Structured Review

Trevigen anti cleaved caspase 3
Pharmacological inhibition of DOT1L affects gene expression and key cellular functions, including proliferation and survival, in MCF-7 cells. ( A ) Left: MA plot showing transcriptome changes, measured by RNA sequencing (RNA-seq), following 6 days of treatment with 6.4 μM EPZ. Right: Bar chart, obtained from KEGG functional enrichment analysis, showing statistically significant deregulated pathways. Green, red, and gray colors represent inhibited, activated, and unaffected signaling pathways, respectively. ( B ) MCF-7 cell proliferation rate in the presence of estrogen (17β-estradiol, E2), the indicated antiestrogens, or increasing concentrations of EPZ, assessed by MTT assays in exponentially growing conditions. Vehicles (EtOH or DMSO) were used as controls. ( C and D ) Cell cycle phase distribution in exponentially growing MCF-7 cell cultures before (−) and after treatment with the indicated concentrations of ICI or EPZ for 3 to 9 days. Percentages of G 1 , S, and G 2 /M (C), and sub-G 1 (D) phase cells were determined by flow cytometry after propidium iodide (PI) staining. ( E ) Bar chart showing accumulation of annexin V–fluorescein isothiocyanate (FITC)/PI–positive cells following treatment with the indicated concentrations of either ICI or EPZ for 3, 6, and 9 days. For (B) to (E), the results shown represent the means ± SD of multiple determinations from a representative experiment performed in octuplicate (B) or triplicate (C to E). ( F ) Western blot showing the extent of activated <t>caspase-3</t> (CASP3) accumulation after 3, 6, or 9 days of treatment with EPZ (+, 6.4 μM) or vehicle (V, DMSO) in MCF-7 cells. β-Actin (ACTB) was used as loading control. ( G ) Left: MCF-7 cell proliferation rate in the presence of estrogen only (17β-estradiol, E2), the indicated antiestrogens, or increasing concentrations of EPZ with E2 coadministration, assessed by MTT assays in hormone-starved cells. Vehicle (EtOH) was used as control. Middle and right: Cell cycle phase distribution in hormone-deprived MCF-7 cell cultures before (−), after E2 only, or treatment with the indicated concentrations of ICI or EPZ for 6 days together with E2. Percentages of G 1 , S, and G 2 /M (middle), and sub-G 1 (right) phase cells were determined by flow cytometry after PI staining. Results shown represent the means ± SD of multiple determinations from a representative experiment performed at least in triplicate. ( H ) ERα transactivating activity in MCF-7 cells stably expressing the ERE-Luc reporter gene (MELN), before and after E2 stimulation, in the presence of either vehicle only (V) or the indicated concentrations of ICI or EPZ. Results shown represent the means ± SD of multiple determinations from a representative experiment performed in triplicate. OD, optical density.
Anti Cleaved Caspase 3, supplied by Trevigen, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Inhibition of histone methyltransferase DOT1L silences ERα gene and blocks proliferation of antiestrogen-resistant breast cancer cells"

Article Title: Inhibition of histone methyltransferase DOT1L silences ERα gene and blocks proliferation of antiestrogen-resistant breast cancer cells

