human breast cancer cell lines bt 549 Search Results


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  • 99
    ATCC bt 549 breast ductal carcinoma cells
    (A) Selected NCI-60 cell lines expressing STING (see ) were treated for 6 or 24 h with 1 μM topotecan (Top) or doxorubicin (Dox) and analyzed by microarray as reported in the NCI Transcriptional Pharmacodynamics Workbench . The heatmap shows the log 2 fold change to NT condition (the values below 0.5 are blue and the values above are purple). Missing values are shown in grey. (B) Indicated cell lines were treated with CPT (see Materials and Methods for dosage used) with or without 3.6 μM H151 for 24 <t>(BT-549,</t> HS-578T, MDA-MB-231, PC-3 and SK-OV-3 cells) or 48 h (MG-63 and HOS cells), and IL-6 levels in supernatants were determined by ELISA. IL-6 levels were normalized to the “CPT only” condition and are shown as percentages. Data shown are averaged from three independent experiments in biological replicate (±s.e.m. and Mann-Whitney U tests are shown). (C) MG-63 and HOS were treated with CPT with or without decreasing concentrations of H151 (3.6, 1.8 and 0.9 μM) for 48 h, and IL-6 levels in supernatants were determined by ELISA. IL-6 levels were normalized to the “CPT only” condition and are shown as percentages. Data shown are averaged from three independent experiments in biological replicate (±s.e.m. and ordinary one-way ANOVA with Dunnett’s multiple comparison tests to the “CPT only” condition). (D) MG-63 and HOS were treated with CPT with or without 3.6 μM H151 for 48 h, and cell lysates were processed for RNA purification and RT-qPCR analyses. IL-6 levels were reported relative to 18S expression and divided further by the mean of the NT condition. Data shown are averaged from three independent experiments (±s.e.m. and ordinary one-way ANOVA with Dunnett’s multiple comparison tests to the “CPT only” condition). (E) MG-63 and HOS were treated with CPT with or without 200 nM WEHI-122 for 48 h and IL-6 levels in supernatants were determined by ELISA. IL-6 levels were normalized to the “CPT only” condition and are shown as percentages. Data shown are averaged from three (MG-63) or two (HOS) independent experiments in biological replicate (±s.e.m. and Mann-Whitney U tests are shown). (F, G) MG-63 were treated overnight with 100 nM GSK#3, with or without 3.6 μM H151 (F) or 200 nM WEHI-112 (G) , and IL-6 levels in supernatants were determined by ELISA. IL-6 levels were normalized to the “GSK only” condition and are shown as percentages. Data shown are averaged from three (F) or two (G) independent experiments in biological replicate (±s.e.m. and Mann-Whitney U tests are shown). (H, I, J) Wild-type (WT) (H) , cGAS -deficient (I) and STING -deficient (J) HaCaT cells were treated with 0.2 μM CPT in the presence of decreasing amounts of H151 (3.6, 1.8 or 0.9 μM) (H) or 3.6 μM (I) for 24 h, and IL-6 levels in supernatants were determined by ELISA. (J) Cells were treated with poly(I:C) [p(I:C)] at 1 μg/ ml, where indicated. IL-6 levels were normalized to the “CPT only” (H, I) or “p(I:C)” (J) condition and are shown as percentages. Data shown are averaged from two (I) or three (H, J) independent experiments in biological replicate [±s.e.m. and ordinary one-way ANOVA with Dunnett’s multiple comparison tests to the “CPT only” condition (H) , and Mann-Whitney U tests are shown (I, J) ]. (K) MG-63 and HOS were transfected with 10 nM of the indicated siRNAs for 24 or 48 h, respectively, prior to CPT treatment for 48 or 24 h, respectively, and IL-6 levels in supernatants were determined by ELISA. IL-6 levels were normalized to the “CPT + siNC5” condition and are shown as percentages (±s.e.m. and ordinary one-way ANOVA with Dunnett’s multiple comparison tests to the “CPT + siNC5” condition). * p ≤0.05, ** p ≤0.01, *** p ≤0.001, **** p ≤0.0001 and “ns” is non-significant.
