Structured Review

Enzo Biochem necrostatin
Ivermectin kills breast cancer cells through a mixed apoptotic and necrotic mechanism. ( A ) Mouse (4T1.2) and human (MDA-MB-231) TNBC cells manifest similar sensitivity to Ivermectin. Viability of cells treated with various doses of Ivermectin for 24 h. ( B ) Extended exposure time reduces IC50 values to as low as 2 μM. 4T1.2 cells were seeded at 100 cells/well and individual colonies were counted after a week. Cancer cells were exposed to Ivermectin during the initial 24 h or during the entire duration of the assay. ( C ) MDA-MB-231 breast cancer cells manifest higher sensitivity to Ivermectin compared to normal non-transformed human foreskin fibroblasts (HFFs). ( D ) Flow cytometry analysis showing that cell death proceeds through two distinct pathways: a directly necrotic 7AAD-single positive or Annexin V/PS-single positive apoptotic pathway. ( E ) Kinetics of necrotic versus apoptotic killing of 4T1.2 breast cancer cells. ( F ) Ivermectin-induced cell death can be reversed by inhibition of various controlled cell death pathways. 4T1.2 cells were treated for 4 h with 32 μM Ivermectin in the presence of μM concentrations of Z-vad-fmk, <t>Necrostatin-1,</t> Digoxin, or VX-765, as indicated. ( G ) Activation of Caspase-1, Caspase-3 and cleavage of PARP in 4T1.2 and MDA-MB-231 cells treated with 32 μM for 4h. Asterisk (*) indicates p
Necrostatin, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/necrostatin/product/Enzo Biochem
Average 94 stars, based on 3 article reviews
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necrostatin - by Bioz Stars, 2022-08
94/100 stars

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1) Product Images from "Modulation of P2X 4/P2X7/Pannexin-1 sensitivity to extracellular ATP via Ivermectin induces a non-apoptotic and inflammatory form of cancer cell death"

Article Title: Modulation of P2X 4/P2X7/Pannexin-1 sensitivity to extracellular ATP via Ivermectin induces a non-apoptotic and inflammatory form of cancer cell death

Journal: Scientific Reports

doi: 10.1038/srep16222

Ivermectin kills breast cancer cells through a mixed apoptotic and necrotic mechanism. ( A ) Mouse (4T1.2) and human (MDA-MB-231) TNBC cells manifest similar sensitivity to Ivermectin. Viability of cells treated with various doses of Ivermectin for 24 h. ( B ) Extended exposure time reduces IC50 values to as low as 2 μM. 4T1.2 cells were seeded at 100 cells/well and individual colonies were counted after a week. Cancer cells were exposed to Ivermectin during the initial 24 h or during the entire duration of the assay. ( C ) MDA-MB-231 breast cancer cells manifest higher sensitivity to Ivermectin compared to normal non-transformed human foreskin fibroblasts (HFFs). ( D ) Flow cytometry analysis showing that cell death proceeds through two distinct pathways: a directly necrotic 7AAD-single positive or Annexin V/PS-single positive apoptotic pathway. ( E ) Kinetics of necrotic versus apoptotic killing of 4T1.2 breast cancer cells. ( F ) Ivermectin-induced cell death can be reversed by inhibition of various controlled cell death pathways. 4T1.2 cells were treated for 4 h with 32 μM Ivermectin in the presence of μM concentrations of Z-vad-fmk, Necrostatin-1, Digoxin, or VX-765, as indicated. ( G ) Activation of Caspase-1, Caspase-3 and cleavage of PARP in 4T1.2 and MDA-MB-231 cells treated with 32 μM for 4h. Asterisk (*) indicates p
Figure Legend Snippet: Ivermectin kills breast cancer cells through a mixed apoptotic and necrotic mechanism. ( A ) Mouse (4T1.2) and human (MDA-MB-231) TNBC cells manifest similar sensitivity to Ivermectin. Viability of cells treated with various doses of Ivermectin for 24 h. ( B ) Extended exposure time reduces IC50 values to as low as 2 μM. 4T1.2 cells were seeded at 100 cells/well and individual colonies were counted after a week. Cancer cells were exposed to Ivermectin during the initial 24 h or during the entire duration of the assay. ( C ) MDA-MB-231 breast cancer cells manifest higher sensitivity to Ivermectin compared to normal non-transformed human foreskin fibroblasts (HFFs). ( D ) Flow cytometry analysis showing that cell death proceeds through two distinct pathways: a directly necrotic 7AAD-single positive or Annexin V/PS-single positive apoptotic pathway. ( E ) Kinetics of necrotic versus apoptotic killing of 4T1.2 breast cancer cells. ( F ) Ivermectin-induced cell death can be reversed by inhibition of various controlled cell death pathways. 4T1.2 cells were treated for 4 h with 32 μM Ivermectin in the presence of μM concentrations of Z-vad-fmk, Necrostatin-1, Digoxin, or VX-765, as indicated. ( G ) Activation of Caspase-1, Caspase-3 and cleavage of PARP in 4T1.2 and MDA-MB-231 cells treated with 32 μM for 4h. Asterisk (*) indicates p

