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  • 94
    Name:
    Etoposide
    Description:
    Molecular Weight
    Catalog Number:
    2200
    Price:
    None
    Category:
    Activators Inhibitors
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    Structured Review

    Cell Signaling Technology Inc etoposide
    p53 induction in response to DNA damage is impaired in TFEB/TFE3 DKO RAW264.7. 7 cells. ( A ) Representative Western blot showing p53 induction, p53 Ser15 phosphorylation, and Mdm2 levels in WT and TFE3/TFEB DKO RAW264.7 cells following <t>etoposide</t> treatment up to 8 hr. ( B ) Quantification of p53 induction from data shown in A. Data represents mean relative p53 level ± standard deviation with n = 3. Significance tested with two-way ANOVA with Sidak’s multiple comparisons test (**p
    Molecular Weight
    https://www.bioz.com/result/etoposide/product/Cell Signaling Technology Inc
    Average 94 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    etoposide - by Bioz Stars, 2020-09
    94/100 stars

    Images

    1) Product Images from "The transcription factors TFE3 and TFEB amplify p53 dependent transcriptional programs in response to DNA damage"

    Article Title: The transcription factors TFE3 and TFEB amplify p53 dependent transcriptional programs in response to DNA damage

    Journal: eLife

    doi: 10.7554/eLife.40856

    p53 induction in response to DNA damage is impaired in TFEB/TFE3 DKO RAW264.7. 7 cells. ( A ) Representative Western blot showing p53 induction, p53 Ser15 phosphorylation, and Mdm2 levels in WT and TFE3/TFEB DKO RAW264.7 cells following etoposide treatment up to 8 hr. ( B ) Quantification of p53 induction from data shown in A. Data represents mean relative p53 level ± standard deviation with n = 3. Significance tested with two-way ANOVA with Sidak’s multiple comparisons test (**p
    Figure Legend Snippet: p53 induction in response to DNA damage is impaired in TFEB/TFE3 DKO RAW264.7. 7 cells. ( A ) Representative Western blot showing p53 induction, p53 Ser15 phosphorylation, and Mdm2 levels in WT and TFE3/TFEB DKO RAW264.7 cells following etoposide treatment up to 8 hr. ( B ) Quantification of p53 induction from data shown in A. Data represents mean relative p53 level ± standard deviation with n = 3. Significance tested with two-way ANOVA with Sidak’s multiple comparisons test (**p

    Techniques Used: Western Blot, Standard Deviation

    ( A ) Representative Western blot showing TFEB and TFE3 gel shifts in response to etoposide in WT MEF, but not in p53 -/- MEF. All the immunoblots are representative of three independent experiments. ( B ) qPCR data showing relative induction of lysosomal-autophagy genes in response to starvation in WT and p53 -/- MEF. Data normalized to untreated cells and represents geometric means ± standard deviation and significance determined with Student’s t-test (*p
    Figure Legend Snippet: ( A ) Representative Western blot showing TFEB and TFE3 gel shifts in response to etoposide in WT MEF, but not in p53 -/- MEF. All the immunoblots are representative of three independent experiments. ( B ) qPCR data showing relative induction of lysosomal-autophagy genes in response to starvation in WT and p53 -/- MEF. Data normalized to untreated cells and represents geometric means ± standard deviation and significance determined with Student’s t-test (*p

    Techniques Used: Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation

    TFE3 and TFEB are necessary for proper execution of apoptosis in response to DNA damage in RAW264.7 cells. ( A ) Representative Western blot showing Caspase-3 cleavage in response to increasing time of etoposide treatment. ( B ) Quantification of data shown in A indicating defects in Caspase-3 cleavage in TFEB/TFE3 DKO RAW264.7 cells. Cleaved Caspase-3 levels are normalized to WT cells after 16 hr etoposide treatment with n = 3. Significance tested with Student’s t-test (#p
    Figure Legend Snippet: TFE3 and TFEB are necessary for proper execution of apoptosis in response to DNA damage in RAW264.7 cells. ( A ) Representative Western blot showing Caspase-3 cleavage in response to increasing time of etoposide treatment. ( B ) Quantification of data shown in A indicating defects in Caspase-3 cleavage in TFEB/TFE3 DKO RAW264.7 cells. Cleaved Caspase-3 levels are normalized to WT cells after 16 hr etoposide treatment with n = 3. Significance tested with Student’s t-test (#p

    Techniques Used: Western Blot

    ( A ) Immunofluorescence images of ARPE-19 cells treated with 100 μM etoposide or 50 μM Cisplatin for 24 hr or 10 hr after UVC irradiation. Scale bar = 10 μm. ( B ) Immunofluorescence images of HeLa cells treated with 100 μM etoposide for 24 hr or 50 μM Cisplatin for 12 hr and 4 hr after UVC irradiation. Scale bar = 10 μm. ( C ) Immunofluorescence images of RAW 264.7 cells treated with 100 μM etoposide for 10 hr or 35 μM Cisplatin for 10 hr or 4 hr after UVC irradiation. Scale bar = 10 μm. ( D ) Representative Western blot showing TFE3 de-phosphorylation at Ser321 and gel shift in TFEB in WT MEFs exposed to 100 μM etoposide for 8 hr or 50 μM Cisplatin for 10 hr or 10 hr after UVC irradiation. ( E ) Representative Western blot showing TFE3 de-phosphorylation at Ser321 and gel shift in TFEB in ARPE19 cells exposed to 100 μM etoposide for 24 hr or 50 μM Cisplatin for 24 hr or 24 hr after UVC irradiation. ( F ) Representative Western blot showing TFE3 de-phosphorylation at Ser321 and gel shift in TFEB in HeLa cells exposed to 100 μM etoposide for 24 hr or 50 μM Cisplatin for 18 hr. ( G ) Representative Western blot showing TFE3 de-phosphorylation at Ser321 and gel shift in TFEB in RAW 264.7 cells exposed to 100 μM etoposide for 8 hr or 50 μM Cisplatin for 8 hr or 4 hr after UVC irradiation. ( H ) Representative Western blot showing TFE3 nuclear distribution by subcellular fractionation of WT MEFs exposed to 50 μM Cisplatin for 10 hr. ( I ) Western blot showing etoposide dependent S6K de-phosphorylation in ARPE19 cells. All the immunoblots are representative of three independent experiments.
    Figure Legend Snippet: ( A ) Immunofluorescence images of ARPE-19 cells treated with 100 μM etoposide or 50 μM Cisplatin for 24 hr or 10 hr after UVC irradiation. Scale bar = 10 μm. ( B ) Immunofluorescence images of HeLa cells treated with 100 μM etoposide for 24 hr or 50 μM Cisplatin for 12 hr and 4 hr after UVC irradiation. Scale bar = 10 μm. ( C ) Immunofluorescence images of RAW 264.7 cells treated with 100 μM etoposide for 10 hr or 35 μM Cisplatin for 10 hr or 4 hr after UVC irradiation. Scale bar = 10 μm. ( D ) Representative Western blot showing TFE3 de-phosphorylation at Ser321 and gel shift in TFEB in WT MEFs exposed to 100 μM etoposide for 8 hr or 50 μM Cisplatin for 10 hr or 10 hr after UVC irradiation. ( E ) Representative Western blot showing TFE3 de-phosphorylation at Ser321 and gel shift in TFEB in ARPE19 cells exposed to 100 μM etoposide for 24 hr or 50 μM Cisplatin for 24 hr or 24 hr after UVC irradiation. ( F ) Representative Western blot showing TFE3 de-phosphorylation at Ser321 and gel shift in TFEB in HeLa cells exposed to 100 μM etoposide for 24 hr or 50 μM Cisplatin for 18 hr. ( G ) Representative Western blot showing TFE3 de-phosphorylation at Ser321 and gel shift in TFEB in RAW 264.7 cells exposed to 100 μM etoposide for 8 hr or 50 μM Cisplatin for 8 hr or 4 hr after UVC irradiation. ( H ) Representative Western blot showing TFE3 nuclear distribution by subcellular fractionation of WT MEFs exposed to 50 μM Cisplatin for 10 hr. ( I ) Western blot showing etoposide dependent S6K de-phosphorylation in ARPE19 cells. All the immunoblots are representative of three independent experiments.

    Techniques Used: Immunofluorescence, Irradiation, Western Blot, De-Phosphorylation Assay, Electrophoretic Mobility Shift Assay, Fractionation

    ( A ) Enriched GO terms in the ‘Biological Process’ category of differentially expressed genes between etoposide-treated WT and TFEB/TFE3 DKO RAW264.7 cells. GO terms are ranked by q value (
    Figure Legend Snippet: ( A ) Enriched GO terms in the ‘Biological Process’ category of differentially expressed genes between etoposide-treated WT and TFEB/TFE3 DKO RAW264.7 cells. GO terms are ranked by q value (

    Techniques Used:

    TFEB and TFE3 are essential for etoposide-induced lysosomal membrane permeabilization in MEFs. ( A ) Immunofluorescence images showing LMP in MEFs. Red galectin-1 puncta appear co-localized or within the lumen of green Lamp1 positive lysosomes. No LMP is detected under basal conditions in either WT or TFEB/TFE3 DKO MEFs. Treatment with etoposide induces profound LMP in WT, but not TFEB/TFE3 DKO cells. No differences in LMP induction were detected in LLOMe treated cells, regardless of genotype. Scale bar = 20 μm, inset = 2 μm. ( B ) WT MEFs exhibit a time-dependent increase in LMP after etoposide treatment. Quantification of data shown in A of galectin-1+/Lamp1 + LMP puncta per WT MEF cell. Data represent mean number of puncta per cell ± standard deviation from randomly selected confocal images, with > 20 cells per counted for each time point over three separate experiments. ( C ) Quantification of total number of galectin-1+/Lamp1+ LMP puncta per cell in WT vs TFE3/TFEB DKO MEFs treated for 8 hr with etoposide. Distribution is representative of one of the three independent experiments performed and shows 29 randomly selected WT MEF cells and 51 randomly selected TFEB/TFE3 DKO MEF cells. Significance determined using Student’s t-test (****p
    Figure Legend Snippet: TFEB and TFE3 are essential for etoposide-induced lysosomal membrane permeabilization in MEFs. ( A ) Immunofluorescence images showing LMP in MEFs. Red galectin-1 puncta appear co-localized or within the lumen of green Lamp1 positive lysosomes. No LMP is detected under basal conditions in either WT or TFEB/TFE3 DKO MEFs. Treatment with etoposide induces profound LMP in WT, but not TFEB/TFE3 DKO cells. No differences in LMP induction were detected in LLOMe treated cells, regardless of genotype. Scale bar = 20 μm, inset = 2 μm. ( B ) WT MEFs exhibit a time-dependent increase in LMP after etoposide treatment. Quantification of data shown in A of galectin-1+/Lamp1 + LMP puncta per WT MEF cell. Data represent mean number of puncta per cell ± standard deviation from randomly selected confocal images, with > 20 cells per counted for each time point over three separate experiments. ( C ) Quantification of total number of galectin-1+/Lamp1+ LMP puncta per cell in WT vs TFE3/TFEB DKO MEFs treated for 8 hr with etoposide. Distribution is representative of one of the three independent experiments performed and shows 29 randomly selected WT MEF cells and 51 randomly selected TFEB/TFE3 DKO MEF cells. Significance determined using Student’s t-test (****p

    Techniques Used: Immunofluorescence, Standard Deviation

    2) Product Images from "The effect of 3-bromopyruvate on human colorectal cancer cells is dependent on glucose concentration but not hexokinase II expression"

    Article Title: The effect of 3-bromopyruvate on human colorectal cancer cells is dependent on glucose concentration but not hexokinase II expression

    Journal: Bioscience Reports

    doi: 10.1042/BSR20150267

    Type of cell death induced by 3BP is dose-dependent Caspase-3 cleavage and DNA fragmentation was assessed to further elucidate the mechanisms of 3BP-induced cell death. Caspase-3 cleavage was examined through western blot analysis ( N =3) ( A ). An image of a longer exposed membrane to better detect cleaved caspase-3 is shown below each full caspase-3 blot. DNA fragmentation was examined through gel electrophoresis in SW480 cells following 48 h 3BP treatment using 0.8% ( B ) and 2% ( C ) gels ( N =2). Etoposide (E; 50 μM) was used as a positive control for caspase-3 cleavage and DNA fragmentation; note nucleosome laddering in etoposide treated cells in ( C ) indicative of apoptosis.
    Figure Legend Snippet: Type of cell death induced by 3BP is dose-dependent Caspase-3 cleavage and DNA fragmentation was assessed to further elucidate the mechanisms of 3BP-induced cell death. Caspase-3 cleavage was examined through western blot analysis ( N =3) ( A ). An image of a longer exposed membrane to better detect cleaved caspase-3 is shown below each full caspase-3 blot. DNA fragmentation was examined through gel electrophoresis in SW480 cells following 48 h 3BP treatment using 0.8% ( B ) and 2% ( C ) gels ( N =2). Etoposide (E; 50 μM) was used as a positive control for caspase-3 cleavage and DNA fragmentation; note nucleosome laddering in etoposide treated cells in ( C ) indicative of apoptosis.