    Bt 549 Breast Ductal Carcinoma Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bt 549 breast ductal carcinoma cells/product/ATCC
    Average 99 stars, based on 1 article reviews
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    86
    Millipore bt 549 human breast epithelial carcinoma cells
    A. Apoptosis was induced in HeLa and immortalized MEFs using STS for the indicated times, and endogenous Bcl-2 was detected by Western blot analysis. B. Apoptosis was induced in HeLa, <t>BT-549</t> and COS-7 cells with etoposide. NT (No Treatment). In primary MEFs apoptosis was induced with 100 µM etoposide for 5 h. A decrease in endogenous Bcl-2 levels was seen upon treatment with both STS and etoposide. C. Apoptosis was induced in HeLa cells using STS in the presence or absence of 20 µM of MG132. Western and densitometry analyses revealed decreased levels of Bcl-2 with STS treatment, and stabilization of Bcl-2 upon MG132 treatment. This suggests that Bcl-2 levels are down-regulated via the UPS. D. WT MEFs and HeLa cells were transiently transfected with Bcl-2, XIAP and ubiquitin and treated with 20 µM MG132 for 6 h and with 1.75 µM STS. IP with anti-Bcl-2 was followed by Western Blotting with anti-ubiquitin antibodies. *Represents the IG heavy chain. Poly-ubiquitylated forms of Bcl-2 appeared in apoptotic cells and correlated with decreased Bcl-2 levels.
    Bt 549 Human Breast Epithelial Carcinoma Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bt 549 human breast epithelial carcinoma cells/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bt 549 human breast epithelial carcinoma cells - by Bioz Stars, 2024-03
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    86
    Galectin Therapeutics 549 human breast cancer cells
    A. Apoptosis was induced in HeLa and immortalized MEFs using STS for the indicated times, and endogenous Bcl-2 was detected by Western blot analysis. B. Apoptosis was induced in HeLa, <t>BT-549</t> and COS-7 cells with etoposide. NT (No Treatment). In primary MEFs apoptosis was induced with 100 µM etoposide for 5 h. A decrease in endogenous Bcl-2 levels was seen upon treatment with both STS and etoposide. C. Apoptosis was induced in HeLa cells using STS in the presence or absence of 20 µM of MG132. Western and densitometry analyses revealed decreased levels of Bcl-2 with STS treatment, and stabilization of Bcl-2 upon MG132 treatment. This suggests that Bcl-2 levels are down-regulated via the UPS. D. WT MEFs and HeLa cells were transiently transfected with Bcl-2, XIAP and ubiquitin and treated with 20 µM MG132 for 6 h and with 1.75 µM STS. IP with anti-Bcl-2 was followed by Western Blotting with anti-ubiquitin antibodies. *Represents the IG heavy chain. Poly-ubiquitylated forms of Bcl-2 appeared in apoptotic cells and correlated with decreased Bcl-2 levels.
    549 Human Breast Cancer Cells, supplied by Galectin Therapeutics, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/549 human breast cancer cells/product/Galectin Therapeutics
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    549 human breast cancer cells - by Bioz Stars, 2024-03