Journal: Science Advances

doi: 10.1126/sciadv.aav5590

Pharmacological inhibition of DOT1L affects gene expression and key cellular functions, including proliferation and survival, in MCF-7 cells. ( A ) Left: MA plot showing transcriptome changes, measured by RNA sequencing (RNA-seq), following 6 days of treatment with 6.4 μM EPZ. Right: Bar chart, obtained from KEGG functional enrichment analysis, showing statistically significant deregulated pathways. Green, red, and gray colors represent inhibited, activated, and unaffected signaling pathways, respectively. ( B ) MCF-7 cell proliferation rate in the presence of estrogen (17β-estradiol, E2), the indicated antiestrogens, or increasing concentrations of EPZ, assessed by MTT assays in exponentially growing conditions. Vehicles (EtOH or DMSO) were used as controls. ( C and D ) Cell cycle phase distribution in exponentially growing MCF-7 cell cultures before (−) and after treatment with the indicated concentrations of ICI or EPZ for 3 to 9 days. Percentages of G 1 , S, and G 2 /M (C), and sub-G 1 (D) phase cells were determined by flow cytometry after propidium iodide (PI) staining. ( E ) Bar chart showing accumulation of annexin V–fluorescein isothiocyanate (FITC)/PI–positive cells following treatment with the indicated concentrations of either ICI or EPZ for 3, 6, and 9 days. For (B) to (E), the results shown represent the means ± SD of multiple determinations from a representative experiment performed in octuplicate (B) or triplicate (C to E). ( F ) Western blot showing the extent of activated caspase-3 (CASP3) accumulation after 3, 6, or 9 days of treatment with EPZ (+, 6.4 μM) or vehicle (V, DMSO) in MCF-7 cells. β-Actin (ACTB) was used as loading control. ( G ) Left: MCF-7 cell proliferation rate in the presence of estrogen only (17β-estradiol, E2), the indicated antiestrogens, or increasing concentrations of EPZ with E2 coadministration, assessed by MTT assays in hormone-starved cells. Vehicle (EtOH) was used as control. Middle and right: Cell cycle phase distribution in hormone-deprived MCF-7 cell cultures before (−), after E2 only, or treatment with the indicated concentrations of ICI or EPZ for 6 days together with E2. Percentages of G 1 , S, and G 2 /M (middle), and sub-G 1 (right) phase cells were determined by flow cytometry after PI staining. Results shown represent the means ± SD of multiple determinations from a representative experiment performed at least in triplicate. ( H ) ERα transactivating activity in MCF-7 cells stably expressing the ERE-Luc reporter gene (MELN), before and after E2 stimulation, in the presence of either vehicle only (V) or the indicated concentrations of ICI or EPZ. Results shown represent the means ± SD of multiple determinations from a representative experiment performed in triplicate. OD, optical density.
Figure Legend Snippet: Pharmacological inhibition of DOT1L affects gene expression and key cellular functions, including proliferation and survival, in MCF-7 cells. ( A ) Left: MA plot showing transcriptome changes, measured by RNA sequencing (RNA-seq), following 6 days of treatment with 6.4 μM EPZ. Right: Bar chart, obtained from KEGG functional enrichment analysis, showing statistically significant deregulated pathways. Green, red, and gray colors represent inhibited, activated, and unaffected signaling pathways, respectively. ( B ) MCF-7 cell proliferation rate in the presence of estrogen (17β-estradiol, E2), the indicated antiestrogens, or increasing concentrations of EPZ, assessed by MTT assays in exponentially growing conditions. Vehicles (EtOH or DMSO) were used as controls. ( C and D ) Cell cycle phase distribution in exponentially growing MCF-7 cell cultures before (−) and after treatment with the indicated concentrations of ICI or EPZ for 3 to 9 days. Percentages of G 1 , S, and G 2 /M (C), and sub-G 1 (D) phase cells were determined by flow cytometry after propidium iodide (PI) staining. ( E ) Bar chart showing accumulation of annexin V–fluorescein isothiocyanate (FITC)/PI–positive cells following treatment with the indicated concentrations of either ICI or EPZ for 3, 6, and 9 days. For (B) to (E), the results shown represent the means ± SD of multiple determinations from a representative experiment performed in octuplicate (B) or triplicate (C to E). ( F ) Western blot showing the extent of activated caspase-3 (CASP3) accumulation after 3, 6, or 9 days of treatment with EPZ (+, 6.4 μM) or vehicle (V, DMSO) in MCF-7 cells. β-Actin (ACTB) was used as loading control. ( G ) Left: MCF-7 cell proliferation rate in the presence of estrogen only (17β-estradiol, E2), the indicated antiestrogens, or increasing concentrations of EPZ with E2 coadministration, assessed by MTT assays in hormone-starved cells. Vehicle (EtOH) was used as control. Middle and right: Cell cycle phase distribution in hormone-deprived MCF-7 cell cultures before (−), after E2 only, or treatment with the indicated concentrations of ICI or EPZ for 6 days together with E2. Percentages of G 1 , S, and G 2 /M (middle), and sub-G 1 (right) phase cells were determined by flow cytometry after PI staining. Results shown represent the means ± SD of multiple determinations from a representative experiment performed at least in triplicate. ( H ) ERα transactivating activity in MCF-7 cells stably expressing the ERE-Luc reporter gene (MELN), before and after E2 stimulation, in the presence of either vehicle only (V) or the indicated concentrations of ICI or EPZ. Results shown represent the means ± SD of multiple determinations from a representative experiment performed in triplicate. OD, optical density.

Techniques Used: Inhibition, Expressing, RNA Sequencing Assay, Functional Assay, MTT Assay, Flow Cytometry, Cytometry, Staining, Western Blot, Activity Assay, Stable Transfection

Related Articles

Immunoprecipitation:

Article Title: Inhibition of histone methyltransferase DOT1L silences ERα gene and blocks proliferation of antiestrogen-resistant breast cancer cells
Article Snippet: .. Antibodies The following antibodies were used for immunoprecipitation and Western blot analyses: C-terminal anti-ERα (sc-543, Santa Cruz Biotechnology), rabbit polyclonal anti-DOT1L (A300-953A, Bethyl Laboratories), β-actin (A1978, Sigma-Aldrich), N-terminal anti-ERα (ab75635), mouse anti-KMT4/DOT1L (ab72454), anti-histone H3 (total; ab1791), anti-H3K79me1 (ab2886), anti-H3k79me2 (ab3594), anti-H3K79me3 (ab2621), anti-H3K4me3 (ab7766), anti-H3K9me3 (ab1220), anti-H3K27me3 (ab24684) from Abcam, anti–cleaved caspase-3 (2305-PC-100, Trevigen), anti-TAP (CAB1001), and rabbit immunoglobulin G (IgG) isotype control (31235) from Thermo Fisher Scientific. .. Protein extraction and coimmunoprecipitation Total and nuclear protein extracts were prepared as described previously ( , ).

BIA-KA:

Article Title: Hepatocyte-specific c-Met Deletion Disrupts Redox Homeostasis and Sensitizes to Fas-mediated Apoptosis *
Article Snippet: .. One hundred μg of total protein or 25 μg of nuclear protein measured using BCA protein assay kit (Pierce) were separated on SDS-polyacrylamide gels (Invitrogen), transferred to polyvinylidene difluoride membranes (Invitrogen), and probed with anti-caspase 3, anti-Bcl-2, anti-Bcl-XL , anti-Mcl-1, anti-cytochrome c , anti-IκB-α, anti-gp91Phox (anti-Nox2) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-cleaved caspase 3 (Trevigen, Gaithersburg, MD), anti-ERK1/2, anti-pERK1/2, anti-Akt, anti-pAkt, anti-PKC α/β II (Thr-638/641), anti-pPKC ζ/λ (Thr-410/403), anti-pPKC ε (Ser-729), anti-STAT3, and anti-pSTAT3 (Cell Signaling, Beverly, MA) antibodies. .. Membranes were incubated with anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibody depending on the origin of the primary antibody.

