Structured Review

Cell Signaling Technology Inc cleaved caspase 3
(A) β-Galactosidase activity assay; HCT116 cells were assayed for β-galactosidase activity after treatment with 20 μM cisplatin or TriplatinNC for 96 h. Shown is a representative of two independent experiments. (B) Confocal microscopy; HCT116 cells were treated with 20 μM TriplatinNC for 24 h. Left, top panel; DAPI stained DNA (blue). Right, top panel; cleaved <t>caspase-3</t> (yellow). Bottom panel; merged DAPI and cleaved caspase-3. Apoptotic cell (white star) containing condensed/fragmented DNA and active caspase-3. Cells with compacted DNA (white arrows) do not contain active caspase-3. (C) Summary; percentage of HCT116 cells undergoing the indicated cellular process after treatment with 20 μM cisplatin or TriplatinNC for 24 h. (D) Schematic; TriplatinNC localizes to the cytoplasm and nucleolus of interphase cells. Cells treated while in S-G 2 proceed through mitosis. During prophase, the DNA (blue) condenses and the nuclear membrane (red) disintegrates allowing cytoplasmic pools of TriplatinNC to interact with condensed DNA. Cells undergo cytokinesis; however, the DNA does not decondense or progress through G 1 . (a) Determined by immunofluorescence: β-tubulin, nucleophosmin/B23, and DAPI DNA staining (Figure 5 A,B). (b) Determined by β-galactosidase staining and light microscopy at 200× magnification (panel A). (c) Determined by immunofluorescence; cleaved caspase-3; and DAPI DNA staining. The percentage of cells undergoing apoptosis was determined as the number of cells positive for cleaved caspase-3 divided by the total number of cells. n > 500 cells each for two repeat experiments (Figure 5 B).
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Images

1) Product Images from "Nucleolar Targeting by Platinum: p53-Independent Apoptosis Follows rRNA Inhibition, Cell-Cycle Arrest, and DNA Compaction"

Article Title: Nucleolar Targeting by Platinum: p53-Independent Apoptosis Follows rRNA Inhibition, Cell-Cycle Arrest, and DNA Compaction

Journal: Molecular Pharmaceutics

doi: 10.1021/mp5006867

(A) β-Galactosidase activity assay; HCT116 cells were assayed for β-galactosidase activity after treatment with 20 μM cisplatin or TriplatinNC for 96 h. Shown is a representative of two independent experiments. (B) Confocal microscopy; HCT116 cells were treated with 20 μM TriplatinNC for 24 h. Left, top panel; DAPI stained DNA (blue). Right, top panel; cleaved caspase-3 (yellow). Bottom panel; merged DAPI and cleaved caspase-3. Apoptotic cell (white star) containing condensed/fragmented DNA and active caspase-3. Cells with compacted DNA (white arrows) do not contain active caspase-3. (C) Summary; percentage of HCT116 cells undergoing the indicated cellular process after treatment with 20 μM cisplatin or TriplatinNC for 24 h. (D) Schematic; TriplatinNC localizes to the cytoplasm and nucleolus of interphase cells. Cells treated while in S-G 2 proceed through mitosis. During prophase, the DNA (blue) condenses and the nuclear membrane (red) disintegrates allowing cytoplasmic pools of TriplatinNC to interact with condensed DNA. Cells undergo cytokinesis; however, the DNA does not decondense or progress through G 1 . (a) Determined by immunofluorescence: β-tubulin, nucleophosmin/B23, and DAPI DNA staining (Figure 5 A,B). (b) Determined by β-galactosidase staining and light microscopy at 200× magnification (panel A). (c) Determined by immunofluorescence; cleaved caspase-3; and DAPI DNA staining. The percentage of cells undergoing apoptosis was determined as the number of cells positive for cleaved caspase-3 divided by the total number of cells. n > 500 cells each for two repeat experiments (Figure 5 B).
Figure Legend Snippet: (A) β-Galactosidase activity assay; HCT116 cells were assayed for β-galactosidase activity after treatment with 20 μM cisplatin or TriplatinNC for 96 h. Shown is a representative of two independent experiments. (B) Confocal microscopy; HCT116 cells were treated with 20 μM TriplatinNC for 24 h. Left, top panel; DAPI stained DNA (blue). Right, top panel; cleaved caspase-3 (yellow). Bottom panel; merged DAPI and cleaved caspase-3. Apoptotic cell (white star) containing condensed/fragmented DNA and active caspase-3. Cells with compacted DNA (white arrows) do not contain active caspase-3. (C) Summary; percentage of HCT116 cells undergoing the indicated cellular process after treatment with 20 μM cisplatin or TriplatinNC for 24 h. (D) Schematic; TriplatinNC localizes to the cytoplasm and nucleolus of interphase cells. Cells treated while in S-G 2 proceed through mitosis. During prophase, the DNA (blue) condenses and the nuclear membrane (red) disintegrates allowing cytoplasmic pools of TriplatinNC to interact with condensed DNA. Cells undergo cytokinesis; however, the DNA does not decondense or progress through G 1 . (a) Determined by immunofluorescence: β-tubulin, nucleophosmin/B23, and DAPI DNA staining (Figure 5 A,B). (b) Determined by β-galactosidase staining and light microscopy at 200× magnification (panel A). (c) Determined by immunofluorescence; cleaved caspase-3; and DAPI DNA staining. The percentage of cells undergoing apoptosis was determined as the number of cells positive for cleaved caspase-3 divided by the total number of cells. n > 500 cells each for two repeat experiments (Figure 5 B).

Techniques Used: Activity Assay, Confocal Microscopy, Staining, Immunofluorescence, Light Microscopy

2) Product Images from "Development of Aortic Valve Disease in Familial Hypercholesterolemic Swine: Implications for Elucidating Disease Etiology"

Article Title: Development of Aortic Valve Disease in Familial Hypercholesterolemic Swine: Implications for Elucidating Disease Etiology

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

doi: 10.1161/JAHA.115.002254

Representative histology of atherosclerotic lesions in the coronary arteries of adult  RFH  swine. A, Hematoxylin and eosin (H  E) staining and Movat's Pentachrome revealed wall thickening, fibrin accumulation, and plaque buildup in coronary arteries. B, α‐ SMA ‐positive cells were detected in the subendothelial region. C, Lipid oxidation was observed by immunostaining of oxApoB and malondialdehyde. D, Apoptotic cells were identified throughout the arteries by immunostaining for caspase 3. E, Leukocyte degranulation ( CD 107a) and the secretion of  MCP ‐1 were also observed in the intima, as expected in an atherosclerotic plaque. Scale bars: 400 μm (A and C); 200 μm (B, D, and E). Arrows indicate areas representative of positive staining. N=4 animals. MCP‐1 indicates monocyte chemoattractant protein‐1; oxApoB, oxidatively modified apolipoprotein B; RFH, Rapacz familial hypercholesterolemic; α‐SMA, alpha‐smooth muscle actin.
Figure Legend Snippet: Representative histology of atherosclerotic lesions in the coronary arteries of adult RFH swine. A, Hematoxylin and eosin (H E) staining and Movat's Pentachrome revealed wall thickening, fibrin accumulation, and plaque buildup in coronary arteries. B, α‐ SMA ‐positive cells were detected in the subendothelial region. C, Lipid oxidation was observed by immunostaining of oxApoB and malondialdehyde. D, Apoptotic cells were identified throughout the arteries by immunostaining for caspase 3. E, Leukocyte degranulation ( CD 107a) and the secretion of MCP ‐1 were also observed in the intima, as expected in an atherosclerotic plaque. Scale bars: 400 μm (A and C); 200 μm (B, D, and E). Arrows indicate areas representative of positive staining. N=4 animals. MCP‐1 indicates monocyte chemoattractant protein‐1; oxApoB, oxidatively modified apolipoprotein B; RFH, Rapacz familial hypercholesterolemic; α‐SMA, alpha‐smooth muscle actin.

Techniques Used: Staining, Immunostaining, Modification

3) Product Images from "Farnesol, a Fungal Quorum-Sensing Molecule Triggers Apoptosis in Human Oral Squamous Carcinoma Cells"

Article Title: Farnesol, a Fungal Quorum-Sensing Molecule Triggers Apoptosis in Human Oral Squamous Carcinoma Cells

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

doi:

Western blot analysis: Assessment of OSCC 9 cell protein expression, all after 48 hours of treatment, demonstrating (A) a decrease in survivin expression and (B) an increase in cleaved-caspase 3 and cleaved-caspase 9 expression in cells exposed to synthetic 30- to 60- µ M farnesol (F30, F60) or  C. albicans  spent culture media grown for 48 to 72 hours (CA48h, CA72h), without a change in expression with the non-farnesol-producing  C. albicans  spent culture media grown for 24 to 72 hours (NF24, NF48h, NF72h). The restoration of caspase expression to baseline levels is seen in the presence of 48 hours of combined DAG and farnesol (DAG-F60) treatment. Actin was used as the loading control.
Figure Legend Snippet: Western blot analysis: Assessment of OSCC 9 cell protein expression, all after 48 hours of treatment, demonstrating (A) a decrease in survivin expression and (B) an increase in cleaved-caspase 3 and cleaved-caspase 9 expression in cells exposed to synthetic 30- to 60- µ M farnesol (F30, F60) or C. albicans spent culture media grown for 48 to 72 hours (CA48h, CA72h), without a change in expression with the non-farnesol-producing C. albicans spent culture media grown for 24 to 72 hours (NF24, NF48h, NF72h). The restoration of caspase expression to baseline levels is seen in the presence of 48 hours of combined DAG and farnesol (DAG-F60) treatment. Actin was used as the loading control.

Techniques Used: Western Blot, Expressing

4) Product Images from "p27 Protein Protects Metabolically Stressed Cardiomyocytes from Apoptosis by Promoting Autophagy *"

Article Title: p27 Protein Protects Metabolically Stressed Cardiomyocytes from Apoptosis by Promoting Autophagy *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.542795

Knockdown of p27 suppresses autophagy and increases apoptosis in glucose-deprived ( GD ) cardiomyocytes. A , Western blot shows lysates from cardiomyocytes treated with and without shRNA to knock down p27 versus control shRNA ( NT ) in the presence or absence of glucose. B , shRNAp27 reduced p27 and LC3-II levels and increased p62 levels. Apoptosis marker cleaved caspase 3 was increased in cardiomyocytes undergoing p27 knockdown. C , representative photomicrographs show cardiomyocytes fixed and stained for LC3 ( green ), α-actinin ( red ), and nuclei ( blue ). D , histogram shows the number of LC3 + dots/cell. E , TUNEL-positive nuclei are quantified. *, p
Figure Legend Snippet: Knockdown of p27 suppresses autophagy and increases apoptosis in glucose-deprived ( GD ) cardiomyocytes. A , Western blot shows lysates from cardiomyocytes treated with and without shRNA to knock down p27 versus control shRNA ( NT ) in the presence or absence of glucose. B , shRNAp27 reduced p27 and LC3-II levels and increased p62 levels. Apoptosis marker cleaved caspase 3 was increased in cardiomyocytes undergoing p27 knockdown. C , representative photomicrographs show cardiomyocytes fixed and stained for LC3 ( green ), α-actinin ( red ), and nuclei ( blue ). D , histogram shows the number of LC3 + dots/cell. E , TUNEL-positive nuclei are quantified. *, p

Techniques Used: Western Blot, shRNA, Marker, Staining, TUNEL Assay

TAT-p27-reduced cardiomyocyte apoptosis following glucose deprivation is autophagy-dependent. Glucose-deprived ( GD ) cardiomyocytes were treated with TAT-p27 in the presence or absence of autophagy flux inhibitor Baf-A1. A and B , TAT-p27-treated glucose-deprived cardiomyocytes showed increased LC3-II levels and reduced p62 and caspase 3 cleavage compared with TAT-β-Gal ( lanes 3 versus 1 ). However, addition of Baf-A1 increased apoptosis. *, p
Figure Legend Snippet: TAT-p27-reduced cardiomyocyte apoptosis following glucose deprivation is autophagy-dependent. Glucose-deprived ( GD ) cardiomyocytes were treated with TAT-p27 in the presence or absence of autophagy flux inhibitor Baf-A1. A and B , TAT-p27-treated glucose-deprived cardiomyocytes showed increased LC3-II levels and reduced p62 and caspase 3 cleavage compared with TAT-β-Gal ( lanes 3 versus 1 ). However, addition of Baf-A1 increased apoptosis. *, p

Techniques Used:

TAT-p27 reduced apoptosis post-MI by promoting autophagy. A and B , homogenates from mice undergoing sham and LAD ligation surgery, with the latter receiving treatments with TAT-p27 or TAT-β-Gal alone or in combination with the autophagy inhibitor CQ, were probed for LC3-II, p62, and cleaved caspase 3. β-Tubulin served as loading control. Compared with sham-operated controls, post-MI groups revealed higher levels of LC3-II and cleaved caspase 3 levels (*, p
Figure Legend Snippet: TAT-p27 reduced apoptosis post-MI by promoting autophagy. A and B , homogenates from mice undergoing sham and LAD ligation surgery, with the latter receiving treatments with TAT-p27 or TAT-β-Gal alone or in combination with the autophagy inhibitor CQ, were probed for LC3-II, p62, and cleaved caspase 3. β-Tubulin served as loading control. Compared with sham-operated controls, post-MI groups revealed higher levels of LC3-II and cleaved caspase 3 levels (*, p

Techniques Used: Mouse Assay, Ligation

TAT-p27 reduced glucose deprivation-induced apoptosis by promoting autophagy. TAT-p27-reduced apoptosis in glucose-deprived cardiomyocytes was abolished by 3-MA or CQ. A and B , 3-MA blocks the initial autophagy step, reducing LC3-II activation and increasing cleaved caspase 3 levels even in the presence of TAT-p27. *, p
Figure Legend Snippet: TAT-p27 reduced glucose deprivation-induced apoptosis by promoting autophagy. TAT-p27-reduced apoptosis in glucose-deprived cardiomyocytes was abolished by 3-MA or CQ. A and B , 3-MA blocks the initial autophagy step, reducing LC3-II activation and increasing cleaved caspase 3 levels even in the presence of TAT-p27. *, p

Techniques Used: Activation Assay

5) Product Images from "Inhibition of COX-2/PGE2 cascade ameliorates cisplatin-induced mesangial cell apoptosis"

Article Title: Inhibition of COX-2/PGE2 cascade ameliorates cisplatin-induced mesangial cell apoptosis

Journal: American Journal of Translational Research

doi:

Inhibition of COX-2 attenuated cisplatin-induced MC apoptosis. (A, B) The MC apoptosis was assessed by Annexin-V/PI staining. (C) The protein levels of caspase-3, cleaved caspase-3, Bax, and Bcl-2 were examined by Western blotting in cisplatin-treated
Figure Legend Snippet: Inhibition of COX-2 attenuated cisplatin-induced MC apoptosis. (A, B) The MC apoptosis was assessed by Annexin-V/PI staining. (C) The protein levels of caspase-3, cleaved caspase-3, Bax, and Bcl-2 were examined by Western blotting in cisplatin-treated

Techniques Used: Inhibition, Staining, Western Blot

6) Product Images from "Reperfusion Differentially Induces Caspase-3 Activation in Ischemic Core and Penumbra After Stroke in Immature Brain"

Article Title: Reperfusion Differentially Induces Caspase-3 Activation in Ischemic Core and Penumbra After Stroke in Immature Brain

Journal: Stroke; a journal of cerebral circulation

doi: 10.1161/01.STR.0000047101.87575.3C

Caspase-3 activation occurs in injured tissue after focal cerebral ischemia in P7 rats and depends on the presence of reperfusion. DEVD-AMC assay (A) and Western blot analysis (B) were performed in brain homogenates obtained from the injured tissue and from tissue in matching contralateral regions. Tissue from the same brains was used for both assays. A, Caspase-3 activity was measured in DEVD-AMC cleavage assay with the use of Ac-DEVD as a substrate. Ac-AMC was used to obtain a standard curve. Enzyme activity is expressed as picomoles per minute per milligram protein. * P
Figure Legend Snippet: Caspase-3 activation occurs in injured tissue after focal cerebral ischemia in P7 rats and depends on the presence of reperfusion. DEVD-AMC assay (A) and Western blot analysis (B) were performed in brain homogenates obtained from the injured tissue and from tissue in matching contralateral regions. Tissue from the same brains was used for both assays. A, Caspase-3 activity was measured in DEVD-AMC cleavage assay with the use of Ac-DEVD as a substrate. Ac-AMC was used to obtain a standard curve. Enzyme activity is expressed as picomoles per minute per milligram protein. * P

Techniques Used: Activation Assay, Ub-AMC Assay, Western Blot, Activity Assay, Cleavage Assay

Temporospatial distribution of cells with cleaved caspase-3 after transient MCA occlusion. A to C, ADC map (A) and CM1-immunoreactive cells (B and C) after 4 hours of reperfusion; D to G and J, ADC map (D) and CM1-immunoreactive cells (E to G, J) 8 hours after reperfusion. C and F are higher-magnification images from boxes shown in B and E. Insert G shows that both cell bodies and processes are intensely stained with CM1 antibody. H and I, ADC map (H) and CM1-immunoreactive cells (I) 24 hours after reperfusion. J, Immunofluorescent double labeling with a neuronal marker, NeuN (red) and CM1 (green). Confocal image from the cortex ipsilateral to MCA occlusion shows that caspase-3 activation occurs predominantly in neurons in injured brain tissue 8 hours after MCA occlusion. Arrows in J point to yellow cells in which CM1 and NeuN staining is colocalized. Bar=20 µm.
Figure Legend Snippet: Temporospatial distribution of cells with cleaved caspase-3 after transient MCA occlusion. A to C, ADC map (A) and CM1-immunoreactive cells (B and C) after 4 hours of reperfusion; D to G and J, ADC map (D) and CM1-immunoreactive cells (E to G, J) 8 hours after reperfusion. C and F are higher-magnification images from boxes shown in B and E. Insert G shows that both cell bodies and processes are intensely stained with CM1 antibody. H and I, ADC map (H) and CM1-immunoreactive cells (I) 24 hours after reperfusion. J, Immunofluorescent double labeling with a neuronal marker, NeuN (red) and CM1 (green). Confocal image from the cortex ipsilateral to MCA occlusion shows that caspase-3 activation occurs predominantly in neurons in injured brain tissue 8 hours after MCA occlusion. Arrows in J point to yellow cells in which CM1 and NeuN staining is colocalized. Bar=20 µm.