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    (A) Selected NCI-60 cell lines expressing STING (see ) were treated for 6 or 24 h with 1 μM topotecan (Top) or doxorubicin (Dox) and analyzed by microarray as reported in the NCI Transcriptional Pharmacodynamics Workbench . The heatmap shows the log 2 fold change to NT condition (the values below 0.5 are blue and the values above are purple). Missing values are shown in grey. (B) Indicated cell lines were treated with CPT (see Materials and Methods for dosage used) with or without 3.6 μM H151 for 24 (BT-549, HS-578T, MDA-MB-231, PC-3 and SK-OV-3 cells) or 48 h (MG-63 and HOS cells), and IL-6 levels in supernatants were determined by ELISA. IL-6 levels were normalized to the “CPT only” condition and are shown as percentages. Data shown are averaged from three independent experiments in biological replicate (±s.e.m. and Mann-Whitney U tests are shown). (C) MG-63 and HOS were treated with CPT with or without decreasing concentrations of H151 (3.6, 1.8 and 0.9 μM) for 48 h, and IL-6 levels in supernatants were determined by ELISA. IL-6 levels were normalized to the “CPT only” condition and are shown as percentages. Data shown are averaged from three independent experiments in biological replicate (±s.e.m. and ordinary one-way ANOVA with Dunnett’s multiple comparison tests to the “CPT only” condition). (D) MG-63 and HOS were treated with CPT with or without 3.6 μM H151 for 48 h, and cell lysates were processed for RNA purification and RT-qPCR analyses. IL-6 levels were reported relative to 18S expression and divided further by the mean of the NT condition. Data shown are averaged from three independent experiments (±s.e.m. and ordinary one-way ANOVA with Dunnett’s multiple comparison tests to the “CPT only” condition). (E) MG-63 and HOS were treated with CPT with or without 200 nM WEHI-122 for 48 h and IL-6 levels in supernatants were determined by ELISA. IL-6 levels were normalized to the “CPT only” condition and are shown as percentages. Data shown are averaged from three (MG-63) or two (HOS) independent experiments in biological replicate (±s.e.m. and Mann-Whitney U tests are shown). (F, G) MG-63 were treated overnight with 100 nM GSK#3, with or without 3.6 μM H151 (F) or 200 nM WEHI-112 (G) , and IL-6 levels in supernatants were determined by ELISA. IL-6 levels were normalized to the “GSK only” condition and are shown as percentages. Data shown are averaged from three (F) or two (G) independent experiments in biological replicate (±s.e.m. and Mann-Whitney U tests are shown). (H, I, J) Wild-type (WT) (H) , cGAS -deficient (I) and STING -deficient (J) HaCaT cells were treated with 0.2 μM CPT in the presence of decreasing amounts of H151 (3.6, 1.8 or 0.9 μM) (H) or 3.6 μM (I) for 24 h, and IL-6 levels in supernatants were determined by ELISA. (J) Cells were treated with poly(I:C) [p(I:C)] at 1 μg/ ml, where indicated. IL-6 levels were normalized to the “CPT only” (H, I) or “p(I:C)” (J) condition and are shown as percentages. Data shown are averaged from two (I) or three (H, J) independent experiments in biological replicate [±s.e.m. and ordinary one-way ANOVA with Dunnett’s multiple comparison tests to the “CPT only” condition (H) , and Mann-Whitney U tests are shown (I, J) ]. (K) MG-63 and HOS were transfected with 10 nM of the indicated siRNAs for 24 or 48 h, respectively, prior to CPT treatment for 48 or 24 h, respectively, and IL-6 levels in supernatants were determined by ELISA. IL-6 levels were normalized to the “CPT + siNC5” condition and are shown as percentages (±s.e.m. and ordinary one-way ANOVA with Dunnett’s multiple comparison tests to the “CPT + siNC5” condition). * p ≤0.05, ** p ≤0.01, *** p ≤0.001, **** p ≤0.0001 and “ns” is non-significant.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Pharmacological Targeting of STING-Dependent IL-6 Production in Cancer Cells