Labeling:

Article Title: DNA double-strand breaks form in bystander cells after microbeam irradiation of three-dimensional human tissue models.
Article Snippet: .. For double labeling, anti-g-H2AX primary antibodies were either rabbit or mouse (Upstate BioTech) with antiRad50, (Novus Biologicals), anti-53bp1 (generously provided by Dr. J. Chen, Department of Oncology, Mayo Clinic and Foundation, Rochester, MN), anti–cleaved caspase-3 (Trevigen), or anti–proliferating cell nuclear antigen (PCNA; Santa Cruz Biotechnology). .. Secondary antibodies labeled with either Alexa 488 or Alexa 546 were from Molecular Probes.

Western Blot:

Article Title: Inhibition of histone methyltransferase DOT1L silences ERα gene and blocks proliferation of antiestrogen-resistant breast cancer cells
Article Snippet: .. Antibodies The following antibodies were used for immunoprecipitation and Western blot analyses: C-terminal anti-ERα (sc-543, Santa Cruz Biotechnology), rabbit polyclonal anti-DOT1L (A300-953A, Bethyl Laboratories), β-actin (A1978, Sigma-Aldrich), N-terminal anti-ERα (ab75635), mouse anti-KMT4/DOT1L (ab72454), anti-histone H3 (total; ab1791), anti-H3K79me1 (ab2886), anti-H3k79me2 (ab3594), anti-H3K79me3 (ab2621), anti-H3K4me3 (ab7766), anti-H3K9me3 (ab1220), anti-H3K27me3 (ab24684) from Abcam, anti–cleaved caspase-3 (2305-PC-100, Trevigen), anti-TAP (CAB1001), and rabbit immunoglobulin G (IgG) isotype control (31235) from Thermo Fisher Scientific. .. Protein extraction and coimmunoprecipitation Total and nuclear protein extracts were prepared as described previously ( , ).

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    Trevigen caspase 3
    Dorsal root ganglion neuronal <t>caspase-3</t> immunohistochemistry at 6 months of diabetes. Examples of dorsal root ganglion sections immunostained for RAGE (a, d, g), NFκ B (b, e, h) and activated caspase-3 (c, f, i). The top panels (a–c) are
    Caspase 3, supplied by Trevigen, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Trevigen anti caspase 3
    Reconstitution of p16 induces senescence and prevents apoptosis in cancer cells in response to ABC294640-mediated SPHK2 inhibition and telomere damage. A, effects of stable expression of p16 on <t>caspase-3</t> activation in A549 cells transfected with Scr- or SPHK2-shRNAs were measured by immunofluorescence. Images were quantified by ImageJ ( lower panel ). Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.001; **, p = 0.0005; ***, p = 0.0004). Expression levels of p16 protein in stable shSPHK2 versus shScr A549 cells were confirmed by Western blotting using anti-p16 antibody ( upper right panel ). Actin was used as a loading control ( lower right panel ). B and C , effects of stable expression of p16 on senescence in response to shRNA knockdown ( B ) or ABC294640-mediated inhibition ( C ) of SPHK2 were measured by SA-β-gal staining in A549 cells compared with Scr-transfected and/or vehicle-treated controls. D and E , effects of stable expression of p16 on telomere damage ( D ). Images were quantified by ImageJ. Data are means ± S.D. from three independent experiments and analyzed by Student's t test ( n = 3, *, p = 0.006; **, p = 0.002) or caspase-3 activation ( E ) in A549 cells, and in the absence/presence of ABC294640 they were measured using TIF assay or caspase-3 activation assay by immunofluorescence and confocal microscopy. Images were quantified by ImageJ ( right panels ). Data are means ± S.D. from three independent experiments and analyzed by Student's t test ( n = 3, *, p = 0.0006; **, p = 0.0005; ***, p = 0.0004). F, shRNA-mediated knockdown of p16 was confirmed using qPCR in H1341 cells compared with Scr-shRNA–transfected controls. Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.00005). G, caspase-3 activation was measured by immunofluorescence using anti-caspase-3 antibody that detects active caspase-3 in H1341 with/without p16 knockdown compared with Scr-shRNA–transfected controls in the absence/presence of ABC294640 ( right panel ). Effects of ectopic expression of p16 on caspase-3 activation in the presence/absence of ABC294640 was measured in A549 cells compared with vector-transfected controls ( left panel ). H, expression levels of p16 protein in different lung cancer cell lines were confirmed by Western blotting using anti-p16 antibody ( upper panel ). Actin was used as a loading control ( lower panel ).
    Anti Caspase 3, supplied by Trevigen, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Trevigen rabbit anti cleaved caspase 3
    Cleaved <t>caspase-3</t> is found in infected areas of the lungs suggesting host cells are undergoing apoptosis. Left lower lobe sections of lungs harvested 1 hour ( A and E ), 24 hours ( B and F ), or 48 hours ( C , D , G , and H ) after infection were perfused in 10%
    Rabbit Anti Cleaved Caspase 3, supplied by Trevigen, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Trevigen mouse anti caspase 3
    Anterior neural tube defect and increased apoptosis in homozygous Trx-2 −/− mice at 9.5 and 10.5 dpc (representative embryos and images). Aberrant anterior neural tube closure in Trx-2 −/− embryo, indicated by arrows, was compared to the wild-type embryo by hemotoxylin and eosin staining of paraffin-embedded embryos at 9.5 dpc (A and B) and at 10.5 dpc (E and F). In panels C and D are shown increased anti-cleaved <t>caspase-3</t> antibody-positive cells from Trx-2 −/− embryos compared to wild-type embryos by immunohistochemical staining of paraffin-embedded 9.5-dpc embryos (adjacent slices of the embryos shown in panels A and B). In panels G and H, massive apoptosis in 10.5-dpc Trx-2 −/− embryos can be seen compared to the wild-type embryos as visualized by anti-caspase-3 antibody staining of paraffin-embedded embryos (adjacent slices of the embryos shown in E and F). Heavy staining in the uterine tissue is not specific to cleaved caspase-3 and is due to endogenous mouse immunoglobulin G interactions with the secondary antibody.
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    Image Search Results