Techniques Used: Multiple Displacement Amplification, Transformation Assay, Flow Cytometry, Cytometry, Inhibition, Activation Assay

2) Product Images from "Persistent Mitochondrial Hyperfusion Promotes G2/M Accumulation and Caspase-Dependent Cell Death"

Article Title: Persistent Mitochondrial Hyperfusion Promotes G2/M Accumulation and Caspase-Dependent Cell Death

Journal: PLoS ONE

doi: 10.1371/journal.pone.0091911

Mitochondrial Hyperfusion Promotes Caspase 8 Dependent Cell Death. (A-C) U2OS cells were transfected with control (black), Drp1 (green), or Mff (red) siRNA for 96 hours. Cells were collected and stained for (A) cleaved caspase 3 or (B) cleaved caspase 8. Positivity of cleaved caspase 3 or 8 is indicated by an increase in fluorescence on the FACS histogram compared to control. (C) U2OS cells transfected with control, Drp1, or Mff siRNAs were treated with pan caspase inhibitor, zVAD (20 μM; dark gray bars), or the necroptosis inhibitor, necrostatin (10 μM; light gray bars), or vehicle (DMSO; black bars) every 24 hours for 96 hours following transfection. Cells were stained with propidium iodide (PI) and percent PI positivity was used as a marker of cell death. Error bars represent standard deviation from two replicate experiments where at least 10,000 events were collected for each treatment.
Figure Legend Snippet: Mitochondrial Hyperfusion Promotes Caspase 8 Dependent Cell Death. (A-C) U2OS cells were transfected with control (black), Drp1 (green), or Mff (red) siRNA for 96 hours. Cells were collected and stained for (A) cleaved caspase 3 or (B) cleaved caspase 8. Positivity of cleaved caspase 3 or 8 is indicated by an increase in fluorescence on the FACS histogram compared to control. (C) U2OS cells transfected with control, Drp1, or Mff siRNAs were treated with pan caspase inhibitor, zVAD (20 μM; dark gray bars), or the necroptosis inhibitor, necrostatin (10 μM; light gray bars), or vehicle (DMSO; black bars) every 24 hours for 96 hours following transfection. Cells were stained with propidium iodide (PI) and percent PI positivity was used as a marker of cell death. Error bars represent standard deviation from two replicate experiments where at least 10,000 events were collected for each treatment.

Techniques Used: Transfection, Staining, Fluorescence, FACS, Marker, Standard Deviation

3) Product Images from "Altered Mitochondria Morphology and Cell Metabolism in Apaf1-Deficient Cells"

Article Title: Altered Mitochondria Morphology and Cell Metabolism in Apaf1-Deficient Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0084666

