduced parasite burden in the presence of ERK inhibitors. (E,F) show nitrite measurements and parasite burden respectively for hMDMs in the presence of ERK/PI3K inhibitors. A minimum of 200 cells was counted for estimating parasite burden. Data are mean ± SEM ( n = 3); * P ≤ 0.05; Mann–Whitney test.

    Article Snippet: Membrane blocking was performed in 5% fat free milk (Santa Cruz Biotechnology, Cat. No. sc-2325) followed by incubation with any of the primary antibodies like, Bcl-2 (1:1000) (BD Biosciences, Cat. No. 51-6511GR), Bcl-xL (1:1000) (Cell Signaling Technologies, Cat. No. 54H6), Mcl-1 (1:1000) (BD Biosciences, Cat. No. 559027), Bad (1:1000) (Santa Cruz Biotechnology, Cat. No. sc-492), p-Bad (1:1000) (Santa Cruz Biotechnology, Cat. No. sc-271963), TLR-2 (1:1000) (Novus Biologicals, Cat. No. NBP1-77843), pERK-1/2 (1:1000) (Cell Signaling Technologies, Cat. No. 9101), p-STAT-3 (1:1000) (Cell Signaling Technologies, Cat. No. D3A7), p-PI3K (1:1000) (Cell Signaling Technologies, Cat. No. 4428), p-Akt (1:1000) (Cell Signaling Technologies, Cat. No. 9271), iNOS (1:1000) (Santa Cruz Biotechnology, Cat. No. sc-8310), and β-tubulin (1:4000) (Thermo, Cat. No. RB-9249-P1).

    Techniques: Activation Assay, Western Blot, Infection, Inhibition, Expressing, Concentration Assay, MANN-WHITNEY

    Expression of Bcl-2 and Mcl-1 increases during L. donovani infection . (A) Bcl-2, Mcl-1, and Bcl-xL protein levels in extracts of THP-1 MDM and hMDM infected with L. donovani parasites in vitro . Note the increase in Bcl-2 from 2 h onward. Blots are representative of three experiments. Densitometric plots are shown below. Data are mean ± SD ( n = 3). (B) Immunocytochemical staining of infected THP-1 MDM using anti-Bcl-2 antibody at different time points post-infection. Note increased Bcl-2 expression at 8 h showing predominantly perinuclear distribution as indicated by white arrows. N = nucleus. (C) Blots showing siRNA mediated downregulation of Bcl-2 and Mcl-1 in THP-1 MDM and hMDM. Control is without any siRNA. Scr, scrambled siRNA; KD, knockdown. Also shown is overexpression of Bcl-2 in THP-1 MDM and hMDM. OE, overexpression. Control is only plasmid, OE, overexpression. Blots are representative of three experiments. The blot below shows control Bcl-2 levels during treatment with only vehicle (DMSO) or ABT-199 or ABT-263. Bcl-2 levels remained unaffected upon these treatments. (D) Plots show parasite uptake and parasite burden during various conditions. Average parasite counts per macrophage have been put on the y -axis. A minimum of 200 cells was counted. Data are mean ± SEM ( n = 3); *** P ≤ 0.001; Mann–Whitney test. Parasite uptake remained unaltered. Note significant reduction of parasite burden in siRNA or ABT-199-treated Bcl-2 downregulated cells. (E) Western blots of lysates of infected mPM stained for Bcl-xL, Bcl-2, and Mcl-1 showing increased expression for Bcl-2 which peaks at 8 h post-infection. Blots are representative of three experiments. Shown below are the densitometric plots. Data are mean ± SD ( n = 3). Alongside are blots showing siRNA0-mediated downregulation of Bcl-2 and Mcl-1 in mPMs. Control is without any siRNA. Scr, scrambled siRNA; KD, knockdown. Shown below are bar graphs expressing parasite uptake and parasite burden during various treatments. Note significantly reduced parasite burden in Bcl-2 KD and ABT-199/ABT-263-treated macrophages. A minimum of 200 cells was counted. Data are mean ± SEM ( n = 3); * P ≤ 0.05; Mann–Whitney test. (F) Western blots from THP-1 MDM and hMDM showing time-dependent degradation of Bad protein during infection. Bad protein undergoes degradation post phosphorylation. The blot below shows Bad phosphorylation upon infection. Blots are representative of three experiments.

    Journal: Frontiers in Immunology

    Article Title: Leishmania donovani-Induced Increase in Macrophage Bcl-2 Favors Parasite Survival