    Dorsal root ganglion neuronal caspase-3 immunohistochemistry at 6 months of diabetes. Examples of dorsal root ganglion sections immunostained for RAGE (a, d, g), NFκ B (b, e, h) and activated caspase-3 (c, f, i). The top panels (a–c) are

    Journal: Neurobiology of Disease

    Article Title: Overexpression of human HSP27 protects sensory neurons from diabetes

    doi: 10.1016/j.nbd.2012.04.017

    Figure Lengend Snippet: Dorsal root ganglion neuronal caspase-3 immunohistochemistry at 6 months of diabetes. Examples of dorsal root ganglion sections immunostained for RAGE (a, d, g), NFκ B (b, e, h) and activated caspase-3 (c, f, i). The top panels (a–c) are

    Article Snippet: Antibodies applied were: NFκb p-50 (H-119), rabbit polyclonal Ig/G from Santa Cruz Biotechnology at the dilution of 1:200, RAGE (ab3611) rabbit polyclonal to RAGE from Abcam (Dilution 1:800), activated Caspase-3 (1:100; Anti-cleaved Caspase-3 Rabbit Polyclonal IgG; Trevigen Inc., Gaithersburg, MD) with Alexa Flour 488 Goat anti-rabbit IgG as secondary antibody from Invitrogen (secondary antibody 1:200).

    Techniques: Immunohistochemistry

    Reconstitution of p16 induces senescence and prevents apoptosis in cancer cells in response to ABC294640-mediated SPHK2 inhibition and telomere damage. A, effects of stable expression of p16 on caspase-3 activation in A549 cells transfected with Scr- or SPHK2-shRNAs were measured by immunofluorescence. Images were quantified by ImageJ ( lower panel ). Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.001; **, p = 0.0005; ***, p = 0.0004). Expression levels of p16 protein in stable shSPHK2 versus shScr A549 cells were confirmed by Western blotting using anti-p16 antibody ( upper right panel ). Actin was used as a loading control ( lower right panel ). B and C , effects of stable expression of p16 on senescence in response to shRNA knockdown ( B ) or ABC294640-mediated inhibition ( C ) of SPHK2 were measured by SA-β-gal staining in A549 cells compared with Scr-transfected and/or vehicle-treated controls. D and E , effects of stable expression of p16 on telomere damage ( D ). Images were quantified by ImageJ. Data are means ± S.D. from three independent experiments and analyzed by Student's t test ( n = 3, *, p = 0.006; **, p = 0.002) or caspase-3 activation ( E ) in A549 cells, and in the absence/presence of ABC294640 they were measured using TIF assay or caspase-3 activation assay by immunofluorescence and confocal microscopy. Images were quantified by ImageJ ( right panels ). Data are means ± S.D. from three independent experiments and analyzed by Student's t test ( n = 3, *, p = 0.0006; **, p = 0.0005; ***, p = 0.0004). F, shRNA-mediated knockdown of p16 was confirmed using qPCR in H1341 cells compared with Scr-shRNA–transfected controls. Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.00005). G, caspase-3 activation was measured by immunofluorescence using anti-caspase-3 antibody that detects active caspase-3 in H1341 with/without p16 knockdown compared with Scr-shRNA–transfected controls in the absence/presence of ABC294640 ( right panel ). Effects of ectopic expression of p16 on caspase-3 activation in the presence/absence of ABC294640 was measured in A549 cells compared with vector-transfected controls ( left panel ). H, expression levels of p16 protein in different lung cancer cell lines were confirmed by Western blotting using anti-p16 antibody ( upper panel ). Actin was used as a loading control ( lower panel ).

    Journal: The Journal of Biological Chemistry

    Article Title: Balance between senescence and apoptosis is regulated by telomere damage–induced association between p16 and caspase-3