    Techniques Used: Western Blot, Nucleic Acid Electrophoresis, Positive Control

    3) Product Images from "DNA damage response defect in Williams-Beuren syndrome"

    Article Title: DNA damage response defect in Williams-Beuren syndrome

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2017.2861

    (A) Kinetics of γ-H2A.X induction in primary fibroblasts from Williams-Beuren syndrome (WBS) patients and healthy donors upon exposure to etoposide or hydroxyurea. (B) Dose effect of γ-H2A.X induction after 6 h of treatment with etoposide or hydroxyurea.
    Figure Legend Snippet: (A) Kinetics of γ-H2A.X induction in primary fibroblasts from Williams-Beuren syndrome (WBS) patients and healthy donors upon exposure to etoposide or hydroxyurea. (B) Dose effect of γ-H2A.X induction after 6 h of treatment with etoposide or hydroxyurea.

    Techniques Used:

    (A) Kinetics of γ-H2A.X induction in the 293T cells stably transfected with siRNAs targeting three Williams-Beuren syndrome genes upon exposure to etoposide or hydroxyurea. (B) Dose effect of γ-H2A.X induction after 6 h of treatment with etoposide or hydroxyurea in the same cells. UT, untransfected cells.
    Figure Legend Snippet: (A) Kinetics of γ-H2A.X induction in the 293T cells stably transfected with siRNAs targeting three Williams-Beuren syndrome genes upon exposure to etoposide or hydroxyurea. (B) Dose effect of γ-H2A.X induction after 6 h of treatment with etoposide or hydroxyurea in the same cells. UT, untransfected cells.

    Techniques Used: Stable Transfection, Transfection

    4) Product Images from "The transcription factors TFE3 and TFEB amplify p53 dependent transcriptional programs in response to DNA damage"

    Article Title: The transcription factors TFE3 and TFEB amplify p53 dependent transcriptional programs in response to DNA damage

    Journal: eLife

    doi: 10.7554/eLife.40856

    Comparative gene expression of etoposide-treated WT and TFEB/TFE3-DKO RAW264.7 cells. ( A ) Principal component analysis of genes with q-value
    Figure Legend Snippet: Comparative gene expression of etoposide-treated WT and TFEB/TFE3-DKO RAW264.7 cells. ( A ) Principal component analysis of genes with q-value

    Techniques Used: Expressing

    DNA damage-induced TFE3 and TFEB activation is a p53 and mTORC1 dependent process. ( A ) Representative Western blot showing p53-dependent inhibition of mTORC1 in response to etoposide treatment in WT and p53 -/- MEFs. EBSS for 2 hr was used as a positive control for mTORC1 inhibition and was relatively unaffected by p53 status. ( B ) Quantification of Western blot data shown in A. Values represent mean ± standard deviation with n = 5. Significance determined with Two-way ANOVA with Sidak’s multiple comparisons test (*p
    Figure Legend Snippet: DNA damage-induced TFE3 and TFEB activation is a p53 and mTORC1 dependent process. ( A ) Representative Western blot showing p53-dependent inhibition of mTORC1 in response to etoposide treatment in WT and p53 -/- MEFs. EBSS for 2 hr was used as a positive control for mTORC1 inhibition and was relatively unaffected by p53 status. ( B ) Quantification of Western blot data shown in A. Values represent mean ± standard deviation with n = 5. Significance determined with Two-way ANOVA with Sidak’s multiple comparisons test (*p

    Techniques Used: Activation Assay, Western Blot, Inhibition, Positive Control, Standard Deviation

    TFE3 and TFEB are necessary for proper execution of apoptosis in response to DNA damage in RAW264.7 cells. ( A ) Representative Western blot showing Caspase-3 cleavage in response to increasing time of etoposide treatment. ( B ) Quantification of data shown in A indicating defects in Caspase-3 cleavage in TFEB/TFE3 DKO RAW264.7 cells. Cleaved Caspase-3 levels are normalized to WT cells after 16 hr etoposide treatment with n = 3. Significance tested with Student’s t-test (#p
    Figure Legend Snippet: TFE3 and TFEB are necessary for proper execution of apoptosis in response to DNA damage in RAW264.7 cells. ( A ) Representative Western blot showing Caspase-3 cleavage in response to increasing time of etoposide treatment. ( B ) Quantification of data shown in A indicating defects in Caspase-3 cleavage in TFEB/TFE3 DKO RAW264.7 cells. Cleaved Caspase-3 levels are normalized to WT cells after 16 hr etoposide treatment with n = 3. Significance tested with Student’s t-test (#p

    Techniques Used: Western Blot

    Differentially regulated genes in WT versus TFEB/TFE3 DKO MEFs undergoing DNA damage. qRT-PCR-based quantification of basal and etoposide induced mRNA levels of Rad9a, Chek2, Trp53inp1, Mdm2, Bbc3, Bax, Sesn1, Sesn2, Dram1, Tp53, Cdkn1a, Laptm5, Ctsd, Wrap53, Egfr and Foxo3 in WT vs TFEB/TFE3 DKO MEFs. All qRT-PCR data represented as geometric mean ± standard deviation and significance tested using Student’s t-test (*p
    Figure Legend Snippet: Differentially regulated genes in WT versus TFEB/TFE3 DKO MEFs undergoing DNA damage. qRT-PCR-based quantification of basal and etoposide induced mRNA levels of Rad9a, Chek2, Trp53inp1, Mdm2, Bbc3, Bax, Sesn1, Sesn2, Dram1, Tp53, Cdkn1a, Laptm5, Ctsd, Wrap53, Egfr and Foxo3 in WT vs TFEB/TFE3 DKO MEFs. All qRT-PCR data represented as geometric mean ± standard deviation and significance tested using Student’s t-test (*p

    Techniques Used: Quantitative RT-PCR, Standard Deviation

    Expression of constitutively active TFEB and TFE3 in HeLa cells increases total p53 protein levels and its extends half-life. ( A ) Representative Western blot showing elevated p53 protein levels in adenovirus infected HeLa cells expressing constitutively active mutants of TFEB and TFE3. Further p53 protein level increases are seen with constitutively active TFEB and TFE3 after treatment with etoposide 8 hr. ( B ) Quantification of basal p53 protein level in HeLa cells expressing constitutively active TFEB and TFE3. Due to the high dynamic range and low detectability of basal endogenous p53 in control cells, values were normalized to intermediate expression samples, TFE3 S321A. Data represents mean relative p53 level ± standard deviation with n = 5 (*p
    Figure Legend Snippet: Expression of constitutively active TFEB and TFE3 in HeLa cells increases total p53 protein levels and its extends half-life. ( A ) Representative Western blot showing elevated p53 protein levels in adenovirus infected HeLa cells expressing constitutively active mutants of TFEB and TFE3. Further p53 protein level increases are seen with constitutively active TFEB and TFE3 after treatment with etoposide 8 hr. ( B ) Quantification of basal p53 protein level in HeLa cells expressing constitutively active TFEB and TFE3. Due to the high dynamic range and low detectability of basal endogenous p53 in control cells, values were normalized to intermediate expression samples, TFE3 S321A. Data represents mean relative p53 level ± standard deviation with n = 5 (*p

    Techniques Used: Expressing, Western Blot, Infection, Standard Deviation

    p53 induction in response to DNA damage is impaired in TFEB/TFE3 DKO RAW264.7. 7 cells. ( A ) Representative Western blot showing p53 induction, p53 Ser15 phosphorylation, and Mdm2 levels in WT and TFE3/TFEB DKO RAW264.7 cells following etoposide treatment up to 8 hr. ( B ) Quantification of p53 induction from data shown in A. Data represents mean relative p53 level ± standard deviation with n = 3. Significance tested with two-way ANOVA with Sidak’s multiple comparisons test (**p
    Figure Legend Snippet: p53 induction in response to DNA damage is impaired in TFEB/TFE3 DKO RAW264.7. 7 cells. ( A ) Representative Western blot showing p53 induction, p53 Ser15 phosphorylation, and Mdm2 levels in WT and TFE3/TFEB DKO RAW264.7 cells following etoposide treatment up to 8 hr. ( B ) Quantification of p53 induction from data shown in A. Data represents mean relative p53 level ± standard deviation with n = 3. Significance tested with two-way ANOVA with Sidak’s multiple comparisons test (**p

    Techniques Used: Western Blot, Standard Deviation

    TFE3 and TFEB translocate to the nucleus in response to genotoxic stress. ( A ) Immunofluorescence images of WT MEFs treated with 100 μM etoposide for up to 16 hr. Scale bar = 20 μm. ( B ) Quantification of TFE3 localization from cells shown in A. Levels represent mean ± standard deviation with n = 3 experiments and > 200 cells counted per trial. Significance determined with Student’s t-test (*p
    Figure Legend Snippet: TFE3 and TFEB translocate to the nucleus in response to genotoxic stress. ( A ) Immunofluorescence images of WT MEFs treated with 100 μM etoposide for up to 16 hr. Scale bar = 20 μm. ( B ) Quantification of TFE3 localization from cells shown in A. Levels represent mean ± standard deviation with n = 3 experiments and > 200 cells counted per trial. Significance determined with Student’s t-test (*p

    Techniques Used: Immunofluorescence, Standard Deviation

    TFEB and TFE3 are essential for etoposide-induced lysosomal membrane permeabilization in MEFs. ( A ) Immunofluorescence images showing LMP in MEFs. Red galectin-1 puncta appear co-localized or within the lumen of green Lamp1 positive lysosomes. No LMP is detected under basal conditions in either WT or TFEB/TFE3 DKO MEFs. Treatment with etoposide induces profound LMP in WT, but not TFEB/TFE3 DKO cells. No differences in LMP induction were detected in LLOMe treated cells, regardless of genotype. Scale bar = 20 μm, inset = 2 μm. ( B ) WT MEFs exhibit a time-dependent increase in LMP after etoposide treatment. Quantification of data shown in A of galectin-1+/Lamp1 + LMP puncta per WT MEF cell. Data represent mean number of puncta per cell ± standard deviation from randomly selected confocal images, with > 20 cells per counted for each time point over three separate experiments. ( C ) Quantification of total number of galectin-1+/Lamp1+ LMP puncta per cell in WT vs TFE3/TFEB DKO MEFs treated for 8 hr with etoposide. Distribution is representative of one of the three independent experiments performed and shows 29 randomly selected WT MEF cells and 51 randomly selected TFEB/TFE3 DKO MEF cells. Significance determined using Student’s t-test (****p
    Figure Legend Snippet: TFEB and TFE3 are essential for etoposide-induced lysosomal membrane permeabilization in MEFs. ( A ) Immunofluorescence images showing LMP in MEFs. Red galectin-1 puncta appear co-localized or within the lumen of green Lamp1 positive lysosomes. No LMP is detected under basal conditions in either WT or TFEB/TFE3 DKO MEFs. Treatment with etoposide induces profound LMP in WT, but not TFEB/TFE3 DKO cells. No differences in LMP induction were detected in LLOMe treated cells, regardless of genotype. Scale bar = 20 μm, inset = 2 μm. ( B ) WT MEFs exhibit a time-dependent increase in LMP after etoposide treatment. Quantification of data shown in A of galectin-1+/Lamp1 + LMP puncta per WT MEF cell. Data represent mean number of puncta per cell ± standard deviation from randomly selected confocal images, with > 20 cells per counted for each time point over three separate experiments. ( C ) Quantification of total number of galectin-1+/Lamp1+ LMP puncta per cell in WT vs TFE3/TFEB DKO MEFs treated for 8 hr with etoposide. Distribution is representative of one of the three independent experiments performed and shows 29 randomly selected WT MEF cells and 51 randomly selected TFEB/TFE3 DKO MEF cells. Significance determined using Student’s t-test (****p