    doi: 10.3389/fimmu.2016.00456

    Figure Lengend Snippet: Expression of Bcl-2 and Mcl-1 increases during L. donovani infection . (A) Bcl-2, Mcl-1, and Bcl-xL protein levels in extracts of THP-1 MDM and hMDM infected with L. donovani parasites in vitro . Note the increase in Bcl-2 from 2 h onward. Blots are representative of three experiments. Densitometric plots are shown below. Data are mean ± SD ( n = 3). (B) Immunocytochemical staining of infected THP-1 MDM using anti-Bcl-2 antibody at different time points post-infection. Note increased Bcl-2 expression at 8 h showing predominantly perinuclear distribution as indicated by white arrows. N = nucleus. (C) Blots showing siRNA mediated downregulation of Bcl-2 and Mcl-1 in THP-1 MDM and hMDM. Control is without any siRNA. Scr, scrambled siRNA; KD, knockdown. Also shown is overexpression of Bcl-2 in THP-1 MDM and hMDM. OE, overexpression. Control is only plasmid, OE, overexpression. Blots are representative of three experiments. The blot below shows control Bcl-2 levels during treatment with only vehicle (DMSO) or ABT-199 or ABT-263. Bcl-2 levels remained unaffected upon these treatments. (D) Plots show parasite uptake and parasite burden during various conditions. Average parasite counts per macrophage have been put on the y -axis. A minimum of 200 cells was counted. Data are mean ± SEM ( n = 3); *** P ≤ 0.001; Mann–Whitney test. Parasite uptake remained unaltered. Note significant reduction of parasite burden in siRNA or ABT-199-treated Bcl-2 downregulated cells. (E) Western blots of lysates of infected mPM stained for Bcl-xL, Bcl-2, and Mcl-1 showing increased expression for Bcl-2 which peaks at 8 h post-infection. Blots are representative of three experiments. Shown below are the densitometric plots. Data are mean ± SD ( n = 3). Alongside are blots showing siRNA0-mediated downregulation of Bcl-2 and Mcl-1 in mPMs. Control is without any siRNA. Scr, scrambled siRNA; KD, knockdown. Shown below are bar graphs expressing parasite uptake and parasite burden during various treatments. Note significantly reduced parasite burden in Bcl-2 KD and ABT-199/ABT-263-treated macrophages. A minimum of 200 cells was counted. Data are mean ± SEM ( n = 3); * P ≤ 0.05; Mann–Whitney test. (F) Western blots from THP-1 MDM and hMDM showing time-dependent degradation of Bad protein during infection. Bad protein undergoes degradation post phosphorylation. The blot below shows Bad phosphorylation upon infection. Blots are representative of three experiments.

    Article Snippet: Membrane blocking was performed in 5% fat free milk (Santa Cruz Biotechnology, Cat. No. sc-2325) followed by incubation with any of the primary antibodies like, Bcl-2 (1:1000) (BD Biosciences, Cat. No. 51-6511GR), Bcl-xL (1:1000) (Cell Signaling Technologies, Cat. No. 54H6), Mcl-1 (1:1000) (BD Biosciences, Cat. No. 559027), Bad (1:1000) (Santa Cruz Biotechnology, Cat. No. sc-492), p-Bad (1:1000) (Santa Cruz Biotechnology, Cat. No. sc-271963), TLR-2 (1:1000) (Novus Biologicals, Cat. No. NBP1-77843), pERK-1/2 (1:1000) (Cell Signaling Technologies, Cat. No. 9101), p-STAT-3 (1:1000) (Cell Signaling Technologies, Cat. No. D3A7), p-PI3K (1:1000) (Cell Signaling Technologies, Cat. No. 4428), p-Akt (1:1000) (Cell Signaling Technologies, Cat. No. 9271), iNOS (1:1000) (Santa Cruz Biotechnology, Cat. No. sc-8310), and β-tubulin (1:4000) (Thermo, Cat. No. RB-9249-P1).

    Techniques: Expressing, Infection, In Vitro, Staining, Over Expression, Plasmid Preparation, MANN-WHITNEY, Western Blot

    Neuroprotective activity of 17-mer peptides. Markers of the cell death pathways were analyzed in rd1 mutant retinas after exposure to vehicle (MOCK) or 17-mer or 17-mer[H105A] (H105A) or 17-mer[R99A] (R99A). a Histogram representing the percentage of photoreceptors labeled by the fluorescent calpain activity assay ( N = 4; ± SD; *** P ≤ 0.001). b αII-spectrin was analyzed by immunoblot in total protein extracts from mouse retinas. The 145–150 kDa fragments resulting from calpain cleavage are marked by an asterisk. The immunoblot was normalized using anti-actin antibodies (lower panel) ( N = 3). c Immunoblotting of BAX protein in mitochondria enriched protein extracts. The immunoblot was normalized with anti-cytochrome c antibodies (lower panel) ( N = 3). d Total protein extracts were analyzed by immunoblot with an anti-BCL2 antibody. The immunoblot was normalized using anti-actin antibodies (lower panel) ( N = 3). e Histogram representing the percentage of photoreceptors labeled with TUNEL (black) or by nuclear localization of AIF (white) ( N = 4; ± SD; *** P ≤ 0.001). f Immunoblotting of AIF protein in nuclear-enriched protein extracts. The immunoblot was normalized with anti-H3 histone antibodies (lower panel) ( N = 3)

    Journal: Cell Death & Disease

    Article Title: Pigment epithelium-derived factor hinders photoreceptor cell death by reducing intracellular calcium in the degenerating retina

    doi: 10.1038/s41419-018-0613-y

    Figure Lengend Snippet: Neuroprotective activity of 17-mer peptides. Markers of the cell death pathways were analyzed in rd1 mutant retinas after exposure to vehicle (MOCK) or 17-mer or 17-mer[H105A] (H105A) or 17-mer[R99A] (R99A). a Histogram representing the percentage of photoreceptors labeled by the fluorescent calpain activity assay ( N = 4; ± SD; *** P ≤ 0.001). b αII-spectrin was analyzed by immunoblot in total protein extracts from mouse retinas. The 145–150 kDa fragments resulting from calpain cleavage are marked by an asterisk. The immunoblot was normalized using anti-actin antibodies (lower panel) ( N = 3). c Immunoblotting of BAX protein in mitochondria enriched protein extracts. The immunoblot was normalized with anti-cytochrome c antibodies (lower panel) ( N = 3). d Total protein extracts were analyzed by immunoblot with an anti-BCL2 antibody. The immunoblot was normalized using anti-actin antibodies (lower panel) ( N = 3). e Histogram representing the percentage of photoreceptors labeled with TUNEL (black) or by nuclear localization of AIF (white) ( N = 4; ± SD; *** P ≤ 0.001). f Immunoblotting of AIF protein in nuclear-enriched protein extracts. The immunoblot was normalized with anti-H3 histone antibodies (lower panel) ( N = 3)

    Article Snippet: Primary antibodies were used as follows: anti-AIF (1:100; Sigma), anti-phosphorylated PERK (1:100, Cell Signaling), anti-RHO (1D4 1:250; Sigma), anti-BAX 6A7 (1:200, BD Biosciences), anti-BCL2 (1:100, Cell Signaling) and anti-calpastatin (1:100, Cell Signaling).