    doi: 10.3389/fcell.2021.709618

    Figure Lengend Snippet: (A) Selected NCI-60 cell lines expressing STING (see ) were treated for 6 or 24 h with 1 μM topotecan (Top) or doxorubicin (Dox) and analyzed by microarray as reported in the NCI Transcriptional Pharmacodynamics Workbench . The heatmap shows the log 2 fold change to NT condition (the values below 0.5 are blue and the values above are purple). Missing values are shown in grey. (B) Indicated cell lines were treated with CPT (see Materials and Methods for dosage used) with or without 3.6 μM H151 for 24 (BT-549, HS-578T, MDA-MB-231, PC-3 and SK-OV-3 cells) or 48 h (MG-63 and HOS cells), and IL-6 levels in supernatants were determined by ELISA. IL-6 levels were normalized to the “CPT only” condition and are shown as percentages. Data shown are averaged from three independent experiments in biological replicate (±s.e.m. and Mann-Whitney U tests are shown). (C) MG-63 and HOS were treated with CPT with or without decreasing concentrations of H151 (3.6, 1.8 and 0.9 μM) for 48 h, and IL-6 levels in supernatants were determined by ELISA. IL-6 levels were normalized to the “CPT only” condition and are shown as percentages. Data shown are averaged from three independent experiments in biological replicate (±s.e.m. and ordinary one-way ANOVA with Dunnett’s multiple comparison tests to the “CPT only” condition). (D) MG-63 and HOS were treated with CPT with or without 3.6 μM H151 for 48 h, and cell lysates were processed for RNA purification and RT-qPCR analyses. IL-6 levels were reported relative to 18S expression and divided further by the mean of the NT condition. Data shown are averaged from three independent experiments (±s.e.m. and ordinary one-way ANOVA with Dunnett’s multiple comparison tests to the “CPT only” condition). (E) MG-63 and HOS were treated with CPT with or without 200 nM WEHI-122 for 48 h and IL-6 levels in supernatants were determined by ELISA. IL-6 levels were normalized to the “CPT only” condition and are shown as percentages. Data shown are averaged from three (MG-63) or two (HOS) independent experiments in biological replicate (±s.e.m. and Mann-Whitney U tests are shown). (F, G) MG-63 were treated overnight with 100 nM GSK#3, with or without 3.6 μM H151 (F) or 200 nM WEHI-112 (G) , and IL-6 levels in supernatants were determined by ELISA. IL-6 levels were normalized to the “GSK only” condition and are shown as percentages. Data shown are averaged from three (F) or two (G) independent experiments in biological replicate (±s.e.m. and Mann-Whitney U tests are shown). (H, I, J) Wild-type (WT) (H) , cGAS -deficient (I) and STING -deficient (J) HaCaT cells were treated with 0.2 μM CPT in the presence of decreasing amounts of H151 (3.6, 1.8 or 0.9 μM) (H) or 3.6 μM (I) for 24 h, and IL-6 levels in supernatants were determined by ELISA. (J) Cells were treated with poly(I:C) [p(I:C)] at 1 μg/ ml, where indicated. IL-6 levels were normalized to the “CPT only” (H, I) or “p(I:C)” (J) condition and are shown as percentages. Data shown are averaged from two (I) or three (H, J) independent experiments in biological replicate [±s.e.m. and ordinary one-way ANOVA with Dunnett’s multiple comparison tests to the “CPT only” condition (H) , and Mann-Whitney U tests are shown (I, J) ]. (K) MG-63 and HOS were transfected with 10 nM of the indicated siRNAs for 24 or 48 h, respectively, prior to CPT treatment for 48 or 24 h, respectively, and IL-6 levels in supernatants were determined by ELISA. IL-6 levels were normalized to the “CPT + siNC5” condition and are shown as percentages (±s.e.m. and ordinary one-way ANOVA with Dunnett’s multiple comparison tests to the “CPT + siNC5” condition). * p ≤0.05, ** p ≤0.01, *** p ≤0.001, **** p ≤0.0001 and “ns” is non-significant.

    Article Snippet: PC-3 cells purchased from ATCC (#CRL-1435) and BT-549 breast ductal carcinoma cells (a kind gift from Prof S. Lakhani) were grown in Roswell Park Memorial Institute (RPMI) 1,640 plus L-glutamine medium (Life Technologies) complemented with 1x antibiotic/antimycotic and 10% heat inactivated fetal bovine serum (referred to as complete RPMI).

    Techniques: Expressing, Microarray, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Purification, Quantitative RT-PCR, Transfection

    (A, B) HOS, MG-63 and TC-1 cells were plated at low density and treated with 3.6 μM H151, 3 μM of the p38 inhibitor SB202190 [SB] or 1 μM of the ERK1/2 inhibitor SCH772984 [SCH] for 10, 12, and 7 days, respectively (see Materials and Methods). The clones formed were stained with crystal violet after fixing (A) and counted manually (B) . (B) The number of colonies were reported to the NT condition. The data shown are representative (A) or averaged (B) from three independent experiments (in biological triplicate) (±s.e.m. and ordinary one-way ANOVA with Dunnett’s multiple comparison tests to the “NT” condition). (C) Proliferation of MG-63 cells treated with 3.6 μM H151 was measured by xCELLigence real-time monitoring over 48 h of treatment. Cell proliferation slopes were calculated and normalized to the NT condition. Data shown are averaged from three independent experiments in biological triplicate (±s.e.m. and Mann-Whitney U tests are shown). (D) MG-63 cells were treated or not with 3.6 μM H151 for 48 h, and cell lysates were processed for RNA purification and RT-qPCR analyses. Expression of the indicated genes was reported relative to 18S expression. Data shown are averaged from three independent experiments in biological duplicate and normalized to the NT condition (±s.e.m. and ordinary two-way ANOVA with Sidak’s multiple comparison tests). (E) Relative confluency of MG-63 cells with or without (NT) increasing concentrations of recombinant IFN (12, 120, 2000 IU/ ml) was assessed over 48 h with Incucyte. Data shown are averaged from 6 wells per condition, and trends are representative of three independent experiments. (F) PC-3, SK-OV-3 and BT-549 cells were treated with CPT for 24 h with or without 1 μM SCH, and IL-6 levels in supernatants were determined by ELISA. IL-6 levels were normalized to the “CPT only” condition and are shown as percentages. Data shown are averaged from a minimum of three independent experiments in biological replicate (±s.e.m. and Mann-Whitney U tests are shown). * p ≤0.05, ** p ≤0.01, *** p ≤0.001, **** p ≤0.0001 and “ns” is non-significant.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Pharmacological Targeting of STING-Dependent IL-6 Production in Cancer Cells