    Techniques Used: Immunofluorescence, Standard Deviation

    5) Product Images from "Survival of midbrain dopamine neurons depends on the Bcl2 factor Mcl1"

    Article Title: Survival of midbrain dopamine neurons depends on the Bcl2 factor Mcl1

    Journal: Cell Death Discovery

    doi: 10.1038/s41420-018-0125-7

    Overexpression of Mcl1 protects against apoptotic stress. a Immunofluorescent staining of cells transfected with empty vector or plasmid encoding for Mcl1. Cell were co-transfected with GFP as a marker for transfected cells and subsequently treated with etoposide for 8 h before treatment with PI. Scale bar represents 50 µm . b Left: blow up of representative example of GFP positive cell and dual GFP and PI positive cell. Scale bar represents 5 µm. Right: quantification of results seen in a . The amount of cleaved caspase 3 positive cells was determined relative to total GFP positive cells. Significance was determined with a Student’s t -test, p value: ** p
    Figure Legend Snippet: Overexpression of Mcl1 protects against apoptotic stress. a Immunofluorescent staining of cells transfected with empty vector or plasmid encoding for Mcl1. Cell were co-transfected with GFP as a marker for transfected cells and subsequently treated with etoposide for 8 h before treatment with PI. Scale bar represents 50 µm . b Left: blow up of representative example of GFP positive cell and dual GFP and PI positive cell. Scale bar represents 5 µm. Right: quantification of results seen in a . The amount of cleaved caspase 3 positive cells was determined relative to total GFP positive cells. Significance was determined with a Student’s t -test, p value: ** p

    Techniques Used: Over Expression, Staining, Transfection, Plasmid Preparation, Marker

    6) Product Images from "Survival of midbrain dopamine neurons depends on the Bcl2 factor Mcl1"

    Article Title: Survival of midbrain dopamine neurons depends on the Bcl2 factor Mcl1

    Journal: Cell Death Discovery

    doi: 10.1038/s41420-018-0125-7

    Overexpression of Mcl1 protects against apoptotic stress. a Immunofluorescent staining of cells transfected with empty vector or plasmid encoding for Mcl1. Cell were co-transfected with GFP as a marker for transfected cells and subsequently treated with etoposide for 8 h before treatment with PI. Scale bar represents 50 µm . b Left: blow up of representative example of GFP positive cell and dual GFP and PI positive cell. Scale bar represents 5 µm. Right: quantification of results seen in a . The amount of cleaved caspase 3 positive cells was determined relative to total GFP positive cells. Significance was determined with a Student’s t -test, p value: ** p
    Figure Legend Snippet: Overexpression of Mcl1 protects against apoptotic stress. a Immunofluorescent staining of cells transfected with empty vector or plasmid encoding for Mcl1. Cell were co-transfected with GFP as a marker for transfected cells and subsequently treated with etoposide for 8 h before treatment with PI. Scale bar represents 50 µm . b Left: blow up of representative example of GFP positive cell and dual GFP and PI positive cell. Scale bar represents 5 µm. Right: quantification of results seen in a . The amount of cleaved caspase 3 positive cells was determined relative to total GFP positive cells. Significance was determined with a Student’s t -test, p value: ** p

    Techniques Used: Over Expression, Staining, Transfection, Plasmid Preparation, Marker

    7) Product Images from "Chronic Intermittent Hypoxia Triggers a Senescence-like Phenotype in Human White Preadipocytes"

    Article Title: Chronic Intermittent Hypoxia Triggers a Senescence-like Phenotype in Human White Preadipocytes

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-63761-7

    Intermittent hypoxia (IH) alters preadipocyte phenotype and replicative capacity. Chronic IH was associated with a characteristic set of phenotypic changes such as flattened and enlarged cells ( a ) and a reduced multiplication ratio (MR) ( b ). Etoposide (ETO) was used as a positive control. Data are presented as mean ± SEM. P -values determined by one-tailed paired t-test compared to the normoxia (NO) control (n = 6 independent experiments).
    Figure Legend Snippet: Intermittent hypoxia (IH) alters preadipocyte phenotype and replicative capacity. Chronic IH was associated with a characteristic set of phenotypic changes such as flattened and enlarged cells ( a ) and a reduced multiplication ratio (MR) ( b ). Etoposide (ETO) was used as a positive control. Data are presented as mean ± SEM. P -values determined by one-tailed paired t-test compared to the normoxia (NO) control (n = 6 independent experiments).

    Techniques Used: Positive Control, One-tailed Test

    Intermittent hypoxia (IH) induces senescence in cultured preadipocytes. Exposure to 7 days of IH was associated with a higher prevalence of senescence associated β-galactosidase (SA-β-gal) positive preadipocytes (n = 6 independent experiments) ( a ). Compared to tissue grown in continuous normoxia (NO), chronic IH exposure was also associated with stronger green coloration of SA-β-gal staining in subcutaneous adipose tissue explants ( ex vivo ) ( b ). Representative images of SA-β-gal staining in cells exposed to 7 days of IH ( c ). Green marks indicate SA-β-gal positive (senescent) cells; Etoposide (ETO) treatment served as a positive control. Data are presented as mean ± SEM. P -values determined by one-tailed paired t-test compared to the NO control.
    Figure Legend Snippet: Intermittent hypoxia (IH) induces senescence in cultured preadipocytes. Exposure to 7 days of IH was associated with a higher prevalence of senescence associated β-galactosidase (SA-β-gal) positive preadipocytes (n = 6 independent experiments) ( a ). Compared to tissue grown in continuous normoxia (NO), chronic IH exposure was also associated with stronger green coloration of SA-β-gal staining in subcutaneous adipose tissue explants ( ex vivo ) ( b ). Representative images of SA-β-gal staining in cells exposed to 7 days of IH ( c ). Green marks indicate SA-β-gal positive (senescent) cells; Etoposide (ETO) treatment served as a positive control. Data are presented as mean ± SEM. P -values determined by one-tailed paired t-test compared to the NO control.

    Techniques Used: Cell Culture, Staining, Ex Vivo, Positive Control, One-tailed Test

    8) Product Images from "Transactivation of human osteoprotegerin promoter by GATA-3"

    Article Title: Transactivation of human osteoprotegerin promoter by GATA-3

    Journal: Scientific Reports

    doi: 10.1038/srep12479

    Regulation of apoptosis by GATA-3 and OPG. ( A ) HEK cells expressing either a scrambled shRNA or a GATA-3 shRNA expressing plasmid were treated with etoposide (10 μM), and apoptosis was assayed by Western blot (left). HEK cells expressing a GATA-3 shRNA expressing plasmid were treated with purified OPG protein for 2 h before treatment with etoposide, and apoptosis was assayed by Western blot (right). Full-length blots are presented in supplementary Figure 1 . ( B ) HEK cells expressing either a control plasmid or a GATA-3 shRNA expressing plasmid were treated with TNF-α (2 μg/mL) with or without pre-treatment with OPG for 2 h. Subsequent cell death was measured using TUNEL assays (left), and quantified with bar graphs (right). *p
    Figure Legend Snippet: Regulation of apoptosis by GATA-3 and OPG. ( A ) HEK cells expressing either a scrambled shRNA or a GATA-3 shRNA expressing plasmid were treated with etoposide (10 μM), and apoptosis was assayed by Western blot (left). HEK cells expressing a GATA-3 shRNA expressing plasmid were treated with purified OPG protein for 2 h before treatment with etoposide, and apoptosis was assayed by Western blot (right). Full-length blots are presented in supplementary Figure 1 . ( B ) HEK cells expressing either a control plasmid or a GATA-3 shRNA expressing plasmid were treated with TNF-α (2 μg/mL) with or without pre-treatment with OPG for 2 h. Subsequent cell death was measured using TUNEL assays (left), and quantified with bar graphs (right). *p

    Techniques Used: Expressing, shRNA, Plasmid Preparation, Western Blot, Purification, TUNEL Assay

    9) Product Images from "Compromised nuclear envelope integrity drives tumor cell invasion"

    Article Title: Compromised nuclear envelope integrity drives tumor cell invasion

    Journal: bioRxiv

    doi: 10.1101/2020.05.22.110122

    Strong confinement induces nuclear envelope rupture and TREX1-dependent DNA damage. (A) RPE1 cells stably expressing 53BP1-EGFP and catalytically inactive cGAS-mCherry were confined at the indicated heights and images were acquired while cells were under confinement (after 2 h). Bar, 5 μm. (B, C) Quantification of DNA damage levels during confinement as assessed by the number of 53BP1 foci in cells displaying or not NE rupture. Data represents the mean ± SEM of 3 independent experiments where 20 cells per experiment and per condition were analyzed. Red vertical line indicates a single time-point, which is represented in “B”. (D, E) Frequency of foci appearance and foci life-time (foci duration) for RPE1 cells confined at 2 μm (with or without NE rupture) or treated with etoposide (25 μM). Data represents the mean ± SD of 3 independent experiments where 20 cells per condition were analyzed. (F) A variety of TREX1-deficient or -proficient RPE1 cells (all stably expressing 53BP1-EGFP and catalytically inactive cGAS-mCherry) were confined at 2 μm height and DNA damage levels were assessed after 2h under confinement by the number of 53BP1 foci in cells displaying or not NE rupture. (G) Same as in “F” but with DCIS cells. (F, G) Data represents the mean ± SEM of 3 independent experiments where 20 cells were analyzed per condition per experiment. Western blots show TREX1 depletion in RPE1 and DCIS cells 48h postknockdown; tubulin is the loading control. Bar, 10 μm. P values were calculated by unpaired Student’s t -test, ***P
    Figure Legend Snippet: Strong confinement induces nuclear envelope rupture and TREX1-dependent DNA damage. (A) RPE1 cells stably expressing 53BP1-EGFP and catalytically inactive cGAS-mCherry were confined at the indicated heights and images were acquired while cells were under confinement (after 2 h). Bar, 5 μm. (B, C) Quantification of DNA damage levels during confinement as assessed by the number of 53BP1 foci in cells displaying or not NE rupture. Data represents the mean ± SEM of 3 independent experiments where 20 cells per experiment and per condition were analyzed. Red vertical line indicates a single time-point, which is represented in “B”. (D, E) Frequency of foci appearance and foci life-time (foci duration) for RPE1 cells confined at 2 μm (with or without NE rupture) or treated with etoposide (25 μM). Data represents the mean ± SD of 3 independent experiments where 20 cells per condition were analyzed. (F) A variety of TREX1-deficient or -proficient RPE1 cells (all stably expressing 53BP1-EGFP and catalytically inactive cGAS-mCherry) were confined at 2 μm height and DNA damage levels were assessed after 2h under confinement by the number of 53BP1 foci in cells displaying or not NE rupture. (G) Same as in “F” but with DCIS cells. (F, G) Data represents the mean ± SEM of 3 independent experiments where 20 cells were analyzed per condition per experiment. Western blots show TREX1 depletion in RPE1 and DCIS cells 48h postknockdown; tubulin is the loading control. Bar, 10 μm. P values were calculated by unpaired Student’s t -test, ***P