Caspase-independent cell death in SV40IM Apaf1 KO cells. (A) Cell survival was measured by the trypan blue exclusion assay in SV40IM and SIM MEFS, WT and Apaf1-depleted cells in the presence of etoposide (5 µM) for 24 h. (B) Caspase-3-like activity was measured under the same etoposide treatment conditions (+; 5 µM) described above. In all cases, bars represent the mean of three experiments ± s.d. (C) Cell survival was measured in the SV40IM cell lines treated with etoposide (5 µM) in the presence or the absence of z-VAD (5 µM), necrostatin (Nec; 100 µM) or SVT016426 (10 µM). (mean ± s.d, n = 3, * p ≤0.05). (D) DAPI staining to analyse the apoptotic features between WT and Apaf1 KO cells in the presence of etoposide (5 µM). In all, 500 nuclei were counted and classified according to apoptotic nuclear bodies (white arrows). Quantification is shown in the right panel. (E) Cell survival was measured by the trypan blue exclusion assay in the HeLa cells transfected with random siRNA (Rsi) or Apaf1 siRNA (Asi) for 24 h and treated with etoposide (+; 5 µM) for another 24-hour period. (F) Caspase-3-like activity was measured under the same conditions described above. Bars represent the mean of three experiments ± s.d. The immunoblotting of the Apaf1 silencer is shown in the right panel.
Figure Legend Snippet: Caspase-independent cell death in SV40IM Apaf1 KO cells. (A) Cell survival was measured by the trypan blue exclusion assay in SV40IM and SIM MEFS, WT and Apaf1-depleted cells in the presence of etoposide (5 µM) for 24 h. (B) Caspase-3-like activity was measured under the same etoposide treatment conditions (+; 5 µM) described above. In all cases, bars represent the mean of three experiments ± s.d. (C) Cell survival was measured in the SV40IM cell lines treated with etoposide (5 µM) in the presence or the absence of z-VAD (5 µM), necrostatin (Nec; 100 µM) or SVT016426 (10 µM). (mean ± s.d, n = 3, * p ≤0.05). (D) DAPI staining to analyse the apoptotic features between WT and Apaf1 KO cells in the presence of etoposide (5 µM). In all, 500 nuclei were counted and classified according to apoptotic nuclear bodies (white arrows). Quantification is shown in the right panel. (E) Cell survival was measured by the trypan blue exclusion assay in the HeLa cells transfected with random siRNA (Rsi) or Apaf1 siRNA (Asi) for 24 h and treated with etoposide (+; 5 µM) for another 24-hour period. (F) Caspase-3-like activity was measured under the same conditions described above. Bars represent the mean of three experiments ± s.d. The immunoblotting of the Apaf1 silencer is shown in the right panel.

Techniques Used: Trypan Blue Exclusion Assay, Activity Assay, Staining, Transfection

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    Enzo Biochem necrostatin 1
    The effects of FAD on colorectal cancer cell lines. ( a and b ) Human colorectal cancer cells HCT116 and SW480 were treated with different concentrations of FAD for 48 h and cell viability was quantified. ( c ) Normal human colon epithelial cells FHC were treated with different concentrations of FAD for 48 h and cell viability was quantified. ( d and e ) HCT116 cells were treated with FAD for 48 h, and apoptosis was quantified by FACS after staining with Annexin V-FITC and propidium iodide. ( f ) Viability of HCT116 cells treated with 6 μ M FAD for 48 h in the presence or absence of 100 μ M Z-VAD-fmk. ( g ) Viability of HCT116 cells treated with 6 μ M FAD for 48 h in the presence or absence of 40 μ M <t>Necrostatin-1</t> (Nec-1). ( h ) Viability of HCT116 cells treated with 3 μ M FAD for 48 h in the presence or absence 20 μ M of chloroquine (CQ). Trypan blue staining was used to determine cell viability. ( i ) HCT116 cells were treated with 6 μ M FAD for the indicated periods, and LC3-I/II protein levels were determined by western blot analysis. ( j ) HCT116 cells expressing EGFP-LC3 were treated with 0 or 3 μ M FAD for 30 h and were imaged with a fluorescence microscope. Serum-free (SF) treatment was used as a positive control for autophagy induction. * P
    Necrostatin 1, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/necrostatin 1/product/Enzo Biochem
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    necrostatin 1 - by Bioz Stars, 2022-08
    94/100 stars
      Buy from Supplier