    doi: 10.3389/fcell.2021.709618

    Figure Lengend Snippet: (A, B) HOS, MG-63 and TC-1 cells were plated at low density and treated with 3.6 μM H151, 3 μM of the p38 inhibitor SB202190 [SB] or 1 μM of the ERK1/2 inhibitor SCH772984 [SCH] for 10, 12, and 7 days, respectively (see Materials and Methods). The clones formed were stained with crystal violet after fixing (A) and counted manually (B) . (B) The number of colonies were reported to the NT condition. The data shown are representative (A) or averaged (B) from three independent experiments (in biological triplicate) (±s.e.m. and ordinary one-way ANOVA with Dunnett’s multiple comparison tests to the “NT” condition). (C) Proliferation of MG-63 cells treated with 3.6 μM H151 was measured by xCELLigence real-time monitoring over 48 h of treatment. Cell proliferation slopes were calculated and normalized to the NT condition. Data shown are averaged from three independent experiments in biological triplicate (±s.e.m. and Mann-Whitney U tests are shown). (D) MG-63 cells were treated or not with 3.6 μM H151 for 48 h, and cell lysates were processed for RNA purification and RT-qPCR analyses. Expression of the indicated genes was reported relative to 18S expression. Data shown are averaged from three independent experiments in biological duplicate and normalized to the NT condition (±s.e.m. and ordinary two-way ANOVA with Sidak’s multiple comparison tests). (E) Relative confluency of MG-63 cells with or without (NT) increasing concentrations of recombinant IFN (12, 120, 2000 IU/ ml) was assessed over 48 h with Incucyte. Data shown are averaged from 6 wells per condition, and trends are representative of three independent experiments. (F) PC-3, SK-OV-3 and BT-549 cells were treated with CPT for 24 h with or without 1 μM SCH, and IL-6 levels in supernatants were determined by ELISA. IL-6 levels were normalized to the “CPT only” condition and are shown as percentages. Data shown are averaged from a minimum of three independent experiments in biological replicate (±s.e.m. and Mann-Whitney U tests are shown). * p ≤0.05, ** p ≤0.01, *** p ≤0.001, **** p ≤0.0001 and “ns” is non-significant.

    Article Snippet: PC-3 cells purchased from ATCC (#CRL-1435) and BT-549 breast ductal carcinoma cells (a kind gift from Prof S. Lakhani) were grown in Roswell Park Memorial Institute (RPMI) 1,640 plus L-glutamine medium (Life Technologies) complemented with 1x antibiotic/antimycotic and 10% heat inactivated fetal bovine serum (referred to as complete RPMI).

    Techniques: Clone Assay, Staining, MANN-WHITNEY, Purification, Quantitative RT-PCR, Expressing, Recombinant, Enzyme-linked Immunosorbent Assay

    A. Apoptosis was induced in HeLa and immortalized MEFs using STS for the indicated times, and endogenous Bcl-2 was detected by Western blot analysis. B. Apoptosis was induced in HeLa, BT-549 and COS-7 cells with etoposide. NT (No Treatment). In primary MEFs apoptosis was induced with 100 µM etoposide for 5 h. A decrease in endogenous Bcl-2 levels was seen upon treatment with both STS and etoposide. C. Apoptosis was induced in HeLa cells using STS in the presence or absence of 20 µM of MG132. Western and densitometry analyses revealed decreased levels of Bcl-2 with STS treatment, and stabilization of Bcl-2 upon MG132 treatment. This suggests that Bcl-2 levels are down-regulated via the UPS. D. WT MEFs and HeLa cells were transiently transfected with Bcl-2, XIAP and ubiquitin and treated with 20 µM MG132 for 6 h and with 1.75 µM STS. IP with anti-Bcl-2 was followed by Western Blotting with anti-ubiquitin antibodies. *Represents the IG heavy chain. Poly-ubiquitylated forms of Bcl-2 appeared in apoptotic cells and correlated with decreased Bcl-2 levels.