    Techniques Used: Stable Transfection, Expressing, Western Blot

    Characterization of nuclear envelope rupture under different confinement heights and of DNA damage following nuclear envelope rupture. (A) RPE1 cells stably expressing catalytically inactive cGAS-EGFP were confined at the indicated heights and images were acquired immediately after (and while cells were under confinement). Bar, 15 μm. (B) Quantification of NE rupture events as assessed by cGAS perinuclear localization. Data represents the mean ± SD of 3 independent experiments where 50 cells per experiment per height were analyzed. (C) MCF10A cells stably expressing 53BP1-EGFP and catalytically inactive cGAS-mCherry were transiently depleted for TREX1 with siRNA and 48 h later cells were confined at 2 μm. (D) Quantification of DNA damage levels during 2 μm confinement as assessed by the number of 53BP1 foci in siControl and siTREX1 cells displaying or not NE rupture. Western blot shows TREX1 depletion 48 h post-knockdown; tubulin is the loading control. Bar, 10 μm. Data represents the mean of 3 independent experiments where 20 cells per experiment per condition were analyzed. (E) RPE1 cells stably expressing catalytically inactive cGAS-EGFP were confined at 2 μm for 2 h; subsequently, the confinement lid was removed, cells were harvested, replated for 30 minutes and fixed for immunostaining with the DNA damage markers RIF1 and γH2AX (red) and DAPI (blue). Arrowheads point to cells with NE rupture, which are also positive for the DNA damage markers. Bar, 20 μm. (F) Quantification of DNA damage foci at the indicated conditions (DMSO, etoposide 25 μM and 2 μm confinement). Images are representative of 2 independent experiments where 30 cells per experiment per condition were analyzed. Bars represent the mean ± SD. (G) RPE1 cells stably expressing 53BP1-EGFP were confined at 2 μm and imaged under spinning disc microscopy. White arrowhead points to a NE bleb while the yellow arrowhead points to a bleb bursting event that is followed by the appearance of DNA damage foci. (H) Graphs showing the absolute number of 53BP1 foci and the events of appearance of new 53BP1 foci (red asterisks) following a bleb bursting event (assessed by an increase in the cytosolic intensity of the probe 53BP1-EGFP, which leaks out of the nucleus upon NE rupture). Blue arrow indicates the instant of NE rupture/bleb bursting. (I) Diagrams illustrating the Cas9/gRNA targeting sequence in the TREX1 gene and the primers used for the sequencing of the TREX1 KO clones generated by CRISPR technology for RPE1 and DCIS cells. (J) Western blots of WT and TREX1 KO clones; tubulin is a loading control. (K) RPE1 cells stably expressing 53BP1-EGFP were transiently depleted for TREX1 using siRNA and treated with etoposide (25 μM) for 2 h. Subsequently cells fixed and stained for endogenous TREX1. Bar, 25 μm. (L) Frequency distribution of the number of nuclear blebs in RPE1 TREX1 KO clones and in parental RPE1 cells. Data represents 3 independent experiments where 60 cells per experiment were analyzed. (M) RPE1 cells transiently transfected with TREX1-EGFP WT were confined at 2 μm using a pressure-controlled dynamic confiner. Images represent a single plane Z slice through the middle section of the nucleus. Bars, 5 μm and 1 μm (magnification). Images are representative of 2 independent experiments.
    Figure Legend Snippet: Characterization of nuclear envelope rupture under different confinement heights and of DNA damage following nuclear envelope rupture. (A) RPE1 cells stably expressing catalytically inactive cGAS-EGFP were confined at the indicated heights and images were acquired immediately after (and while cells were under confinement). Bar, 15 μm. (B) Quantification of NE rupture events as assessed by cGAS perinuclear localization. Data represents the mean ± SD of 3 independent experiments where 50 cells per experiment per height were analyzed. (C) MCF10A cells stably expressing 53BP1-EGFP and catalytically inactive cGAS-mCherry were transiently depleted for TREX1 with siRNA and 48 h later cells were confined at 2 μm. (D) Quantification of DNA damage levels during 2 μm confinement as assessed by the number of 53BP1 foci in siControl and siTREX1 cells displaying or not NE rupture. Western blot shows TREX1 depletion 48 h post-knockdown; tubulin is the loading control. Bar, 10 μm. Data represents the mean of 3 independent experiments where 20 cells per experiment per condition were analyzed. (E) RPE1 cells stably expressing catalytically inactive cGAS-EGFP were confined at 2 μm for 2 h; subsequently, the confinement lid was removed, cells were harvested, replated for 30 minutes and fixed for immunostaining with the DNA damage markers RIF1 and γH2AX (red) and DAPI (blue). Arrowheads point to cells with NE rupture, which are also positive for the DNA damage markers. Bar, 20 μm. (F) Quantification of DNA damage foci at the indicated conditions (DMSO, etoposide 25 μM and 2 μm confinement). Images are representative of 2 independent experiments where 30 cells per experiment per condition were analyzed. Bars represent the mean ± SD. (G) RPE1 cells stably expressing 53BP1-EGFP were confined at 2 μm and imaged under spinning disc microscopy. White arrowhead points to a NE bleb while the yellow arrowhead points to a bleb bursting event that is followed by the appearance of DNA damage foci. (H) Graphs showing the absolute number of 53BP1 foci and the events of appearance of new 53BP1 foci (red asterisks) following a bleb bursting event (assessed by an increase in the cytosolic intensity of the probe 53BP1-EGFP, which leaks out of the nucleus upon NE rupture). Blue arrow indicates the instant of NE rupture/bleb bursting. (I) Diagrams illustrating the Cas9/gRNA targeting sequence in the TREX1 gene and the primers used for the sequencing of the TREX1 KO clones generated by CRISPR technology for RPE1 and DCIS cells. (J) Western blots of WT and TREX1 KO clones; tubulin is a loading control. (K) RPE1 cells stably expressing 53BP1-EGFP were transiently depleted for TREX1 using siRNA and treated with etoposide (25 μM) for 2 h. Subsequently cells fixed and stained for endogenous TREX1. Bar, 25 μm. (L) Frequency distribution of the number of nuclear blebs in RPE1 TREX1 KO clones and in parental RPE1 cells. Data represents 3 independent experiments where 60 cells per experiment were analyzed. (M) RPE1 cells transiently transfected with TREX1-EGFP WT were confined at 2 μm using a pressure-controlled dynamic confiner. Images represent a single plane Z slice through the middle section of the nucleus. Bars, 5 μm and 1 μm (magnification). Images are representative of 2 independent experiments.

    Techniques Used: Stable Transfection, Expressing, Western Blot, Immunostaining, Microscopy, Sequencing, Clone Assay, Generated, CRISPR, Staining, Transfection

    Long-term, strong confinement causes cell senescence in non-transformed cells but not in cancer cells. (A) RPE1, MCF10A and DCIS cells were treated with doxorubicin (40 nM), etoposide (25 μM) or DMSO (vehicle) for the indicated lengths of time. Cells were then fixed and processed for β-gal staining. The percentage of β-gal-positive cells was plotted. Graph: data represents mean ± SD of 3 independent experiments where 200 cells were scored per condition per experiment. (B) Frequency distribution (percentage) of the designated categories for cells under the indicated confinement heights. Data represents 3 independent experiments where 37 non-confined cells, 40 confined cells with non-ruptured nuclei and 60 confined cells with ruptured nuclei were scored per condition per experiment. (C) RPE1 cells were confined at 2 μm in the presence of propidium iodide and imaged overnight. Graph: data represents the mean ± SD of 2 independent experiments where 5 random fields (100 cells scored per field) were analyzed per time-point per experiment. Bar, 50μm. (D) Epifluorescence and phase images of RPE1 cells stably expressing catalytically inactive cGAS-EGFP harvested from 2 μm confinement and replated for cell cycle duration measurement over a 72 h period. Bar, 25 μm. Inset shows a cell with ruptured NE, as evidenced by cGAS perinuclear accumulation. (E) MCF10A cells or (F) DCIS cells were confined for 12 h at 10 or 2 μm, harvested from confinement and replated for 72 h before fixation for immunostaining with heterochromatin foci (HP1γ) or lysis for western blot analysis of lamin B1 and p21. GAPDH is the loading control; western blot images are representative of 2 independent experiments. Graph: box and whisker plot showing the median value and 10-90 percentiles of the number of HP1γ foci per nucleus. Data represents 3 independent experiments where 100 cells per condition per experiment were analyzed. Bars, 10 μm. (G) RPE1 cells were confined for 12 h at 4 or 2 μm; cells were then harvested from confinement and replated for 72 h before lysis for western blot analysis of lamin B1 and p21. Tubulin is the loading control; western blot images are representative of 2 independent experiments. (H) Western blot analysis of whole cell extracts of human fibroblasts, RPE1 cells and HeLa cells. GAPDH is the loading control. P values were calculated by unpaired Student’s t -test, ***P
    Figure Legend Snippet: Long-term, strong confinement causes cell senescence in non-transformed cells but not in cancer cells. (A) RPE1, MCF10A and DCIS cells were treated with doxorubicin (40 nM), etoposide (25 μM) or DMSO (vehicle) for the indicated lengths of time. Cells were then fixed and processed for β-gal staining. The percentage of β-gal-positive cells was plotted. Graph: data represents mean ± SD of 3 independent experiments where 200 cells were scored per condition per experiment. (B) Frequency distribution (percentage) of the designated categories for cells under the indicated confinement heights. Data represents 3 independent experiments where 37 non-confined cells, 40 confined cells with non-ruptured nuclei and 60 confined cells with ruptured nuclei were scored per condition per experiment. (C) RPE1 cells were confined at 2 μm in the presence of propidium iodide and imaged overnight. Graph: data represents the mean ± SD of 2 independent experiments where 5 random fields (100 cells scored per field) were analyzed per time-point per experiment. Bar, 50μm. (D) Epifluorescence and phase images of RPE1 cells stably expressing catalytically inactive cGAS-EGFP harvested from 2 μm confinement and replated for cell cycle duration measurement over a 72 h period. Bar, 25 μm. Inset shows a cell with ruptured NE, as evidenced by cGAS perinuclear accumulation. (E) MCF10A cells or (F) DCIS cells were confined for 12 h at 10 or 2 μm, harvested from confinement and replated for 72 h before fixation for immunostaining with heterochromatin foci (HP1γ) or lysis for western blot analysis of lamin B1 and p21. GAPDH is the loading control; western blot images are representative of 2 independent experiments. Graph: box and whisker plot showing the median value and 10-90 percentiles of the number of HP1γ foci per nucleus. Data represents 3 independent experiments where 100 cells per condition per experiment were analyzed. Bars, 10 μm. (G) RPE1 cells were confined for 12 h at 4 or 2 μm; cells were then harvested from confinement and replated for 72 h before lysis for western blot analysis of lamin B1 and p21. Tubulin is the loading control; western blot images are representative of 2 independent experiments. (H) Western blot analysis of whole cell extracts of human fibroblasts, RPE1 cells and HeLa cells. GAPDH is the loading control. P values were calculated by unpaired Student’s t -test, ***P

    Techniques Used: Transformation Assay, Staining, Stable Transfection, Expressing, Immunostaining, Lysis, Western Blot, Whisker Assay

    10) Product Images from "Highly activated p53 contributes to selectively increased apoptosis of latently HIV-1 infected cells upon treatment of anticancer drugs"

    Article Title: Highly activated p53 contributes to selectively increased apoptosis of latently HIV-1 infected cells upon treatment of anticancer drugs

    Journal: Virology Journal

    doi: 10.1186/s12985-016-0595-2

    Anticancer drugs increased the death of cells latently infected with HIV-1. a Cells were treated with etoposide (5 μM) and doxycycline (0.5 μM) for 36 h. The sub-G1 population (dead cells) of cells was determined by flow cytometry after PI staining ( left panel). The data are shown graphically with statistical analysis ( right panel). The data are shown as mean ± SD ( n = 3). * p
    Figure Legend Snippet: Anticancer drugs increased the death of cells latently infected with HIV-1. a Cells were treated with etoposide (5 μM) and doxycycline (0.5 μM) for 36 h. The sub-G1 population (dead cells) of cells was determined by flow cytometry after PI staining ( left panel). The data are shown graphically with statistical analysis ( right panel). The data are shown as mean ± SD ( n = 3). * p