    94
    Enzo Biochem necroptosis inhibitor necrostatin 1
    DAMP release and monocyte attraction occur during secondary necrosis upon irradiation in combination with HSP90i treatment. (A) Induction of cell death, THP-1 cell attraction, and DAMP release in HCT116 subclones with or without functional Bax and/or hyperactive Kras G 13 D . Parental HCT116 Kras +/ G 13 D Bax +/+ cells (upper row), HCT116 Kras +/ G 13 D Bax –/– cells (middle row), and HCT116 Kras +/– Bax +/+ cells (lower panel) were treated with 5 Gy plus 625 nM HSP90i or left untreated. At the indicated time points, induction of apoptosis (first column) and necrosis (second column), THP-1 cell transwell migration (third column), and release of HSP70 (fourth column) and ATP (last column) were measured as in Figure 1 . Data points of n = 3–5 independent experiments (apoptosis, necrosis, transwell migration, ATP release) or means ± SD of triplicates of one representative experiment (HSP70 release) are shown. (B) Impact of different cell death inhibitors on apoptosis and necrosis induction, THP-1 cell attraction, and DAMP release in HCT116 cells upon treatment with radiotherapy and HSP90i. HCT116 cells were treated with 5 Gy plus 625 nM HSP90i in the presence of the poly-caspase inhibitor zVAD-fmk or the <t>necroptosis</t> inhibitor necrostatin-1, respectively (upper panel). Untreated cells served as controls (lower panel). Induction of apoptosis (first column) and necrosis (second column), THP-1 cell transwell migration (third column), and release of HSP70 (fourth column) and ATP (last column) were measured as in Figure 1 . Data points of n = 3–5 independent experiments (apoptosis, necrosis, transwell migration) or means ± SD of triplicates of one representative experiment (release of HSP70 and ATP) are shown.
    Necroptosis Inhibitor Necrostatin 1, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/necroptosis inhibitor necrostatin 1/product/Enzo Biochem
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    necroptosis inhibitor necrostatin 1 - by Bioz Stars, 2022-08
    94/100 stars
      Buy from Supplier

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    The effects of FAD on colorectal cancer cell lines. ( a and b ) Human colorectal cancer cells HCT116 and SW480 were treated with different concentrations of FAD for 48 h and cell viability was quantified. ( c ) Normal human colon epithelial cells FHC were treated with different concentrations of FAD for 48 h and cell viability was quantified. ( d and e ) HCT116 cells were treated with FAD for 48 h, and apoptosis was quantified by FACS after staining with Annexin V-FITC and propidium iodide. ( f ) Viability of HCT116 cells treated with 6 μ M FAD for 48 h in the presence or absence of 100 μ M Z-VAD-fmk. ( g ) Viability of HCT116 cells treated with 6 μ M FAD for 48 h in the presence or absence of 40 μ M Necrostatin-1 (Nec-1). ( h ) Viability of HCT116 cells treated with 3 μ M FAD for 48 h in the presence or absence 20 μ M of chloroquine (CQ). Trypan blue staining was used to determine cell viability. ( i ) HCT116 cells were treated with 6 μ M FAD for the indicated periods, and LC3-I/II protein levels were determined by western blot analysis. ( j ) HCT116 cells expressing EGFP-LC3 were treated with 0 or 3 μ M FAD for 30 h and were imaged with a fluorescence microscope. Serum-free (SF) treatment was used as a positive control for autophagy induction. * P

    Journal: Cell Death & Disease

    Article Title: The antitumor natural compound falcarindiol promotes cancer cell death by inducing endoplasmic reticulum stress