    Techniques: Activity Assay, Mutagenesis, Labeling, TUNEL Assay

    Scheme on the mechanism mediating PEDF promotion of photoreceptor survival. a Cell death pathway in rd1 photoreceptors is associated with high [Ca 2+ ] i , activation of calpain (Calpain a ), Cathepsin D (Cat D a ) and BAX, as well as of translocation of AIF from the mitochondria to the nucleus. b PEDF, or the 17-mer peptide, binds to PEDF-R and stimulates its PLA2 activity to release DHA from phospholipids (P1 peptide can interfere with the binding of PEDF to PEDF-R). DHA decreases [Ca 2+ ] i through PMCA activity (Ca 2+ extrusion). Decreased [Ca 2+ ] i is associated with inactivation of calpain (Calpain i ) and Cathepsin D (Cat D i ), blockage of mitochondrial translocation of BAX and nuclear translocation of AIF, as well as increase in BCL2 levels, all of which contribute to cell survival in the rd1 mouse photoreceptors

    Journal: Cell Death & Disease

    Article Title: Pigment epithelium-derived factor hinders photoreceptor cell death by reducing intracellular calcium in the degenerating retina

    doi: 10.1038/s41419-018-0613-y

    Figure Lengend Snippet: Scheme on the mechanism mediating PEDF promotion of photoreceptor survival. a Cell death pathway in rd1 photoreceptors is associated with high [Ca 2+ ] i , activation of calpain (Calpain a ), Cathepsin D (Cat D a ) and BAX, as well as of translocation of AIF from the mitochondria to the nucleus. b PEDF, or the 17-mer peptide, binds to PEDF-R and stimulates its PLA2 activity to release DHA from phospholipids (P1 peptide can interfere with the binding of PEDF to PEDF-R). DHA decreases [Ca 2+ ] i through PMCA activity (Ca 2+ extrusion). Decreased [Ca 2+ ] i is associated with inactivation of calpain (Calpain i ) and Cathepsin D (Cat D i ), blockage of mitochondrial translocation of BAX and nuclear translocation of AIF, as well as increase in BCL2 levels, all of which contribute to cell survival in the rd1 mouse photoreceptors

    Article Snippet: Primary antibodies were used as follows: anti-AIF (1:100; Sigma), anti-phosphorylated PERK (1:100, Cell Signaling), anti-RHO (1D4 1:250; Sigma), anti-BAX 6A7 (1:200, BD Biosciences), anti-BCL2 (1:100, Cell Signaling) and anti-calpastatin (1:100, Cell Signaling).

    Techniques: Activation Assay, Translocation Assay, Activity Assay, Binding Assay

    PEDF effects on Cathepsin D, BAX and BCL2. Protein analyses on wild-type (WT) and rd1 mutant mouse retinas treated with either vehicle (MOCK) or PEDF or PEDF and P1 blocking peptide (PEDF + P1). a Total protein extracts were analyzed by immunoblot with an anti-cathepsin D antibody. The antibody recognizes both uncleaved cathepsin D (arrow) and cleaved/activated cathepsin D (asterisk). The immunoblot was normalized using anti-actin antibodies (lower panel). b Quantification by ImageJ of three immunoblots from biological replicates as the one shown in panel ( a ) (±SD; N = 3). White bars show values for uncleaved cathepsin D and gray bars show values for activated/cleaved cathepsin D. c Analysis of the BAX protein in mitochondria enriched extracts. The immunoblot was normalized with anti-cytochrome c antibodies (lower panel). d Quantification by ImageJ of three immunoblots from biological replicates as the one shown in panel ( c ) (±SD; N = 3). e Total protein extracts were analyzed by immunoblot with an anti-BCL2 antibody. The immunoblot was normalized using anti-actin antibodies (lower panel). f Quantification by ImageJ of three immunoblots from biological replicates as the one shown in panel ( a ) (±SD; N = 3). MW molecular weight markers are shown in kDa. *** P ≤ 0.001; * P ≤ 0.05

    Journal: Cell Death & Disease

    Article Title: Pigment epithelium-derived factor hinders photoreceptor cell death by reducing intracellular calcium in the degenerating retina

    doi: 10.1038/s41419-018-0613-y

    Figure Lengend Snippet: PEDF effects on Cathepsin D, BAX and BCL2. Protein analyses on wild-type (WT) and rd1 mutant mouse retinas treated with either vehicle (MOCK) or PEDF or PEDF and P1 blocking peptide (PEDF + P1). a Total protein extracts were analyzed by immunoblot with an anti-cathepsin D antibody. The antibody recognizes both uncleaved cathepsin D (arrow) and cleaved/activated cathepsin D (asterisk). The immunoblot was normalized using anti-actin antibodies (lower panel). b Quantification by ImageJ of three immunoblots from biological replicates as the one shown in panel ( a ) (±SD; N = 3). White bars show values for uncleaved cathepsin D and gray bars show values for activated/cleaved cathepsin D. c Analysis of the BAX protein in mitochondria enriched extracts. The immunoblot was normalized with anti-cytochrome c antibodies (lower panel). d Quantification by ImageJ of three immunoblots from biological replicates as the one shown in panel ( c ) (±SD; N = 3). e Total protein extracts were analyzed by immunoblot with an anti-BCL2 antibody. The immunoblot was normalized using anti-actin antibodies (lower panel). f Quantification by ImageJ of three immunoblots from biological replicates as the one shown in panel ( a ) (±SD; N = 3). MW molecular weight markers are shown in kDa. *** P ≤ 0.001; * P ≤ 0.05

    Article Snippet: Primary antibodies were used as follows: anti-AIF (1:100; Sigma), anti-phosphorylated PERK (1:100, Cell Signaling), anti-RHO (1D4 1:250; Sigma), anti-BAX 6A7 (1:200, BD Biosciences), anti-BCL2 (1:100, Cell Signaling) and anti-calpastatin (1:100, Cell Signaling).