Techniques Used: Staining, Labeling, Marker, Activation Assay

Activation of caspase-3 in immature brain after focal cerebral ischemia differs in the ischemic core and penumbra. Quantitative analysis of CM1-immunoreactive (CM1-IR) cells in the core and penumbra after transient MCA occlusion in P7 rat is shown. * P
Figure Legend Snippet: Activation of caspase-3 in immature brain after focal cerebral ischemia differs in the ischemic core and penumbra. Quantitative analysis of CM1-immunoreactive (CM1-IR) cells in the core and penumbra after transient MCA occlusion in P7 rat is shown. * P

Techniques Used: Activation Assay

7) Product Images from "Infiltrating macrophages in diabetic nephropathy promote podocytes apoptosis via TNF-α-ROS-p38MAPK pathway"

Article Title: Infiltrating macrophages in diabetic nephropathy promote podocytes apoptosis via TNF-α-ROS-p38MAPK pathway

Journal: Oncotarget

doi: 10.18632/oncotarget.18394

Macrophages promoted podocytes apoptosis in the condition of high glucose Flow cytometry analysis of podocytes apoptosis with Annexin V-FITC/PI staining  (A) . Percentage of apoptotic podocytes  (B) . Representative western blotting analysis of cleaved caspase 3 in podocytes  (C) . β-actin was used as an internal control. Quantification of cleaved caspase 3 protein expression  (D) . NC+P alone: podocytes treated with normal PRMI 1640 media. Man+P alone: Podocytes treated with 25 mM mannitol. HG+P alone: Podocytes treated with 25 mM high glucose. NC+P+M : Transwell co-culture of podocytes (P) and RAW 264.7 cells (M) in the absence of 25 mM high glucose at indicated ratios of P to M. Man+P+M: Transwell co-culture of P and M in 25 mM mannitol at indicated ratios of P to M. HG+P+M: Transwell co-culture of P and M in the presence of 25 mM high glucose at indicated ratios of P to M. Data were presented as mean ± SD from three independent experiments. *P
Figure Legend Snippet: Macrophages promoted podocytes apoptosis in the condition of high glucose Flow cytometry analysis of podocytes apoptosis with Annexin V-FITC/PI staining (A) . Percentage of apoptotic podocytes (B) . Representative western blotting analysis of cleaved caspase 3 in podocytes (C) . β-actin was used as an internal control. Quantification of cleaved caspase 3 protein expression (D) . NC+P alone: podocytes treated with normal PRMI 1640 media. Man+P alone: Podocytes treated with 25 mM mannitol. HG+P alone: Podocytes treated with 25 mM high glucose. NC+P+M : Transwell co-culture of podocytes (P) and RAW 264.7 cells (M) in the absence of 25 mM high glucose at indicated ratios of P to M. Man+P+M: Transwell co-culture of P and M in 25 mM mannitol at indicated ratios of P to M. HG+P+M: Transwell co-culture of P and M in the presence of 25 mM high glucose at indicated ratios of P to M. Data were presented as mean ± SD from three independent experiments. *P

Techniques Used: Flow Cytometry, Cytometry, Staining, Western Blot, Expressing, Co-Culture Assay

8) Product Images from "A Novel Chimeric Oncolytic Virus Vector for Improved Safety and Efficacy as a Platform for the Treatment of Hepatocellular Carcinoma"

Article Title: A Novel Chimeric Oncolytic Virus Vector for Improved Safety and Efficacy as a Platform for the Treatment of Hepatocellular Carcinoma

Journal: Journal of Virology

doi: 10.1128/JVI.01386-18

The maximum tolerated dose (MTD) of rVSV-NDV is elevated by 3 logs compared to that of rVSV in immunodeficient mice. Immunodeficient male NOD-SCID mice were treated by tail vein injection with increasing doses of rVSV-GFP or rVSV-NDV in a classic 3-plus-3 dosing scheme. Mice were monitored daily for the onset of dose-limiting toxicities and euthanized at humane endpoints. (A) A summary of the administered doses and of the occurrence of toxic events for each virus is shown in tabular form, as well as by a Kaplan-Meier survival curve. Red boxes in the graph legend indicate the respective MTD values for the viruses. (B) Hematoxylin-eosin staining of liver tissue revealed small-group necrosis of hepatocytes after rVSV treatment, marked by hepatocellular degeneration with karyolysis, as indicated by short black arrows (top left panel). Acute necrosis in the brain stem was observed after rVSV application, with degenerating glia cells exhibiting pyknosis and karyorrhexis, as indicated by long black arrows (top right panel) and by positive immunohistochemical staining (brown) for cleaved caspase-3 (bottom right). Representative images are shown; scale bars equal 50 µm. Viral titers were quantified in brain and liver tissue lysate obtained from mice receiving rVSV after demonstrating signs of toxicity (bottom left). Means + standard errors of the means (SEM) are shown.
Figure Legend Snippet: The maximum tolerated dose (MTD) of rVSV-NDV is elevated by 3 logs compared to that of rVSV in immunodeficient mice. Immunodeficient male NOD-SCID mice were treated by tail vein injection with increasing doses of rVSV-GFP or rVSV-NDV in a classic 3-plus-3 dosing scheme. Mice were monitored daily for the onset of dose-limiting toxicities and euthanized at humane endpoints. (A) A summary of the administered doses and of the occurrence of toxic events for each virus is shown in tabular form, as well as by a Kaplan-Meier survival curve. Red boxes in the graph legend indicate the respective MTD values for the viruses. (B) Hematoxylin-eosin staining of liver tissue revealed small-group necrosis of hepatocytes after rVSV treatment, marked by hepatocellular degeneration with karyolysis, as indicated by short black arrows (top left panel). Acute necrosis in the brain stem was observed after rVSV application, with degenerating glia cells exhibiting pyknosis and karyorrhexis, as indicated by long black arrows (top right panel) and by positive immunohistochemical staining (brown) for cleaved caspase-3 (bottom right). Representative images are shown; scale bars equal 50 µm. Viral titers were quantified in brain and liver tissue lysate obtained from mice receiving rVSV after demonstrating signs of toxicity (bottom left). Means + standard errors of the means (SEM) are shown.

Techniques Used: Mouse Assay, Injection, Staining, Immunohistochemistry

9) Product Images from "Tropism of and Innate Immune Responses to the Novel Human Betacoronavirus Lineage C Virus in Human Ex Vivo Respiratory Organ Cultures"

Article Title: Tropism of and Innate Immune Responses to the Novel Human Betacoronavirus Lineage C Virus in Human Ex Vivo Respiratory Organ Cultures

Journal: Journal of Virology

doi: 10.1128/JVI.00009-13

Apoptotic cells identified in human lung tissue ex vivo culture upon HCoV-EMC and SARS-CoV infection. (A to C) Ex vivo culture of lung tissue mock infected (A) or infected with HCoV-EMC (B) or SARS-CoV (C) at 48 hpi. The reddish brown stain identifies the presence of cleaved caspase 3. (D and E) Costaining of HCoV-EMC (D) and SARS-CoV (E) antigen (pink stain) with cleaved caspase 3 (reddish brown stain).
Figure Legend Snippet: Apoptotic cells identified in human lung tissue ex vivo culture upon HCoV-EMC and SARS-CoV infection. (A to C) Ex vivo culture of lung tissue mock infected (A) or infected with HCoV-EMC (B) or SARS-CoV (C) at 48 hpi. The reddish brown stain identifies the presence of cleaved caspase 3. (D and E) Costaining of HCoV-EMC (D) and SARS-CoV (E) antigen (pink stain) with cleaved caspase 3 (reddish brown stain).

Techniques Used: Ex Vivo, Infection, Staining

10) Product Images from "Oral application of a periodontal pathogen impacts SerpinE1 expression and pancreatic islet architecture in prediabetes"

Article Title: Oral application of a periodontal pathogen impacts SerpinE1 expression and pancreatic islet architecture in prediabetes

Journal: Journal of periodontal research

doi: 10.1111/jre.12474

P. gingivalis induces apoptosis of MIN6 cells. A. a: TUNEL histochemical staining at time 0, 8 and 24 h after the addition of Pg (MOI of 1:200). B. % of TUNEL positive cells/total number of cells counted. Cells were counted in 5 random fields. Four independent experiments were performed. Data presented are mean ± SD. B. Results from Western blot analyses indicating cleaved caspase 3 (a), 8 (b) and 9 (c) in MIN6 cells in response to Pg. a. caspase 3 cleavage was monitored for 3, 8, and 17 h following addition of Pg (MOI 1:200). For b and c, caspase cleavage shown at 17 h post Pg addition. Four independent experiments were performed. Data are presented as mean ± SD. C. Pg suppresses AKT phosphorylation in MIN6 cells. Western blot probed for AKT and p-AKT. Four independent experiments were performed. Data are presented as mean ± SD. D. MIN6 cells with SerpinE1 knockdown are less susceptible to Pg induced apoptosis. Y-axis: % apoptosis determined by % TUNEL positive cells. C: control scrambled shRNA (clone C), 23: SerpinE1 shRNA transduced clone (clone 23). Cells were incubated with Pg or control media for 24 h. Four independent TUNEL experiments were performed. Data are presented as mean ± SD.
Figure Legend Snippet: P. gingivalis induces apoptosis of MIN6 cells. A. a: TUNEL histochemical staining at time 0, 8 and 24 h after the addition of Pg (MOI of 1:200). B. % of TUNEL positive cells/total number of cells counted. Cells were counted in 5 random fields. Four independent experiments were performed. Data presented are mean ± SD. B. Results from Western blot analyses indicating cleaved caspase 3 (a), 8 (b) and 9 (c) in MIN6 cells in response to Pg. a. caspase 3 cleavage was monitored for 3, 8, and 17 h following addition of Pg (MOI 1:200). For b and c, caspase cleavage shown at 17 h post Pg addition. Four independent experiments were performed. Data are presented as mean ± SD. C. Pg suppresses AKT phosphorylation in MIN6 cells. Western blot probed for AKT and p-AKT. Four independent experiments were performed. Data are presented as mean ± SD. D. MIN6 cells with SerpinE1 knockdown are less susceptible to Pg induced apoptosis. Y-axis: % apoptosis determined by % TUNEL positive cells. C: control scrambled shRNA (clone C), 23: SerpinE1 shRNA transduced clone (clone 23). Cells were incubated with Pg or control media for 24 h. Four independent TUNEL experiments were performed. Data are presented as mean ± SD.

Techniques Used: TUNEL Assay, Staining, Western Blot, shRNA, Incubation

11) Product Images from "The Role of IL-6RA in UHMWPE Promotes Proliferation in Fibro-Like Synovial Cells"

Article Title: The Role of IL-6RA in UHMWPE Promotes Proliferation in Fibro-Like Synovial Cells

Journal: BioMed Research International

doi: 10.1155/2018/3928915

Effects of UHMWPE on apoptosis of FLS cells. FLS were incubated with 0-1.0 g/L UHMWPE for 0, 1, and 7 d or incubated with 1.0 g/L UHMWPE for 0, 1, and 7 d. Apoptotic and necrotic cell populations were analyzed by flow cytometry; quantitative analysis of apoptotic cells after UHMWPE treatment, n=5 (a and b). The expression of apoptosis-related proteins in FLS cells was detected by Western blot assay and the changes of Bcl-2, Bax, caspase-3, and cleaved-caspase-3 were statistically analyzed, n=5 (c). Data was obtained from three independent experiments; results were shown as means ± standard deviation. ∗ p
Figure Legend Snippet: Effects of UHMWPE on apoptosis of FLS cells. FLS were incubated with 0-1.0 g/L UHMWPE for 0, 1, and 7 d or incubated with 1.0 g/L UHMWPE for 0, 1, and 7 d. Apoptotic and necrotic cell populations were analyzed by flow cytometry; quantitative analysis of apoptotic cells after UHMWPE treatment, n=5 (a and b). The expression of apoptosis-related proteins in FLS cells was detected by Western blot assay and the changes of Bcl-2, Bax, caspase-3, and cleaved-caspase-3 were statistically analyzed, n=5 (c). Data was obtained from three independent experiments; results were shown as means ± standard deviation. ∗ p

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

The antagonistic effect of IL-6RA on FLS cells treated with UHMWPE for 7 d. The experiment was divided into 3 groups, including UHMWPE (1 g/L), UHMWPE (1 g/L) + IL-6RA (50 μ g/ml), and IL-6RA(50 μ g/ml). Apoptotic rate was detected by flow cytometry after treatment IL-6RA, n=5 (a). The expression levels of apoptosis-related proteins including Bax, Bcl2, caspase-3, and cleaved-caspase-3 were analyzed by Western blot after treatment IL-6RA, n=5 (b). Data was obtained from three independent experiments; results were shown as means ± standard deviation. ∗ p
Figure Legend Snippet: The antagonistic effect of IL-6RA on FLS cells treated with UHMWPE for 7 d. The experiment was divided into 3 groups, including UHMWPE (1 g/L), UHMWPE (1 g/L) + IL-6RA (50 μ g/ml), and IL-6RA(50 μ g/ml). Apoptotic rate was detected by flow cytometry after treatment IL-6RA, n=5 (a). The expression levels of apoptosis-related proteins including Bax, Bcl2, caspase-3, and cleaved-caspase-3 were analyzed by Western blot after treatment IL-6RA, n=5 (b). Data was obtained from three independent experiments; results were shown as means ± standard deviation. ∗ p

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

12) Product Images from "Differential induction of apoptosis in human breast cancer cell lines by phenethyl isothiocyanate, a glutathione depleting agent"

Article Title: Differential induction of apoptosis in human breast cancer cell lines by phenethyl isothiocyanate, a glutathione depleting agent

Journal: Cell Stress & Chaperones

doi: 10.1007/s12192-012-0329-3

Caspase expression in PEITC-treated cells. MDA-MB-231 or MCF7 cells were treated with PEITC (20 μM) for the indicated times or DMSO for 6 h as a control. Expression of caspase 9, caspase 8, caspase 3 (MDA-MB-231 cells) and caspase 7 (MCF7 cells) was analysed by immunoblotting. β-actin was analysed as a loading control. a Representative immunoblots. The positions of migration of pro- and cleaved forms of caspases are indicated. b Quantitation. Data shown are means ± SD derived from two independent experiments for pro-caspase 9, caspase 9, caspase 3 and pro-caspase 8 in MDA-MB-231 cells (i–iv, respectively) and pro-caspase 9, pro-caspase 7 and pro-caspase 8 in MCF7 cells (v–vii, respectively). Expression values were normalized to that of β-actin. Statistically significant differences between DMSO and PEITC treated cells are indicated (* p
Figure Legend Snippet: Caspase expression in PEITC-treated cells. MDA-MB-231 or MCF7 cells were treated with PEITC (20 μM) for the indicated times or DMSO for 6 h as a control. Expression of caspase 9, caspase 8, caspase 3 (MDA-MB-231 cells) and caspase 7 (MCF7 cells) was analysed by immunoblotting. β-actin was analysed as a loading control. a Representative immunoblots. The positions of migration of pro- and cleaved forms of caspases are indicated. b Quantitation. Data shown are means ± SD derived from two independent experiments for pro-caspase 9, caspase 9, caspase 3 and pro-caspase 8 in MDA-MB-231 cells (i–iv, respectively) and pro-caspase 9, pro-caspase 7 and pro-caspase 8 in MCF7 cells (v–vii, respectively). Expression values were normalized to that of β-actin. Statistically significant differences between DMSO and PEITC treated cells are indicated (* p

Techniques Used: Expressing, Multiple Displacement Amplification, Western Blot, Migration, Quantitation Assay, Derivative Assay

13) Product Images from "Identification of Unprecedented Anticancer Properties of High Molecular Weight Biomacromolecular Complex Containing Bovine Lactoferrin (HMW-bLf)"

Article Title: Identification of Unprecedented Anticancer Properties of High Molecular Weight Biomacromolecular Complex Containing Bovine Lactoferrin (HMW-bLf)

Journal: PLoS ONE

doi: 10.1371/journal.pone.0106568

Caspase-3 activation. A and B represent the increased caspase-3 activity measurements upon treatment with HMW-bLf in MDA-MB-231 and SW480 cells, respectively. (* P
Figure Legend Snippet: Caspase-3 activation. A and B represent the increased caspase-3 activity measurements upon treatment with HMW-bLf in MDA-MB-231 and SW480 cells, respectively. (* P

Techniques Used: Activation Assay, Activity Assay, Multiple Displacement Amplification

14) Product Images from "The dual PI3K/mTOR inhibitor NVP-BEZ235 enhances nab-paclitaxel antitumor response in experimental gastric cancer"

Article Title: The dual PI3K/mTOR inhibitor NVP-BEZ235 enhances nab-paclitaxel antitumor response in experimental gastric cancer

Journal: International Journal of Oncology

doi: 10.3892/ijo.2013.2099

BEZ235 and nab-paclitaxel effects on the PI3K-mTOR signaling pathway and apoptosis-related proteins. Subconfluent monolayers of human gastric cancer cells SNU16, NCI-N87 and AGS were treated with PBS control (C), BEZ235 (10 μ M), nab-paclitaxel (10 μ M), or a combination for 16 h. Total cell extracts were analyzed by immunoblotting for p-Akt (Ser473), total Akt, p-mTOR (Ser2448), total mTOR, p-p70 S6K (Thr389), total p70 S6K, p-4E-BP1 (Thr37/46) and total 4E-BP1, cleaved PARP-1, cleaved caspase-3 and β-actin (loading control). Data are representative of two independent experiments with similar results.
Figure Legend Snippet: BEZ235 and nab-paclitaxel effects on the PI3K-mTOR signaling pathway and apoptosis-related proteins. Subconfluent monolayers of human gastric cancer cells SNU16, NCI-N87 and AGS were treated with PBS control (C), BEZ235 (10 μ M), nab-paclitaxel (10 μ M), or a combination for 16 h. Total cell extracts were analyzed by immunoblotting for p-Akt (Ser473), total Akt, p-mTOR (Ser2448), total mTOR, p-p70 S6K (Thr389), total p70 S6K, p-4E-BP1 (Thr37/46) and total 4E-BP1, cleaved PARP-1, cleaved caspase-3 and β-actin (loading control). Data are representative of two independent experiments with similar results.

Techniques Used:

15) Product Images from "The dual PI3K/mTOR inhibitor NVP-BEZ235 enhances nab-paclitaxel antitumor response in experimental gastric cancer"

Article Title: The dual PI3K/mTOR inhibitor NVP-BEZ235 enhances nab-paclitaxel antitumor response in experimental gastric cancer

Journal: International Journal of Oncology

doi: 10.3892/ijo.2013.2099

BEZ235 and nab-paclitaxel effects on the PI3K-mTOR signaling pathway and apoptosis-related proteins. Subconfluent monolayers of human gastric cancer cells SNU16, NCI-N87 and AGS were treated with PBS control (C), BEZ235 (10 μ M), nab-paclitaxel (10 μ M), or a combination for 16 h. Total cell extracts were analyzed by immunoblotting for p-Akt (Ser473), total Akt, p-mTOR (Ser2448), total mTOR, p-p70 S6K (Thr389), total p70 S6K, p-4E-BP1 (Thr37/46) and total 4E-BP1, cleaved PARP-1, cleaved caspase-3 and β-actin (loading control). Data are representative of two independent experiments with similar results.
Figure Legend Snippet: BEZ235 and nab-paclitaxel effects on the PI3K-mTOR signaling pathway and apoptosis-related proteins. Subconfluent monolayers of human gastric cancer cells SNU16, NCI-N87 and AGS were treated with PBS control (C), BEZ235 (10 μ M), nab-paclitaxel (10 μ M), or a combination for 16 h. Total cell extracts were analyzed by immunoblotting for p-Akt (Ser473), total Akt, p-mTOR (Ser2448), total mTOR, p-p70 S6K (Thr389), total p70 S6K, p-4E-BP1 (Thr37/46) and total 4E-BP1, cleaved PARP-1, cleaved caspase-3 and β-actin (loading control). Data are representative of two independent experiments with similar results.

Techniques Used:

16) Product Images from "BMPR1a Signaling Determines Numbers of Oligodendrocytes and Calbindin-Expressing Interneurons in the Cortex"

Article Title: BMPR1a Signaling Determines Numbers of Oligodendrocytes and Calbindin-Expressing Interneurons in the Cortex

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.1434-07.2007

BMPR1a mutation does not alter apoptosis in the ventral forebrain. A , B , Immunohistochemistry was done for cleaved caspase 3 (Cl. Caspase 3) in the ventral forebrain of P0 BMPR1a mutant and wild-type mice. Inset, Area of ventral forebrain in which cell count was done. DAPI, 4′,6′-Diamidino-2-phenylindole dihydrochloride.
Figure Legend Snippet: BMPR1a mutation does not alter apoptosis in the ventral forebrain. A , B , Immunohistochemistry was done for cleaved caspase 3 (Cl. Caspase 3) in the ventral forebrain of P0 BMPR1a mutant and wild-type mice. Inset, Area of ventral forebrain in which cell count was done. DAPI, 4′,6′-Diamidino-2-phenylindole dihydrochloride.