    Techniques Used: Infection, Flow Cytometry, Cytometry, Staining

    11) Product Images from "Targeting survivin as a potential new treatment for chondrosarcoma of bone"

    Article Title: Targeting survivin as a potential new treatment for chondrosarcoma of bone

    Journal: Oncogenesis

    doi: 10.1038/oncsis.2016.33

    YM155 does not cause caspase 3/7 and PARP-dependent apoptosis, but cell-cycle deregulation in chondrosarcoma cell lines. ( a , b ) Caspase 3/7 activity ( a ) and viability ( b ) after 24 h as percentage to untreated control measured in nine chondrosarcoma cell lines by caspase-glo assay and presto blue viability assay. No caspase 3/7 activity is seen after YM155 treatment in all cell lines. Pan Caspase inhibitor z-vad was not able to rescue the YM155-dependent reduction in viability. MCS170 cells treated with doxorubicin and ABT-737 were used as a positive control. Error bars are shown for two independent experiments performed in duplicate. ( c ) Western blot analysis for PARP and cleaved PARP expression in four chondrosarcoma cell lines. No differences are seen between treated (+) and untreated (–) samples. Alpha tubulin was used as a loading control. Jurkat cell lysates treated with 25 μ m etoposide obtained from Cell Signaling were used as a positive control. ( d ) FACS analysis of three chondrosarcoma cell lines treated with YM155 for 48 h. JJ012 and CH2879 show a reduction in G1 and an increase in S phase after treatment with YM155. NDCS1 is not showing a difference in cell-cycle distribution.
    Figure Legend Snippet: YM155 does not cause caspase 3/7 and PARP-dependent apoptosis, but cell-cycle deregulation in chondrosarcoma cell lines. ( a , b ) Caspase 3/7 activity ( a ) and viability ( b ) after 24 h as percentage to untreated control measured in nine chondrosarcoma cell lines by caspase-glo assay and presto blue viability assay. No caspase 3/7 activity is seen after YM155 treatment in all cell lines. Pan Caspase inhibitor z-vad was not able to rescue the YM155-dependent reduction in viability. MCS170 cells treated with doxorubicin and ABT-737 were used as a positive control. Error bars are shown for two independent experiments performed in duplicate. ( c ) Western blot analysis for PARP and cleaved PARP expression in four chondrosarcoma cell lines. No differences are seen between treated (+) and untreated (–) samples. Alpha tubulin was used as a loading control. Jurkat cell lysates treated with 25 μ m etoposide obtained from Cell Signaling were used as a positive control. ( d ) FACS analysis of three chondrosarcoma cell lines treated with YM155 for 48 h. JJ012 and CH2879 show a reduction in G1 and an increase in S phase after treatment with YM155. NDCS1 is not showing a difference in cell-cycle distribution.

    Techniques Used: Activity Assay, Caspase-Glo Assay, Viability Assay, Positive Control, Western Blot, Expressing, FACS

    12) Product Images from "Chronic Intermittent Hypoxia Triggers a Senescence-like Phenotype in Human White Preadipocytes"

    Article Title: Chronic Intermittent Hypoxia Triggers a Senescence-like Phenotype in Human White Preadipocytes

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-63761-7

    Intermittent hypoxia (IH) alters preadipocyte phenotype and replicative capacity. Chronic IH was associated with a characteristic set of phenotypic changes such as flattened and enlarged cells ( a ) and a reduced multiplication ratio (MR) ( b ). Etoposide (ETO) was used as a positive control. Data are presented as mean ± SEM. P -values determined by one-tailed paired t-test compared to the normoxia (NO) control (n = 6 independent experiments).
    Figure Legend Snippet: Intermittent hypoxia (IH) alters preadipocyte phenotype and replicative capacity. Chronic IH was associated with a characteristic set of phenotypic changes such as flattened and enlarged cells ( a ) and a reduced multiplication ratio (MR) ( b ). Etoposide (ETO) was used as a positive control. Data are presented as mean ± SEM. P -values determined by one-tailed paired t-test compared to the normoxia (NO) control (n = 6 independent experiments).

    Techniques Used: Positive Control, One-tailed Test

    Intermittent hypoxia (IH) induces senescence in cultured preadipocytes. Exposure to 7 days of IH was associated with a higher prevalence of senescence associated β-galactosidase (SA-β-gal) positive preadipocytes (n = 6 independent experiments) ( a ). Compared to tissue grown in continuous normoxia (NO), chronic IH exposure was also associated with stronger green coloration of SA-β-gal staining in subcutaneous adipose tissue explants ( ex vivo ) ( b ). Representative images of SA-β-gal staining in cells exposed to 7 days of IH ( c ). Green marks indicate SA-β-gal positive (senescent) cells; Etoposide (ETO) treatment served as a positive control. Data are presented as mean ± SEM. P -values determined by one-tailed paired t-test compared to the NO control.
    Figure Legend Snippet: Intermittent hypoxia (IH) induces senescence in cultured preadipocytes. Exposure to 7 days of IH was associated with a higher prevalence of senescence associated β-galactosidase (SA-β-gal) positive preadipocytes (n = 6 independent experiments) ( a ). Compared to tissue grown in continuous normoxia (NO), chronic IH exposure was also associated with stronger green coloration of SA-β-gal staining in subcutaneous adipose tissue explants ( ex vivo ) ( b ). Representative images of SA-β-gal staining in cells exposed to 7 days of IH ( c ). Green marks indicate SA-β-gal positive (senescent) cells; Etoposide (ETO) treatment served as a positive control. Data are presented as mean ± SEM. P -values determined by one-tailed paired t-test compared to the NO control.

    Techniques Used: Cell Culture, Staining, Ex Vivo, Positive Control, One-tailed Test

    13) Product Images from "The exocyst gene Sec10 regulates renal epithelial monolayer homeostasis and apoptotic sensitivity"

    Article Title: The exocyst gene Sec10 regulates renal epithelial monolayer homeostasis and apoptotic sensitivity

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00011.2015

    Sec10-KD cells demonstrate an increased sensitivity to apoptotic stimuli. A : treatment of control and Sec10-KD cells grown in 2D on Transwells with the topoisomerase II inhibitor, etoposide, led to an increase in cells positive for activated caspase-3
    Figure Legend Snippet: Sec10-KD cells demonstrate an increased sensitivity to apoptotic stimuli. A : treatment of control and Sec10-KD cells grown in 2D on Transwells with the topoisomerase II inhibitor, etoposide, led to an increase in cells positive for activated caspase-3

    Techniques Used:

    14) Product Images from "Repeat‐associated non‐ AUG translation in C9orf72‐ ALS/ FTD is driven by neuronal excitation and stress"

    Article Title: Repeat‐associated non‐ AUG translation in C9orf72‐ ALS/ FTD is driven by neuronal excitation and stress

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201809423

    Cellular stress or neuronal excitotoxic stress increases DPR levels for all C9orf72 NRE ORFs in rat cortical neurons and in patient‐derived iPS spinal motor neurons Quantitative fluorescent microscopy imaging shows a dose‐dependent increase in the number of C9 DPR reporter‐positive cells with increasing concentrations of an apoptotic stimulator, staurosporine (SSP). The C9 DPR reporter in the GP frame was utilized for these experiments. n = 4. Filter‐trap binding assays using DPR‐specific antibodies show all DPR levels are increased for the sense‐coding C9 DPR reporter when NSC34 cells are challenged with stress‐inducing compounds. These stress‐inducing compounds broadly cover distinct or overlapping cellular pathways (see Table EV2 ). Antibodies specific for the C9orf72 NRE DPRs, poly‐GA, ‐GP, or ‐GR, were used to quantify DPR levels normalized to GAPDH 24 h post‐transient transfection of the C9 DPR reporter constructs. n = 4. Quantitative fluorescent microscopy imaging shows that NSC34, rat primary cortical neurons, have increased relative DPR fluorescent intensity levels following compound induced cellular stress. In NES‐mIFP cotransfection‐positive NSC34 and rat primary cortical neurons, there is a significant increase in C9 DPR reporter fluorescent intensity levels 24 post‐treatment with most stress‐inducing compounds. Fluorescent intensity was not normalized to NES‐mIFP fluorescent levels. NSC34: n = 10 with m > 500 cells analyzed per n . Cortical neuron: n = 6 with m > 40 cells per n . Endogenously expression of DPRs in iPS spinal motor neurons (iPS sMN) derived from C9orf72 NRE patients increases following induction of cellular stress. DPR fluorescent intensity in iPS sMN was measured using the DPR‐specific antibodies described above. IPS sMN: n = 4 with m > 20 cells per n . Statistical comparisons were performed using an uncorrected Fisher's exact test. Data information: All changes in DPR levels are shown relative to non‐stressed or DMSO‐only (vehicle) treated CTRL cells unless otherwise noted. Control (CTRL), thapsigargin (TG), menadione (Mena), staurosporine (SSP), diamide (Daim), cytochalasin D (Cyto D), etoposide (Etop), leptomycin B (Lepto), homocystine (HC), sodium arsenite (NaArs), tunicamycin (TM), glutamate (Glut). Statistical comparisons in (A) were calculated using t ‐tests per concentration, and (B) and (C) were calculated using an uncorrected Fisher LSD two‐way ANOVA for CTRL versus treatment (**** P
    Figure Legend Snippet: Cellular stress or neuronal excitotoxic stress increases DPR levels for all C9orf72 NRE ORFs in rat cortical neurons and in patient‐derived iPS spinal motor neurons Quantitative fluorescent microscopy imaging shows a dose‐dependent increase in the number of C9 DPR reporter‐positive cells with increasing concentrations of an apoptotic stimulator, staurosporine (SSP). The C9 DPR reporter in the GP frame was utilized for these experiments. n = 4. Filter‐trap binding assays using DPR‐specific antibodies show all DPR levels are increased for the sense‐coding C9 DPR reporter when NSC34 cells are challenged with stress‐inducing compounds. These stress‐inducing compounds broadly cover distinct or overlapping cellular pathways (see Table EV2 ). Antibodies specific for the C9orf72 NRE DPRs, poly‐GA, ‐GP, or ‐GR, were used to quantify DPR levels normalized to GAPDH 24 h post‐transient transfection of the C9 DPR reporter constructs. n = 4. Quantitative fluorescent microscopy imaging shows that NSC34, rat primary cortical neurons, have increased relative DPR fluorescent intensity levels following compound induced cellular stress. In NES‐mIFP cotransfection‐positive NSC34 and rat primary cortical neurons, there is a significant increase in C9 DPR reporter fluorescent intensity levels 24 post‐treatment with most stress‐inducing compounds. Fluorescent intensity was not normalized to NES‐mIFP fluorescent levels. NSC34: n = 10 with m > 500 cells analyzed per n . Cortical neuron: n = 6 with m > 40 cells per n . Endogenously expression of DPRs in iPS spinal motor neurons (iPS sMN) derived from C9orf72 NRE patients increases following induction of cellular stress. DPR fluorescent intensity in iPS sMN was measured using the DPR‐specific antibodies described above. IPS sMN: n = 4 with m > 20 cells per n . Statistical comparisons were performed using an uncorrected Fisher's exact test. Data information: All changes in DPR levels are shown relative to non‐stressed or DMSO‐only (vehicle) treated CTRL cells unless otherwise noted. Control (CTRL), thapsigargin (TG), menadione (Mena), staurosporine (SSP), diamide (Daim), cytochalasin D (Cyto D), etoposide (Etop), leptomycin B (Lepto), homocystine (HC), sodium arsenite (NaArs), tunicamycin (TM), glutamate (Glut). Statistical comparisons in (A) were calculated using t ‐tests per concentration, and (B) and (C) were calculated using an uncorrected Fisher LSD two‐way ANOVA for CTRL versus treatment (**** P

    Techniques Used: Derivative Assay, Microscopy, Imaging, Binding Assay, Transfection, Construct, Cotransfection, Expressing, Concentration Assay

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    Article Title: The exocyst gene Sec10 regulates renal epithelial monolayer homeostasis and apoptotic sensitivity
    Article Snippet: .. For the etoposide treatment, upon removal of the medium, fresh complete medium was supplemented with etoposide (10 μM) or DMSO alone (0.1%) as control, and the cells were incubated for 6 h before fixation and processing for immunofluorescent staining with cleaved caspase-3 antibody (Cell Signaling Technology). ..