    doi: 10.1074/jbc.RA118.003506

    Figure Lengend Snippet: Reconstitution of p16 induces senescence and prevents apoptosis in cancer cells in response to ABC294640-mediated SPHK2 inhibition and telomere damage. A, effects of stable expression of p16 on caspase-3 activation in A549 cells transfected with Scr- or SPHK2-shRNAs were measured by immunofluorescence. Images were quantified by ImageJ ( lower panel ). Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.001; **, p = 0.0005; ***, p = 0.0004). Expression levels of p16 protein in stable shSPHK2 versus shScr A549 cells were confirmed by Western blotting using anti-p16 antibody ( upper right panel ). Actin was used as a loading control ( lower right panel ). B and C , effects of stable expression of p16 on senescence in response to shRNA knockdown ( B ) or ABC294640-mediated inhibition ( C ) of SPHK2 were measured by SA-β-gal staining in A549 cells compared with Scr-transfected and/or vehicle-treated controls. D and E , effects of stable expression of p16 on telomere damage ( D ). Images were quantified by ImageJ. Data are means ± S.D. from three independent experiments and analyzed by Student's t test ( n = 3, *, p = 0.006; **, p = 0.002) or caspase-3 activation ( E ) in A549 cells, and in the absence/presence of ABC294640 they were measured using TIF assay or caspase-3 activation assay by immunofluorescence and confocal microscopy. Images were quantified by ImageJ ( right panels ). Data are means ± S.D. from three independent experiments and analyzed by Student's t test ( n = 3, *, p = 0.0006; **, p = 0.0005; ***, p = 0.0004). F, shRNA-mediated knockdown of p16 was confirmed using qPCR in H1341 cells compared with Scr-shRNA–transfected controls. Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.00005). G, caspase-3 activation was measured by immunofluorescence using anti-caspase-3 antibody that detects active caspase-3 in H1341 with/without p16 knockdown compared with Scr-shRNA–transfected controls in the absence/presence of ABC294640 ( right panel ). Effects of ectopic expression of p16 on caspase-3 activation in the presence/absence of ABC294640 was measured in A549 cells compared with vector-transfected controls ( left panel ). H, expression levels of p16 protein in different lung cancer cell lines were confirmed by Western blotting using anti-p16 antibody ( upper panel ). Actin was used as a loading control ( lower panel ).

    Article Snippet: Fixed and permeabilized cells were incubated with S1P-specific antibody (Sphingomab, LT1002; 20 μg/ml) and hTERT antibody (ab32020, Abcam) or anti-p16 (A301-267A, Bethyl Laboratories) and anti-caspase-3 (2305-PC-020, Trevigen) antibodies for 18 h. PLA was performed and visualized by IF-CM using Duolink in situ hybridization kit as described by the manufacturer (Olink Biosciences).

    Techniques: Inhibition, Expressing, Activation Assay, Transfection, Immunofluorescence, Western Blot, shRNA, Staining, Caspase-3 Activity Assay, Confocal Microscopy, Real-time Polymerase Chain Reaction, Plasmid Preparation

    S1P-hTERT binding protects caspase-3 activation in response to ABC294640 by phosphomimicking of hTERT by S1P at Ser 921 . A and B , effects of ectopic expression of WT-hTERT, hTERT D684A , hTERT S921D , and hTERT S921D/D684A on caspase-3 activation in the absence/presence of ABC294640 were measured in GM847 cells using immunofluorescence ( A ). Images were quantified by ImageJ ( B ). Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.052; **, p = 0.010). Scale bars represent 100 μm. C, association of S1P with S921D-hTERT and S921D-D684A-hTERT compared with vector-transfected GM847 cells was measured by PLA using anti-S1P (Sphingomab) and anti-hTERT antibodies. Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.00064; **, p = 0.0028; ***, p = 0.0028). Scale bars represent 100 μm. D, ectopic expression of WT–, D684A–, S921A–, S921A/D684–, S921D–, and S921D/D684A–hTERT proteins in the presence/absence of ABC294640 in GM847 cells was confirmed by Western blotting using anti-hTERT antibody. Vector-transfected cell extracts were used as negative controls ( right panel ). Actin was used as a loading control ( lower panel ). Images represent at least three independent studies.

    Journal: The Journal of Biological Chemistry

    Article Title: Balance between senescence and apoptosis is regulated by telomere damage–induced association between p16 and caspase-3

    doi: 10.1074/jbc.RA118.003506

    Figure Lengend Snippet: S1P-hTERT binding protects caspase-3 activation in response to ABC294640 by phosphomimicking of hTERT by S1P at Ser 921 . A and B , effects of ectopic expression of WT-hTERT, hTERT D684A , hTERT S921D , and hTERT S921D/D684A on caspase-3 activation in the absence/presence of ABC294640 were measured in GM847 cells using immunofluorescence ( A ). Images were quantified by ImageJ ( B ). Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.052; **, p = 0.010). Scale bars represent 100 μm. C, association of S1P with S921D-hTERT and S921D-D684A-hTERT compared with vector-transfected GM847 cells was measured by PLA using anti-S1P (Sphingomab) and anti-hTERT antibodies. Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.00064; **, p = 0.0028; ***, p = 0.0028). Scale bars represent 100 μm. D, ectopic expression of WT–, D684A–, S921A–, S921A/D684–, S921D–, and S921D/D684A–hTERT proteins in the presence/absence of ABC294640 in GM847 cells was confirmed by Western blotting using anti-hTERT antibody. Vector-transfected cell extracts were used as negative controls ( right panel ). Actin was used as a loading control ( lower panel ). Images represent at least three independent studies.

    Article Snippet: Fixed and permeabilized cells were incubated with S1P-specific antibody (Sphingomab, LT1002; 20 μg/ml) and hTERT antibody (ab32020, Abcam) or anti-p16 (A301-267A, Bethyl Laboratories) and anti-caspase-3 (2305-PC-020, Trevigen) antibodies for 18 h. PLA was performed and visualized by IF-CM using Duolink in situ hybridization kit as described by the manufacturer (Olink Biosciences).