Techniques Used: Mutagenesis, Immunohistochemistry, Mouse Assay, Cell Counting

17) Product Images from "Prevention of Wogonin on Colorectal Cancer Tumorigenesis by Regulating p53 Nuclear Translocation"

Article Title: Prevention of Wogonin on Colorectal Cancer Tumorigenesis by Regulating p53 Nuclear Translocation

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2018.01356

Wogonin caused apoptotic cell death through increased ER stress in human CRC HCT-116 cell.  (A)  IRE-1α, CHOP, PDI, and CALNEXIN protein levels in HCT-116 cells treated with wogonin and vesicle (0.1% DMSO) for 72 h were detected using western blot assay. The band intensities were quantified by Quantity One and represented as bar graph in the right panel. GAPDH was used as an internal control.  (B)  Gene expression of  DDIT3  and  XBP1  were determined by real-time PCR after treatments with indicated concentrations of wogonin for 72 h.  GAPDH  served as housekeeping gene.  (C)  Apoptotic effects in HCT-116 cells after wogonin treatment (10 μM) with/without TUDCA (500 μM) for 48 h, and the proportion of apoptotic cells was determined by flow cytometry using Annexin V/propidium iodide (PI) double staining.  (D)  Protein expression levels of cleaved caspase 3, cleaved caspase 9, IRE-1α, PDI, LC3-II to LC3-I in HCT-116 cells after wogonin treatment (10 μM) with/without TUDCA (500 μM) for 72 h. GAPDH was used as an equal loading control. Experiments were performed in triplicate and data represent as mean ± SD, significant difference versus control group,  ∗ P
Figure Legend Snippet: Wogonin caused apoptotic cell death through increased ER stress in human CRC HCT-116 cell. (A) IRE-1α, CHOP, PDI, and CALNEXIN protein levels in HCT-116 cells treated with wogonin and vesicle (0.1% DMSO) for 72 h were detected using western blot assay. The band intensities were quantified by Quantity One and represented as bar graph in the right panel. GAPDH was used as an internal control. (B) Gene expression of DDIT3 and XBP1 were determined by real-time PCR after treatments with indicated concentrations of wogonin for 72 h. GAPDH served as housekeeping gene. (C) Apoptotic effects in HCT-116 cells after wogonin treatment (10 μM) with/without TUDCA (500 μM) for 48 h, and the proportion of apoptotic cells was determined by flow cytometry using Annexin V/propidium iodide (PI) double staining. (D) Protein expression levels of cleaved caspase 3, cleaved caspase 9, IRE-1α, PDI, LC3-II to LC3-I in HCT-116 cells after wogonin treatment (10 μM) with/without TUDCA (500 μM) for 72 h. GAPDH was used as an equal loading control. Experiments were performed in triplicate and data represent as mean ± SD, significant difference versus control group, ∗ P

Techniques Used: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Double Staining

Wogonin induced cell death via caspase-dependent apoptotic pathway in human CRC cells. (A) Wogonin inhibited human CRC HCT-116 cells viability of in a concentration dependent manner. HCT-116 cells were treated with various concentrations (0–100 μM) of wogonin for 72 h as measured by MTT assay. (B) Effects of wogonin on the colony formation abilities of HCT-116 cells. Colony formation rate was showed in histogram. (C) Effects of wogonin on apoptotic cell death in HCT-116 cells. HCT-116 cells treated with wogonin for 48 h, and the proportion of apoptotic cells was determined by flow cytometry using Annexin V/propidium iodide (PI) double staining. (D) The expression levels of cleaved caspase 3 and 9 in9 in HCT-116 cells after wogonin treatment were measured using Western blot assay. GAPDH was used as an equal loading control. (E) Western blot analysis of autophagy-related proteins including LC3-II and LC3-I in HCT-116 cells after wogonin treatment for 72 h. GAPDH was used as an equal loading control. The results represent the mean of three independent experiments. Values represent mean ± SD, significant difference versus control group, ∗∗∗ P
Figure Legend Snippet: Wogonin induced cell death via caspase-dependent apoptotic pathway in human CRC cells. (A) Wogonin inhibited human CRC HCT-116 cells viability of in a concentration dependent manner. HCT-116 cells were treated with various concentrations (0–100 μM) of wogonin for 72 h as measured by MTT assay. (B) Effects of wogonin on the colony formation abilities of HCT-116 cells. Colony formation rate was showed in histogram. (C) Effects of wogonin on apoptotic cell death in HCT-116 cells. HCT-116 cells treated with wogonin for 48 h, and the proportion of apoptotic cells was determined by flow cytometry using Annexin V/propidium iodide (PI) double staining. (D) The expression levels of cleaved caspase 3 and 9 in9 in HCT-116 cells after wogonin treatment were measured using Western blot assay. GAPDH was used as an equal loading control. (E) Western blot analysis of autophagy-related proteins including LC3-II and LC3-I in HCT-116 cells after wogonin treatment for 72 h. GAPDH was used as an equal loading control. The results represent the mean of three independent experiments. Values represent mean ± SD, significant difference versus control group, ∗∗∗ P

Techniques Used: Concentration Assay, MTT Assay, Flow Cytometry, Cytometry, Double Staining, Expressing, Western Blot

18) Product Images from "Endurance exercise attenuates ventilator-induced diaphragm dysfunction"

Article Title: Endurance exercise attenuates ventilator-induced diaphragm dysfunction

Journal: Journal of Applied Physiology

doi: 10.1152/japplphysiol.01086.2011

Calpain and caspase-3 activation in diaphragm was determined via Western blotting. Representative Western blots are shown at top . Values are mean percent change ± SE and normalized to α-tubulin. Assessment of calpain activity via assessment
Figure Legend Snippet: Calpain and caspase-3 activation in diaphragm was determined via Western blotting. Representative Western blots are shown at top . Values are mean percent change ± SE and normalized to α-tubulin. Assessment of calpain activity via assessment

Techniques Used: Activation Assay, Western Blot, Activity Assay

19) Product Images from "Pycnogenol Induces Nuclear Translocation of Apoptosis-inducing Factor and Caspase-independent Apoptosis in MC-3 Human Mucoepidermoid Carcinoma Cell Line"

Article Title: Pycnogenol Induces Nuclear Translocation of Apoptosis-inducing Factor and Caspase-independent Apoptosis in MC-3 Human Mucoepidermoid Carcinoma Cell Line

Journal: Journal of Cancer Prevention

doi: 10.15430/JCP.2014.19.4.265

Caspase 3-independent apoptosis in pycnogenol-treated MC-3 cells. (A) Expression levels of cleaved poly(ADP-ribose) polymerase (PARP) and cleaved caspase 3 were investigated by Western blotting and actin was used as loading control. 1 μM Withaferin A-treated human oral squamous carcinoma (HSC-3) cells were used as a positive control (P.C). (B) benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone (Z-VAD), pan caspase inhibitor) was used to estimate the connection of caspase 3 in pycnogenol-induced apoptosis.
Figure Legend Snippet: Caspase 3-independent apoptosis in pycnogenol-treated MC-3 cells. (A) Expression levels of cleaved poly(ADP-ribose) polymerase (PARP) and cleaved caspase 3 were investigated by Western blotting and actin was used as loading control. 1 μM Withaferin A-treated human oral squamous carcinoma (HSC-3) cells were used as a positive control (P.C). (B) benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone (Z-VAD), pan caspase inhibitor) was used to estimate the connection of caspase 3 in pycnogenol-induced apoptosis.

Techniques Used: Expressing, Western Blot, Positive Control

20) Product Images from "NADH-dehydrogenase Type-2 Suppresses Irreversible Visual Loss and Neurodegeneration in the EAE Animal Model of MS"

Article Title: NADH-dehydrogenase Type-2 Suppresses Irreversible Visual Loss and Neurodegeneration in the EAE Animal Model of MS

Journal: Molecular Therapy

doi: 10.1038/mt.2013.104

Apoptosis. Immunofluorescence of cleaved caspase 3 and Thy 1.2 in longitudinal retinal cryosections of mice sensitized for EAE 6 months earlier whose eyes were injected with scAAV- mCherry and stained for ( a ) DAPI. ( b ) Cleaved caspase 3 is shown in the
Figure Legend Snippet: Apoptosis. Immunofluorescence of cleaved caspase 3 and Thy 1.2 in longitudinal retinal cryosections of mice sensitized for EAE 6 months earlier whose eyes were injected with scAAV- mCherry and stained for ( a ) DAPI. ( b ) Cleaved caspase 3 is shown in the

Techniques Used: Immunofluorescence, Mouse Assay, Injection, Staining

21) Product Images from "The Deleted in Liver Cancer 1 (Dlc1) tumor suppressor is haploinsufficient for mammary gland development and epithelial cell polarity"

Article Title: The Deleted in Liver Cancer 1 (Dlc1) tumor suppressor is haploinsufficient for mammary gland development and epithelial cell polarity

Journal: BMC Cancer

doi: 10.1186/s12885-015-1642-x

Histological sections of mouse mammary glands stained with cytokeratin lineage markers, cleaved caspase-3 and Ki67. a – b Immunostaining of the mouse mammary glands with lineage markers cytokeratins 14 (Basal/myoepithelial) and 18 (Luminal) from 10-week old WT mice ( a ) and Dlc1 gt/+ mice ( b ). Cleaved caspase-3 staining in 10-week old Dlc1 gt/+ ( d ) and WT mice ( c ). Ki67 staining of mouse mammary glands from WT ( e ) and Dlc1 gt/+ ( f ). The orange arrows indicate cells with Ki67 positive nuclei. The bar graph is the percentage of Ki67 positive cells ( g ). Data represent the mean ± SEM from a total number of 3 different glands ( N = 3) from 3 different mice for each group p = 0.2. Scale bar 50 μm
Figure Legend Snippet: Histological sections of mouse mammary glands stained with cytokeratin lineage markers, cleaved caspase-3 and Ki67. a – b Immunostaining of the mouse mammary glands with lineage markers cytokeratins 14 (Basal/myoepithelial) and 18 (Luminal) from 10-week old WT mice ( a ) and Dlc1 gt/+ mice ( b ). Cleaved caspase-3 staining in 10-week old Dlc1 gt/+ ( d ) and WT mice ( c ). Ki67 staining of mouse mammary glands from WT ( e ) and Dlc1 gt/+ ( f ). The orange arrows indicate cells with Ki67 positive nuclei. The bar graph is the percentage of Ki67 positive cells ( g ). Data represent the mean ± SEM from a total number of 3 different glands ( N = 3) from 3 different mice for each group p = 0.2. Scale bar 50 μm

Techniques Used: Staining, Immunostaining, Mouse Assay

Luminal filling of the acinar structures formed from mammary epithelial cells of Dlc1 gt/+ mice grown in 3D Matrigel culture is not due to defects in apoptosis. Representative images of acinar structures from mammary epithelial cells from 10 week old wild type mice ( a e ) at days 6 12 after culturing in Matrigel or treated with etoposide ( b f ) and stained with cleaved caspase-3 antibody and To-Pro-3. Acinar structures of mammary epithelial cells from Dlc1 gt/+ mice at day 6 ( c ) and day 12 ( g ) or treated with etoposide ( d h ). Scale bar 100 μm
Figure Legend Snippet: Luminal filling of the acinar structures formed from mammary epithelial cells of Dlc1 gt/+ mice grown in 3D Matrigel culture is not due to defects in apoptosis. Representative images of acinar structures from mammary epithelial cells from 10 week old wild type mice ( a e ) at days 6 12 after culturing in Matrigel or treated with etoposide ( b f ) and stained with cleaved caspase-3 antibody and To-Pro-3. Acinar structures of mammary epithelial cells from Dlc1 gt/+ mice at day 6 ( c ) and day 12 ( g ) or treated with etoposide ( d h ). Scale bar 100 μm

Techniques Used: Mouse Assay, Staining

22) Product Images from "The Deleted in Liver Cancer 1 (Dlc1) tumor suppressor is haploinsufficient for mammary gland development and epithelial cell polarity"

Article Title: The Deleted in Liver Cancer 1 (Dlc1) tumor suppressor is haploinsufficient for mammary gland development and epithelial cell polarity

Journal: BMC Cancer

doi: 10.1186/s12885-015-1642-x

Histological sections of mouse mammary glands stained with cytokeratin lineage markers, cleaved caspase-3 and Ki67. a – b Immunostaining of the mouse mammary glands with lineage markers cytokeratins 14 (Basal/myoepithelial) and 18 (Luminal) from 10-week old WT mice ( a ) and Dlc1 gt/+ mice ( b ). Cleaved caspase-3 staining in 10-week old Dlc1 gt/+ ( d ) and WT mice ( c ). Ki67 staining of mouse mammary glands from WT ( e ) and Dlc1 gt/+ ( f ). The orange arrows indicate cells with Ki67 positive nuclei. The bar graph is the percentage of Ki67 positive cells ( g ). Data represent the mean ± SEM from a total number of 3 different glands ( N = 3) from 3 different mice for each group p = 0.2. Scale bar 50 μm
Figure Legend Snippet: Histological sections of mouse mammary glands stained with cytokeratin lineage markers, cleaved caspase-3 and Ki67. a – b Immunostaining of the mouse mammary glands with lineage markers cytokeratins 14 (Basal/myoepithelial) and 18 (Luminal) from 10-week old WT mice ( a ) and Dlc1 gt/+ mice ( b ). Cleaved caspase-3 staining in 10-week old Dlc1 gt/+ ( d ) and WT mice ( c ). Ki67 staining of mouse mammary glands from WT ( e ) and Dlc1 gt/+ ( f ). The orange arrows indicate cells with Ki67 positive nuclei. The bar graph is the percentage of Ki67 positive cells ( g ). Data represent the mean ± SEM from a total number of 3 different glands ( N = 3) from 3 different mice for each group p = 0.2. Scale bar 50 μm

Techniques Used: Staining, Immunostaining, Mouse Assay

Luminal filling of the acinar structures formed from mammary epithelial cells of Dlc1 gt/+ mice grown in 3D Matrigel culture is not due to defects in apoptosis. Representative images of acinar structures from mammary epithelial cells from 10 week old wild type mice ( a e ) at days 6 12 after culturing in Matrigel or treated with etoposide ( b f ) and stained with cleaved caspase-3 antibody and To-Pro-3. Acinar structures of mammary epithelial cells from Dlc1 gt/+ mice at day 6 ( c ) and day 12 ( g ) or treated with etoposide ( d h ). Scale bar 100 μm
Figure Legend Snippet: Luminal filling of the acinar structures formed from mammary epithelial cells of Dlc1 gt/+ mice grown in 3D Matrigel culture is not due to defects in apoptosis. Representative images of acinar structures from mammary epithelial cells from 10 week old wild type mice ( a e ) at days 6 12 after culturing in Matrigel or treated with etoposide ( b f ) and stained with cleaved caspase-3 antibody and To-Pro-3. Acinar structures of mammary epithelial cells from Dlc1 gt/+ mice at day 6 ( c ) and day 12 ( g ) or treated with etoposide ( d h ). Scale bar 100 μm

Techniques Used: Mouse Assay, Staining

23) Product Images from "The Impact of Growth Hormone Therapy on the Apoptosis Assessment in CD34+ Hematopoietic Cells from Children with Growth Hormone Deficiency"

Article Title: The Impact of Growth Hormone Therapy on the Apoptosis Assessment in CD34+ Hematopoietic Cells from Children with Growth Hormone Deficiency

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms18010111

The expression profile of selected apoptosis-related molecules, BCL-2, BCL-XL, BAX and caspase-3 in CD34+ cells from GHD patients. The expression of selected apoptosis-related gene transcripts and proteins was evaluated in CD34+ cells of controls and GHD patients at different time points (before GH-TS and in the 3rd and 6th month of GH-TS). The mRNA expression of BCL-2 ( A ); BCL-XL ( D ), and BAX ( E ) was determined by the quantitative PCR. The mRNA levels are expressed in arbitrary units as the mean value ± S.D. The protein expression of BCL-2 ( B , C ); BAX ( F , G ); caspase-3 and procaspase-3 ( J , K ) was determined by western blot. Beta-2-microglobulin (BMG) served as loading control. Protein bands were analyzed by densitometry. Bars represent mean ± S.D. of selected protein to BMG ratio calculated in all examined groups. The ratio between the optical densities measured in 11-, 17- and 20-kDa cleaved caspase-3 and 32-kDa procaspase-3 bands was calculated as an index of caspase-3 activation. In addition, the BAX/BCL-2 mRNA ( H ) and protein ( I ) ratio was calculated. * p
Figure Legend Snippet: The expression profile of selected apoptosis-related molecules, BCL-2, BCL-XL, BAX and caspase-3 in CD34+ cells from GHD patients. The expression of selected apoptosis-related gene transcripts and proteins was evaluated in CD34+ cells of controls and GHD patients at different time points (before GH-TS and in the 3rd and 6th month of GH-TS). The mRNA expression of BCL-2 ( A ); BCL-XL ( D ), and BAX ( E ) was determined by the quantitative PCR. The mRNA levels are expressed in arbitrary units as the mean value ± S.D. The protein expression of BCL-2 ( B , C ); BAX ( F , G ); caspase-3 and procaspase-3 ( J , K ) was determined by western blot. Beta-2-microglobulin (BMG) served as loading control. Protein bands were analyzed by densitometry. Bars represent mean ± S.D. of selected protein to BMG ratio calculated in all examined groups. The ratio between the optical densities measured in 11-, 17- and 20-kDa cleaved caspase-3 and 32-kDa procaspase-3 bands was calculated as an index of caspase-3 activation. In addition, the BAX/BCL-2 mRNA ( H ) and protein ( I ) ratio was calculated. * p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Activation Assay

24) Product Images from "cAMP signaling increases histone deacetylase 8 expression via the Epac2–Rap1A–Akt pathway in H1299 lung cancer cells"

Article Title: cAMP signaling increases histone deacetylase 8 expression via the Epac2–Rap1A–Akt pathway in H1299 lung cancer cells

Journal: Experimental & Molecular Medicine

doi: 10.1038/emm.2016.152

ISO augmented cisplatin-induced apoptosis by increasing HDAC8 expression. ( a ) Effects of ISO and HDAC8 on cisplatin-induced cleavage of caspase-3 and PARP. ( b ) Effects of ISO and HDAC8 on cisplatin-induced annexin V-staining. ( c ) Effects of ISO and HDAC8 on γ-ray-induced cleavage of caspase-3 and PARP. ( d ) Effects of HDAC8 on cisplatin-induced TIPRL expression. ( e ) Effects of TIPRL knockdown on cisplatin-induced cleavage of caspase-3 and PARP. H1299 cells were transfected with wild-type HDAC8, HDAC8 shRNA, scrambled shRNA (S), TIPRL siRNA, control siRNA (C) or the corresponding control vectors (V) and incubated for 24 h. The cells were pretreated with ISO for 30 min (if necessary) and treated with 50 μ M cisplatin. Asterisks (*) on the bar graphs indicate a statistically significant difference compared with the corresponding control ( P
Figure Legend Snippet: ISO augmented cisplatin-induced apoptosis by increasing HDAC8 expression. ( a ) Effects of ISO and HDAC8 on cisplatin-induced cleavage of caspase-3 and PARP. ( b ) Effects of ISO and HDAC8 on cisplatin-induced annexin V-staining. ( c ) Effects of ISO and HDAC8 on γ-ray-induced cleavage of caspase-3 and PARP. ( d ) Effects of HDAC8 on cisplatin-induced TIPRL expression. ( e ) Effects of TIPRL knockdown on cisplatin-induced cleavage of caspase-3 and PARP. H1299 cells were transfected with wild-type HDAC8, HDAC8 shRNA, scrambled shRNA (S), TIPRL siRNA, control siRNA (C) or the corresponding control vectors (V) and incubated for 24 h. The cells were pretreated with ISO for 30 min (if necessary) and treated with 50 μ M cisplatin. Asterisks (*) on the bar graphs indicate a statistically significant difference compared with the corresponding control ( P

Techniques Used: Expressing, Staining, Transfection, shRNA, Incubation

25) Product Images from "InsR/IGF1R pathway mediates resistance to EGFR inhibitors in glioblastoma"

Article Title: InsR/IGF1R pathway mediates resistance to EGFR inhibitors in glioblastoma

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

doi: 10.1158/1078-0432.CCR-15-1677

Inhibition of the InsR/IGF1R pathway synergistically increases sensitivity to EGFR inhibitors (A) GBM39 or (B) GBM76 cells were plated in 96-well plates and treated with gefitinib ± OSI-906 following a 2-fold serial dilution. The combination contained a fixed equal molar ratio of gefitinib and OSI-906. Cell viability was determined 5 days after treatment. The combination index values were calculated for each data point by the Chou-Talalay method and presented as green crosses. (C) GBM39 or (D) GBM76 cells were treated with gefitinib ± OSI-906 at indicated concentrations. Three days after treatment, activities of caspase 3 were determined by Promega Caspase3/7-Glo assay and normalized to the corresponding cell titers.
Figure Legend Snippet: Inhibition of the InsR/IGF1R pathway synergistically increases sensitivity to EGFR inhibitors (A) GBM39 or (B) GBM76 cells were plated in 96-well plates and treated with gefitinib ± OSI-906 following a 2-fold serial dilution. The combination contained a fixed equal molar ratio of gefitinib and OSI-906. Cell viability was determined 5 days after treatment. The combination index values were calculated for each data point by the Chou-Talalay method and presented as green crosses. (C) GBM39 or (D) GBM76 cells were treated with gefitinib ± OSI-906 at indicated concentrations. Three days after treatment, activities of caspase 3 were determined by Promega Caspase3/7-Glo assay and normalized to the corresponding cell titers.