    Incubation:

    Article Title: The exocyst gene Sec10 regulates renal epithelial monolayer homeostasis and apoptotic sensitivity
    Article Snippet: .. For the etoposide treatment, upon removal of the medium, fresh complete medium was supplemented with etoposide (10 μM) or DMSO alone (0.1%) as control, and the cells were incubated for 6 h before fixation and processing for immunofluorescent staining with cleaved caspase-3 antibody (Cell Signaling Technology). ..

    other:

    Article Title: Survival of midbrain dopamine neurons depends on the Bcl2 factor Mcl1
    Article Snippet: Chemical inhibitors and stressors UMI-77 (APExBIO); Bax-inhibiting peptide V5 (BIP-V5; Sigma Aldrich); MG132 (Enzo Life Sciences), etoposide (Cell Signaling Technologies); PAC1, Bam7, Birinapant, Lexibulin (CYT997), Nutlin-3a, YM155, A1210477, Navitoclax (ABT-263), Venetoclax (ABT-199) and Obatoclax mesylate were all purchased from Selleckchem.

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    Proliferation Assay:

    Article Title: DNA damage response defect in Williams-Beuren syndrome
    Article Snippet: .. Drug treatments and cell proliferation assay Cells were treated with 0.05, 0.5 and 5 mM of HU (no. H8627, 500 mM stock solution in ddH2 O; Sigma, St. Louis, MO, USA) or 1, 5 and 25 µM of etoposide (ETP, no. 2200, 50 mM stock solution in DMSO; Cell Signaling Technology, Inc., Danvers, MA, USA) for the indicated times. .. For the cell proliferation assay, 10 μ l of Premix WST-1 cell proliferation assay reagent (no. MK400; Takara Bio, Dalian, China) was added to the cultured cells in triplicate in a 96-well plate at the indicated times and conditions with 100 μ l of culture media and then the cells were incubated for 2 h before measuring the optical density (OD) following the manufacturer's recommendations.

    Lysis:

    Article Title: The effect of 3-bromopyruvate on human colorectal cancer cells is dependent on glucose concentration but not hexokinase II expression
    Article Snippet: .. Following treatment with 3BP or 50 μM etoposide, cells were lysed on ice with lysis buffer (Cell Signaling Technology) freshly supplemented with 2 μg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride (PMSF) and phosphatase inhibitor cocktail II (Sigma–Aldrich). .. Protein was quantified using the Bio-Rad DC Protein Assay Kit (Hercules, CA).

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    Cell Signaling Technology Inc etoposide
    p53 induction in response to DNA damage is impaired in TFEB/TFE3 DKO RAW264.7. 7 cells. ( A ) Representative Western blot showing p53 induction, p53 Ser15 phosphorylation, and Mdm2 levels in WT and TFE3/TFEB DKO RAW264.7 cells following <t>etoposide</t> treatment up to 8 hr. ( B ) Quantification of p53 induction from data shown in A. Data represents mean relative p53 level ± standard deviation with n = 3. Significance tested with two-way ANOVA with Sidak’s multiple comparisons test (**p
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    p53 induction in response to DNA damage is impaired in TFEB/TFE3 DKO RAW264.7. 7 cells. ( A ) Representative Western blot showing p53 induction, p53 Ser15 phosphorylation, and Mdm2 levels in WT and TFE3/TFEB DKO RAW264.7 cells following etoposide treatment up to 8 hr. ( B ) Quantification of p53 induction from data shown in A. Data represents mean relative p53 level ± standard deviation with n = 3. Significance tested with two-way ANOVA with Sidak’s multiple comparisons test (**p

    Journal: eLife

    Article Title: The transcription factors TFE3 and TFEB amplify p53 dependent transcriptional programs in response to DNA damage

    doi: 10.7554/eLife.40856

    Figure Lengend Snippet: p53 induction in response to DNA damage is impaired in TFEB/TFE3 DKO RAW264.7. 7 cells. ( A ) Representative Western blot showing p53 induction, p53 Ser15 phosphorylation, and Mdm2 levels in WT and TFE3/TFEB DKO RAW264.7 cells following etoposide treatment up to 8 hr. ( B ) Quantification of p53 induction from data shown in A. Data represents mean relative p53 level ± standard deviation with n = 3. Significance tested with two-way ANOVA with Sidak’s multiple comparisons test (**p

    Article Snippet: For drug treatments, cells were treated for the indicated times with the following reagents: DMSO (Sigma-Aldrich), 100 μM Etoposide (Cell Signaling Technology), dimethylformamide (Sigma-Aldrich D4551), 50 μM Cisplatin (Sigma-Aldrich 479306), Ethanol (Werner Graham Company), 2 mM LLOMe (Sigma-Aldrich L7393).

    Techniques: Western Blot, Standard Deviation

    ( A ) Representative Western blot showing TFEB and TFE3 gel shifts in response to etoposide in WT MEF, but not in p53 -/- MEF. All the immunoblots are representative of three independent experiments. ( B ) qPCR data showing relative induction of lysosomal-autophagy genes in response to starvation in WT and p53 -/- MEF. Data normalized to untreated cells and represents geometric means ± standard deviation and significance determined with Student’s t-test (*p

    Journal: eLife

    Article Title: The transcription factors TFE3 and TFEB amplify p53 dependent transcriptional programs in response to DNA damage

    doi: 10.7554/eLife.40856

    Figure Lengend Snippet: ( A ) Representative Western blot showing TFEB and TFE3 gel shifts in response to etoposide in WT MEF, but not in p53 -/- MEF. All the immunoblots are representative of three independent experiments. ( B ) qPCR data showing relative induction of lysosomal-autophagy genes in response to starvation in WT and p53 -/- MEF. Data normalized to untreated cells and represents geometric means ± standard deviation and significance determined with Student’s t-test (*p

    Article Snippet: For drug treatments, cells were treated for the indicated times with the following reagents: DMSO (Sigma-Aldrich), 100 μM Etoposide (Cell Signaling Technology), dimethylformamide (Sigma-Aldrich D4551), 50 μM Cisplatin (Sigma-Aldrich 479306), Ethanol (Werner Graham Company), 2 mM LLOMe (Sigma-Aldrich L7393).

    Techniques: Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation

    TFE3 and TFEB are necessary for proper execution of apoptosis in response to DNA damage in RAW264.7 cells. ( A ) Representative Western blot showing Caspase-3 cleavage in response to increasing time of etoposide treatment. ( B ) Quantification of data shown in A indicating defects in Caspase-3 cleavage in TFEB/TFE3 DKO RAW264.7 cells. Cleaved Caspase-3 levels are normalized to WT cells after 16 hr etoposide treatment with n = 3. Significance tested with Student’s t-test (#p

    Journal: eLife

    Article Title: The transcription factors TFE3 and TFEB amplify p53 dependent transcriptional programs in response to DNA damage

    doi: 10.7554/eLife.40856

    Figure Lengend Snippet: TFE3 and TFEB are necessary for proper execution of apoptosis in response to DNA damage in RAW264.7 cells. ( A ) Representative Western blot showing Caspase-3 cleavage in response to increasing time of etoposide treatment. ( B ) Quantification of data shown in A indicating defects in Caspase-3 cleavage in TFEB/TFE3 DKO RAW264.7 cells. Cleaved Caspase-3 levels are normalized to WT cells after 16 hr etoposide treatment with n = 3. Significance tested with Student’s t-test (#p

    Article Snippet: For drug treatments, cells were treated for the indicated times with the following reagents: DMSO (Sigma-Aldrich), 100 μM Etoposide (Cell Signaling Technology), dimethylformamide (Sigma-Aldrich D4551), 50 μM Cisplatin (Sigma-Aldrich 479306), Ethanol (Werner Graham Company), 2 mM LLOMe (Sigma-Aldrich L7393).

    Techniques: Western Blot

    ( A ) Immunofluorescence images of ARPE-19 cells treated with 100 μM etoposide or 50 μM Cisplatin for 24 hr or 10 hr after UVC irradiation. Scale bar = 10 μm. ( B ) Immunofluorescence images of HeLa cells treated with 100 μM etoposide for 24 hr or 50 μM Cisplatin for 12 hr and 4 hr after UVC irradiation. Scale bar = 10 μm. ( C ) Immunofluorescence images of RAW 264.7 cells treated with 100 μM etoposide for 10 hr or 35 μM Cisplatin for 10 hr or 4 hr after UVC irradiation. Scale bar = 10 μm. ( D ) Representative Western blot showing TFE3 de-phosphorylation at Ser321 and gel shift in TFEB in WT MEFs exposed to 100 μM etoposide for 8 hr or 50 μM Cisplatin for 10 hr or 10 hr after UVC irradiation. ( E ) Representative Western blot showing TFE3 de-phosphorylation at Ser321 and gel shift in TFEB in ARPE19 cells exposed to 100 μM etoposide for 24 hr or 50 μM Cisplatin for 24 hr or 24 hr after UVC irradiation. ( F ) Representative Western blot showing TFE3 de-phosphorylation at Ser321 and gel shift in TFEB in HeLa cells exposed to 100 μM etoposide for 24 hr or 50 μM Cisplatin for 18 hr. ( G ) Representative Western blot showing TFE3 de-phosphorylation at Ser321 and gel shift in TFEB in RAW 264.7 cells exposed to 100 μM etoposide for 8 hr or 50 μM Cisplatin for 8 hr or 4 hr after UVC irradiation. ( H ) Representative Western blot showing TFE3 nuclear distribution by subcellular fractionation of WT MEFs exposed to 50 μM Cisplatin for 10 hr. ( I ) Western blot showing etoposide dependent S6K de-phosphorylation in ARPE19 cells. All the immunoblots are representative of three independent experiments.

    Journal: eLife

    Article Title: The transcription factors TFE3 and TFEB amplify p53 dependent transcriptional programs in response to DNA damage

    doi: 10.7554/eLife.40856

    Figure Lengend Snippet: ( A ) Immunofluorescence images of ARPE-19 cells treated with 100 μM etoposide or 50 μM Cisplatin for 24 hr or 10 hr after UVC irradiation. Scale bar = 10 μm. ( B ) Immunofluorescence images of HeLa cells treated with 100 μM etoposide for 24 hr or 50 μM Cisplatin for 12 hr and 4 hr after UVC irradiation. Scale bar = 10 μm. ( C ) Immunofluorescence images of RAW 264.7 cells treated with 100 μM etoposide for 10 hr or 35 μM Cisplatin for 10 hr or 4 hr after UVC irradiation. Scale bar = 10 μm. ( D ) Representative Western blot showing TFE3 de-phosphorylation at Ser321 and gel shift in TFEB in WT MEFs exposed to 100 μM etoposide for 8 hr or 50 μM Cisplatin for 10 hr or 10 hr after UVC irradiation. ( E ) Representative Western blot showing TFE3 de-phosphorylation at Ser321 and gel shift in TFEB in ARPE19 cells exposed to 100 μM etoposide for 24 hr or 50 μM Cisplatin for 24 hr or 24 hr after UVC irradiation. ( F ) Representative Western blot showing TFE3 de-phosphorylation at Ser321 and gel shift in TFEB in HeLa cells exposed to 100 μM etoposide for 24 hr or 50 μM Cisplatin for 18 hr. ( G ) Representative Western blot showing TFE3 de-phosphorylation at Ser321 and gel shift in TFEB in RAW 264.7 cells exposed to 100 μM etoposide for 8 hr or 50 μM Cisplatin for 8 hr or 4 hr after UVC irradiation. ( H ) Representative Western blot showing TFE3 nuclear distribution by subcellular fractionation of WT MEFs exposed to 50 μM Cisplatin for 10 hr. ( I ) Western blot showing etoposide dependent S6K de-phosphorylation in ARPE19 cells. All the immunoblots are representative of three independent experiments.