    Journal: Cell reports

    Article Title: Degradation of Bcl-2 by XIAP and ARTS promotes apoptosis

    doi: 10.1016/j.celrep.2017.09.052

    Figure Lengend Snippet: A. Apoptosis was induced in HeLa and immortalized MEFs using STS for the indicated times, and endogenous Bcl-2 was detected by Western blot analysis. B. Apoptosis was induced in HeLa, BT-549 and COS-7 cells with etoposide. NT (No Treatment). In primary MEFs apoptosis was induced with 100 µM etoposide for 5 h. A decrease in endogenous Bcl-2 levels was seen upon treatment with both STS and etoposide. C. Apoptosis was induced in HeLa cells using STS in the presence or absence of 20 µM of MG132. Western and densitometry analyses revealed decreased levels of Bcl-2 with STS treatment, and stabilization of Bcl-2 upon MG132 treatment. This suggests that Bcl-2 levels are down-regulated via the UPS. D. WT MEFs and HeLa cells were transiently transfected with Bcl-2, XIAP and ubiquitin and treated with 20 µM MG132 for 6 h and with 1.75 µM STS. IP with anti-Bcl-2 was followed by Western Blotting with anti-ubiquitin antibodies. *Represents the IG heavy chain. Poly-ubiquitylated forms of Bcl-2 appeared in apoptotic cells and correlated with decreased Bcl-2 levels.

    Article Snippet: To induce apoptosis COS-7, HeLa, MEFs, BT-549 (human breast epithelial carcinoma) cells were incubated with staurosporine (STS) (Sigma-Aldrich) (1.75 µM for HeLa, 0.6 µM for BT-549, 1.75 µM for MEFs and 1.25µM for COS-7) for different time periods or with Etoposide (Sigma Aldrich) (200µM for HeLa, BT-549, COS-7, and immortalized MEFs and 100µM for primary MEFs) for different time periods.

    Techniques: Western Blot, Transfection

    AI. HeLa ARTS knockdown (ARTS KD) cells and Sept4/ARTS KO MEFs show significantly higher levels of steady-state Bcl-2 protein when compared with WT cells. This suggests that ARTS plays an important role in regulating Bcl-2 levels. B. WT and ARTS KD HeLa cells were treated with 1.75 µM STS. Western Blot analyses demonstrate that while decreased Bcl-2 levels were seen in apoptotic WT HeLa cells, Bcl-2 levels in ARTS KD HeLa cells remained unchanged. C. Western Blot analyses of cytosolic fractions of BT-549, HeLa WT and HeLa ARTS KD cells reveal that endogenous Bcl-2 is found in the cytosol of WT STS-treated cells. In contrast, a strong inhibition in translocation of Bcl-2 to the cytosol was seen in ARTS KD HeLa cells. This suggests that ARTS is required for the proper translocation of Bcl-2 from mitochondria to the cytosol upon apoptotic induction. D. Immuno-fluorescence (IF) was performed on HeLa and a stable Bcl-2 knockdown (Bcl-2 KD) cells. The fraction of cells with cytosolic staining of ARTS is represented in the bar chart. While only a small portion of WT untreated (NT) HeLa cells show the presence of ARTS in the cytosol, a significant increase in cells containing cytosolic ARTS was seen following STS treatment. In contrast, the majority of HeLa Bcl-2 KD NT cells exhibit cytosolic ARTS (four fold higher than WT HeLa cells), and, only a slight increase in cells with cytosolic ARTS is seen after STS treatment. (* * p-value ≤ 0.01). See also supplemental Figure S1. E. Cytosolic and mitochondrial fractions of WT MEFs and Bcl-2 KO MEFs were analyzed by WB analysis with COX IV as a mitochondrial and GAPDH as a cytosolic marker. In Bcl-2 KO MEFs, the majority of ARTS was in the cytosol. This suggests that Bcl-2 is involved in localizing ARTS to mitochondria. F. IF of WT MEFs and Sept4/ARTS KO MEFs transiently transfected with GFP-Bcl-2. Cellular localization of Bcl-2 was quantified and the fraction of cells with cytosolic Bcl-2 is shown in the bar charts. While a significant increase in cytosolic Bcl-2 was seen in apoptotic WT MEFs, the levels of cytosolic Bcl-2 in Sept4/ARTS KO MEFs remained unchanged. See also supplemental Figure S1. G. Subcellular fractionation of HeLa cells was followed by in vivo ubiquitylation of each fraction. IgG represents the control cells incubated with non-specific IgG. Poly-ubiquitylated forms of Bcl-2 were seen only in the cytosolic fraction. * Represents the IG heavy chain. This indicates that ubiquitylation of Bcl-2 occurs in the cytosol and that ARTS is required for translocation of Bcl-2 to the cytosol during apoptosis.