    doi: 10.1038/cddis.2012.122

    Figure Lengend Snippet: The effects of FAD on colorectal cancer cell lines. ( a and b ) Human colorectal cancer cells HCT116 and SW480 were treated with different concentrations of FAD for 48 h and cell viability was quantified. ( c ) Normal human colon epithelial cells FHC were treated with different concentrations of FAD for 48 h and cell viability was quantified. ( d and e ) HCT116 cells were treated with FAD for 48 h, and apoptosis was quantified by FACS after staining with Annexin V-FITC and propidium iodide. ( f ) Viability of HCT116 cells treated with 6 μ M FAD for 48 h in the presence or absence of 100 μ M Z-VAD-fmk. ( g ) Viability of HCT116 cells treated with 6 μ M FAD for 48 h in the presence or absence of 40 μ M Necrostatin-1 (Nec-1). ( h ) Viability of HCT116 cells treated with 3 μ M FAD for 48 h in the presence or absence 20 μ M of chloroquine (CQ). Trypan blue staining was used to determine cell viability. ( i ) HCT116 cells were treated with 6 μ M FAD for the indicated periods, and LC3-I/II protein levels were determined by western blot analysis. ( j ) HCT116 cells expressing EGFP-LC3 were treated with 0 or 3 μ M FAD for 30 h and were imaged with a fluorescence microscope. Serum-free (SF) treatment was used as a positive control for autophagy induction. * P

    Article Snippet: Chemicals and reagents Z-VAD-fmk and Necrostatin-1 were obtained from Enzo Life Sciences (Farmingdale, NY, USA).

    Techniques: FACS, Staining, Western Blot, Expressing, Fluorescence, Microscopy, Positive Control

    Necrostatin-1 attenuates cell death by necroptosis resulting from either Plk1 depletion or Plk1 inhibition in LNCaP-AI cells. ( a ) LNCaP-AI cells were transfected with either siLuciferase or Plk1 RNAi oligos for 5 days. Cell lysates (20 μg) were analyzed for Plk1 levels. α-tubulin was used as a loading control. ( b ) Cells prepared as in (a) or treated with 0.8 nM BI2536 were analyzed after 5 days by an MTS assay. Triplicate samples (mean ± SD) from one of n = 2 experiments are shown. ( c ) Cells were treated as in (b) and counterstained with DAPI to visualize DNA, shown here in black and white for contrast. Arrows, cells in mitosis in control cultures. Single giant cells containing clusters of nuclear vesicles are shown for siPlk1 and BI2536-treated LNCaP-AI cells. Bars, 10 μm. Note differences in scale. ( d ) Cells were transfected with siPlk1 for 5 days. 7.7 μM Necrostatin-1 (Nec-1), the necroptosis inhibitor, was added 3 h prior to siPlk1 transfection and remained throughout the experiment. Triplicate samples (mean ± SD) from MTS assays from one of n = 2 experiments are shown. ( e ) Cells were treated with 0.4 or 0.8 nM BI2536 for 5 days. 5 μM Nec-1 was added 3 h prior to BI2536 addition and remained throughout the experiment. Triplicate samples (mean ± SD) from MTS assays from one of n = 3 experiments are shown. ( f ) Working model of necroptosis induction by Plk1 inhibition in androgen-insensitive PCa cells. See text.

    Journal: Oncogene

    Article Title: Plk1 is upregulated in androgen-insensitive prostate cancer cells and its inhibition leads to necroptosis

    doi: 10.1038/onc.2012.309

    Figure Lengend Snippet: Necrostatin-1 attenuates cell death by necroptosis resulting from either Plk1 depletion or Plk1 inhibition in LNCaP-AI cells. ( a ) LNCaP-AI cells were transfected with either siLuciferase or Plk1 RNAi oligos for 5 days. Cell lysates (20 μg) were analyzed for Plk1 levels. α-tubulin was used as a loading control. ( b ) Cells prepared as in (a) or treated with 0.8 nM BI2536 were analyzed after 5 days by an MTS assay. Triplicate samples (mean ± SD) from one of n = 2 experiments are shown. ( c ) Cells were treated as in (b) and counterstained with DAPI to visualize DNA, shown here in black and white for contrast. Arrows, cells in mitosis in control cultures. Single giant cells containing clusters of nuclear vesicles are shown for siPlk1 and BI2536-treated LNCaP-AI cells. Bars, 10 μm. Note differences in scale. ( d ) Cells were transfected with siPlk1 for 5 days. 7.7 μM Necrostatin-1 (Nec-1), the necroptosis inhibitor, was added 3 h prior to siPlk1 transfection and remained throughout the experiment. Triplicate samples (mean ± SD) from MTS assays from one of n = 2 experiments are shown. ( e ) Cells were treated with 0.4 or 0.8 nM BI2536 for 5 days. 5 μM Nec-1 was added 3 h prior to BI2536 addition and remained throughout the experiment. Triplicate samples (mean ± SD) from MTS assays from one of n = 3 experiments are shown. ( f ) Working model of necroptosis induction by Plk1 inhibition in androgen-insensitive PCa cells. See text.