    Techniques: Mutagenesis, Blocking Assay, Western Blot, Molecular Weight

    Mir223 deficiency increased autophagy and resting microglia in the brains of EAE mice. ( A ) Flow cytometric analysis of microglia, demonstrating PTPRC and ITGAM cells isolated from the CNS of EAE mice (n = 6 mice per group), detected on the 15th day after the induction of EAE. The data are shown in a representative plot. ( B ) The absolute numbers of the cell subpopulations are shown; the black column is for mir223 −/- mice. Rest, ITGAM + PTPRC low ; mc, ITGAM + PTPRC hi and low . ( C ) Autophagy was measured in the brains of the mice. LC3 puncta were visualized by confocal imaging of microglia immunostained for LC3 and AIF1, followed by Alexa Fluor 488/555-conjugated secondary antibodies (green/red); nuclei were stained with DAPI. Representative images are shown. Scale bars: 20 µm. ( D ) and ( E ) BCL2 and BECN1 expression were visualized by fluorescence imaging of microglia. Scale bars: 50 µm.

    Journal: Autophagy

    Article Title: Mir223 restrains autophagy and promotes CNS inflammation by targeting ATG16L1

    doi: 10.1080/15548627.2018.1522467

    Figure Lengend Snippet: Mir223 deficiency increased autophagy and resting microglia in the brains of EAE mice. ( A ) Flow cytometric analysis of microglia, demonstrating PTPRC and ITGAM cells isolated from the CNS of EAE mice (n = 6 mice per group), detected on the 15th day after the induction of EAE. The data are shown in a representative plot. ( B ) The absolute numbers of the cell subpopulations are shown; the black column is for mir223 −/- mice. Rest, ITGAM + PTPRC low ; mc, ITGAM + PTPRC hi and low . ( C ) Autophagy was measured in the brains of the mice. LC3 puncta were visualized by confocal imaging of microglia immunostained for LC3 and AIF1, followed by Alexa Fluor 488/555-conjugated secondary antibodies (green/red); nuclei were stained with DAPI. Representative images are shown. Scale bars: 20 µm. ( D ) and ( E ) BCL2 and BECN1 expression were visualized by fluorescence imaging of microglia. Scale bars: 50 µm.

    Article Snippet: The brains from the wild-type C57BL/6 mice and mir223 −/- mice (n = 6) were perfused transcardially with 4% (weight:volume) paraformaldehyde and then were dissected and post-fixed for 48 h. The brain paraffin sections (7 μm) were stained with DAPI (Thermo Fisher Scientific, 00–4959-52), LC3A/B (Cell Signaling Technology, 12,741), BCL2 (Abcam, ab32124), BECN1 (Cell Signaling Technology, 3495), AIF1/IBA1 (allograft inflammatory factor 1) (Abcam, ab107159) and Alexa Fluor 488 (Proteintech, SA00006-2) and Alexa Fluor 546 (Thermo Fisher Scientific, A11056) secondary antibody conjugates (green and red, respectively).

    Techniques: Mouse Assay, Flow Cytometry, Isolation, Imaging, Staining, Expressing, Fluorescence

    M14 xenografts carrying bcl-2 protein lacking the BH4 domain show decreased proliferation and increased autophagy. (A) Representative images and (B) quantification of Ki67 immunostaining in tumor xenografts from M14 control (puro) and its derivatives

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Removal of the BH4 Domain from Bcl-2 Protein Triggers an Autophagic Process that Impairs Tumor Growth 1Removal of the BH4 Domain from Bcl-2 Protein Triggers an Autophagic Process that Impairs Tumor Growth 1 2

    doi:

    Figure Lengend Snippet: M14 xenografts carrying bcl-2 protein lacking the BH4 domain show decreased proliferation and increased autophagy. (A) Representative images and (B) quantification of Ki67 immunostaining in tumor xenografts from M14 control (puro) and its derivatives

    Article Snippet: Antibodies against bcl-2, p62/SQSTM1, HA epitope (Santa Cruz Biotechnology), Beclin-1 (Cell Signaling Technology), poly (ADP-ribose) polymerase (PARP; BD Pharmingen International), β-actin, LC3B, bromodeoxyuridine (BrdU; Sigma-Aldrich), Ki67 (Novus Biologicals, Littleton, CO), and HSP72/73 (Calbiochem, EMD Biosciences, La Jolla, CA) were used.

    Techniques: Immunostaining

    Beclin-1 down-regulation enhances cell proliferation of M14 cells expressing bcl-2 protein lacking the BH4 domain. (A) Western blot analysis of LC3B-I/II and Beclin-1 protein expression and (B) AVOs in cells grown in complete media or under serum starvation

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Removal of the BH4 Domain from Bcl-2 Protein Triggers an Autophagic Process that Impairs Tumor Growth 1Removal of the BH4 Domain from Bcl-2 Protein Triggers an Autophagic Process that Impairs Tumor Growth 1 2

    doi:

    Figure Lengend Snippet: Beclin-1 down-regulation enhances cell proliferation of M14 cells expressing bcl-2 protein lacking the BH4 domain. (A) Western blot analysis of LC3B-I/II and Beclin-1 protein expression and (B) AVOs in cells grown in complete media or under serum starvation

    Article Snippet: Antibodies against bcl-2, p62/SQSTM1, HA epitope (Santa Cruz Biotechnology), Beclin-1 (Cell Signaling Technology), poly (ADP-ribose) polymerase (PARP; BD Pharmingen International), β-actin, LC3B, bromodeoxyuridine (BrdU; Sigma-Aldrich), Ki67 (Novus Biologicals, Littleton, CO), and HSP72/73 (Calbiochem, EMD Biosciences, La Jolla, CA) were used.