    Techniques: Binding Assay, Activation Assay, Expressing, Immunofluorescence, Plasmid Preparation, Transfection, Proximity Ligation Assay, Western Blot

    Graphical summary. Our data suggest that SPHK2-generated S1P binds and stabilizes TERT involving its Asp 684 residue via mimicking TERT phosp horylation at Ser 921 , which protects telomere damage and results in delayed senescence and protection from apoptosis. Our data also suggest that targeting/inhibition of the SPHK2/S1P/telomerase axis results in telomere damage, which signals TCF21-dependent caspase-3 activation in cancer cells or immortalized MEFs, or p16-dependent senescence and accelerated aging phenotype in noncancerous primary lung fibroblasts, primary MEFs, or noncancerous tissues, such as testes, of subsequent generations of SphK2 −/− mice. Thus, these data reveal that p16 abundance induces senescence and prevents caspase-3–dependent apoptosis in response to telomere damage via p16–caspase-3 interaction in response to targeting the SPHK2/S1P/telomerase axis.

    Journal: The Journal of Biological Chemistry

    Article Title: Balance between senescence and apoptosis is regulated by telomere damage–induced association between p16 and caspase-3

    doi: 10.1074/jbc.RA118.003506

    Figure Lengend Snippet: Graphical summary. Our data suggest that SPHK2-generated S1P binds and stabilizes TERT involving its Asp 684 residue via mimicking TERT phosp horylation at Ser 921 , which protects telomere damage and results in delayed senescence and protection from apoptosis. Our data also suggest that targeting/inhibition of the SPHK2/S1P/telomerase axis results in telomere damage, which signals TCF21-dependent caspase-3 activation in cancer cells or immortalized MEFs, or p16-dependent senescence and accelerated aging phenotype in noncancerous primary lung fibroblasts, primary MEFs, or noncancerous tissues, such as testes, of subsequent generations of SphK2 −/− mice. Thus, these data reveal that p16 abundance induces senescence and prevents caspase-3–dependent apoptosis in response to telomere damage via p16–caspase-3 interaction in response to targeting the SPHK2/S1P/telomerase axis.

    Article Snippet: Fixed and permeabilized cells were incubated with S1P-specific antibody (Sphingomab, LT1002; 20 μg/ml) and hTERT antibody (ab32020, Abcam) or anti-p16 (A301-267A, Bethyl Laboratories) and anti-caspase-3 (2305-PC-020, Trevigen) antibodies for 18 h. PLA was performed and visualized by IF-CM using Duolink in situ hybridization kit as described by the manufacturer (Olink Biosciences).

    Techniques: Generated, Inhibition, Activation Assay, Mouse Assay

    Genetic loss of SphK2 results in accelerated p16-dependent senescence but not apoptosis via increased telomere damage in noncancerous fibroblasts. A, senescence in testes tissues obtained from WT and SphK2 −/− mice at generations 4–6 was measured by SA-β-gal staining. Scale bars represent 100 μm. B, abundance of p16 mRNA was measured by qPCR in MEFs isolated from WT, SphK1 −/− , and SphK2 −/− mice at generation 6 in the absence/presence of Scr- or p16-shRNAs. Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.0402; **, p = 0.0019). C and D, effects of p16 knockdown on telomere damage ( C ). Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.006; **, p = 0.013) or caspase-3 activation ( D ), and were measured using immunofluorescence in MEFs obtained from WT, SphK1 −/− , and SphK2 −/− mice at generation 6. Scr-shRNA transfected SphK2 −/− MEFs were used as controls. Images were quantified using ImageJ ( right panels ). Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.001).

    Journal: The Journal of Biological Chemistry

    Article Title: Balance between senescence and apoptosis is regulated by telomere damage–induced association between p16 and caspase-3

    doi: 10.1074/jbc.RA118.003506

    Figure Lengend Snippet: Genetic loss of SphK2 results in accelerated p16-dependent senescence but not apoptosis via increased telomere damage in noncancerous fibroblasts. A, senescence in testes tissues obtained from WT and SphK2 −/− mice at generations 4–6 was measured by SA-β-gal staining. Scale bars represent 100 μm. B, abundance of p16 mRNA was measured by qPCR in MEFs isolated from WT, SphK1 −/− , and SphK2 −/− mice at generation 6 in the absence/presence of Scr- or p16-shRNAs. Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.0402; **, p = 0.0019). C and D, effects of p16 knockdown on telomere damage ( C ). Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.006; **, p = 0.013) or caspase-3 activation ( D ), and were measured using immunofluorescence in MEFs obtained from WT, SphK1 −/− , and SphK2 −/− mice at generation 6. Scr-shRNA transfected SphK2 −/− MEFs were used as controls. Images were quantified using ImageJ ( right panels ). Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.001).

    Article Snippet: Fixed and permeabilized cells were incubated with S1P-specific antibody (Sphingomab, LT1002; 20 μg/ml) and hTERT antibody (ab32020, Abcam) or anti-p16 (A301-267A, Bethyl Laboratories) and anti-caspase-3 (2305-PC-020, Trevigen) antibodies for 18 h. PLA was performed and visualized by IF-CM using Duolink in situ hybridization kit as described by the manufacturer (Olink Biosciences).