    Article Snippet: For drug treatments, cells were treated for the indicated times with the following reagents: DMSO (Sigma-Aldrich), 100 μM Etoposide (Cell Signaling Technology), dimethylformamide (Sigma-Aldrich D4551), 50 μM Cisplatin (Sigma-Aldrich 479306), Ethanol (Werner Graham Company), 2 mM LLOMe (Sigma-Aldrich L7393).

    Techniques: Immunofluorescence, Irradiation, Western Blot, De-Phosphorylation Assay, Electrophoretic Mobility Shift Assay, Fractionation

    ( A ) Enriched GO terms in the ‘Biological Process’ category of differentially expressed genes between etoposide-treated WT and TFEB/TFE3 DKO RAW264.7 cells. GO terms are ranked by q value (

    Journal: eLife

    Article Title: The transcription factors TFE3 and TFEB amplify p53 dependent transcriptional programs in response to DNA damage

    doi: 10.7554/eLife.40856

    Figure Lengend Snippet: ( A ) Enriched GO terms in the ‘Biological Process’ category of differentially expressed genes between etoposide-treated WT and TFEB/TFE3 DKO RAW264.7 cells. GO terms are ranked by q value (

    Article Snippet: For drug treatments, cells were treated for the indicated times with the following reagents: DMSO (Sigma-Aldrich), 100 μM Etoposide (Cell Signaling Technology), dimethylformamide (Sigma-Aldrich D4551), 50 μM Cisplatin (Sigma-Aldrich 479306), Ethanol (Werner Graham Company), 2 mM LLOMe (Sigma-Aldrich L7393).

    Techniques:

    TFEB and TFE3 are essential for etoposide-induced lysosomal membrane permeabilization in MEFs. ( A ) Immunofluorescence images showing LMP in MEFs. Red galectin-1 puncta appear co-localized or within the lumen of green Lamp1 positive lysosomes. No LMP is detected under basal conditions in either WT or TFEB/TFE3 DKO MEFs. Treatment with etoposide induces profound LMP in WT, but not TFEB/TFE3 DKO cells. No differences in LMP induction were detected in LLOMe treated cells, regardless of genotype. Scale bar = 20 μm, inset = 2 μm. ( B ) WT MEFs exhibit a time-dependent increase in LMP after etoposide treatment. Quantification of data shown in A of galectin-1+/Lamp1 + LMP puncta per WT MEF cell. Data represent mean number of puncta per cell ± standard deviation from randomly selected confocal images, with > 20 cells per counted for each time point over three separate experiments. ( C ) Quantification of total number of galectin-1+/Lamp1+ LMP puncta per cell in WT vs TFE3/TFEB DKO MEFs treated for 8 hr with etoposide. Distribution is representative of one of the three independent experiments performed and shows 29 randomly selected WT MEF cells and 51 randomly selected TFEB/TFE3 DKO MEF cells. Significance determined using Student’s t-test (****p

    Journal: eLife

    Article Title: The transcription factors TFE3 and TFEB amplify p53 dependent transcriptional programs in response to DNA damage

    doi: 10.7554/eLife.40856

    Figure Lengend Snippet: TFEB and TFE3 are essential for etoposide-induced lysosomal membrane permeabilization in MEFs. ( A ) Immunofluorescence images showing LMP in MEFs. Red galectin-1 puncta appear co-localized or within the lumen of green Lamp1 positive lysosomes. No LMP is detected under basal conditions in either WT or TFEB/TFE3 DKO MEFs. Treatment with etoposide induces profound LMP in WT, but not TFEB/TFE3 DKO cells. No differences in LMP induction were detected in LLOMe treated cells, regardless of genotype. Scale bar = 20 μm, inset = 2 μm. ( B ) WT MEFs exhibit a time-dependent increase in LMP after etoposide treatment. Quantification of data shown in A of galectin-1+/Lamp1 + LMP puncta per WT MEF cell. Data represent mean number of puncta per cell ± standard deviation from randomly selected confocal images, with > 20 cells per counted for each time point over three separate experiments. ( C ) Quantification of total number of galectin-1+/Lamp1+ LMP puncta per cell in WT vs TFE3/TFEB DKO MEFs treated for 8 hr with etoposide. Distribution is representative of one of the three independent experiments performed and shows 29 randomly selected WT MEF cells and 51 randomly selected TFEB/TFE3 DKO MEF cells. Significance determined using Student’s t-test (****p

    Article Snippet: For drug treatments, cells were treated for the indicated times with the following reagents: DMSO (Sigma-Aldrich), 100 μM Etoposide (Cell Signaling Technology), dimethylformamide (Sigma-Aldrich D4551), 50 μM Cisplatin (Sigma-Aldrich 479306), Ethanol (Werner Graham Company), 2 mM LLOMe (Sigma-Aldrich L7393).

    Techniques: Immunofluorescence, Standard Deviation

    Type of cell death induced by 3BP is dose-dependent Caspase-3 cleavage and DNA fragmentation was assessed to further elucidate the mechanisms of 3BP-induced cell death. Caspase-3 cleavage was examined through western blot analysis ( N =3) ( A ). An image of a longer exposed membrane to better detect cleaved caspase-3 is shown below each full caspase-3 blot. DNA fragmentation was examined through gel electrophoresis in SW480 cells following 48 h 3BP treatment using 0.8% ( B ) and 2% ( C ) gels ( N =2). Etoposide (E; 50 μM) was used as a positive control for caspase-3 cleavage and DNA fragmentation; note nucleosome laddering in etoposide treated cells in ( C ) indicative of apoptosis.

    Journal: Bioscience Reports

    Article Title: The effect of 3-bromopyruvate on human colorectal cancer cells is dependent on glucose concentration but not hexokinase II expression

    doi: 10.1042/BSR20150267

    Figure Lengend Snippet: Type of cell death induced by 3BP is dose-dependent Caspase-3 cleavage and DNA fragmentation was assessed to further elucidate the mechanisms of 3BP-induced cell death. Caspase-3 cleavage was examined through western blot analysis ( N =3) ( A ). An image of a longer exposed membrane to better detect cleaved caspase-3 is shown below each full caspase-3 blot. DNA fragmentation was examined through gel electrophoresis in SW480 cells following 48 h 3BP treatment using 0.8% ( B ) and 2% ( C ) gels ( N =2). Etoposide (E; 50 μM) was used as a positive control for caspase-3 cleavage and DNA fragmentation; note nucleosome laddering in etoposide treated cells in ( C ) indicative of apoptosis.

    Article Snippet: Following treatment with 3BP or 50 μM etoposide, cells were lysed on ice with lysis buffer (Cell Signaling Technology) freshly supplemented with 2 μg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride (PMSF) and phosphatase inhibitor cocktail II (Sigma–Aldrich).

    Techniques: Western Blot, Nucleic Acid Electrophoresis, Positive Control

    (A) Kinetics of γ-H2A.X induction in primary fibroblasts from Williams-Beuren syndrome (WBS) patients and healthy donors upon exposure to etoposide or hydroxyurea. (B) Dose effect of γ-H2A.X induction after 6 h of treatment with etoposide or hydroxyurea.

    Journal: International Journal of Molecular Medicine

    Article Title: DNA damage response defect in Williams-Beuren syndrome

    doi: 10.3892/ijmm.2017.2861

    Figure Lengend Snippet: (A) Kinetics of γ-H2A.X induction in primary fibroblasts from Williams-Beuren syndrome (WBS) patients and healthy donors upon exposure to etoposide or hydroxyurea. (B) Dose effect of γ-H2A.X induction after 6 h of treatment with etoposide or hydroxyurea.

    Article Snippet: Drug treatments and cell proliferation assay Cells were treated with 0.05, 0.5 and 5 mM of HU (no. H8627, 500 mM stock solution in ddH2 O; Sigma, St. Louis, MO, USA) or 1, 5 and 25 µM of etoposide (ETP, no. 2200, 50 mM stock solution in DMSO; Cell Signaling Technology, Inc., Danvers, MA, USA) for the indicated times.

    Techniques:

    (A) Kinetics of γ-H2A.X induction in the 293T cells stably transfected with siRNAs targeting three Williams-Beuren syndrome genes upon exposure to etoposide or hydroxyurea. (B) Dose effect of γ-H2A.X induction after 6 h of treatment with etoposide or hydroxyurea in the same cells. UT, untransfected cells.

    Journal: International Journal of Molecular Medicine

    Article Title: DNA damage response defect in Williams-Beuren syndrome

    doi: 10.3892/ijmm.2017.2861

    Figure Lengend Snippet: (A) Kinetics of γ-H2A.X induction in the 293T cells stably transfected with siRNAs targeting three Williams-Beuren syndrome genes upon exposure to etoposide or hydroxyurea. (B) Dose effect of γ-H2A.X induction after 6 h of treatment with etoposide or hydroxyurea in the same cells. UT, untransfected cells.

    Article Snippet: Drug treatments and cell proliferation assay Cells were treated with 0.05, 0.5 and 5 mM of HU (no. H8627, 500 mM stock solution in ddH2 O; Sigma, St. Louis, MO, USA) or 1, 5 and 25 µM of etoposide (ETP, no. 2200, 50 mM stock solution in DMSO; Cell Signaling Technology, Inc., Danvers, MA, USA) for the indicated times.

    Techniques: Stable Transfection, Transfection

    Comparative gene expression of etoposide-treated WT and TFEB/TFE3-DKO RAW264.7 cells. ( A ) Principal component analysis of genes with q-value

    Journal: eLife

    Article Title: The transcription factors TFE3 and TFEB amplify p53 dependent transcriptional programs in response to DNA damage

    doi: 10.7554/eLife.40856

    Figure Lengend Snippet: Comparative gene expression of etoposide-treated WT and TFEB/TFE3-DKO RAW264.7 cells. ( A ) Principal component analysis of genes with q-value

    Article Snippet: For drug treatments, cells were treated for the indicated times with the following reagents: DMSO (Sigma-Aldrich), 100 μM Etoposide (Cell Signaling Technology), dimethylformamide (Sigma-Aldrich D4551), 50 μM Cisplatin (Sigma-Aldrich 479306), Ethanol (Werner Graham Company), 2 mM LLOMe (Sigma-Aldrich L7393).

    Techniques: Expressing

    DNA damage-induced TFE3 and TFEB activation is a p53 and mTORC1 dependent process. ( A ) Representative Western blot showing p53-dependent inhibition of mTORC1 in response to etoposide treatment in WT and p53 -/- MEFs. EBSS for 2 hr was used as a positive control for mTORC1 inhibition and was relatively unaffected by p53 status. ( B ) Quantification of Western blot data shown in A. Values represent mean ± standard deviation with n = 5. Significance determined with Two-way ANOVA with Sidak’s multiple comparisons test (*p

    Journal: eLife

    Article Title: The transcription factors TFE3 and TFEB amplify p53 dependent transcriptional programs in response to DNA damage

    doi: 10.7554/eLife.40856

    Figure Lengend Snippet: DNA damage-induced TFE3 and TFEB activation is a p53 and mTORC1 dependent process. ( A ) Representative Western blot showing p53-dependent inhibition of mTORC1 in response to etoposide treatment in WT and p53 -/- MEFs. EBSS for 2 hr was used as a positive control for mTORC1 inhibition and was relatively unaffected by p53 status. ( B ) Quantification of Western blot data shown in A. Values represent mean ± standard deviation with n = 5. Significance determined with Two-way ANOVA with Sidak’s multiple comparisons test (*p

    Article Snippet: For drug treatments, cells were treated for the indicated times with the following reagents: DMSO (Sigma-Aldrich), 100 μM Etoposide (Cell Signaling Technology), dimethylformamide (Sigma-Aldrich D4551), 50 μM Cisplatin (Sigma-Aldrich 479306), Ethanol (Werner Graham Company), 2 mM LLOMe (Sigma-Aldrich L7393).