    Techniques: Expressing, Western Blot

    Bcl-2 protein lacking the BH4 domain strongly impairs the binding between bcl-2 and bax or Beclin-1. (A) Western blot analysis of bcl-2, bax, and Beclin-1 proteins in total lysates of M14 control cells (puro) and its derivatives overexpressing bcl-2 wt

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Removal of the BH4 Domain from Bcl-2 Protein Triggers an Autophagic Process that Impairs Tumor Growth 1Removal of the BH4 Domain from Bcl-2 Protein Triggers an Autophagic Process that Impairs Tumor Growth 1 2

    doi:

    Figure Lengend Snippet: Bcl-2 protein lacking the BH4 domain strongly impairs the binding between bcl-2 and bax or Beclin-1. (A) Western blot analysis of bcl-2, bax, and Beclin-1 proteins in total lysates of M14 control cells (puro) and its derivatives overexpressing bcl-2 wt

    Article Snippet: Antibodies against bcl-2, p62/SQSTM1, HA epitope (Santa Cruz Biotechnology), Beclin-1 (Cell Signaling Technology), poly (ADP-ribose) polymerase (PARP; BD Pharmingen International), β-actin, LC3B, bromodeoxyuridine (BrdU; Sigma-Aldrich), Ki67 (Novus Biologicals, Littleton, CO), and HSP72/73 (Calbiochem, EMD Biosciences, La Jolla, CA) were used.

    Techniques: Binding Assay, Western Blot

    Deletion of BH4 domain from bcl-2 protein in HT29 colon carcinoma and JR8 melanoma cells does not affect tumorigenicity. (A) Western blot analysis of bcl-2 and Beclin-1 proteins, (C, E) in vitro cell proliferation, and (D, F) in vivo tumor growth of HT29

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Removal of the BH4 Domain from Bcl-2 Protein Triggers an Autophagic Process that Impairs Tumor Growth 1Removal of the BH4 Domain from Bcl-2 Protein Triggers an Autophagic Process that Impairs Tumor Growth 1 2

    doi:

    Figure Lengend Snippet: Deletion of BH4 domain from bcl-2 protein in HT29 colon carcinoma and JR8 melanoma cells does not affect tumorigenicity. (A) Western blot analysis of bcl-2 and Beclin-1 proteins, (C, E) in vitro cell proliferation, and (D, F) in vivo tumor growth of HT29

    Article Snippet: Antibodies against bcl-2, p62/SQSTM1, HA epitope (Santa Cruz Biotechnology), Beclin-1 (Cell Signaling Technology), poly (ADP-ribose) polymerase (PARP; BD Pharmingen International), β-actin, LC3B, bromodeoxyuridine (BrdU; Sigma-Aldrich), Ki67 (Novus Biologicals, Littleton, CO), and HSP72/73 (Calbiochem, EMD Biosciences, La Jolla, CA) were used.

    Techniques: Western Blot, In Vitro, In Vivo

    Deletion of BH4 domain from bcl-2 protein enhances autophagic flux. (A) Immunofluorescence staining of p62/SQSTM1 protein in M14 control clone (puro) and its derivatives overexpressing bcl-2 wt (Bcl-2wt) or deleted of BH4 domain (BH4del) stably transfected

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Removal of the BH4 Domain from Bcl-2 Protein Triggers an Autophagic Process that Impairs Tumor Growth 1Removal of the BH4 Domain from Bcl-2 Protein Triggers an Autophagic Process that Impairs Tumor Growth 1 2

    doi:

    Figure Lengend Snippet: Deletion of BH4 domain from bcl-2 protein enhances autophagic flux. (A) Immunofluorescence staining of p62/SQSTM1 protein in M14 control clone (puro) and its derivatives overexpressing bcl-2 wt (Bcl-2wt) or deleted of BH4 domain (BH4del) stably transfected

    Article Snippet: Antibodies against bcl-2, p62/SQSTM1, HA epitope (Santa Cruz Biotechnology), Beclin-1 (Cell Signaling Technology), poly (ADP-ribose) polymerase (PARP; BD Pharmingen International), β-actin, LC3B, bromodeoxyuridine (BrdU; Sigma-Aldrich), Ki67 (Novus Biologicals, Littleton, CO), and HSP72/73 (Calbiochem, EMD Biosciences, La Jolla, CA) were used.

    Techniques: Immunofluorescence, Staining, Stable Transfection, Transfection

    Deletion of BH4 domain from bcl-2 protein in M14 cells reduces tumorigenicity. In vitro (A) cell proliferation and (B) clonogenic ability and (C) in vivo tumor growth of M14 control (puro) and its derivatives overexpressing bcl-2 wt (Bcl-2wt) or deleted

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Removal of the BH4 Domain from Bcl-2 Protein Triggers an Autophagic Process that Impairs Tumor Growth 1Removal of the BH4 Domain from Bcl-2 Protein Triggers an Autophagic Process that Impairs Tumor Growth 1 2

    doi:

    Figure Lengend Snippet: Deletion of BH4 domain from bcl-2 protein in M14 cells reduces tumorigenicity. In vitro (A) cell proliferation and (B) clonogenic ability and (C) in vivo tumor growth of M14 control (puro) and its derivatives overexpressing bcl-2 wt (Bcl-2wt) or deleted

    Article Snippet: Antibodies against bcl-2, p62/SQSTM1, HA epitope (Santa Cruz Biotechnology), Beclin-1 (Cell Signaling Technology), poly (ADP-ribose) polymerase (PARP; BD Pharmingen International), β-actin, LC3B, bromodeoxyuridine (BrdU; Sigma-Aldrich), Ki67 (Novus Biologicals, Littleton, CO), and HSP72/73 (Calbiochem, EMD Biosciences, La Jolla, CA) were used.