    Article Snippet: Reagents used were as follows: Protease inhibitors, nocodazole, TNF-α, cycloheximide, rapamycin and insulin (Sigma-Aldrich); Q-VD-OPh (MP Biomedicals); necrostatin-1 (Enzo Life Sciences) and BI2536 (ChemieTek).

    Techniques: Inhibition, Transfection, MTS Assay

    Morphological changes in PANC-1 cells after treatment with GEF and macrolides. (A) May-Giemsa staining: May-Giemsa staining was performed after treatment with either GEF (25 μM) or gemcitabine (100 μM) in the presence or absence of EM900 (50 μM) for 48 h. Scale bars, 10 μm. Arrows indicate nuclear chromatin condensations. (B) Electron microscopy: PANC-1 cells were processed for electron microscopy after treatment with GEF (25 μM), AZM (50 μM), GEF+AZM, and gemcitabine (100 μM) for 48 h. Lower panels are at higher magnifications of the squared areas in each upper panel. Scale bars, 2 μm. (C) PANC-1 cells were treated with GEF (25 μM), AZM (50 μM), EM900 (50 μM), GEF+AZM, and gemcitabine (10 and 100 μM) for 48 h. Cellular proteins were separated by 15% SDS-PAGE for caspase-3 and 7.5% for PARP and immunoblotted with either anti-cleaved caspase-3 Ab or anti-PARP Ab. Cell lysate derived from HL-60 cells treated with vitamin K2 was used as a positive control for apoptosis induction (42). (D) PANC-1 cells were treated with various concentrations of either necrostatin-1 or Z-VAD-FMK in the presence or absence of GEF (50 μM), GEF (50 μM) plus EM900 (50 μM), or gem (300 μM) for 48 h. The number of viable cells was assessed using CellTiter Blue.

    Journal: International Journal of Oncology

    Article Title: Macrolides sensitize EGFR-TKI-induced non-apoptotic cell death via blocking autophagy flux in pancreatic cancer cell lines

    doi: 10.3892/ijo.2015.3237

    Figure Lengend Snippet: Morphological changes in PANC-1 cells after treatment with GEF and macrolides. (A) May-Giemsa staining: May-Giemsa staining was performed after treatment with either GEF (25 μM) or gemcitabine (100 μM) in the presence or absence of EM900 (50 μM) for 48 h. Scale bars, 10 μm. Arrows indicate nuclear chromatin condensations. (B) Electron microscopy: PANC-1 cells were processed for electron microscopy after treatment with GEF (25 μM), AZM (50 μM), GEF+AZM, and gemcitabine (100 μM) for 48 h. Lower panels are at higher magnifications of the squared areas in each upper panel. Scale bars, 2 μm. (C) PANC-1 cells were treated with GEF (25 μM), AZM (50 μM), EM900 (50 μM), GEF+AZM, and gemcitabine (10 and 100 μM) for 48 h. Cellular proteins were separated by 15% SDS-PAGE for caspase-3 and 7.5% for PARP and immunoblotted with either anti-cleaved caspase-3 Ab or anti-PARP Ab. Cell lysate derived from HL-60 cells treated with vitamin K2 was used as a positive control for apoptosis induction (42). (D) PANC-1 cells were treated with various concentrations of either necrostatin-1 or Z-VAD-FMK in the presence or absence of GEF (50 μM), GEF (50 μM) plus EM900 (50 μM), or gem (300 μM) for 48 h. The number of viable cells was assessed using CellTiter Blue.

    Article Snippet: Necrostatin-1, a specific inhibitor of RIPK1, was purchased from Enzo Life Sciences (Farmingdale, NY, USA).