Techniques Used: Inhibition, Serial Dilution, Glo Assay

26) Product Images from "CD4+ CD25+ FoxP3+ regulatory T cells suppress cytotoxicity of CD8+ effector T cells: implications for their capacity to limit inflammatory central nervous system damage at the parenchymal level"

Article Title: CD4+ CD25+ FoxP3+ regulatory T cells suppress cytotoxicity of CD8+ effector T cells: implications for their capacity to limit inflammatory central nervous system damage at the parenchymal level

Journal: Journal of Neuroinflammation

doi: 10.1186/1742-2094-9-41

Suppression of cytotoxicity by OT-II T reg cells depends on the strength of the antigen stimulation of OT-I T cells in a cell coculture system . (A) Ratio of apoptotic cleaved caspase-3 + EG.7 and EL-4 cells after 6 h of cocultivation with activated OT-I T cells at different ratios in the absence of externally applied OVA 257-264 (n = 6, respectively). (B) Representative flow cytometry analysis of cleaved caspase-3 levels in EG.7 cells cocultured for 6 h at a ratio of 1:1 with activated OT-I T cells in the absence (left panel) and the presence (right panel) of OT-II T reg cells at a ratio of 2:1 (cleaved caspase-3 + EG.7 cells: no OT-I T cells: 6.5 ± 1.3%; OT-I T cells: 18.9 ± 2.7%; OT-I T cells + OT-II T reg cells: 18.3 ± 3.4%; P = 0.864; n = 4). (C) OVA 257-264 concentration dependence of the OT-II T reg and wild-type T reg cell-mediated reduction of cleaved caspase-3 + EL-4 cells induced by activated OT-I T cells (ratio OT-I to T reg: 2:1; ratio OT-I to EL-4: 1:1). At low OVA 257-264 concentrations (0.001 to 1 nM) OT-II T reg cells and wild-type T reg cells caused significant reduction of activated OT-I T cell-induced cleaved caspase-3 + EL-4 cells (n = 6, respectively). At high OVA 257-264 concentrations (10 to 1,000 nM) neither OT-II T reg cells nor wild-type T reg cells exerted any suppressive effect (n = 6, respectively). (D) OVA 257-264 peptide concentration dependence of intracellular ATP levels (detected following cell lysis) as a measure of cell viability in EL-4 cells following 4 h of cocultivation with activated OT-I T cells (ratio 1:1) in the absence and presence of OT-II T reg cells (ratio 2:1). At low OVA 257-264 concentrations (0.001 to 0.1 nM) significantly higher intracellular ATP levels in EL-4 cells (ratio 1:1) were detected in the presence of OT-II T reg cells as compared to their absence (OVA 257-264 0.1 nM: P = 0.0271; n = 8). At high OVA concentrations (1 to 10 nM) OT-II T reg cells did not exert any suppressive effect (OVA 257-264 10 nM: P = 0.734; n = 8). P values
Figure Legend Snippet: Suppression of cytotoxicity by OT-II T reg cells depends on the strength of the antigen stimulation of OT-I T cells in a cell coculture system . (A) Ratio of apoptotic cleaved caspase-3 + EG.7 and EL-4 cells after 6 h of cocultivation with activated OT-I T cells at different ratios in the absence of externally applied OVA 257-264 (n = 6, respectively). (B) Representative flow cytometry analysis of cleaved caspase-3 levels in EG.7 cells cocultured for 6 h at a ratio of 1:1 with activated OT-I T cells in the absence (left panel) and the presence (right panel) of OT-II T reg cells at a ratio of 2:1 (cleaved caspase-3 + EG.7 cells: no OT-I T cells: 6.5 ± 1.3%; OT-I T cells: 18.9 ± 2.7%; OT-I T cells + OT-II T reg cells: 18.3 ± 3.4%; P = 0.864; n = 4). (C) OVA 257-264 concentration dependence of the OT-II T reg and wild-type T reg cell-mediated reduction of cleaved caspase-3 + EL-4 cells induced by activated OT-I T cells (ratio OT-I to T reg: 2:1; ratio OT-I to EL-4: 1:1). At low OVA 257-264 concentrations (0.001 to 1 nM) OT-II T reg cells and wild-type T reg cells caused significant reduction of activated OT-I T cell-induced cleaved caspase-3 + EL-4 cells (n = 6, respectively). At high OVA 257-264 concentrations (10 to 1,000 nM) neither OT-II T reg cells nor wild-type T reg cells exerted any suppressive effect (n = 6, respectively). (D) OVA 257-264 peptide concentration dependence of intracellular ATP levels (detected following cell lysis) as a measure of cell viability in EL-4 cells following 4 h of cocultivation with activated OT-I T cells (ratio 1:1) in the absence and presence of OT-II T reg cells (ratio 2:1). At low OVA 257-264 concentrations (0.001 to 0.1 nM) significantly higher intracellular ATP levels in EL-4 cells (ratio 1:1) were detected in the presence of OT-II T reg cells as compared to their absence (OVA 257-264 0.1 nM: P = 0.0271; n = 8). At high OVA concentrations (1 to 10 nM) OT-II T reg cells did not exert any suppressive effect (OVA 257-264 10 nM: P = 0.734; n = 8). P values

Techniques Used: Flow Cytometry, Cytometry, Concentration Assay, Lysis

OT-II regulatory T (T reg) cells reduce OT-I T cell-induced oligodendroglial (and collateral neuronal) apoptosis in acute brain slices form ODC-OVA mice . (A, B) Representative immunofluorescence images of the cortical gray matter of ODC-OVA slices incubated for 6 h with (A) or without (B) activated OT-I T cells. Apoptotic NogoA + ODCs (left) and neuronal nuclear antigen (NeuN) + neurons (right) were detected by staining for cleaved caspase-3. (C) Densities of cleaved caspase-3 + NogoA + ODCs (left) and NeuN + neurons (right) in slices from naïve ODC-OVA mice under the conditions indicated (n = 6, respectively). P values
Figure Legend Snippet: OT-II regulatory T (T reg) cells reduce OT-I T cell-induced oligodendroglial (and collateral neuronal) apoptosis in acute brain slices form ODC-OVA mice . (A, B) Representative immunofluorescence images of the cortical gray matter of ODC-OVA slices incubated for 6 h with (A) or without (B) activated OT-I T cells. Apoptotic NogoA + ODCs (left) and neuronal nuclear antigen (NeuN) + neurons (right) were detected by staining for cleaved caspase-3. (C) Densities of cleaved caspase-3 + NogoA + ODCs (left) and NeuN + neurons (right) in slices from naïve ODC-OVA mice under the conditions indicated (n = 6, respectively). P values

Techniques Used: Mouse Assay, Immunofluorescence, Incubation, Staining

27) Product Images from "Drug screening and grouping by sensitivity with a panel of primary cultured cancer spheroids derived from endometrial cancer"

Article Title: Drug screening and grouping by sensitivity with a panel of primary cultured cancer spheroids derived from endometrial cancer

Journal: Cancer Science

doi: 10.1111/cas.12898

Sensitivity assay and intracellular signaling with a mammalian target of rapamycin complex 1 inhibitor in panels of endometrial cancer tissue‐originated spheroids ( CTOS ). (a) Dose–response curve for everolimus. Twelve CTOS from different patients’ tumors were examined. CTOS were prepared from original tumors or xenograft tumors. xp1, mouse tumor derived from primary CTOS line. Markers in the graph indicate the median value at each dose. (b) Double staining of cleaved caspase‐3 (red) and proliferating cell nuclear antigen ( PCNA ; green) in cp22 CTOS after exposure to 1 μM everolimus at the indicated time point. (c) Western blotting of CTOS exposed to 1 μM everolimus. CTOS , time after exposure, and antibodies are indicated. pAKT antibody is against S473. Sensitivity of each CTOS in (a) are shown at the top.
Figure Legend Snippet: Sensitivity assay and intracellular signaling with a mammalian target of rapamycin complex 1 inhibitor in panels of endometrial cancer tissue‐originated spheroids ( CTOS ). (a) Dose–response curve for everolimus. Twelve CTOS from different patients’ tumors were examined. CTOS were prepared from original tumors or xenograft tumors. xp1, mouse tumor derived from primary CTOS line. Markers in the graph indicate the median value at each dose. (b) Double staining of cleaved caspase‐3 (red) and proliferating cell nuclear antigen ( PCNA ; green) in cp22 CTOS after exposure to 1 μM everolimus at the indicated time point. (c) Western blotting of CTOS exposed to 1 μM everolimus. CTOS , time after exposure, and antibodies are indicated. pAKT antibody is against S473. Sensitivity of each CTOS in (a) are shown at the top.

Techniques Used: Sensitive Assay, Derivative Assay, Double Staining, Western Blot

28) Product Images from "Magnetic multiwalled carbon nanotubes with controlled release of epirubicin: an intravesical instillation system for bladder cancer"

Article Title: Magnetic multiwalled carbon nanotubes with controlled release of epirubicin: an intravesical instillation system for bladder cancer

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S189688

Immunohistochemical staining of exemplary tissue sections of bladders in different groups and statistical analysis of the IOD/area data. Notes:  ( A ) Representative light-microscopy sections of BCL2, BAX, and cleaved caspase 3-stained bladder tissue; ( B ) statistical analysis of IOD/area data of bladder-tissue sections for BCL2, BAX, and cleaved caspase 3. * P
Figure Legend Snippet: Immunohistochemical staining of exemplary tissue sections of bladders in different groups and statistical analysis of the IOD/area data. Notes: ( A ) Representative light-microscopy sections of BCL2, BAX, and cleaved caspase 3-stained bladder tissue; ( B ) statistical analysis of IOD/area data of bladder-tissue sections for BCL2, BAX, and cleaved caspase 3. * P

Techniques Used: Immunohistochemistry, Staining, Light Microscopy

29) Product Images from "Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Interacts with Multifunctional Angiogenin To Utilize Its Antiapoptotic Functions"

Article Title: Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Interacts with Multifunctional Angiogenin To Utilize Its Antiapoptotic Functions

Journal: Journal of Virology

doi: 10.1128/JVI.00070-12

Apoptotic effects of neomycin and antiapoptotic effects of angiogenin. (A) Serum-starved (8 h) HMVEC-d cells in 8-well chamber slides were either left untreated, treated with 1 μg/ml ANG, infected with 20 KSHV DNA/cell, or pretreated with 200 μM neomycin (N) for 1 h and then infected with the virus or treated with 1 μg/ml ANG. At 24 h, the slides were stained with anti-cleaved caspase-3 antibody and visualized under the fluorescence microscope. (B) HMVEC-d cells were treated similarly, and the samples were separated with a 10% gel and subjected to Western blotting for cleaved caspase-3.
Figure Legend Snippet: Apoptotic effects of neomycin and antiapoptotic effects of angiogenin. (A) Serum-starved (8 h) HMVEC-d cells in 8-well chamber slides were either left untreated, treated with 1 μg/ml ANG, infected with 20 KSHV DNA/cell, or pretreated with 200 μM neomycin (N) for 1 h and then infected with the virus or treated with 1 μg/ml ANG. At 24 h, the slides were stained with anti-cleaved caspase-3 antibody and visualized under the fluorescence microscope. (B) HMVEC-d cells were treated similarly, and the samples were separated with a 10% gel and subjected to Western blotting for cleaved caspase-3.

Techniques Used: Infection, Staining, Fluorescence, Microscopy, Western Blot

Reduction of cell survival upon angiogenin knockdown. (A) 293T cells were transfected with control sh-GFP and sh-Renilla (RL) plasmids or two sh-ANG (sh-ANG1 and sh-ANG2) plasmids, and ANG knockdown was checked by separating the lysates with a 12.5% gel and performing Western blotting for ANG. (B) ANG knockdown was also checked by real-time RT-PCR in sh-GFP- and sh-ANG1-transduced BCBL-1 cells. (C to E) BCBL-1 cells transduced with either sh-GFP or sh-ANG1 were subjected to Western blotting for p53, p-p53, p21, Bcl-2, Bax, caspase 3, and β-actin. Fold changes are indicated. (F) BCBL cells transduced with either sh-GFP or sh-ANG1 were stained with YO-PRO dye and analyzed by FACS for the percentage of live cells. Values shown represent the means ± standard deviations from three independent experiments. ***,  P
Figure Legend Snippet: Reduction of cell survival upon angiogenin knockdown. (A) 293T cells were transfected with control sh-GFP and sh-Renilla (RL) plasmids or two sh-ANG (sh-ANG1 and sh-ANG2) plasmids, and ANG knockdown was checked by separating the lysates with a 12.5% gel and performing Western blotting for ANG. (B) ANG knockdown was also checked by real-time RT-PCR in sh-GFP- and sh-ANG1-transduced BCBL-1 cells. (C to E) BCBL-1 cells transduced with either sh-GFP or sh-ANG1 were subjected to Western blotting for p53, p-p53, p21, Bcl-2, Bax, caspase 3, and β-actin. Fold changes are indicated. (F) BCBL cells transduced with either sh-GFP or sh-ANG1 were stained with YO-PRO dye and analyzed by FACS for the percentage of live cells. Values shown represent the means ± standard deviations from three independent experiments. ***, P

Techniques Used: Transfection, Western Blot, Quantitative RT-PCR, Transduction, Staining, FACS

30) Product Images from "Protective effect of DA-9401 in finasteride-induced apoptosis in rat testis: inositol requiring kinase 1 and c-Jun N-terminal kinase pathway"

Article Title: Protective effect of DA-9401 in finasteride-induced apoptosis in rat testis: inositol requiring kinase 1 and c-Jun N-terminal kinase pathway

Journal: Drug Design, Development and Therapy

doi: 10.2147/DDDT.S140543

Correlation between finasteride-induced oxidative injury and endoplasmic reticulum (ER) stress and apoptosis. Abbreviations: GRP-78, glucose-regulated protein-78; p-JNK, phosphorylated c-jun-N-terminal kinase; p-IRE1, phosphorylated inositol-requiring transmembrane kinase/endoribonuclease; Clev.Casp.-3, cleaved caspase-3; ROS, reactive oxygen species.
Figure Legend Snippet: Correlation between finasteride-induced oxidative injury and endoplasmic reticulum (ER) stress and apoptosis. Abbreviations: GRP-78, glucose-regulated protein-78; p-JNK, phosphorylated c-jun-N-terminal kinase; p-IRE1, phosphorylated inositol-requiring transmembrane kinase/endoribonuclease; Clev.Casp.-3, cleaved caspase-3; ROS, reactive oxygen species.