    Techniques: In Vitro, In Vivo

    Deletion of BH4 domain from bcl-2 protein in A375SM-SC1 cells reduces tumorigenicity. (A) Western blot analysis of bcl-2 protein expression in human A375SM-SC1 melanoma cells stably transfected with empty vector (puro) or with vectors encoding bcl-2 wt

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Removal of the BH4 Domain from Bcl-2 Protein Triggers an Autophagic Process that Impairs Tumor Growth 1Removal of the BH4 Domain from Bcl-2 Protein Triggers an Autophagic Process that Impairs Tumor Growth 1 2

    doi:

    Figure Lengend Snippet: Deletion of BH4 domain from bcl-2 protein in A375SM-SC1 cells reduces tumorigenicity. (A) Western blot analysis of bcl-2 protein expression in human A375SM-SC1 melanoma cells stably transfected with empty vector (puro) or with vectors encoding bcl-2 wt

    Article Snippet: Antibodies against bcl-2, p62/SQSTM1, HA epitope (Santa Cruz Biotechnology), Beclin-1 (Cell Signaling Technology), poly (ADP-ribose) polymerase (PARP; BD Pharmingen International), β-actin, LC3B, bromodeoxyuridine (BrdU; Sigma-Aldrich), Ki67 (Novus Biologicals, Littleton, CO), and HSP72/73 (Calbiochem, EMD Biosciences, La Jolla, CA) were used.

    Techniques: Western Blot, Expressing, Stable Transfection, Transfection, Plasmid Preparation

    Deletion of α1 helix of bcl-2 does not affect in vivo tumor growth of M14 cells. (A) AVOs in cells grown in completemedia or under serum starvation (hours) and (B) in vivo tumor growth of M14 control (empty) and its derivatives overexpressing

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Removal of the BH4 Domain from Bcl-2 Protein Triggers an Autophagic Process that Impairs Tumor Growth 1Removal of the BH4 Domain from Bcl-2 Protein Triggers an Autophagic Process that Impairs Tumor Growth 1 2

    doi:

    Figure Lengend Snippet: Deletion of α1 helix of bcl-2 does not affect in vivo tumor growth of M14 cells. (A) AVOs in cells grown in completemedia or under serum starvation (hours) and (B) in vivo tumor growth of M14 control (empty) and its derivatives overexpressing

    Article Snippet: Antibodies against bcl-2, p62/SQSTM1, HA epitope (Santa Cruz Biotechnology), Beclin-1 (Cell Signaling Technology), poly (ADP-ribose) polymerase (PARP; BD Pharmingen International), β-actin, LC3B, bromodeoxyuridine (BrdU; Sigma-Aldrich), Ki67 (Novus Biologicals, Littleton, CO), and HSP72/73 (Calbiochem, EMD Biosciences, La Jolla, CA) were used.

    Techniques: In Vivo

    Deletion of BH4 domain from bcl-2 protein in M14 and H1299 cells increases autophagosome formation in response to autophagic stimuli. (A) Western blot analysis of bcl-2 protein expression in M14 control clone (puro) and its derivatives overexpressing

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Removal of the BH4 Domain from Bcl-2 Protein Triggers an Autophagic Process that Impairs Tumor Growth 1Removal of the BH4 Domain from Bcl-2 Protein Triggers an Autophagic Process that Impairs Tumor Growth 1 2

    doi:

    Figure Lengend Snippet: Deletion of BH4 domain from bcl-2 protein in M14 and H1299 cells increases autophagosome formation in response to autophagic stimuli. (A) Western blot analysis of bcl-2 protein expression in M14 control clone (puro) and its derivatives overexpressing

    Article Snippet: Antibodies against bcl-2, p62/SQSTM1, HA epitope (Santa Cruz Biotechnology), Beclin-1 (Cell Signaling Technology), poly (ADP-ribose) polymerase (PARP; BD Pharmingen International), β-actin, LC3B, bromodeoxyuridine (BrdU; Sigma-Aldrich), Ki67 (Novus Biologicals, Littleton, CO), and HSP72/73 (Calbiochem, EMD Biosciences, La Jolla, CA) were used.

    Techniques: Western Blot, Expressing

    Loss of Bak does not prevent thrombocytopaenia in BCL-2 tg mice. ( a ) Blood platelet counts in WT (white), Bak − / − (light grey), BCL-2 tg (dark grey) and Bak − / − BCL-2 tg (black) mice at 6–8 weeks ( n =4–7 per genotype), 12 weeks ( n =9–14) and 24 weeks ( n =15–20). Bars represent mean±S.E.M. Statistical significance is shown only for 12-week-old mice; *** P

    Journal: Cell Death and Differentiation

    Article Title: Loss of Bak enhances lymphocytosis but does not ameliorate thrombocytopaenia in BCL-2 transgenic mice

    doi: 10.1038/cdd.2013.201

    Figure Lengend Snippet: Loss of Bak does not prevent thrombocytopaenia in BCL-2 tg mice. ( a ) Blood platelet counts in WT (white), Bak − / − (light grey), BCL-2 tg (dark grey) and Bak − / − BCL-2 tg (black) mice at 6–8 weeks ( n =4–7 per genotype), 12 weeks ( n =9–14) and 24 weeks ( n =15–20). Bars represent mean±S.E.M. Statistical significance is shown only for 12-week-old mice; *** P

    Article Snippet: Blots were probed with: anti-Mcl-1 (clone 19C4-15; WEHI mAb facility), anti-human Bcl-2 (clone Bcl-2-100; WEHI mAb facility), anti-Bcl-2 (clone 7; BD Biosciences), anti-Bim (polyclonal; Enzo Lifesciences, Farmingdale, NY, USA), anti-Bcl-xL (polyclonal; BD Biosciences), anti-Bak (polyclonal; Sigma-Aldrich) and anti-β -actin (clone AC-74; Sigma-Aldrich).