    Techniques: Staining, Electron Microscopy, SDS Page, Derivative Assay, Positive Control

    DAMP release and monocyte attraction occur during secondary necrosis upon irradiation in combination with HSP90i treatment. (A) Induction of cell death, THP-1 cell attraction, and DAMP release in HCT116 subclones with or without functional Bax and/or hyperactive Kras G 13 D . Parental HCT116 Kras +/ G 13 D Bax +/+ cells (upper row), HCT116 Kras +/ G 13 D Bax –/– cells (middle row), and HCT116 Kras +/– Bax +/+ cells (lower panel) were treated with 5 Gy plus 625 nM HSP90i or left untreated. At the indicated time points, induction of apoptosis (first column) and necrosis (second column), THP-1 cell transwell migration (third column), and release of HSP70 (fourth column) and ATP (last column) were measured as in Figure 1 . Data points of n = 3–5 independent experiments (apoptosis, necrosis, transwell migration, ATP release) or means ± SD of triplicates of one representative experiment (HSP70 release) are shown. (B) Impact of different cell death inhibitors on apoptosis and necrosis induction, THP-1 cell attraction, and DAMP release in HCT116 cells upon treatment with radiotherapy and HSP90i. HCT116 cells were treated with 5 Gy plus 625 nM HSP90i in the presence of the poly-caspase inhibitor zVAD-fmk or the necroptosis inhibitor necrostatin-1, respectively (upper panel). Untreated cells served as controls (lower panel). Induction of apoptosis (first column) and necrosis (second column), THP-1 cell transwell migration (third column), and release of HSP70 (fourth column) and ATP (last column) were measured as in Figure 1 . Data points of n = 3–5 independent experiments (apoptosis, necrosis, transwell migration) or means ± SD of triplicates of one representative experiment (release of HSP70 and ATP) are shown.

    Journal: Frontiers in Oncology

    Article Title: Priming of Anti-tumor Immune Mechanisms by Radiotherapy Is Augmented by Inhibition of Heat Shock Protein 90

    doi: 10.3389/fonc.2020.01668

    Figure Lengend Snippet: DAMP release and monocyte attraction occur during secondary necrosis upon irradiation in combination with HSP90i treatment. (A) Induction of cell death, THP-1 cell attraction, and DAMP release in HCT116 subclones with or without functional Bax and/or hyperactive Kras G 13 D . Parental HCT116 Kras +/ G 13 D Bax +/+ cells (upper row), HCT116 Kras +/ G 13 D Bax –/– cells (middle row), and HCT116 Kras +/– Bax +/+ cells (lower panel) were treated with 5 Gy plus 625 nM HSP90i or left untreated. At the indicated time points, induction of apoptosis (first column) and necrosis (second column), THP-1 cell transwell migration (third column), and release of HSP70 (fourth column) and ATP (last column) were measured as in Figure 1 . Data points of n = 3–5 independent experiments (apoptosis, necrosis, transwell migration, ATP release) or means ± SD of triplicates of one representative experiment (HSP70 release) are shown. (B) Impact of different cell death inhibitors on apoptosis and necrosis induction, THP-1 cell attraction, and DAMP release in HCT116 cells upon treatment with radiotherapy and HSP90i. HCT116 cells were treated with 5 Gy plus 625 nM HSP90i in the presence of the poly-caspase inhibitor zVAD-fmk or the necroptosis inhibitor necrostatin-1, respectively (upper panel). Untreated cells served as controls (lower panel). Induction of apoptosis (first column) and necrosis (second column), THP-1 cell transwell migration (third column), and release of HSP70 (fourth column) and ATP (last column) were measured as in Figure 1 . Data points of n = 3–5 independent experiments (apoptosis, necrosis, transwell migration) or means ± SD of triplicates of one representative experiment (release of HSP70 and ATP) are shown.

    Article Snippet: For analyses of secondary necrosis and necroptosis, the poly-caspase inhibitor zVAD-fmk (50 μM; Bachem, Bubendorf, Switzerland) or the necroptosis inhibitor necrostatin-1 (50 μM; Enzo Life Sciences, Loerrach, Germany) were used in addition to irradiation and HSP90i treatment.

    Techniques: Irradiation, Functional Assay, Migration