Techniques Used:

Effect of DA-9401 on the finasteride-induced endoplasmic reticulum (ER) stress response. Notes: ( A , B ) GRP-78; ( C , D ) p-IRE1; ( E , F ) p-JNK54; ( G , H ) procaspase-3; ( I , J ) cleaved caspase-3. Data are presented in mean ± SE. * P
Figure Legend Snippet: Effect of DA-9401 on the finasteride-induced endoplasmic reticulum (ER) stress response. Notes: ( A , B ) GRP-78; ( C , D ) p-IRE1; ( E , F ) p-JNK54; ( G , H ) procaspase-3; ( I , J ) cleaved caspase-3. Data are presented in mean ± SE. * P

Techniques Used:

31) Product Images from "MFGE8/Integrin β3 Pathway Alleviates Apoptosis and Inflammation in Early Brain Injury after Subarachnoid Hemorrhage in Rats"

Article Title: MFGE8/Integrin β3 Pathway Alleviates Apoptosis and Inflammation in Early Brain Injury after Subarachnoid Hemorrhage in Rats

Journal: Experimental neurology

doi: 10.1016/j.expneurol.2015.04.016

The adverse effects of silencing endogenous MFGE8 by siRNA at 24 hours after SAH Administration of MFGE8 siRNA decreased the modified Garcia Score (A) and Beam Balance Testing Score (B). Representative Western blots bands (C) and quantitative analysis of MFGE8 (D), CC3 (E), IL-1β (F) showed that MFGE8 siRNA decreased the protein level of endogenous MFGE8 and upregulated Capase3 and IL-1β expressions. MFGE8: Milk fat globule-epidermal growth factor-factor 8; rhMFGE8: Recombinant human milk fat globule-epidermal growth factor-factor 8; CC3: Cleaved caspase 3; n=6 for each group; * P
Figure Legend Snippet: The adverse effects of silencing endogenous MFGE8 by siRNA at 24 hours after SAH Administration of MFGE8 siRNA decreased the modified Garcia Score (A) and Beam Balance Testing Score (B). Representative Western blots bands (C) and quantitative analysis of MFGE8 (D), CC3 (E), IL-1β (F) showed that MFGE8 siRNA decreased the protein level of endogenous MFGE8 and upregulated Capase3 and IL-1β expressions. MFGE8: Milk fat globule-epidermal growth factor-factor 8; rhMFGE8: Recombinant human milk fat globule-epidermal growth factor-factor 8; CC3: Cleaved caspase 3; n=6 for each group; * P

Techniques Used: Modification, Western Blot, Recombinant

Integrinβ3 siRNA reversed the protective effects of rhMFGE8 treatment at 24 hours after SAH Integrinβ3 siRNA decreased Modified Garcia Score (A) and Beam Balance Testing Score (B) in the presence of rhMFGE8 treatment. Representative Western blots bands (C) and quantitative analysis of MFGE8 (D), CC3 (E) and IL-1β (F) showed that integrinβ3 siRNA decreased the protein level of MFGE8 and attenuated Capase3 and IL-1β expressions in the presence of rhMFGE8 treatment. MFGE8: Milk fat globule-epidermal growth factor-factor 8; rhMFGE8: Recombinant human milk fat globule-epidermal growth factor-factor 8; CC3: Cleaved caspase 3; n=6 for each group; * P
Figure Legend Snippet: Integrinβ3 siRNA reversed the protective effects of rhMFGE8 treatment at 24 hours after SAH Integrinβ3 siRNA decreased Modified Garcia Score (A) and Beam Balance Testing Score (B) in the presence of rhMFGE8 treatment. Representative Western blots bands (C) and quantitative analysis of MFGE8 (D), CC3 (E) and IL-1β (F) showed that integrinβ3 siRNA decreased the protein level of MFGE8 and attenuated Capase3 and IL-1β expressions in the presence of rhMFGE8 treatment. MFGE8: Milk fat globule-epidermal growth factor-factor 8; rhMFGE8: Recombinant human milk fat globule-epidermal growth factor-factor 8; CC3: Cleaved caspase 3; n=6 for each group; * P

Techniques Used: Modification, Western Blot, Recombinant

The protective effects of rhMFGE8 treatment at 24 hours after SAH Administration of rhMFGE8 increased the Modified Garcia Score (A) and Beam Balance Testing Score (B). Representative Western blots bands (C) and quantitative analysis of MFGE8 (D), CC3 (E) and IL-1β (F) showed that rhMFGE8 treatment increased the protein level of MFGE8 and decreased Capase3 and IL-1β expressions. MFGE8: Milk fat globule-epidermal growth factor-factor 8; rhMFGE8: Recombinant human milk fat globule-epidermal growth factor-factor 8; CC3: Cleaved caspase 3; n=6 for each group; * P
Figure Legend Snippet: The protective effects of rhMFGE8 treatment at 24 hours after SAH Administration of rhMFGE8 increased the Modified Garcia Score (A) and Beam Balance Testing Score (B). Representative Western blots bands (C) and quantitative analysis of MFGE8 (D), CC3 (E) and IL-1β (F) showed that rhMFGE8 treatment increased the protein level of MFGE8 and decreased Capase3 and IL-1β expressions. MFGE8: Milk fat globule-epidermal growth factor-factor 8; rhMFGE8: Recombinant human milk fat globule-epidermal growth factor-factor 8; CC3: Cleaved caspase 3; n=6 for each group; * P

Techniques Used: Modification, Western Blot, Recombinant

32) Product Images from "MFGE8/Integrin β3 Pathway Alleviates Apoptosis and Inflammation in Early Brain Injury after Subarachnoid Hemorrhage in Rats"

Article Title: MFGE8/Integrin β3 Pathway Alleviates Apoptosis and Inflammation in Early Brain Injury after Subarachnoid Hemorrhage in Rats

Journal: Experimental neurology

doi: 10.1016/j.expneurol.2015.04.016

The adverse effects of silencing endogenous MFGE8 by siRNA at 24 hours after SAH Administration of MFGE8 siRNA decreased the modified Garcia Score (A) and Beam Balance Testing Score (B). Representative Western blots bands (C) and quantitative analysis of MFGE8 (D), CC3 (E), IL-1β (F) showed that MFGE8 siRNA decreased the protein level of endogenous MFGE8 and upregulated Capase3 and IL-1β expressions. MFGE8: Milk fat globule-epidermal growth factor-factor 8; rhMFGE8: Recombinant human milk fat globule-epidermal growth factor-factor 8; CC3: Cleaved caspase 3; n=6 for each group; * P
Figure Legend Snippet: The adverse effects of silencing endogenous MFGE8 by siRNA at 24 hours after SAH Administration of MFGE8 siRNA decreased the modified Garcia Score (A) and Beam Balance Testing Score (B). Representative Western blots bands (C) and quantitative analysis of MFGE8 (D), CC3 (E), IL-1β (F) showed that MFGE8 siRNA decreased the protein level of endogenous MFGE8 and upregulated Capase3 and IL-1β expressions. MFGE8: Milk fat globule-epidermal growth factor-factor 8; rhMFGE8: Recombinant human milk fat globule-epidermal growth factor-factor 8; CC3: Cleaved caspase 3; n=6 for each group; * P

Techniques Used: Modification, Western Blot, Recombinant

Integrinβ3 siRNA reversed the protective effects of rhMFGE8 treatment at 24 hours after SAH Integrinβ3 siRNA decreased Modified Garcia Score (A) and Beam Balance Testing Score (B) in the presence of rhMFGE8 treatment. Representative Western blots bands (C) and quantitative analysis of MFGE8 (D), CC3 (E) and IL-1β (F) showed that integrinβ3 siRNA decreased the protein level of MFGE8 and attenuated Capase3 and IL-1β expressions in the presence of rhMFGE8 treatment. MFGE8: Milk fat globule-epidermal growth factor-factor 8; rhMFGE8: Recombinant human milk fat globule-epidermal growth factor-factor 8; CC3: Cleaved caspase 3; n=6 for each group; * P
Figure Legend Snippet: Integrinβ3 siRNA reversed the protective effects of rhMFGE8 treatment at 24 hours after SAH Integrinβ3 siRNA decreased Modified Garcia Score (A) and Beam Balance Testing Score (B) in the presence of rhMFGE8 treatment. Representative Western blots bands (C) and quantitative analysis of MFGE8 (D), CC3 (E) and IL-1β (F) showed that integrinβ3 siRNA decreased the protein level of MFGE8 and attenuated Capase3 and IL-1β expressions in the presence of rhMFGE8 treatment. MFGE8: Milk fat globule-epidermal growth factor-factor 8; rhMFGE8: Recombinant human milk fat globule-epidermal growth factor-factor 8; CC3: Cleaved caspase 3; n=6 for each group; * P

Techniques Used: Modification, Western Blot, Recombinant

The protective effects of rhMFGE8 treatment at 24 hours after SAH Administration of rhMFGE8 increased the Modified Garcia Score (A) and Beam Balance Testing Score (B). Representative Western blots bands (C) and quantitative analysis of MFGE8 (D), CC3 (E) and IL-1β (F) showed that rhMFGE8 treatment increased the protein level of MFGE8 and decreased Capase3 and IL-1β expressions. MFGE8: Milk fat globule-epidermal growth factor-factor 8; rhMFGE8: Recombinant human milk fat globule-epidermal growth factor-factor 8; CC3: Cleaved caspase 3; n=6 for each group; * P
Figure Legend Snippet: The protective effects of rhMFGE8 treatment at 24 hours after SAH Administration of rhMFGE8 increased the Modified Garcia Score (A) and Beam Balance Testing Score (B). Representative Western blots bands (C) and quantitative analysis of MFGE8 (D), CC3 (E) and IL-1β (F) showed that rhMFGE8 treatment increased the protein level of MFGE8 and decreased Capase3 and IL-1β expressions. MFGE8: Milk fat globule-epidermal growth factor-factor 8; rhMFGE8: Recombinant human milk fat globule-epidermal growth factor-factor 8; CC3: Cleaved caspase 3; n=6 for each group; * P

Techniques Used: Modification, Western Blot, Recombinant

33) Product Images from "Efficient suilysin-mediated invasion and apoptosis in porcine respiratory epithelial cells after streptococcal infection under air-liquid interface conditions"

Article Title: Efficient suilysin-mediated invasion and apoptosis in porcine respiratory epithelial cells after streptococcal infection under air-liquid interface conditions

Journal: Scientific Reports

doi: 10.1038/srep26748

Detection of apoptosis in S. suis infected porcine bronchial epithelial cells. PBEC were apically infected with approximately 1 × 10 7 CFU of S. suis wt, 10Δsly, cW461F or cS148 respectively. After 4 h, cells were washed thoroughly to remove non-adherent bacteria, and further incubated until 48 hpi under air-liquid interface conditions. Colocalization of S. suis and apoptotic cells in PBEC (A). Analysis by confocal laser scanning microscopy, cells were stained for cleaved caspase-3 (red) and streptococci (green). Nuclei were stained by DAPI (blue). Bars represent 25 μm. Colocalization of suilysin and apoptotic cells of PBEC ( B). For analysis by confocal laser scanning microscopy, cells were immunostained for cleaved caspase-3 (red) and suilysin (green). Nuclei were stained by DAPI (blue). Bars represent 25 μm. Quantification of apoptotic cells in PBEC (C). To quantify the apoptotic cells on PBEC, the areas containing red-fluorescent cells were determined from panels A and B. Results are expressed as x-fold increase compared to mock-infected cells (mock). Results are expressed as means ± SEM and significance, indicated by ***(P
Figure Legend Snippet: Detection of apoptosis in S. suis infected porcine bronchial epithelial cells. PBEC were apically infected with approximately 1 × 10 7 CFU of S. suis wt, 10Δsly, cW461F or cS148 respectively. After 4 h, cells were washed thoroughly to remove non-adherent bacteria, and further incubated until 48 hpi under air-liquid interface conditions. Colocalization of S. suis and apoptotic cells in PBEC (A). Analysis by confocal laser scanning microscopy, cells were stained for cleaved caspase-3 (red) and streptococci (green). Nuclei were stained by DAPI (blue). Bars represent 25 μm. Colocalization of suilysin and apoptotic cells of PBEC ( B). For analysis by confocal laser scanning microscopy, cells were immunostained for cleaved caspase-3 (red) and suilysin (green). Nuclei were stained by DAPI (blue). Bars represent 25 μm. Quantification of apoptotic cells in PBEC (C). To quantify the apoptotic cells on PBEC, the areas containing red-fluorescent cells were determined from panels A and B. Results are expressed as x-fold increase compared to mock-infected cells (mock). Results are expressed as means ± SEM and significance, indicated by ***(P

Techniques Used: Infection, Incubation, Confocal Laser Scanning Microscopy, Staining

34) Product Images from "Non-thermal plasma-induced apoptosis is modulated by ATR- and PARP1-mediated DNA damage responses and circadian clock"

Article Title: Non-thermal plasma-induced apoptosis is modulated by ATR- and PARP1-mediated DNA damage responses and circadian clock

Journal: Oncotarget

doi: 10.18632/oncotarget.9087

Inhibition of PARP1 augments apoptosis during NTP and NTPO treatment ( A ) A549 and SK-MEL2 cells pretreated with a PARP inhibitor during NTP and NTPO treatment were stained for cleaved-caspase 3, and Hoechst-stained nuclei were depicted as dotted lines. ( B ) The quantitative analysis for (A). The bars and error bars represent the mean ± SD from three independent experiments. ( C ) A TUNEL assay was performed in A549 cells pretreated with ATR, PARP, or ATR + PARP inhibitor during NTP and NTPO treatment. Bars and error bars represent the mean ± SD from three independent experiments (* =  p
Figure Legend Snippet: Inhibition of PARP1 augments apoptosis during NTP and NTPO treatment ( A ) A549 and SK-MEL2 cells pretreated with a PARP inhibitor during NTP and NTPO treatment were stained for cleaved-caspase 3, and Hoechst-stained nuclei were depicted as dotted lines. ( B ) The quantitative analysis for (A). The bars and error bars represent the mean ± SD from three independent experiments. ( C ) A TUNEL assay was performed in A549 cells pretreated with ATR, PARP, or ATR + PARP inhibitor during NTP and NTPO treatment. Bars and error bars represent the mean ± SD from three independent experiments (* = p

Techniques Used: Inhibition, Staining, TUNEL Assay

PARP1 activity dictates circadian toxicity of NTP and NTPO in normal cells ( A, B ) WT-MEF and CRY DKO -MEF cells exposed to either NTP or NTPO at the indicated ZTs were analyzed for cleaved-caspase 3 (A) and TUNEL signal (B). Bars and error bars represent the mean ± SD from three independent experiments (* =  p
Figure Legend Snippet: PARP1 activity dictates circadian toxicity of NTP and NTPO in normal cells ( A, B ) WT-MEF and CRY DKO -MEF cells exposed to either NTP or NTPO at the indicated ZTs were analyzed for cleaved-caspase 3 (A) and TUNEL signal (B). Bars and error bars represent the mean ± SD from three independent experiments (* = p

Techniques Used: Activity Assay, TUNEL Assay

Enhanced apoptosis by the addition of oxygen gas flow during NTP treatment in human cancer cells ( A ) A549 and SK-MEL2 cells treated with NTP supplemented with or without the flow of oxygen gas were fixed and immunostained for cleaved-caspase 3, and Hoechst-stained nuclei were depicted as dotted lines. ( B ) A TUNEL assay was performed to detect DNA fragmentation. Bars and error bars represent mean ± SD from three independent experiments (* =  p
Figure Legend Snippet: Enhanced apoptosis by the addition of oxygen gas flow during NTP treatment in human cancer cells ( A ) A549 and SK-MEL2 cells treated with NTP supplemented with or without the flow of oxygen gas were fixed and immunostained for cleaved-caspase 3, and Hoechst-stained nuclei were depicted as dotted lines. ( B ) A TUNEL assay was performed to detect DNA fragmentation. Bars and error bars represent mean ± SD from three independent experiments (* = p

Techniques Used: Flow Cytometry, Staining, TUNEL Assay

35) Product Images from "IAP antagonists Birinapant and AT-406 efficiently synergise with either TRAIL, BRAF, or BCL-2 inhibitors to sensitise BRAFV600E colorectal tumour cells to apoptosis"

Article Title: IAP antagonists Birinapant and AT-406 efficiently synergise with either TRAIL, BRAF, or BCL-2 inhibitors to sensitise BRAFV600E colorectal tumour cells to apoptosis

Journal: BMC Cancer

doi: 10.1186/s12885-016-2606-5

Co-treatment of Birinapant with TRAIL can synergistically increase their efficiency and induce apoptosis in colorectal adenocarcinoma cells in 2D and 3D. 5a: Cell viability of cell line HT29 after co-treatment with the SMAC-mimetic Birinapant and the apoptotic agent TRAIL. Cells were either left untreated (ctr = control) or treated with Birinapant, TRAIL and their combination for 48 and 72 h and the % percentage cell viability was measured. The average of three independent experiments is presented. Columns = % percentage of cell viability, bars = SD. 5a-RKO: Respectively for cell line RKO (upper panel). To check for the reversibility of treatment effects, RKO cells were either left untreated (ctr = control), treated with either Birinapant, TRAIL or their combination for 48 h. Cells were then incubated with additional 48 h without treatments (NoT) and the % percentage cell viability was measured. The average of three independent experiments is presented. 5b-I. Protein levels of cIAP-1 and XIAP after 0.5, 1 and 5 μM AT-406 (lanes 3–5) or Birinapant (lanes 6–8) treatments respectively for 48 h, compared to control lanes 1 (no treatment) or 2 (DMSO treatment). 5b-II and 5b-III. Protein levels of PARP-1 and total Caspase-3 in HT29 cell line after co-treatment with SMAC-mimetic Birinapant and TRAIL for 48 and 72 h. Cells were either left untreated (lane 1) or treated with DMSO (lane 2), with 5 μM Birinapant (lane 3), with 10, 50 and 100 ng/mL TRAIL (lanes 4–6), or with 5 μM Birinapant and combination with TRAIL (lanes 7–9) for 48 and 72 h. Sensitive cell line HCT116 treated with TRAIL is used as positive control (lane 10). Using W. B., protein levels of PARP-1 (Figure 5b-II) and of total Caspase 3 (Figure 5b-III) were analyzed after the corresponding treatments. Data are representative for three independent experiments. 5b-I-RKO: Protein levels of cIAP-1 and XIAP after mono-treatments (lanes 3 and 4 for AT-406 and Birinapant respectively and lanes 5–7 for TRAIL 10, 50 and 100 ng/mL respectively) and co-treatments (lanes 8–13 as shown) for 24 h. Untreated (lane 1) and treated with DMSO (lane 2), cells are also presented. 5b-II-RKO and 5b-III-RKO: Protein levels of PARP-1 and total Caspase-3 after respective mono-treatments and co-treatments (same lines as 5-I-RKO). Data are representative of three independent experiments. 5C: Light microscopy images from HT29 culture after combined treatment with Birinapant and TRAIL. Detached (apoptotic) cells are shown in supernatant of co-treated groups for 48 and 72 h. Representative images. 5d: Confocal microscope images were taken after co-treatment with Birinapant, TRAIL and their combination. Nuclei were detected with HOECHST staining (blue color), cleaved Caspase-3 (green color). Representative images.. Scale bar: 9.9 μm. 5e. Light microscopy of three-dimensional culture of HT29 cells after co-treatment with Birinapant, TRAIL and their combinations in 3D for 6 days. Representative images. 5f: Confocal microscope images were taken after co-treatment with Birinapant, TRAIL and their combinations in 3D culture for 6 days. The nuclei were detected with HOECHST staining (blue color), cleaved Caspase-3 (green color). Representative images. Scale bar: 57.4 μm
Figure Legend Snippet: Co-treatment of Birinapant with TRAIL can synergistically increase their efficiency and induce apoptosis in colorectal adenocarcinoma cells in 2D and 3D. 5a: Cell viability of cell line HT29 after co-treatment with the SMAC-mimetic Birinapant and the apoptotic agent TRAIL. Cells were either left untreated (ctr = control) or treated with Birinapant, TRAIL and their combination for 48 and 72 h and the % percentage cell viability was measured. The average of three independent experiments is presented. Columns = % percentage of cell viability, bars = SD. 5a-RKO: Respectively for cell line RKO (upper panel). To check for the reversibility of treatment effects, RKO cells were either left untreated (ctr = control), treated with either Birinapant, TRAIL or their combination for 48 h. Cells were then incubated with additional 48 h without treatments (NoT) and the % percentage cell viability was measured. The average of three independent experiments is presented. 5b-I. Protein levels of cIAP-1 and XIAP after 0.5, 1 and 5 μM AT-406 (lanes 3–5) or Birinapant (lanes 6–8) treatments respectively for 48 h, compared to control lanes 1 (no treatment) or 2 (DMSO treatment). 5b-II and 5b-III. Protein levels of PARP-1 and total Caspase-3 in HT29 cell line after co-treatment with SMAC-mimetic Birinapant and TRAIL for 48 and 72 h. Cells were either left untreated (lane 1) or treated with DMSO (lane 2), with 5 μM Birinapant (lane 3), with 10, 50 and 100 ng/mL TRAIL (lanes 4–6), or with 5 μM Birinapant and combination with TRAIL (lanes 7–9) for 48 and 72 h. Sensitive cell line HCT116 treated with TRAIL is used as positive control (lane 10). Using W. B., protein levels of PARP-1 (Figure 5b-II) and of total Caspase 3 (Figure 5b-III) were analyzed after the corresponding treatments. Data are representative for three independent experiments. 5b-I-RKO: Protein levels of cIAP-1 and XIAP after mono-treatments (lanes 3 and 4 for AT-406 and Birinapant respectively and lanes 5–7 for TRAIL 10, 50 and 100 ng/mL respectively) and co-treatments (lanes 8–13 as shown) for 24 h. Untreated (lane 1) and treated with DMSO (lane 2), cells are also presented. 5b-II-RKO and 5b-III-RKO: Protein levels of PARP-1 and total Caspase-3 after respective mono-treatments and co-treatments (same lines as 5-I-RKO). Data are representative of three independent experiments. 5C: Light microscopy images from HT29 culture after combined treatment with Birinapant and TRAIL. Detached (apoptotic) cells are shown in supernatant of co-treated groups for 48 and 72 h. Representative images. 5d: Confocal microscope images were taken after co-treatment with Birinapant, TRAIL and their combination. Nuclei were detected with HOECHST staining (blue color), cleaved Caspase-3 (green color). Representative images.. Scale bar: 9.9 μm. 5e. Light microscopy of three-dimensional culture of HT29 cells after co-treatment with Birinapant, TRAIL and their combinations in 3D for 6 days. Representative images. 5f: Confocal microscope images were taken after co-treatment with Birinapant, TRAIL and their combinations in 3D culture for 6 days. The nuclei were detected with HOECHST staining (blue color), cleaved Caspase-3 (green color). Representative images. Scale bar: 57.4 μm