    Techniques: Mouse Assay

    Thrombocytopaenia in BCL-2 tg mice is platelet extrinsic. ( a – d ) Analysis of red blood cell (RBC) ( a ), white blood cell (WBC) ( b ) and platelets ( c , d ) in the blood of mice 9 weeks following lethal irradiation and reconstitution with GFP tg, BCL-2 tg or a 50 : 50 mix of GFP tg and BCL-2 tg bone marrow cells. Data represent mean±S.E.M., n =6; * P

    Journal: Cell Death and Differentiation

    Article Title: Loss of Bak enhances lymphocytosis but does not ameliorate thrombocytopaenia in BCL-2 transgenic mice

    doi: 10.1038/cdd.2013.201

    Figure Lengend Snippet: Thrombocytopaenia in BCL-2 tg mice is platelet extrinsic. ( a – d ) Analysis of red blood cell (RBC) ( a ), white blood cell (WBC) ( b ) and platelets ( c , d ) in the blood of mice 9 weeks following lethal irradiation and reconstitution with GFP tg, BCL-2 tg or a 50 : 50 mix of GFP tg and BCL-2 tg bone marrow cells. Data represent mean±S.E.M., n =6; * P

    Article Snippet: Blots were probed with: anti-Mcl-1 (clone 19C4-15; WEHI mAb facility), anti-human Bcl-2 (clone Bcl-2-100; WEHI mAb facility), anti-Bcl-2 (clone 7; BD Biosciences), anti-Bim (polyclonal; Enzo Lifesciences, Farmingdale, NY, USA), anti-Bcl-xL (polyclonal; BD Biosciences), anti-Bak (polyclonal; Sigma-Aldrich) and anti-β -actin (clone AC-74; Sigma-Aldrich).

    Techniques: Mouse Assay, Irradiation

    Kinetic analysis of T lymphopoiesis in BCL-2 tg and Bak − / − BCL-2 tg mice. Enumeration of total cellularity and indicated T-lymphoid populations in the ( a ) thymus and ( b ) spleen of 6- to 8-, 12- and 24-week-old male mice ( n =6–8 per genotype: WT, white; Bak − / − , light grey; BCL-2 tg, dark grey; Bak − / − BCL-2 tg, black). Bars represent mean±S.E.M.; see also Supplementary Table 4 . Statistical significance is shown only for BCL-2 tg versus Bak − / − BCL-2 tg; * P

    Journal: Cell Death and Differentiation

    Article Title: Loss of Bak enhances lymphocytosis but does not ameliorate thrombocytopaenia in BCL-2 transgenic mice

    doi: 10.1038/cdd.2013.201

    Figure Lengend Snippet: Kinetic analysis of T lymphopoiesis in BCL-2 tg and Bak − / − BCL-2 tg mice. Enumeration of total cellularity and indicated T-lymphoid populations in the ( a ) thymus and ( b ) spleen of 6- to 8-, 12- and 24-week-old male mice ( n =6–8 per genotype: WT, white; Bak − / − , light grey; BCL-2 tg, dark grey; Bak − / − BCL-2 tg, black). Bars represent mean±S.E.M.; see also Supplementary Table 4 . Statistical significance is shown only for BCL-2 tg versus Bak − / − BCL-2 tg; * P

    Article Snippet: Blots were probed with: anti-Mcl-1 (clone 19C4-15; WEHI mAb facility), anti-human Bcl-2 (clone Bcl-2-100; WEHI mAb facility), anti-Bcl-2 (clone 7; BD Biosciences), anti-Bim (polyclonal; Enzo Lifesciences, Farmingdale, NY, USA), anti-Bcl-xL (polyclonal; BD Biosciences), anti-Bak (polyclonal; Sigma-Aldrich) and anti-β -actin (clone AC-74; Sigma-Aldrich).

    Techniques: Mouse Assay

    Loss of Bak further enhances survival of thymocytes overexpressing Bcl-2. ( a ) DP thymocytes isolated by FACS were cultured in medium lacking cytokines (untreated), or following exposure to 10 Gy γ -irradiation, or in the presence of the indicated concentrations of etoposide, dexamethasone, PMA or ionomycin. Cell viability was determined by propidium iodide and Annexin V staining followed by flow cytometry. Stimulus-specific viability was calculated relative to viability of untreated cells at each time point (see Materials and Methods). n =4 from two independent experiments; values are mean±S.E.M. Statistical significance (Student's t -test) is only indicated for Bak − / − BCL-2 tg versus BCL-2 tg (* P

    Journal: Cell Death and Differentiation

    Article Title: Loss of Bak enhances lymphocytosis but does not ameliorate thrombocytopaenia in BCL-2 transgenic mice

    doi: 10.1038/cdd.2013.201

    Figure Lengend Snippet: Loss of Bak further enhances survival of thymocytes overexpressing Bcl-2. ( a ) DP thymocytes isolated by FACS were cultured in medium lacking cytokines (untreated), or following exposure to 10 Gy γ -irradiation, or in the presence of the indicated concentrations of etoposide, dexamethasone, PMA or ionomycin. Cell viability was determined by propidium iodide and Annexin V staining followed by flow cytometry. Stimulus-specific viability was calculated relative to viability of untreated cells at each time point (see Materials and Methods). n =4 from two independent experiments; values are mean±S.E.M. Statistical significance (Student's t -test) is only indicated for Bak − / − BCL-2 tg versus BCL-2 tg (* P

    Article Snippet: Blots were probed with: anti-Mcl-1 (clone 19C4-15; WEHI mAb facility), anti-human Bcl-2 (clone Bcl-2-100; WEHI mAb facility), anti-Bcl-2 (clone 7; BD Biosciences), anti-Bim (polyclonal; Enzo Lifesciences, Farmingdale, NY, USA), anti-Bcl-xL (polyclonal; BD Biosciences), anti-Bak (polyclonal; Sigma-Aldrich) and anti-β -actin (clone AC-74; Sigma-Aldrich).