    Techniques: Mouse Assay, Staining, Real-time Polymerase Chain Reaction, Isolation, Activation Assay, Immunofluorescence, shRNA, Transfection

    Interaction between p16 and caspase-3 prevents apoptosis and induces senescence in response to telomere damage. A, association between endogenous p16 and caspase-3 was measured using PLA in H1341 cells in the absence/presence of Scr- or p16-shRNAs. Quantification of PLA images was performed using the PLA software as described by the manufacturer. Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.0349). Successful knockdown of p16 using shRNA was confirmed by qPCR in H1341 cells compared with Scr-shRNA–transfected cells ( right panel ). Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.004). B and C , interaction and colocalization of p16 and caspase-3 in response to SPHK2 inhibitor ABC294640 ( ABC ) in the absence/presence of ectopic expression of WT-p16 (p16) and/or shRNA-dependent knockdown of SPHK2 ( shSPHK2 ) were measured using PLA. Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.045; **, p = 0.094; ***, p = 0.005) ( B ) or confocal microscopy and immunofluorescence ( C ) in A549 cells. Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.484; **, p = 0.001; ***, p = 0.0009, ****, p = 0.001). Vehicle-treated, vector-only, or Scr-shRNA–transfected cells were used as controls. Data represent at least three independent studies. Quantification of PLA images were performed using the PLA software as described by the manufacturer. Quantification of colocalization was performed using ImageJ. shRNA-mediated knockdown of SPHK2 and ectopic expression of WT-p16 were confirmed using qPCR ( upper panels ) and Western blotting, respectively ( lower panels ). Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.049). D, molecular modeling for the predicted interaction between p16 and caspase-3 was generated using the already existing NMR structure of p16 and crystal structure caspase-3 as described under “Materials and methods.” Ser 152 of p16 ( turquoise ) and Gly 251 of caspase-3 ( gray ) were among key residues for the interaction between these two proteins. E, association between p16 and caspase-3 was measured by PLA in A549 cells expressing WT-p16 or p16 S152A in the absence/presence of ABC294640. Vector-only–transfected and vehicle-treated cells were used as controls. Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.010; **, p = 0.009; ***, p = 0.009). Quantification of PLA images was performed using the PLA software as described by the manufacturer. F and G , effects of ABC294640 exposure on caspase-3 activation were measured by confocal microscopy and immunofluorescence using anti-caspase-3 antibody that detects activated caspase-3 in A549 cells expressing WT-p16 or p16 S152A ( F ). Data are means ± S.D. from three independent experiments and analyzed by Student's t test ( n = 3, *, p = 0.0001; **, p = 0.643; ***, p = 0.0006). Vector-only–transfected and vehicle-treated cells were used as controls. Quantification of colocalization was performed using ImageJ. Expression of WT-p16 and p16 S152A was confirmed by Western blotting ( G ). Vector-transfected cell extracts were used as controls. H , association between p16 and caspase-3 was measured by PLA in testes tissues obtained from WT (SphK1 +/+ /SphK2 +/+ ), SphK1-deficient (SphK1 −/− ), or SphK2-deficient (SphK2 −/− ) mice at generation 6. Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.002; **, p = 0.001). Quantification of PLA images was performed using the PLA software as described by the manufacturer. Scale bars represent 100 μm.

    Journal: The Journal of Biological Chemistry

    Article Title: Balance between senescence and apoptosis is regulated by telomere damage–induced association between p16 and caspase-3

    doi: 10.1074/jbc.RA118.003506

    Figure Lengend Snippet: Interaction between p16 and caspase-3 prevents apoptosis and induces senescence in response to telomere damage. A, association between endogenous p16 and caspase-3 was measured using PLA in H1341 cells in the absence/presence of Scr- or p16-shRNAs. Quantification of PLA images was performed using the PLA software as described by the manufacturer. Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.0349). Successful knockdown of p16 using shRNA was confirmed by qPCR in H1341 cells compared with Scr-shRNA–transfected cells ( right panel ). Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.004). B and C , interaction and colocalization of p16 and caspase-3 in response to SPHK2 inhibitor ABC294640 ( ABC ) in the absence/presence of ectopic expression of WT-p16 (p16) and/or shRNA-dependent knockdown of SPHK2 ( shSPHK2 ) were measured using PLA. Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.045; **, p = 0.094; ***, p = 0.005) ( B ) or confocal microscopy and immunofluorescence ( C ) in A549 cells. Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.484; **, p = 0.001; ***, p = 0.0009, ****, p = 0.001). Vehicle-treated, vector-only, or Scr-shRNA–transfected cells were used as controls. Data represent at least three independent studies. Quantification of PLA images were performed using the PLA software as described by the manufacturer. Quantification of colocalization was performed using ImageJ. shRNA-mediated knockdown of SPHK2 and ectopic expression of WT-p16 were confirmed using qPCR ( upper panels ) and Western blotting, respectively ( lower panels ). Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.049). D, molecular modeling for the predicted interaction between p16 and caspase-3 was generated using the already existing NMR structure of p16 and crystal structure caspase-3 as described under “Materials and methods.” Ser 152 of p16 ( turquoise ) and Gly 251 of caspase-3 ( gray ) were among key residues for the interaction between these two proteins. E, association between p16 and caspase-3 was measured by PLA in A549 cells expressing WT-p16 or p16 S152A in the absence/presence of ABC294640. Vector-only–transfected and vehicle-treated cells were used as controls. Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.010; **, p = 0.009; ***, p = 0.009). Quantification of PLA images was performed using the PLA software as described by the manufacturer. F and G , effects of ABC294640 exposure on caspase-3 activation were measured by confocal microscopy and immunofluorescence using anti-caspase-3 antibody that detects activated caspase-3 in A549 cells expressing WT-p16 or p16 S152A ( F ). Data are means ± S.D. from three independent experiments and analyzed by Student's t test ( n = 3, *, p = 0.0001; **, p = 0.643; ***, p = 0.0006). Vector-only–transfected and vehicle-treated cells were used as controls. Quantification of colocalization was performed using ImageJ. Expression of WT-p16 and p16 S152A was confirmed by Western blotting ( G ). Vector-transfected cell extracts were used as controls. H , association between p16 and caspase-3 was measured by PLA in testes tissues obtained from WT (SphK1 +/+ /SphK2 +/+ ), SphK1-deficient (SphK1 −/− ), or SphK2-deficient (SphK2 −/− ) mice at generation 6. Data are means ± S.D. from three independent experiments, analyzed by Student's t test ( n = 3, *, p = 0.002; **, p = 0.001). Quantification of PLA images was performed using the PLA software as described by the manufacturer. Scale bars represent 100 μm.