Techniques Used: Incubation, Positive Control, Light Microscopy, Microscopy, Staining

SMAC-mimetic AT-406 and TRAIL synergistically kill resistant tumour cells. 6a Cell viability of HT29 cell line after combined treatment with SMAC-mimetic AT-406 and the apoptotic agent TRAIL. Cells were either left untreated (ctr = control) or treated with AT-406 combined with TRAIL for 48 and 72 h and the % percentage cell viability was measured by SRB staining. The values are the average of three independent experiments and are presented as fold change of the absorbance of treated/untreated cells, for each condition. Columns = % percentage of cell viability, bars = SD. 6a-RKO: Respectively for cell line RKO. 6b. Confocal microscope images were taken after co-treatment with AT-406, TRAIL and their combinations for 48 and 72 h. The nuclei were detected with HOECHST staining (blue color), cleaved Caspase-3 (green color). Representative images. Scale bar: 9.9 μM
Figure Legend Snippet: SMAC-mimetic AT-406 and TRAIL synergistically kill resistant tumour cells. 6a Cell viability of HT29 cell line after combined treatment with SMAC-mimetic AT-406 and the apoptotic agent TRAIL. Cells were either left untreated (ctr = control) or treated with AT-406 combined with TRAIL for 48 and 72 h and the % percentage cell viability was measured by SRB staining. The values are the average of three independent experiments and are presented as fold change of the absorbance of treated/untreated cells, for each condition. Columns = % percentage of cell viability, bars = SD. 6a-RKO: Respectively for cell line RKO. 6b. Confocal microscope images were taken after co-treatment with AT-406, TRAIL and their combinations for 48 and 72 h. The nuclei were detected with HOECHST staining (blue color), cleaved Caspase-3 (green color). Representative images. Scale bar: 9.9 μM

Techniques Used: Sulforhodamine B Assay, Staining, Microscopy

Birinapant treatment results in reduced cell viability and appearance of apoptosis in selected colorectal adenocarcinoma cells. a : Cell viability of cell lines after treatment with SMAC-mimetics. Cells were either left untreated (ctr = control) or treated with 0.5 μM, 1 mM or 5 mM AT-406 and Birinapant for 48 h (1) and 72 h (2) and the % percentage cell viability was measured by SRB. Average of three independent experiments are presented as fold change of the absorbance of treated/untreated cells for each condition. Columns = % percentage of cell viability, bars = SD. b : Protein levels of XIAP, cIAP-1, PARP-1 and total caspase-3 in RKO and HCT116 were analysed by W.B. after treatment with 0.5, 1 and 5 μM SMAC-mimetics AT-406 (lanes 3–5) and Birinapant (lanes 6–8) for 48 and 72 h. Untreated (lane 1) or treated with DMSO cells (lane 2) were used as control. Proteins are quantified against α-Tubulin. Data are representative for three independent experiments. c - d : Confocal microscope images and Hoechst staining of RKO (2C) and HCT116 (2D) cell lines two-dimensional culture, after treatment with SMAC-mimetics AT-406 (A) and Birinapant (B). Crescent nuclei of RKO cells present after cell treatments are shown by arrows. Confocal microscope images were taken after treatment with SMAC-mimetics AT-406 and Birinapant in RKO (2c) and HCT116 (2d) 48 and 72 h. The nuclei were detected with HOECHST staining. Representative images are presented
Figure Legend Snippet: Birinapant treatment results in reduced cell viability and appearance of apoptosis in selected colorectal adenocarcinoma cells. a : Cell viability of cell lines after treatment with SMAC-mimetics. Cells were either left untreated (ctr = control) or treated with 0.5 μM, 1 mM or 5 mM AT-406 and Birinapant for 48 h (1) and 72 h (2) and the % percentage cell viability was measured by SRB. Average of three independent experiments are presented as fold change of the absorbance of treated/untreated cells for each condition. Columns = % percentage of cell viability, bars = SD. b : Protein levels of XIAP, cIAP-1, PARP-1 and total caspase-3 in RKO and HCT116 were analysed by W.B. after treatment with 0.5, 1 and 5 μM SMAC-mimetics AT-406 (lanes 3–5) and Birinapant (lanes 6–8) for 48 and 72 h. Untreated (lane 1) or treated with DMSO cells (lane 2) were used as control. Proteins are quantified against α-Tubulin. Data are representative for three independent experiments. c - d : Confocal microscope images and Hoechst staining of RKO (2C) and HCT116 (2D) cell lines two-dimensional culture, after treatment with SMAC-mimetics AT-406 (A) and Birinapant (B). Crescent nuclei of RKO cells present after cell treatments are shown by arrows. Confocal microscope images were taken after treatment with SMAC-mimetics AT-406 and Birinapant in RKO (2c) and HCT116 (2d) 48 and 72 h. The nuclei were detected with HOECHST staining. Representative images are presented

Techniques Used: Sulforhodamine B Assay, Microscopy, Staining

Pre-treatment with Birinapant and then co-treatment with Birinapant/ BRAF inhibitor PLX4720 synergistically induce apoptosis of colorectal adenocarcinoma cells in 2D and 3D. a : Cell viability after co-treatment with the SMAC-mimetics Birinapant or AT-406 in combination with the BRAF inhibitor PLX4720. Cells were either left untreated (ctr = control) or treated with 5 μM Birinapant, 5 μM AT-406 and 1 μM PLX4720 and their combinations for 48 and 72 h. The average of three independent experiments is presented as fold change of the absorbance of treated/untreated cells, for each condition. Columns = % percentage of cell viability, bars = SD. b : Cell viability after pre-treatment with the SMAC-mimetic Birinapant and then co-treatment with BRAF inhibitor PLX4720 and Birinapant. Cells were either left untreated (ctr = control) or treated with 0.5 or 1 μM Birinapant and 0.2 or 0.5 μM PLX4720. For the pre-treatment testing, cells were first incubated for 24 h with either 0.5 or 1 μM of Birinapant and then co-treated with 0.2 and 0.5 μM PLX4720 for another 24 or 48 h. c : Protein levels of PARP-1, total Caspase-3, XIAP, cIAP-1 and p-ERK1/2 in RKO by W.B., after pre-treatment with 0.5 or 1 μM Birinapant and then co-treatment with 0.2 or 0.5 μM PLX4720. Untreated cells were used as control. Proteins are quantified against α-Tubulin. Data are representative for three independent experiments. d : Light microscopy images from RKO culture after pre-treatment with Birinapant and then co-treatment with PLX4720. Detached cells are shown in supernatant of co-treated group. Several images were taken from untreated and treated with Birinapant (0.5 or 1 μM) and PLX4720 (0.2 or 0.5 μM) RKO cells while been cultured in 6-well plates for 48 and 72 h. Representative images are presented. e : Light microscopy of three-dimensional culture of RKO cells after co-treatment with 0.5 μM, 1 μM Birinapant and 0.2 μM, 0.5 μM PLX4720 and their combination in 3D culture for 6 d. Representative images. f : Confocal microscope images were taken after co-treatment with 0.5 μM, 1 μM Birinapant and 0.2 μM, 0.5 μM PLX4720 and combinations in 3D cultures for 6 days. The nuclei were detected with HOECHST staining (blue color), cleaved Caspase-3 (green color). Representative images. Scale bar: 20 μm
Figure Legend Snippet: Pre-treatment with Birinapant and then co-treatment with Birinapant/ BRAF inhibitor PLX4720 synergistically induce apoptosis of colorectal adenocarcinoma cells in 2D and 3D. a : Cell viability after co-treatment with the SMAC-mimetics Birinapant or AT-406 in combination with the BRAF inhibitor PLX4720. Cells were either left untreated (ctr = control) or treated with 5 μM Birinapant, 5 μM AT-406 and 1 μM PLX4720 and their combinations for 48 and 72 h. The average of three independent experiments is presented as fold change of the absorbance of treated/untreated cells, for each condition. Columns = % percentage of cell viability, bars = SD. b : Cell viability after pre-treatment with the SMAC-mimetic Birinapant and then co-treatment with BRAF inhibitor PLX4720 and Birinapant. Cells were either left untreated (ctr = control) or treated with 0.5 or 1 μM Birinapant and 0.2 or 0.5 μM PLX4720. For the pre-treatment testing, cells were first incubated for 24 h with either 0.5 or 1 μM of Birinapant and then co-treated with 0.2 and 0.5 μM PLX4720 for another 24 or 48 h. c : Protein levels of PARP-1, total Caspase-3, XIAP, cIAP-1 and p-ERK1/2 in RKO by W.B., after pre-treatment with 0.5 or 1 μM Birinapant and then co-treatment with 0.2 or 0.5 μM PLX4720. Untreated cells were used as control. Proteins are quantified against α-Tubulin. Data are representative for three independent experiments. d : Light microscopy images from RKO culture after pre-treatment with Birinapant and then co-treatment with PLX4720. Detached cells are shown in supernatant of co-treated group. Several images were taken from untreated and treated with Birinapant (0.5 or 1 μM) and PLX4720 (0.2 or 0.5 μM) RKO cells while been cultured in 6-well plates for 48 and 72 h. Representative images are presented. e : Light microscopy of three-dimensional culture of RKO cells after co-treatment with 0.5 μM, 1 μM Birinapant and 0.2 μM, 0.5 μM PLX4720 and their combination in 3D culture for 6 d. Representative images. f : Confocal microscope images were taken after co-treatment with 0.5 μM, 1 μM Birinapant and 0.2 μM, 0.5 μM PLX4720 and combinations in 3D cultures for 6 days. The nuclei were detected with HOECHST staining (blue color), cleaved Caspase-3 (green color). Representative images. Scale bar: 20 μm

Techniques Used: Incubation, Light Microscopy, Cell Culture, Microscopy, Staining

36) Product Images from "IAP antagonists Birinapant and AT-406 efficiently synergise with either TRAIL, BRAF, or BCL-2 inhibitors to sensitise BRAFV600E colorectal tumour cells to apoptosis"

Article Title: IAP antagonists Birinapant and AT-406 efficiently synergise with either TRAIL, BRAF, or BCL-2 inhibitors to sensitise BRAFV600E colorectal tumour cells to apoptosis

Journal: BMC Cancer

doi: 10.1186/s12885-016-2606-5

Co-treatment of Birinapant with TRAIL can synergistically increase their efficiency and induce apoptosis in colorectal adenocarcinoma cells in 2D and 3D. 5a: Cell viability of cell line HT29 after co-treatment with the SMAC-mimetic Birinapant and the apoptotic agent TRAIL. Cells were either left untreated (ctr = control) or treated with Birinapant, TRAIL and their combination for 48 and 72 h and the % percentage cell viability was measured. The average of three independent experiments is presented. Columns = % percentage of cell viability, bars = SD. 5a-RKO: Respectively for cell line RKO (upper panel). To check for the reversibility of treatment effects, RKO cells were either left untreated (ctr = control), treated with either Birinapant, TRAIL or their combination for 48 h. Cells were then incubated with additional 48 h without treatments (NoT) and the % percentage cell viability was measured. The average of three independent experiments is presented. 5b-I. Protein levels of cIAP-1 and XIAP after 0.5, 1 and 5 μM AT-406 (lanes 3–5) or Birinapant (lanes 6–8) treatments respectively for 48 h, compared to control lanes 1 (no treatment) or 2 (DMSO treatment). 5b-II and 5b-III. Protein levels of PARP-1 and total Caspase-3 in HT29 cell line after co-treatment with SMAC-mimetic Birinapant and TRAIL for 48 and 72 h. Cells were either left untreated (lane 1) or treated with DMSO (lane 2), with 5 μM Birinapant (lane 3), with 10, 50 and 100 ng/mL TRAIL (lanes 4–6), or with 5 μM Birinapant and combination with TRAIL (lanes 7–9) for 48 and 72 h. Sensitive cell line HCT116 treated with TRAIL is used as positive control (lane 10). Using W. B., protein levels of PARP-1 (Figure 5b-II) and of total Caspase 3 (Figure 5b-III) were analyzed after the corresponding treatments. Data are representative for three independent experiments. 5b-I-RKO: Protein levels of cIAP-1 and XIAP after mono-treatments (lanes 3 and 4 for AT-406 and Birinapant respectively and lanes 5–7 for TRAIL 10, 50 and 100 ng/mL respectively) and co-treatments (lanes 8–13 as shown) for 24 h. Untreated (lane 1) and treated with DMSO (lane 2), cells are also presented. 5b-II-RKO and 5b-III-RKO: Protein levels of PARP-1 and total Caspase-3 after respective mono-treatments and co-treatments (same lines as 5-I-RKO). Data are representative of three independent experiments. 5C: Light microscopy images from HT29 culture after combined treatment with Birinapant and TRAIL. Detached (apoptotic) cells are shown in supernatant of co-treated groups for 48 and 72 h. Representative images. 5d: Confocal microscope images were taken after co-treatment with Birinapant, TRAIL and their combination. Nuclei were detected with HOECHST staining (blue color), cleaved Caspase-3 (green color). Representative images.. Scale bar: 9.9 μm. 5e. Light microscopy of three-dimensional culture of HT29 cells after co-treatment with Birinapant, TRAIL and their combinations in 3D for 6 days. Representative images. 5f: Confocal microscope images were taken after co-treatment with Birinapant, TRAIL and their combinations in 3D culture for 6 days. The nuclei were detected with HOECHST staining (blue color), cleaved Caspase-3 (green color). Representative images. Scale bar: 57.4 μm
Figure Legend Snippet: Co-treatment of Birinapant with TRAIL can synergistically increase their efficiency and induce apoptosis in colorectal adenocarcinoma cells in 2D and 3D. 5a: Cell viability of cell line HT29 after co-treatment with the SMAC-mimetic Birinapant and the apoptotic agent TRAIL. Cells were either left untreated (ctr = control) or treated with Birinapant, TRAIL and their combination for 48 and 72 h and the % percentage cell viability was measured. The average of three independent experiments is presented. Columns = % percentage of cell viability, bars = SD. 5a-RKO: Respectively for cell line RKO (upper panel). To check for the reversibility of treatment effects, RKO cells were either left untreated (ctr = control), treated with either Birinapant, TRAIL or their combination for 48 h. Cells were then incubated with additional 48 h without treatments (NoT) and the % percentage cell viability was measured. The average of three independent experiments is presented. 5b-I. Protein levels of cIAP-1 and XIAP after 0.5, 1 and 5 μM AT-406 (lanes 3–5) or Birinapant (lanes 6–8) treatments respectively for 48 h, compared to control lanes 1 (no treatment) or 2 (DMSO treatment). 5b-II and 5b-III. Protein levels of PARP-1 and total Caspase-3 in HT29 cell line after co-treatment with SMAC-mimetic Birinapant and TRAIL for 48 and 72 h. Cells were either left untreated (lane 1) or treated with DMSO (lane 2), with 5 μM Birinapant (lane 3), with 10, 50 and 100 ng/mL TRAIL (lanes 4–6), or with 5 μM Birinapant and combination with TRAIL (lanes 7–9) for 48 and 72 h. Sensitive cell line HCT116 treated with TRAIL is used as positive control (lane 10). Using W. B., protein levels of PARP-1 (Figure 5b-II) and of total Caspase 3 (Figure 5b-III) were analyzed after the corresponding treatments. Data are representative for three independent experiments. 5b-I-RKO: Protein levels of cIAP-1 and XIAP after mono-treatments (lanes 3 and 4 for AT-406 and Birinapant respectively and lanes 5–7 for TRAIL 10, 50 and 100 ng/mL respectively) and co-treatments (lanes 8–13 as shown) for 24 h. Untreated (lane 1) and treated with DMSO (lane 2), cells are also presented. 5b-II-RKO and 5b-III-RKO: Protein levels of PARP-1 and total Caspase-3 after respective mono-treatments and co-treatments (same lines as 5-I-RKO). Data are representative of three independent experiments. 5C: Light microscopy images from HT29 culture after combined treatment with Birinapant and TRAIL. Detached (apoptotic) cells are shown in supernatant of co-treated groups for 48 and 72 h. Representative images. 5d: Confocal microscope images were taken after co-treatment with Birinapant, TRAIL and their combination. Nuclei were detected with HOECHST staining (blue color), cleaved Caspase-3 (green color). Representative images.. Scale bar: 9.9 μm. 5e. Light microscopy of three-dimensional culture of HT29 cells after co-treatment with Birinapant, TRAIL and their combinations in 3D for 6 days. Representative images. 5f: Confocal microscope images were taken after co-treatment with Birinapant, TRAIL and their combinations in 3D culture for 6 days. The nuclei were detected with HOECHST staining (blue color), cleaved Caspase-3 (green color). Representative images. Scale bar: 57.4 μm

Techniques Used: Incubation, Positive Control, Light Microscopy, Microscopy, Staining

SMAC-mimetic AT-406 and TRAIL synergistically kill resistant tumour cells. 6a Cell viability of HT29 cell line after combined treatment with SMAC-mimetic AT-406 and the apoptotic agent TRAIL. Cells were either left untreated (ctr = control) or treated with AT-406 combined with TRAIL for 48 and 72 h and the % percentage cell viability was measured by SRB staining. The values are the average of three independent experiments and are presented as fold change of the absorbance of treated/untreated cells, for each condition. Columns = % percentage of cell viability, bars = SD. 6a-RKO: Respectively for cell line RKO. 6b. Confocal microscope images were taken after co-treatment with AT-406, TRAIL and their combinations for 48 and 72 h. The nuclei were detected with HOECHST staining (blue color), cleaved Caspase-3 (green color). Representative images. Scale bar: 9.9 μM
Figure Legend Snippet: SMAC-mimetic AT-406 and TRAIL synergistically kill resistant tumour cells. 6a Cell viability of HT29 cell line after combined treatment with SMAC-mimetic AT-406 and the apoptotic agent TRAIL. Cells were either left untreated (ctr = control) or treated with AT-406 combined with TRAIL for 48 and 72 h and the % percentage cell viability was measured by SRB staining. The values are the average of three independent experiments and are presented as fold change of the absorbance of treated/untreated cells, for each condition. Columns = % percentage of cell viability, bars = SD. 6a-RKO: Respectively for cell line RKO. 6b. Confocal microscope images were taken after co-treatment with AT-406, TRAIL and their combinations for 48 and 72 h. The nuclei were detected with HOECHST staining (blue color), cleaved Caspase-3 (green color). Representative images. Scale bar: 9.9 μM