    Techniques: Isolation, FACS, Cell Culture, Irradiation, Staining, Flow Cytometry, Cytometry

    Loss of Bak exacerbates lymphocytosis in BCL-2 tg mice. Enumeration of total leukocytes and indicated lymphoid populations in the ( a ) spleen and ( b ) thymus of 12- to 14-week-old male mice ( n =6–10 per genotype: WT, white; Bak − / − , light grey; BCL-2 tg, dark grey; Bak − / − BCL-2 tg, black). B220 + IgM/D + indicates B220 + cells that are IgM + and/or IgD + . Bars represent mean±S.E.M.; see also Supplementary Table 2 . Statistical significance is shown only for BCL-2 tg versus Bak − / − BCL-2 tg; * P

    Journal: Cell Death and Differentiation

    Article Title: Loss of Bak enhances lymphocytosis but does not ameliorate thrombocytopaenia in BCL-2 transgenic mice

    doi: 10.1038/cdd.2013.201

    Figure Lengend Snippet: Loss of Bak exacerbates lymphocytosis in BCL-2 tg mice. Enumeration of total leukocytes and indicated lymphoid populations in the ( a ) spleen and ( b ) thymus of 12- to 14-week-old male mice ( n =6–10 per genotype: WT, white; Bak − / − , light grey; BCL-2 tg, dark grey; Bak − / − BCL-2 tg, black). B220 + IgM/D + indicates B220 + cells that are IgM + and/or IgD + . Bars represent mean±S.E.M.; see also Supplementary Table 2 . Statistical significance is shown only for BCL-2 tg versus Bak − / − BCL-2 tg; * P

    Article Snippet: Blots were probed with: anti-Mcl-1 (clone 19C4-15; WEHI mAb facility), anti-human Bcl-2 (clone Bcl-2-100; WEHI mAb facility), anti-Bcl-2 (clone 7; BD Biosciences), anti-Bim (polyclonal; Enzo Lifesciences, Farmingdale, NY, USA), anti-Bcl-xL (polyclonal; BD Biosciences), anti-Bak (polyclonal; Sigma-Aldrich) and anti-β -actin (clone AC-74; Sigma-Aldrich).

    Techniques: Mouse Assay

    Thrombocytopaenia in BCL-2tg mice is provoked by the external milieu. ( a ) Blood platelets increase following splenectomy in WT and BCL-2 tg mice. Platelet counts were determined before and 4 weeks after surgery on 8-week-old female mice ( n =9–10). ( b ) Recovery from anti-platelet serum (APS)-induced thrombocytopaenia is impaired in splenectomised BCL-2 tg mice. Mice from a were injected with APS 5 weeks after splenectomy and platelet counts determined on d0 ( n =9–10), d1 ( n =5) and d5 ( n =4–5). ( c ) Lymphocyte counts in BCL-2 tg mice are significantly reduced on a Rag1 − / − background, to around WT numbers. BCL-2 tg mice were crossed to Rag1 −/− mice and blood counts determined in offspring of the indicated genotypes ( n =7–9). ( d ) Platelet counts are normal in BCL-2 tg mice that lack mature lymphocytes. Data represent mean±S.E.M.; ** P

    Journal: Cell Death and Differentiation

    Article Title: Loss of Bak enhances lymphocytosis but does not ameliorate thrombocytopaenia in BCL-2 transgenic mice

    doi: 10.1038/cdd.2013.201

    Figure Lengend Snippet: Thrombocytopaenia in BCL-2tg mice is provoked by the external milieu. ( a ) Blood platelets increase following splenectomy in WT and BCL-2 tg mice. Platelet counts were determined before and 4 weeks after surgery on 8-week-old female mice ( n =9–10). ( b ) Recovery from anti-platelet serum (APS)-induced thrombocytopaenia is impaired in splenectomised BCL-2 tg mice. Mice from a were injected with APS 5 weeks after splenectomy and platelet counts determined on d0 ( n =9–10), d1 ( n =5) and d5 ( n =4–5). ( c ) Lymphocyte counts in BCL-2 tg mice are significantly reduced on a Rag1 − / − background, to around WT numbers. BCL-2 tg mice were crossed to Rag1 −/− mice and blood counts determined in offspring of the indicated genotypes ( n =7–9). ( d ) Platelet counts are normal in BCL-2 tg mice that lack mature lymphocytes. Data represent mean±S.E.M.; ** P

    Article Snippet: Blots were probed with: anti-Mcl-1 (clone 19C4-15; WEHI mAb facility), anti-human Bcl-2 (clone Bcl-2-100; WEHI mAb facility), anti-Bcl-2 (clone 7; BD Biosciences), anti-Bim (polyclonal; Enzo Lifesciences, Farmingdale, NY, USA), anti-Bcl-xL (polyclonal; BD Biosciences), anti-Bak (polyclonal; Sigma-Aldrich) and anti-β -actin (clone AC-74; Sigma-Aldrich).

    Techniques: Mouse Assay, Injection