    Journal: Cell reports

    Article Title: Degradation of Bcl-2 by XIAP and ARTS promotes apoptosis

    doi: 10.1016/j.celrep.2017.09.052

    Figure Lengend Snippet: AI. HeLa ARTS knockdown (ARTS KD) cells and Sept4/ARTS KO MEFs show significantly higher levels of steady-state Bcl-2 protein when compared with WT cells. This suggests that ARTS plays an important role in regulating Bcl-2 levels. B. WT and ARTS KD HeLa cells were treated with 1.75 µM STS. Western Blot analyses demonstrate that while decreased Bcl-2 levels were seen in apoptotic WT HeLa cells, Bcl-2 levels in ARTS KD HeLa cells remained unchanged. C. Western Blot analyses of cytosolic fractions of BT-549, HeLa WT and HeLa ARTS KD cells reveal that endogenous Bcl-2 is found in the cytosol of WT STS-treated cells. In contrast, a strong inhibition in translocation of Bcl-2 to the cytosol was seen in ARTS KD HeLa cells. This suggests that ARTS is required for the proper translocation of Bcl-2 from mitochondria to the cytosol upon apoptotic induction. D. Immuno-fluorescence (IF) was performed on HeLa and a stable Bcl-2 knockdown (Bcl-2 KD) cells. The fraction of cells with cytosolic staining of ARTS is represented in the bar chart. While only a small portion of WT untreated (NT) HeLa cells show the presence of ARTS in the cytosol, a significant increase in cells containing cytosolic ARTS was seen following STS treatment. In contrast, the majority of HeLa Bcl-2 KD NT cells exhibit cytosolic ARTS (four fold higher than WT HeLa cells), and, only a slight increase in cells with cytosolic ARTS is seen after STS treatment. (* * p-value ≤ 0.01). See also supplemental Figure S1. E. Cytosolic and mitochondrial fractions of WT MEFs and Bcl-2 KO MEFs were analyzed by WB analysis with COX IV as a mitochondrial and GAPDH as a cytosolic marker. In Bcl-2 KO MEFs, the majority of ARTS was in the cytosol. This suggests that Bcl-2 is involved in localizing ARTS to mitochondria. F. IF of WT MEFs and Sept4/ARTS KO MEFs transiently transfected with GFP-Bcl-2. Cellular localization of Bcl-2 was quantified and the fraction of cells with cytosolic Bcl-2 is shown in the bar charts. While a significant increase in cytosolic Bcl-2 was seen in apoptotic WT MEFs, the levels of cytosolic Bcl-2 in Sept4/ARTS KO MEFs remained unchanged. See also supplemental Figure S1. G. Subcellular fractionation of HeLa cells was followed by in vivo ubiquitylation of each fraction. IgG represents the control cells incubated with non-specific IgG. Poly-ubiquitylated forms of Bcl-2 were seen only in the cytosolic fraction. * Represents the IG heavy chain. This indicates that ubiquitylation of Bcl-2 occurs in the cytosol and that ARTS is required for translocation of Bcl-2 to the cytosol during apoptosis.

    Article Snippet: To induce apoptosis COS-7, HeLa, MEFs, BT-549 (human breast epithelial carcinoma) cells were incubated with staurosporine (STS) (Sigma-Aldrich) (1.75 µM for HeLa, 0.6 µM for BT-549, 1.75 µM for MEFs and 1.25µM for COS-7) for different time periods or with Etoposide (Sigma Aldrich) (200µM for HeLa, BT-549, COS-7, and immortalized MEFs and 100µM for primary MEFs) for different time periods.

    Techniques: Western Blot, Inhibition, Translocation Assay, Fluorescence, Staining, Marker, Transfection, Fractionation, In Vivo, Incubation