Techniques Used: Sulforhodamine B Assay, Staining, Microscopy

Birinapant treatment results in reduced cell viability and appearance of apoptosis in selected colorectal adenocarcinoma cells. a : Cell viability of cell lines after treatment with SMAC-mimetics. Cells were either left untreated (ctr = control) or treated with 0.5 μM, 1 mM or 5 mM AT-406 and Birinapant for 48 h (1) and 72 h (2) and the % percentage cell viability was measured by SRB. Average of three independent experiments are presented as fold change of the absorbance of treated/untreated cells for each condition. Columns = % percentage of cell viability, bars = SD. b : Protein levels of XIAP, cIAP-1, PARP-1 and total caspase-3 in RKO and HCT116 were analysed by W.B. after treatment with 0.5, 1 and 5 μM SMAC-mimetics AT-406 (lanes 3–5) and Birinapant (lanes 6–8) for 48 and 72 h. Untreated (lane 1) or treated with DMSO cells (lane 2) were used as control. Proteins are quantified against α-Tubulin. Data are representative for three independent experiments. c - d : Confocal microscope images and Hoechst staining of RKO (2C) and HCT116 (2D) cell lines two-dimensional culture, after treatment with SMAC-mimetics AT-406 (A) and Birinapant (B). Crescent nuclei of RKO cells present after cell treatments are shown by arrows. Confocal microscope images were taken after treatment with SMAC-mimetics AT-406 and Birinapant in RKO (2c) and HCT116 (2d) 48 and 72 h. The nuclei were detected with HOECHST staining. Representative images are presented
Figure Legend Snippet: Birinapant treatment results in reduced cell viability and appearance of apoptosis in selected colorectal adenocarcinoma cells. a : Cell viability of cell lines after treatment with SMAC-mimetics. Cells were either left untreated (ctr = control) or treated with 0.5 μM, 1 mM or 5 mM AT-406 and Birinapant for 48 h (1) and 72 h (2) and the % percentage cell viability was measured by SRB. Average of three independent experiments are presented as fold change of the absorbance of treated/untreated cells for each condition. Columns = % percentage of cell viability, bars = SD. b : Protein levels of XIAP, cIAP-1, PARP-1 and total caspase-3 in RKO and HCT116 were analysed by W.B. after treatment with 0.5, 1 and 5 μM SMAC-mimetics AT-406 (lanes 3–5) and Birinapant (lanes 6–8) for 48 and 72 h. Untreated (lane 1) or treated with DMSO cells (lane 2) were used as control. Proteins are quantified against α-Tubulin. Data are representative for three independent experiments. c - d : Confocal microscope images and Hoechst staining of RKO (2C) and HCT116 (2D) cell lines two-dimensional culture, after treatment with SMAC-mimetics AT-406 (A) and Birinapant (B). Crescent nuclei of RKO cells present after cell treatments are shown by arrows. Confocal microscope images were taken after treatment with SMAC-mimetics AT-406 and Birinapant in RKO (2c) and HCT116 (2d) 48 and 72 h. The nuclei were detected with HOECHST staining. Representative images are presented

Techniques Used: Sulforhodamine B Assay, Microscopy, Staining

Pre-treatment with Birinapant and then co-treatment with Birinapant/ BRAF inhibitor PLX4720 synergistically induce apoptosis of colorectal adenocarcinoma cells in 2D and 3D. a : Cell viability after co-treatment with the SMAC-mimetics Birinapant or AT-406 in combination with the BRAF inhibitor PLX4720. Cells were either left untreated (ctr = control) or treated with 5 μM Birinapant, 5 μM AT-406 and 1 μM PLX4720 and their combinations for 48 and 72 h. The average of three independent experiments is presented as fold change of the absorbance of treated/untreated cells, for each condition. Columns = % percentage of cell viability, bars = SD. b : Cell viability after pre-treatment with the SMAC-mimetic Birinapant and then co-treatment with BRAF inhibitor PLX4720 and Birinapant. Cells were either left untreated (ctr = control) or treated with 0.5 or 1 μM Birinapant and 0.2 or 0.5 μM PLX4720. For the pre-treatment testing, cells were first incubated for 24 h with either 0.5 or 1 μM of Birinapant and then co-treated with 0.2 and 0.5 μM PLX4720 for another 24 or 48 h. c : Protein levels of PARP-1, total Caspase-3, XIAP, cIAP-1 and p-ERK1/2 in RKO by W.B., after pre-treatment with 0.5 or 1 μM Birinapant and then co-treatment with 0.2 or 0.5 μM PLX4720. Untreated cells were used as control. Proteins are quantified against α-Tubulin. Data are representative for three independent experiments. d : Light microscopy images from RKO culture after pre-treatment with Birinapant and then co-treatment with PLX4720. Detached cells are shown in supernatant of co-treated group. Several images were taken from untreated and treated with Birinapant (0.5 or 1 μM) and PLX4720 (0.2 or 0.5 μM) RKO cells while been cultured in 6-well plates for 48 and 72 h. Representative images are presented. e : Light microscopy of three-dimensional culture of RKO cells after co-treatment with 0.5 μM, 1 μM Birinapant and 0.2 μM, 0.5 μM PLX4720 and their combination in 3D culture for 6 d. Representative images. f : Confocal microscope images were taken after co-treatment with 0.5 μM, 1 μM Birinapant and 0.2 μM, 0.5 μM PLX4720 and combinations in 3D cultures for 6 days. The nuclei were detected with HOECHST staining (blue color), cleaved Caspase-3 (green color). Representative images. Scale bar: 20 μm
Figure Legend Snippet: Pre-treatment with Birinapant and then co-treatment with Birinapant/ BRAF inhibitor PLX4720 synergistically induce apoptosis of colorectal adenocarcinoma cells in 2D and 3D. a : Cell viability after co-treatment with the SMAC-mimetics Birinapant or AT-406 in combination with the BRAF inhibitor PLX4720. Cells were either left untreated (ctr = control) or treated with 5 μM Birinapant, 5 μM AT-406 and 1 μM PLX4720 and their combinations for 48 and 72 h. The average of three independent experiments is presented as fold change of the absorbance of treated/untreated cells, for each condition. Columns = % percentage of cell viability, bars = SD. b : Cell viability after pre-treatment with the SMAC-mimetic Birinapant and then co-treatment with BRAF inhibitor PLX4720 and Birinapant. Cells were either left untreated (ctr = control) or treated with 0.5 or 1 μM Birinapant and 0.2 or 0.5 μM PLX4720. For the pre-treatment testing, cells were first incubated for 24 h with either 0.5 or 1 μM of Birinapant and then co-treated with 0.2 and 0.5 μM PLX4720 for another 24 or 48 h. c : Protein levels of PARP-1, total Caspase-3, XIAP, cIAP-1 and p-ERK1/2 in RKO by W.B., after pre-treatment with 0.5 or 1 μM Birinapant and then co-treatment with 0.2 or 0.5 μM PLX4720. Untreated cells were used as control. Proteins are quantified against α-Tubulin. Data are representative for three independent experiments. d : Light microscopy images from RKO culture after pre-treatment with Birinapant and then co-treatment with PLX4720. Detached cells are shown in supernatant of co-treated group. Several images were taken from untreated and treated with Birinapant (0.5 or 1 μM) and PLX4720 (0.2 or 0.5 μM) RKO cells while been cultured in 6-well plates for 48 and 72 h. Representative images are presented. e : Light microscopy of three-dimensional culture of RKO cells after co-treatment with 0.5 μM, 1 μM Birinapant and 0.2 μM, 0.5 μM PLX4720 and their combination in 3D culture for 6 d. Representative images. f : Confocal microscope images were taken after co-treatment with 0.5 μM, 1 μM Birinapant and 0.2 μM, 0.5 μM PLX4720 and combinations in 3D cultures for 6 days. The nuclei were detected with HOECHST staining (blue color), cleaved Caspase-3 (green color). Representative images. Scale bar: 20 μm

Techniques Used: Incubation, Light Microscopy, Cell Culture, Microscopy, Staining

37) Product Images from "IAP antagonists Birinapant and AT-406 efficiently synergise with either TRAIL, BRAF, or BCL-2 inhibitors to sensitise BRAFV600E colorectal tumour cells to apoptosis"

Article Title: IAP antagonists Birinapant and AT-406 efficiently synergise with either TRAIL, BRAF, or BCL-2 inhibitors to sensitise BRAFV600E colorectal tumour cells to apoptosis

Journal: BMC Cancer

doi: 10.1186/s12885-016-2606-5

Co-treatment of Birinapant with TRAIL can synergistically increase their efficiency and induce apoptosis in colorectal adenocarcinoma cells in 2D and 3D. 5a: Cell viability of cell line HT29 after co-treatment with the SMAC-mimetic Birinapant and the apoptotic agent TRAIL. Cells were either left untreated (ctr = control) or treated with Birinapant, TRAIL and their combination for 48 and 72 h and the % percentage cell viability was measured. The average of three independent experiments is presented. Columns = % percentage of cell viability, bars = SD. 5a-RKO: Respectively for cell line RKO (upper panel). To check for the reversibility of treatment effects, RKO cells were either left untreated (ctr = control), treated with either Birinapant, TRAIL or their combination for 48 h. Cells were then incubated with additional 48 h without treatments (NoT) and the % percentage cell viability was measured. The average of three independent experiments is presented. 5b-I. Protein levels of cIAP-1 and XIAP after 0.5, 1 and 5 μM AT-406 (lanes 3–5) or Birinapant (lanes 6–8) treatments respectively for 48 h, compared to control lanes 1 (no treatment) or 2 (DMSO treatment). 5b-II and 5b-III. Protein levels of PARP-1 and total Caspase-3 in HT29 cell line after co-treatment with SMAC-mimetic Birinapant and TRAIL for 48 and 72 h. Cells were either left untreated (lane 1) or treated with DMSO (lane 2), with 5 μM Birinapant (lane 3), with 10, 50 and 100 ng/mL TRAIL (lanes 4–6), or with 5 μM Birinapant and combination with TRAIL (lanes 7–9) for 48 and 72 h. Sensitive cell line HCT116 treated with TRAIL is used as positive control (lane 10). Using W. B., protein levels of PARP-1 (Figure 5b-II) and of total Caspase 3 (Figure 5b-III) were analyzed after the corresponding treatments. Data are representative for three independent experiments. 5b-I-RKO: Protein levels of cIAP-1 and XIAP after mono-treatments (lanes 3 and 4 for AT-406 and Birinapant respectively and lanes 5–7 for TRAIL 10, 50 and 100 ng/mL respectively) and co-treatments (lanes 8–13 as shown) for 24 h. Untreated (lane 1) and treated with DMSO (lane 2), cells are also presented. 5b-II-RKO and 5b-III-RKO: Protein levels of PARP-1 and total Caspase-3 after respective mono-treatments and co-treatments (same lines as 5-I-RKO). Data are representative of three independent experiments. 5C: Light microscopy images from HT29 culture after combined treatment with Birinapant and TRAIL. Detached (apoptotic) cells are shown in supernatant of co-treated groups for 48 and 72 h. Representative images. 5d: Confocal microscope images were taken after co-treatment with Birinapant, TRAIL and their combination. Nuclei were detected with HOECHST staining (blue color), cleaved Caspase-3 (green color). Representative images.. Scale bar: 9.9 μm. 5e. Light microscopy of three-dimensional culture of HT29 cells after co-treatment with Birinapant, TRAIL and their combinations in 3D for 6 days. Representative images. 5f: Confocal microscope images were taken after co-treatment with Birinapant, TRAIL and their combinations in 3D culture for 6 days. The nuclei were detected with HOECHST staining (blue color), cleaved Caspase-3 (green color). Representative images. Scale bar: 57.4 μm
Figure Legend Snippet: Co-treatment of Birinapant with TRAIL can synergistically increase their efficiency and induce apoptosis in colorectal adenocarcinoma cells in 2D and 3D. 5a: Cell viability of cell line HT29 after co-treatment with the SMAC-mimetic Birinapant and the apoptotic agent TRAIL. Cells were either left untreated (ctr = control) or treated with Birinapant, TRAIL and their combination for 48 and 72 h and the % percentage cell viability was measured. The average of three independent experiments is presented. Columns = % percentage of cell viability, bars = SD. 5a-RKO: Respectively for cell line RKO (upper panel). To check for the reversibility of treatment effects, RKO cells were either left untreated (ctr = control), treated with either Birinapant, TRAIL or their combination for 48 h. Cells were then incubated with additional 48 h without treatments (NoT) and the % percentage cell viability was measured. The average of three independent experiments is presented. 5b-I. Protein levels of cIAP-1 and XIAP after 0.5, 1 and 5 μM AT-406 (lanes 3–5) or Birinapant (lanes 6–8) treatments respectively for 48 h, compared to control lanes 1 (no treatment) or 2 (DMSO treatment). 5b-II and 5b-III. Protein levels of PARP-1 and total Caspase-3 in HT29 cell line after co-treatment with SMAC-mimetic Birinapant and TRAIL for 48 and 72 h. Cells were either left untreated (lane 1) or treated with DMSO (lane 2), with 5 μM Birinapant (lane 3), with 10, 50 and 100 ng/mL TRAIL (lanes 4–6), or with 5 μM Birinapant and combination with TRAIL (lanes 7–9) for 48 and 72 h. Sensitive cell line HCT116 treated with TRAIL is used as positive control (lane 10). Using W. B., protein levels of PARP-1 (Figure 5b-II) and of total Caspase 3 (Figure 5b-III) were analyzed after the corresponding treatments. Data are representative for three independent experiments. 5b-I-RKO: Protein levels of cIAP-1 and XIAP after mono-treatments (lanes 3 and 4 for AT-406 and Birinapant respectively and lanes 5–7 for TRAIL 10, 50 and 100 ng/mL respectively) and co-treatments (lanes 8–13 as shown) for 24 h. Untreated (lane 1) and treated with DMSO (lane 2), cells are also presented. 5b-II-RKO and 5b-III-RKO: Protein levels of PARP-1 and total Caspase-3 after respective mono-treatments and co-treatments (same lines as 5-I-RKO). Data are representative of three independent experiments. 5C: Light microscopy images from HT29 culture after combined treatment with Birinapant and TRAIL. Detached (apoptotic) cells are shown in supernatant of co-treated groups for 48 and 72 h. Representative images. 5d: Confocal microscope images were taken after co-treatment with Birinapant, TRAIL and their combination. Nuclei were detected with HOECHST staining (blue color), cleaved Caspase-3 (green color). Representative images.. Scale bar: 9.9 μm. 5e. Light microscopy of three-dimensional culture of HT29 cells after co-treatment with Birinapant, TRAIL and their combinations in 3D for 6 days. Representative images. 5f: Confocal microscope images were taken after co-treatment with Birinapant, TRAIL and their combinations in 3D culture for 6 days. The nuclei were detected with HOECHST staining (blue color), cleaved Caspase-3 (green color). Representative images. Scale bar: 57.4 μm

Techniques Used: Incubation, Positive Control, Light Microscopy, Microscopy, Staining

SMAC-mimetic AT-406 and TRAIL synergistically kill resistant tumour cells. 6a Cell viability of HT29 cell line after combined treatment with SMAC-mimetic AT-406 and the apoptotic agent TRAIL. Cells were either left untreated (ctr = control) or treated with AT-406 combined with TRAIL for 48 and 72 h and the % percentage cell viability was measured by SRB staining. The values are the average of three independent experiments and are presented as fold change of the absorbance of treated/untreated cells, for each condition. Columns = % percentage of cell viability, bars = SD. 6a-RKO: Respectively for cell line RKO. 6b. Confocal microscope images were taken after co-treatment with AT-406, TRAIL and their combinations for 48 and 72 h. The nuclei were detected with HOECHST staining (blue color), cleaved Caspase-3 (green color). Representative images. Scale bar: 9.9 μM
Figure Legend Snippet: SMAC-mimetic AT-406 and TRAIL synergistically kill resistant tumour cells. 6a Cell viability of HT29 cell line after combined treatment with SMAC-mimetic AT-406 and the apoptotic agent TRAIL. Cells were either left untreated (ctr = control) or treated with AT-406 combined with TRAIL for 48 and 72 h and the % percentage cell viability was measured by SRB staining. The values are the average of three independent experiments and are presented as fold change of the absorbance of treated/untreated cells, for each condition. Columns = % percentage of cell viability, bars = SD. 6a-RKO: Respectively for cell line RKO. 6b. Confocal microscope images were taken after co-treatment with AT-406, TRAIL and their combinations for 48 and 72 h. The nuclei were detected with HOECHST staining (blue color), cleaved Caspase-3 (green color). Representative images. Scale bar: 9.9 μM

Techniques Used: Sulforhodamine B Assay, Staining, Microscopy

Birinapant treatment results in reduced cell viability and appearance of apoptosis in selected colorectal adenocarcinoma cells. a : Cell viability of cell lines after treatment with SMAC-mimetics. Cells were either left untreated (ctr = control) or treated with 0.5 μM, 1 mM or 5 mM AT-406 and Birinapant for 48 h (1) and 72 h (2) and the % percentage cell viability was measured by SRB. Average of three independent experiments are presented as fold change of the absorbance of treated/untreated cells for each condition. Columns = % percentage of cell viability, bars = SD. b : Protein levels of XIAP, cIAP-1, PARP-1 and total caspase-3 in RKO and HCT116 were analysed by W.B. after treatment with 0.5, 1 and 5 μM SMAC-mimetics AT-406 (lanes 3–5) and Birinapant (lanes 6–8) for 48 and 72 h. Untreated (lane 1) or treated with DMSO cells (lane 2) were used as control. Proteins are quantified against α-Tubulin. Data are representative for three independent experiments. c - d : Confocal microscope images and Hoechst staining of RKO (2C) and HCT116 (2D) cell lines two-dimensional culture, after treatment with SMAC-mimetics AT-406 (A) and Birinapant (B). Crescent nuclei of RKO cells present after cell treatments are shown by arrows. Confocal microscope images were taken after treatment with SMAC-mimetics AT-406 and Birinapant in RKO (2c) and HCT116 (2d) 48 and 72 h. The nuclei were detected with HOECHST staining. Representative images are presented
Figure Legend Snippet: Birinapant treatment results in reduced cell viability and appearance of apoptosis in selected colorectal adenocarcinoma cells. a : Cell viability of cell lines after treatment with SMAC-mimetics. Cells were either left untreated (ctr = control) or treated with 0.5 μM, 1 mM or 5 mM AT-406 and Birinapant for 48 h (1) and 72 h (2) and the % percentage cell viability was measured by SRB. Average of three independent experiments are presented as fold change of the absorbance of treated/untreated cells for each condition. Columns = % percentage of cell viability, bars = SD. b : Protein levels of XIAP, cIAP-1, PARP-1 and total caspase-3 in RKO and HCT116 were analysed by W.B. after treatment with 0.5, 1 and 5 μM SMAC-mimetics AT-406 (lanes 3–5) and Birinapant (lanes 6–8) for 48 and 72 h. Untreated (lane 1) or treated with DMSO cells (lane 2) were used as control. Proteins are quantified against α-Tubulin. Data are representative for three independent experiments. c - d : Confocal microscope images and Hoechst staining of RKO (2C) and HCT116 (2D) cell lines two-dimensional culture, after treatment with SMAC-mimetics AT-406 (A) and Birinapant (B). Crescent nuclei of RKO cells present after cell treatments are shown by arrows. Confocal microscope images were taken after treatment with SMAC-mimetics AT-406 and Birinapant in RKO (2c) and HCT116 (2d) 48 and 72 h. The nuclei were detected with HOECHST staining. Representative images are presented