    Techniques: Activation Assay, Western Blot, Inhibition, Positive Control, Standard Deviation

    TFE3 and TFEB are necessary for proper execution of apoptosis in response to DNA damage in RAW264.7 cells. ( A ) Representative Western blot showing Caspase-3 cleavage in response to increasing time of etoposide treatment. ( B ) Quantification of data shown in A indicating defects in Caspase-3 cleavage in TFEB/TFE3 DKO RAW264.7 cells. Cleaved Caspase-3 levels are normalized to WT cells after 16 hr etoposide treatment with n = 3. Significance tested with Student’s t-test (#p

    Journal: eLife

    Article Title: The transcription factors TFE3 and TFEB amplify p53 dependent transcriptional programs in response to DNA damage

    doi: 10.7554/eLife.40856

    Figure Lengend Snippet: TFE3 and TFEB are necessary for proper execution of apoptosis in response to DNA damage in RAW264.7 cells. ( A ) Representative Western blot showing Caspase-3 cleavage in response to increasing time of etoposide treatment. ( B ) Quantification of data shown in A indicating defects in Caspase-3 cleavage in TFEB/TFE3 DKO RAW264.7 cells. Cleaved Caspase-3 levels are normalized to WT cells after 16 hr etoposide treatment with n = 3. Significance tested with Student’s t-test (#p

    Article Snippet: For drug treatments, cells were treated for the indicated times with the following reagents: DMSO (Sigma-Aldrich), 100 μM Etoposide (Cell Signaling Technology), dimethylformamide (Sigma-Aldrich D4551), 50 μM Cisplatin (Sigma-Aldrich 479306), Ethanol (Werner Graham Company), 2 mM LLOMe (Sigma-Aldrich L7393).

    Techniques: Western Blot

    Differentially regulated genes in WT versus TFEB/TFE3 DKO MEFs undergoing DNA damage. qRT-PCR-based quantification of basal and etoposide induced mRNA levels of Rad9a, Chek2, Trp53inp1, Mdm2, Bbc3, Bax, Sesn1, Sesn2, Dram1, Tp53, Cdkn1a, Laptm5, Ctsd, Wrap53, Egfr and Foxo3 in WT vs TFEB/TFE3 DKO MEFs. All qRT-PCR data represented as geometric mean ± standard deviation and significance tested using Student’s t-test (*p

    Journal: eLife

    Article Title: The transcription factors TFE3 and TFEB amplify p53 dependent transcriptional programs in response to DNA damage

    doi: 10.7554/eLife.40856

    Figure Lengend Snippet: Differentially regulated genes in WT versus TFEB/TFE3 DKO MEFs undergoing DNA damage. qRT-PCR-based quantification of basal and etoposide induced mRNA levels of Rad9a, Chek2, Trp53inp1, Mdm2, Bbc3, Bax, Sesn1, Sesn2, Dram1, Tp53, Cdkn1a, Laptm5, Ctsd, Wrap53, Egfr and Foxo3 in WT vs TFEB/TFE3 DKO MEFs. All qRT-PCR data represented as geometric mean ± standard deviation and significance tested using Student’s t-test (*p

    Article Snippet: For drug treatments, cells were treated for the indicated times with the following reagents: DMSO (Sigma-Aldrich), 100 μM Etoposide (Cell Signaling Technology), dimethylformamide (Sigma-Aldrich D4551), 50 μM Cisplatin (Sigma-Aldrich 479306), Ethanol (Werner Graham Company), 2 mM LLOMe (Sigma-Aldrich L7393).

    Techniques: Quantitative RT-PCR, Standard Deviation

    Expression of constitutively active TFEB and TFE3 in HeLa cells increases total p53 protein levels and its extends half-life. ( A ) Representative Western blot showing elevated p53 protein levels in adenovirus infected HeLa cells expressing constitutively active mutants of TFEB and TFE3. Further p53 protein level increases are seen with constitutively active TFEB and TFE3 after treatment with etoposide 8 hr. ( B ) Quantification of basal p53 protein level in HeLa cells expressing constitutively active TFEB and TFE3. Due to the high dynamic range and low detectability of basal endogenous p53 in control cells, values were normalized to intermediate expression samples, TFE3 S321A. Data represents mean relative p53 level ± standard deviation with n = 5 (*p

    Journal: eLife

    Article Title: The transcription factors TFE3 and TFEB amplify p53 dependent transcriptional programs in response to DNA damage

    doi: 10.7554/eLife.40856

    Figure Lengend Snippet: Expression of constitutively active TFEB and TFE3 in HeLa cells increases total p53 protein levels and its extends half-life. ( A ) Representative Western blot showing elevated p53 protein levels in adenovirus infected HeLa cells expressing constitutively active mutants of TFEB and TFE3. Further p53 protein level increases are seen with constitutively active TFEB and TFE3 after treatment with etoposide 8 hr. ( B ) Quantification of basal p53 protein level in HeLa cells expressing constitutively active TFEB and TFE3. Due to the high dynamic range and low detectability of basal endogenous p53 in control cells, values were normalized to intermediate expression samples, TFE3 S321A. Data represents mean relative p53 level ± standard deviation with n = 5 (*p

    Article Snippet: For drug treatments, cells were treated for the indicated times with the following reagents: DMSO (Sigma-Aldrich), 100 μM Etoposide (Cell Signaling Technology), dimethylformamide (Sigma-Aldrich D4551), 50 μM Cisplatin (Sigma-Aldrich 479306), Ethanol (Werner Graham Company), 2 mM LLOMe (Sigma-Aldrich L7393).

    Techniques: Expressing, Western Blot, Infection, Standard Deviation

    p53 induction in response to DNA damage is impaired in TFEB/TFE3 DKO RAW264.7. 7 cells. ( A ) Representative Western blot showing p53 induction, p53 Ser15 phosphorylation, and Mdm2 levels in WT and TFE3/TFEB DKO RAW264.7 cells following etoposide treatment up to 8 hr. ( B ) Quantification of p53 induction from data shown in A. Data represents mean relative p53 level ± standard deviation with n = 3. Significance tested with two-way ANOVA with Sidak’s multiple comparisons test (**p

    Journal: eLife

    Article Title: The transcription factors TFE3 and TFEB amplify p53 dependent transcriptional programs in response to DNA damage

    doi: 10.7554/eLife.40856

    Figure Lengend Snippet: p53 induction in response to DNA damage is impaired in TFEB/TFE3 DKO RAW264.7. 7 cells. ( A ) Representative Western blot showing p53 induction, p53 Ser15 phosphorylation, and Mdm2 levels in WT and TFE3/TFEB DKO RAW264.7 cells following etoposide treatment up to 8 hr. ( B ) Quantification of p53 induction from data shown in A. Data represents mean relative p53 level ± standard deviation with n = 3. Significance tested with two-way ANOVA with Sidak’s multiple comparisons test (**p

    Article Snippet: For drug treatments, cells were treated for the indicated times with the following reagents: DMSO (Sigma-Aldrich), 100 μM Etoposide (Cell Signaling Technology), dimethylformamide (Sigma-Aldrich D4551), 50 μM Cisplatin (Sigma-Aldrich 479306), Ethanol (Werner Graham Company), 2 mM LLOMe (Sigma-Aldrich L7393).

    Techniques: Western Blot, Standard Deviation

    TFE3 and TFEB translocate to the nucleus in response to genotoxic stress. ( A ) Immunofluorescence images of WT MEFs treated with 100 μM etoposide for up to 16 hr. Scale bar = 20 μm. ( B ) Quantification of TFE3 localization from cells shown in A. Levels represent mean ± standard deviation with n = 3 experiments and > 200 cells counted per trial. Significance determined with Student’s t-test (*p

    Journal: eLife

    Article Title: The transcription factors TFE3 and TFEB amplify p53 dependent transcriptional programs in response to DNA damage

    doi: 10.7554/eLife.40856

    Figure Lengend Snippet: TFE3 and TFEB translocate to the nucleus in response to genotoxic stress. ( A ) Immunofluorescence images of WT MEFs treated with 100 μM etoposide for up to 16 hr. Scale bar = 20 μm. ( B ) Quantification of TFE3 localization from cells shown in A. Levels represent mean ± standard deviation with n = 3 experiments and > 200 cells counted per trial. Significance determined with Student’s t-test (*p

    Article Snippet: For drug treatments, cells were treated for the indicated times with the following reagents: DMSO (Sigma-Aldrich), 100 μM Etoposide (Cell Signaling Technology), dimethylformamide (Sigma-Aldrich D4551), 50 μM Cisplatin (Sigma-Aldrich 479306), Ethanol (Werner Graham Company), 2 mM LLOMe (Sigma-Aldrich L7393).

    Techniques: Immunofluorescence, Standard Deviation

    TFEB and TFE3 are essential for etoposide-induced lysosomal membrane permeabilization in MEFs. ( A ) Immunofluorescence images showing LMP in MEFs. Red galectin-1 puncta appear co-localized or within the lumen of green Lamp1 positive lysosomes. No LMP is detected under basal conditions in either WT or TFEB/TFE3 DKO MEFs. Treatment with etoposide induces profound LMP in WT, but not TFEB/TFE3 DKO cells. No differences in LMP induction were detected in LLOMe treated cells, regardless of genotype. Scale bar = 20 μm, inset = 2 μm. ( B ) WT MEFs exhibit a time-dependent increase in LMP after etoposide treatment. Quantification of data shown in A of galectin-1+/Lamp1 + LMP puncta per WT MEF cell. Data represent mean number of puncta per cell ± standard deviation from randomly selected confocal images, with > 20 cells per counted for each time point over three separate experiments. ( C ) Quantification of total number of galectin-1+/Lamp1+ LMP puncta per cell in WT vs TFE3/TFEB DKO MEFs treated for 8 hr with etoposide. Distribution is representative of one of the three independent experiments performed and shows 29 randomly selected WT MEF cells and 51 randomly selected TFEB/TFE3 DKO MEF cells. Significance determined using Student’s t-test (****p

    Journal: eLife

    Article Title: The transcription factors TFE3 and TFEB amplify p53 dependent transcriptional programs in response to DNA damage

    doi: 10.7554/eLife.40856

    Figure Lengend Snippet: TFEB and TFE3 are essential for etoposide-induced lysosomal membrane permeabilization in MEFs. ( A ) Immunofluorescence images showing LMP in MEFs. Red galectin-1 puncta appear co-localized or within the lumen of green Lamp1 positive lysosomes. No LMP is detected under basal conditions in either WT or TFEB/TFE3 DKO MEFs. Treatment with etoposide induces profound LMP in WT, but not TFEB/TFE3 DKO cells. No differences in LMP induction were detected in LLOMe treated cells, regardless of genotype. Scale bar = 20 μm, inset = 2 μm. ( B ) WT MEFs exhibit a time-dependent increase in LMP after etoposide treatment. Quantification of data shown in A of galectin-1+/Lamp1 + LMP puncta per WT MEF cell. Data represent mean number of puncta per cell ± standard deviation from randomly selected confocal images, with > 20 cells per counted for each time point over three separate experiments. ( C ) Quantification of total number of galectin-1+/Lamp1+ LMP puncta per cell in WT vs TFE3/TFEB DKO MEFs treated for 8 hr with etoposide. Distribution is representative of one of the three independent experiments performed and shows 29 randomly selected WT MEF cells and 51 randomly selected TFEB/TFE3 DKO MEF cells. Significance determined using Student’s t-test (****p

    Article Snippet: For drug treatments, cells were treated for the indicated times with the following reagents: DMSO (Sigma-Aldrich), 100 μM Etoposide (Cell Signaling Technology), dimethylformamide (Sigma-Aldrich D4551), 50 μM Cisplatin (Sigma-Aldrich 479306), Ethanol (Werner Graham Company), 2 mM LLOMe (Sigma-Aldrich L7393).

    Techniques: Immunofluorescence, Standard Deviation