    AI. IP of ARTS in COS-7 cells transiently transfected with 6-Myc-ARTS using a monoclonal anti-ARTS antibody (Sigma-Aldrich). Endogenous ARTS was immunoprecipitated from HeLa and BT-549 cells. Western blot analyses show that in COS-7, HeLa and BT-549 cells, Bcl-2 and XIAP co- precipitate with ARTS. AII. HeLa cells were treated with 1.75 µM STS and IP of ARTS was performed as described in AI. While binding of ARTS to Bcl-2 was seen in non-treated cells (NT), binding of ARTS to XIAP and Bcl-2 is increased in STS treated cells. B. Recombinant His-ARTS, Bcl-2 and GST-XIAP were incubated overnight at 4 °C. Pulldown of GST-XIAP shows that binding of Bcl-2 to XIAP depends on ARTS. C. To assess proximity of proteins, consistent with complex formation, we used BiFC. HeLa cells were transiently transfected with ARTS, Bcl-2 and XIAP fused to parts of YFP YFP-Venus. Jun and bFos, known to form heterodimers, served as a positive control (p.c.). Jun and bFosdelZIP lacking the carboxyl-terminal half of the bFos were used as a negative control (n.c.). The fluorescent signal indicating the proximity of each pair of proteins was measured by flow cytometry. Mean fluorescence intensity (MFI). FACS results were normalized to the readings of transfection efficiency reporter (pdsRED). The values represent mean values ± SE of three independent experiments (*, p ≤ 0.05; **, p ≤ 0.01). The Y-axis represents the ratio between YFP fluorescence (reflecting binding of a pair of proteins) and the red fluorescence (marking the transfected cells). FACS analyses reveal that ARTS can bind to both Bcl-2 and to XIAP. Yet, only background levels of fluorescence were seen with XIAP and Bcl2, suggesting that these two proteins do not bind each other. These results indicate that ARTS, XIAP and Bcl-2 form a ternary complex, and that ARTS is required for the formation of this complex. See also supplemental Figure S2.

    Journal: Cell reports

    Article Title: Degradation of Bcl-2 by XIAP and ARTS promotes apoptosis

    doi: 10.1016/j.celrep.2017.09.052

    Figure Lengend Snippet: AI. IP of ARTS in COS-7 cells transiently transfected with 6-Myc-ARTS using a monoclonal anti-ARTS antibody (Sigma-Aldrich). Endogenous ARTS was immunoprecipitated from HeLa and BT-549 cells. Western blot analyses show that in COS-7, HeLa and BT-549 cells, Bcl-2 and XIAP co- precipitate with ARTS. AII. HeLa cells were treated with 1.75 µM STS and IP of ARTS was performed as described in AI. While binding of ARTS to Bcl-2 was seen in non-treated cells (NT), binding of ARTS to XIAP and Bcl-2 is increased in STS treated cells. B. Recombinant His-ARTS, Bcl-2 and GST-XIAP were incubated overnight at 4 °C. Pulldown of GST-XIAP shows that binding of Bcl-2 to XIAP depends on ARTS. C. To assess proximity of proteins, consistent with complex formation, we used BiFC. HeLa cells were transiently transfected with ARTS, Bcl-2 and XIAP fused to parts of YFP YFP-Venus. Jun and bFos, known to form heterodimers, served as a positive control (p.c.). Jun and bFosdelZIP lacking the carboxyl-terminal half of the bFos were used as a negative control (n.c.). The fluorescent signal indicating the proximity of each pair of proteins was measured by flow cytometry. Mean fluorescence intensity (MFI). FACS results were normalized to the readings of transfection efficiency reporter (pdsRED). The values represent mean values ± SE of three independent experiments (*, p ≤ 0.05; **, p ≤ 0.01). The Y-axis represents the ratio between YFP fluorescence (reflecting binding of a pair of proteins) and the red fluorescence (marking the transfected cells). FACS analyses reveal that ARTS can bind to both Bcl-2 and to XIAP. Yet, only background levels of fluorescence were seen with XIAP and Bcl2, suggesting that these two proteins do not bind each other. These results indicate that ARTS, XIAP and Bcl-2 form a ternary complex, and that ARTS is required for the formation of this complex. See also supplemental Figure S2.

    Article Snippet: To induce apoptosis COS-7, HeLa, MEFs, BT-549 (human breast epithelial carcinoma) cells were incubated with staurosporine (STS) (Sigma-Aldrich) (1.75 µM for HeLa, 0.6 µM for BT-549, 1.75 µM for MEFs and 1.25µM for COS-7) for different time periods or with Etoposide (Sigma Aldrich) (200µM for HeLa, BT-549, COS-7, and immortalized MEFs and 100µM for primary MEFs) for different time periods.

    Techniques: Transfection, Immunoprecipitation, Western Blot, Binding Assay, Recombinant, Incubation, Positive Control, Negative Control, Flow Cytometry, Fluorescence