    Article Snippet: Fixed and permeabilized cells were incubated with S1P-specific antibody (Sphingomab, LT1002; 20 μg/ml) and hTERT antibody (ab32020, Abcam) or anti-p16 (A301-267A, Bethyl Laboratories) and anti-caspase-3 (2305-PC-020, Trevigen) antibodies for 18 h. PLA was performed and visualized by IF-CM using Duolink in situ hybridization kit as described by the manufacturer (Olink Biosciences).

    Techniques: Proximity Ligation Assay, Software, shRNA, Real-time Polymerase Chain Reaction, Transfection, Expressing, Confocal Microscopy, Immunofluorescence, Plasmid Preparation, Western Blot, Generated, Nuclear Magnetic Resonance, Activation Assay, Mouse Assay

    Cleaved caspase-3 is found in infected areas of the lungs suggesting host cells are undergoing apoptosis. Left lower lobe sections of lungs harvested 1 hour ( A and E ), 24 hours ( B and F ), or 48 hours ( C , D , G , and H ) after infection were perfused in 10%

    Journal:

    Article Title: Pneumonic Plague Pathogenesis and Immunity in Brown Norway Rats

    doi: 10.2353/ajpath.2009.071168

    Figure Lengend Snippet: Cleaved caspase-3 is found in infected areas of the lungs suggesting host cells are undergoing apoptosis. Left lower lobe sections of lungs harvested 1 hour ( A and E ), 24 hours ( B and F ), or 48 hours ( C , D , G , and H ) after infection were perfused in 10%

    Article Snippet: Slides were stained with mouse anti-rat CD68 (MorphoSys, Oxford, UK) or rabbit anti-cleaved caspase-3 (Trevigen, Gaithersburg, MD) according to the manufacturer’s protocol.

    Techniques: Infection

    Anterior neural tube defect and increased apoptosis in homozygous Trx-2 −/− mice at 9.5 and 10.5 dpc (representative embryos and images). Aberrant anterior neural tube closure in Trx-2 −/− embryo, indicated by arrows, was compared to the wild-type embryo by hemotoxylin and eosin staining of paraffin-embedded embryos at 9.5 dpc (A and B) and at 10.5 dpc (E and F). In panels C and D are shown increased anti-cleaved caspase-3 antibody-positive cells from Trx-2 −/− embryos compared to wild-type embryos by immunohistochemical staining of paraffin-embedded 9.5-dpc embryos (adjacent slices of the embryos shown in panels A and B). In panels G and H, massive apoptosis in 10.5-dpc Trx-2 −/− embryos can be seen compared to the wild-type embryos as visualized by anti-caspase-3 antibody staining of paraffin-embedded embryos (adjacent slices of the embryos shown in E and F). Heavy staining in the uterine tissue is not specific to cleaved caspase-3 and is due to endogenous mouse immunoglobulin G interactions with the secondary antibody.

    Journal: Molecular and Cellular Biology

    Article Title: The Absence of Mitochondrial Thioredoxin 2 Causes Massive Apoptosis, Exencephaly, and Early Embryonic Lethality in Homozygous Mice

    doi: 10.1128/MCB.23.3.916-922.2003

    Figure Lengend Snippet: Anterior neural tube defect and increased apoptosis in homozygous Trx-2 −/− mice at 9.5 and 10.5 dpc (representative embryos and images). Aberrant anterior neural tube closure in Trx-2 −/− embryo, indicated by arrows, was compared to the wild-type embryo by hemotoxylin and eosin staining of paraffin-embedded embryos at 9.5 dpc (A and B) and at 10.5 dpc (E and F). In panels C and D are shown increased anti-cleaved caspase-3 antibody-positive cells from Trx-2 −/− embryos compared to wild-type embryos by immunohistochemical staining of paraffin-embedded 9.5-dpc embryos (adjacent slices of the embryos shown in panels A and B). In panels G and H, massive apoptosis in 10.5-dpc Trx-2 −/− embryos can be seen compared to the wild-type embryos as visualized by anti-caspase-3 antibody staining of paraffin-embedded embryos (adjacent slices of the embryos shown in E and F). Heavy staining in the uterine tissue is not specific to cleaved caspase-3 and is due to endogenous mouse immunoglobulin G interactions with the secondary antibody.

    Article Snippet: Sections were incubated with mouse anti-caspase-3 (Trevigen, Gaithersburg Md.) for 32 min at 42°C on a Ventana ES (Ventana Medical Systems, Tucson, Ariz.) automated slide stainer and then visualized by using the basic DAB detection kit (Ventana Medical Systems).

    Techniques: Mouse Assay, Staining, Immunohistochemistry