Techniques Used: Sulforhodamine B Assay, Microscopy, Staining

Pre-treatment with Birinapant and then co-treatment with Birinapant/ BRAF inhibitor PLX4720 synergistically induce apoptosis of colorectal adenocarcinoma cells in 2D and 3D. a : Cell viability after co-treatment with the SMAC-mimetics Birinapant or AT-406 in combination with the BRAF inhibitor PLX4720. Cells were either left untreated (ctr = control) or treated with 5 μM Birinapant, 5 μM AT-406 and 1 μM PLX4720 and their combinations for 48 and 72 h. The average of three independent experiments is presented as fold change of the absorbance of treated/untreated cells, for each condition. Columns = % percentage of cell viability, bars = SD. b : Cell viability after pre-treatment with the SMAC-mimetic Birinapant and then co-treatment with BRAF inhibitor PLX4720 and Birinapant. Cells were either left untreated (ctr = control) or treated with 0.5 or 1 μM Birinapant and 0.2 or 0.5 μM PLX4720. For the pre-treatment testing, cells were first incubated for 24 h with either 0.5 or 1 μM of Birinapant and then co-treated with 0.2 and 0.5 μM PLX4720 for another 24 or 48 h. c : Protein levels of PARP-1, total Caspase-3, XIAP, cIAP-1 and p-ERK1/2 in RKO by W.B., after pre-treatment with 0.5 or 1 μM Birinapant and then co-treatment with 0.2 or 0.5 μM PLX4720. Untreated cells were used as control. Proteins are quantified against α-Tubulin. Data are representative for three independent experiments. d : Light microscopy images from RKO culture after pre-treatment with Birinapant and then co-treatment with PLX4720. Detached cells are shown in supernatant of co-treated group. Several images were taken from untreated and treated with Birinapant (0.5 or 1 μM) and PLX4720 (0.2 or 0.5 μM) RKO cells while been cultured in 6-well plates for 48 and 72 h. Representative images are presented. e : Light microscopy of three-dimensional culture of RKO cells after co-treatment with 0.5 μM, 1 μM Birinapant and 0.2 μM, 0.5 μM PLX4720 and their combination in 3D culture for 6 d. Representative images. f : Confocal microscope images were taken after co-treatment with 0.5 μM, 1 μM Birinapant and 0.2 μM, 0.5 μM PLX4720 and combinations in 3D cultures for 6 days. The nuclei were detected with HOECHST staining (blue color), cleaved Caspase-3 (green color). Representative images. Scale bar: 20 μm
Figure Legend Snippet: Pre-treatment with Birinapant and then co-treatment with Birinapant/ BRAF inhibitor PLX4720 synergistically induce apoptosis of colorectal adenocarcinoma cells in 2D and 3D. a : Cell viability after co-treatment with the SMAC-mimetics Birinapant or AT-406 in combination with the BRAF inhibitor PLX4720. Cells were either left untreated (ctr = control) or treated with 5 μM Birinapant, 5 μM AT-406 and 1 μM PLX4720 and their combinations for 48 and 72 h. The average of three independent experiments is presented as fold change of the absorbance of treated/untreated cells, for each condition. Columns = % percentage of cell viability, bars = SD. b : Cell viability after pre-treatment with the SMAC-mimetic Birinapant and then co-treatment with BRAF inhibitor PLX4720 and Birinapant. Cells were either left untreated (ctr = control) or treated with 0.5 or 1 μM Birinapant and 0.2 or 0.5 μM PLX4720. For the pre-treatment testing, cells were first incubated for 24 h with either 0.5 or 1 μM of Birinapant and then co-treated with 0.2 and 0.5 μM PLX4720 for another 24 or 48 h. c : Protein levels of PARP-1, total Caspase-3, XIAP, cIAP-1 and p-ERK1/2 in RKO by W.B., after pre-treatment with 0.5 or 1 μM Birinapant and then co-treatment with 0.2 or 0.5 μM PLX4720. Untreated cells were used as control. Proteins are quantified against α-Tubulin. Data are representative for three independent experiments. d : Light microscopy images from RKO culture after pre-treatment with Birinapant and then co-treatment with PLX4720. Detached cells are shown in supernatant of co-treated group. Several images were taken from untreated and treated with Birinapant (0.5 or 1 μM) and PLX4720 (0.2 or 0.5 μM) RKO cells while been cultured in 6-well plates for 48 and 72 h. Representative images are presented. e : Light microscopy of three-dimensional culture of RKO cells after co-treatment with 0.5 μM, 1 μM Birinapant and 0.2 μM, 0.5 μM PLX4720 and their combination in 3D culture for 6 d. Representative images. f : Confocal microscope images were taken after co-treatment with 0.5 μM, 1 μM Birinapant and 0.2 μM, 0.5 μM PLX4720 and combinations in 3D cultures for 6 days. The nuclei were detected with HOECHST staining (blue color), cleaved Caspase-3 (green color). Representative images. Scale bar: 20 μm

Techniques Used: Incubation, Light Microscopy, Cell Culture, Microscopy, Staining

38) Product Images from "YAP/TAZ and Hedgehog coordinate growth and patterning in gastrointestinal mesenchyme"

Article Title: YAP/TAZ and Hedgehog coordinate growth and patterning in gastrointestinal mesenchyme

Journal: Developmental cell

doi: 10.1016/j.devcel.2017.08.019

YAP and TAZ are required for gastrointestinal mesenchymal development (A) Histology of forestomach, corpus, and intestine of control and YAP/TAZ deficient mesenchyme ( Nkx3.2 Cre Yap flox/flox Taz flox/flox ) animals at E18.5. (B) Relative mesenchyme thickness quantification. Data are mean ± S.D., ** = p value ≤0.01; *** = p value ≤0.001. (C) Immunohistochemical βcatenin, CD44, and H/K-ATPase staining in stomach mesenchyme of control and Nkx3.2 Cre Yap flox/flox Taz flox/flox animals at E18.5. (D) Immunohistochemical α-smooth muscle actin (α-SMA), desmin, and β-Tubulin III staining in stomach mesenchyme of control and Nkx3.2 Cre Yap flox/flox Taz flox/flox animals at E18.5. (E) αSMA, β-Tubulin III, CD31, Ki67, and cleaved caspase 3 immunofluorescence staining in stomach mesenchyme of control and Nkx3.2 Cre Yap flox/flox Taz flox/flox animals at E14.5. Ep: Epithelium; Me: Mesenchyme. Scale bar = 20µM. (F) Quantification of fold change of Ki67 + cells in stomach mesenchyme of control and Nkx3.2 Cre Yap flox/flox Taz flox/flox .
Figure Legend Snippet: YAP and TAZ are required for gastrointestinal mesenchymal development (A) Histology of forestomach, corpus, and intestine of control and YAP/TAZ deficient mesenchyme ( Nkx3.2 Cre Yap flox/flox Taz flox/flox ) animals at E18.5. (B) Relative mesenchyme thickness quantification. Data are mean ± S.D., ** = p value ≤0.01; *** = p value ≤0.001. (C) Immunohistochemical βcatenin, CD44, and H/K-ATPase staining in stomach mesenchyme of control and Nkx3.2 Cre Yap flox/flox Taz flox/flox animals at E18.5. (D) Immunohistochemical α-smooth muscle actin (α-SMA), desmin, and β-Tubulin III staining in stomach mesenchyme of control and Nkx3.2 Cre Yap flox/flox Taz flox/flox animals at E18.5. (E) αSMA, β-Tubulin III, CD31, Ki67, and cleaved caspase 3 immunofluorescence staining in stomach mesenchyme of control and Nkx3.2 Cre Yap flox/flox Taz flox/flox animals at E14.5. Ep: Epithelium; Me: Mesenchyme. Scale bar = 20µM. (F) Quantification of fold change of Ki67 + cells in stomach mesenchyme of control and Nkx3.2 Cre Yap flox/flox Taz flox/flox .

Techniques Used: Immunohistochemistry, Staining, Immunofluorescence

39) Product Images from "Magnolol and honokiol exert a synergistic anti-tumor effect through autophagy and apoptosis in human glioblastomas"

Article Title: Magnolol and honokiol exert a synergistic anti-tumor effect through autophagy and apoptosis in human glioblastomas

Journal: Oncotarget

doi: 10.18632/oncotarget.8674

Hono-Mag promoted apoptosis and autophagy-associated proteins in GBM cells LN229 and U87MG cells were treated with Hono, Mag or Hono-Mag (20–40 μM) for 24 hours. The apoptotic cells of ( A ) LN229 and ( C ) U87MG were determined using Annexin V staining and detected by flow cytometry analysis. The right panels show the percentage of the apoptotic cells. The cell lysates of ( B ) LN229 and ( D ) U87MG cells were then analyzed for cleaved caspase 3, cleaved PARP, Bcl-2 and LC3β by Western blotting. GAPDH was used as the loading control. The right panels show the quantitative analyses of the proteins. * p
Figure Legend Snippet: Hono-Mag promoted apoptosis and autophagy-associated proteins in GBM cells LN229 and U87MG cells were treated with Hono, Mag or Hono-Mag (20–40 μM) for 24 hours. The apoptotic cells of ( A ) LN229 and ( C ) U87MG were determined using Annexin V staining and detected by flow cytometry analysis. The right panels show the percentage of the apoptotic cells. The cell lysates of ( B ) LN229 and ( D ) U87MG cells were then analyzed for cleaved caspase 3, cleaved PARP, Bcl-2 and LC3β by Western blotting. GAPDH was used as the loading control. The right panels show the quantitative analyses of the proteins. * p

Techniques Used: Staining, Flow Cytometry, Cytometry, Western Blot

Suppression of p-ERK or autophagy enhanced Hono-Mag-stimulated apoptosis in human GBM Cells The 40 μM of Hono, Mag, or Hono-Mag groups were combined with PD98059 or 3-MA in LN229 cells. After 24 hours of treatment, these cells were analyzed for populations in different stages of the cell cycle and protein expression. ( A ) The percentage of the cell population in the sub-G1, G0/G1, S, and G2/M phases of the cell cycle was determined using BD FACSuite analysis software. The right panels show quantitative analyses. ( B ) The protein expression levels of p-ERK, ERK, LC3β, Bcl-2, cleaved caspase 3 and PARP in LN229 cells were detected by Western blotting. GAPDH was used as the loading control. The lower panels show the quantitative analyses of the proteins. * p
Figure Legend Snippet: Suppression of p-ERK or autophagy enhanced Hono-Mag-stimulated apoptosis in human GBM Cells The 40 μM of Hono, Mag, or Hono-Mag groups were combined with PD98059 or 3-MA in LN229 cells. After 24 hours of treatment, these cells were analyzed for populations in different stages of the cell cycle and protein expression. ( A ) The percentage of the cell population in the sub-G1, G0/G1, S, and G2/M phases of the cell cycle was determined using BD FACSuite analysis software. The right panels show quantitative analyses. ( B ) The protein expression levels of p-ERK, ERK, LC3β, Bcl-2, cleaved caspase 3 and PARP in LN229 cells were detected by Western blotting. GAPDH was used as the loading control. The lower panels show the quantitative analyses of the proteins. * p

Techniques Used: Expressing, Software, Western Blot

40) Product Images from "Antiproliferative and apoptotic effect of LY2090314, a GSK-3 inhibitor, in neuroblastoma in vitro"

Article Title: Antiproliferative and apoptotic effect of LY2090314, a GSK-3 inhibitor, in neuroblastoma in vitro

Journal: BMC Cancer

doi: 10.1186/s12885-018-4474-7

Caspase-3 and-7 activities were measured by Caspase glo assay and showed a dose dependent significant increase of luminescence in all three cell lines treated with LY2090314, indicating increased apoptotic activity. **, P > 0.001. Experiments were repeated three times in triplicates and “the averages are shown here”
Figure Legend Snippet: Caspase-3 and-7 activities were measured by Caspase glo assay and showed a dose dependent significant increase of luminescence in all three cell lines treated with LY2090314, indicating increased apoptotic activity. **, P > 0.001. Experiments were repeated three times in triplicates and “the averages are shown here”

Techniques Used: Caspase-Glo Assay, Activity Assay

LY2090314 decreased GSK-3 phosphorylation and attenuate apoptosis inhibitor expression. a Western blots of all 3 cell lines (NGP, SK-N-AS, SH-SY-5Y), showing decreased phosphorylation of GSK-3α at Tyr279 compared to GSK-3β phosphorylation at Tyr216 as well as decrease in inactive phosphorylation ser 9 and 21. However, there is minimal to no decrease in phosphorylation of active Akt at ser 473. b Western blot analysis showed there is an increase in pro-apoptotic markers such as cleaved PARP and cleaved caspase-3, and a decrease in anti-apoptotic markers, cyclin D1, Mcl-1, and survivin. GAPDH was used as loading control. Experiments were repeated at least three times and the best pictures are shown here
Figure Legend Snippet: LY2090314 decreased GSK-3 phosphorylation and attenuate apoptosis inhibitor expression. a Western blots of all 3 cell lines (NGP, SK-N-AS, SH-SY-5Y), showing decreased phosphorylation of GSK-3α at Tyr279 compared to GSK-3β phosphorylation at Tyr216 as well as decrease in inactive phosphorylation ser 9 and 21. However, there is minimal to no decrease in phosphorylation of active Akt at ser 473. b Western blot analysis showed there is an increase in pro-apoptotic markers such as cleaved PARP and cleaved caspase-3, and a decrease in anti-apoptotic markers, cyclin D1, Mcl-1, and survivin. GAPDH was used as loading control. Experiments were repeated at least three times and the best pictures are shown here

Techniques Used: Expressing, Western Blot

41) Product Images from "Potential role of insulin receptor isoforms and IGF receptors in plaque instability of human and experimental atherosclerosis"

Article Title: Potential role of insulin receptor isoforms and IGF receptors in plaque instability of human and experimental atherosclerosis

Journal: Cardiovascular Diabetology

doi: 10.1186/s12933-018-0675-2

Antiapoptotic effect of IGF-IR on VSMCs. a Effect of IGF-IR inhibition by PPP (1 µmol/L) on cleaved caspase 3 levels analyzed by Western blot in IRLoxP +/+ and IR −/− VSMCs stimulated with IGF-I (10 nmol/L) or IGF-II (10 nmol/L) for 24 h. b Dose–response effect of thapsigargin (0-100 nmol/L, 18 h) on cleaved caspase 3 levels in IRLoxP +/+ and IR −/− VSMCs. c Effect of IGF-I or IGF-I pretreatment (10 nmol/L, 1 h) in IRLoxP +/+ and IR −/− VSMCs treated with thapsigargin (100 nmol/L) for 18 h. Experiments were performed 4 or 5 times. *p
Figure Legend Snippet: Antiapoptotic effect of IGF-IR on VSMCs. a Effect of IGF-IR inhibition by PPP (1 µmol/L) on cleaved caspase 3 levels analyzed by Western blot in IRLoxP +/+ and IR −/− VSMCs stimulated with IGF-I (10 nmol/L) or IGF-II (10 nmol/L) for 24 h. b Dose–response effect of thapsigargin (0-100 nmol/L, 18 h) on cleaved caspase 3 levels in IRLoxP +/+ and IR −/− VSMCs. c Effect of IGF-I or IGF-I pretreatment (10 nmol/L, 1 h) in IRLoxP +/+ and IR −/− VSMCs treated with thapsigargin (100 nmol/L) for 18 h. Experiments were performed 4 or 5 times. *p

Techniques Used: Inhibition, Western Blot

Differential contribution of IR isoforms to VSMCs apoptosis. a Effect of thapsigargin (100 nmol/L, 18 h) on cleaved caspase 3 levels analyzed by Western blot in IRA and IRB VSMCs. b Effect of IGF-IR inhibition by PPP (1 µmol/L) on cleaved caspase 3 levels in IRB and IRLoxP +/+ VSMCs treated with thapsigargin (100 nmol/L) for 18 h. Experiments were performed 4 times. *p
Figure Legend Snippet: Differential contribution of IR isoforms to VSMCs apoptosis. a Effect of thapsigargin (100 nmol/L, 18 h) on cleaved caspase 3 levels analyzed by Western blot in IRA and IRB VSMCs. b Effect of IGF-IR inhibition by PPP (1 µmol/L) on cleaved caspase 3 levels in IRB and IRLoxP +/+ VSMCs treated with thapsigargin (100 nmol/L) for 18 h. Experiments were performed 4 times. *p

Techniques Used: Western Blot, Inhibition

42) Product Images from "Downregulation of microRNA-21 inhibited radiation-resistance of esophageal squamous cell carcinoma"

Article Title: Downregulation of microRNA-21 inhibited radiation-resistance of esophageal squamous cell carcinoma

Journal: Cancer Cell International

doi: 10.1186/s12935-018-0502-6

Expression of cleaved-caspase-3 and cleaved-PARP in miR-21 mimics and inhibitor transfected-TE-1 cells after exposure of 6 Gy for 48 h. After transfection with miR-21 mimics and inhibitor, cells were irradiated with 6 Gy for 48 h. And then, the cells were collected and the levels of cleaved-caspase-3 and cleaved-PARP were detected by western blot
Figure Legend Snippet: Expression of cleaved-caspase-3 and cleaved-PARP in miR-21 mimics and inhibitor transfected-TE-1 cells after exposure of 6 Gy for 48 h. After transfection with miR-21 mimics and inhibitor, cells were irradiated with 6 Gy for 48 h. And then, the cells were collected and the levels of cleaved-caspase-3 and cleaved-PARP were detected by western blot

Techniques Used: Expressing, Transfection, Irradiation, Western Blot

43) Product Images from "MMP-7 mediates cleavage of N-cadherin and promotes smooth muscle cell apoptosis"

Article Title: MMP-7 mediates cleavage of N-cadherin and promotes smooth muscle cell apoptosis

Journal: Cardiovascular Research

doi: 10.1093/cvr/cvq042

MMP-7 and apoptosis in atherosclerotic plaques. In situ zymography of coronary atherosclerotic plaque in the absence ( A ) and presence ( B ) of 10 nM MMP-7 inhibitor. Green colour indicates MMP activity and nuclei are stained blue with DAPI. Dual detection of cleaved caspase-3 (red) and MMP activity (green) in coronary plaque ( C ) and IMA ( D ). ( E ) High power image of marked area in ( C ), arrows indicate co-location of apoptosis and MMP activity. ( F ) Quantification of the percentage of apoptotic (ISEL positive) cells in atherosclerotic plaques from ApoE −/− /MMP-7 +/+ and ApoE −/− /MMP-7 −/− . * indicates a significant difference from ApoE −/− /MMP-7 +/+ . ISEL staining of atherosclerotic plaques from ApoE −/− /MMP-7 +/+ ( G ) and ApoE −/− /MMP-7 −/− ( H ) mice. ISEL positive apoptotic cells are green and nuclei are stained blue (DAPI).
Figure Legend Snippet: MMP-7 and apoptosis in atherosclerotic plaques. In situ zymography of coronary atherosclerotic plaque in the absence ( A ) and presence ( B ) of 10 nM MMP-7 inhibitor. Green colour indicates MMP activity and nuclei are stained blue with DAPI. Dual detection of cleaved caspase-3 (red) and MMP activity (green) in coronary plaque ( C ) and IMA ( D ). ( E ) High power image of marked area in ( C ), arrows indicate co-location of apoptosis and MMP activity. ( F ) Quantification of the percentage of apoptotic (ISEL positive) cells in atherosclerotic plaques from ApoE −/− /MMP-7 +/+ and ApoE −/− /MMP-7 −/− . * indicates a significant difference from ApoE −/− /MMP-7 +/+ . ISEL staining of atherosclerotic plaques from ApoE −/− /MMP-7 +/+ ( G ) and ApoE −/− /MMP-7 −/− ( H ) mice. ISEL positive apoptotic cells are green and nuclei are stained blue (DAPI).

Techniques Used: In Situ, Zymography, Activity Assay, Staining, Mouse Assay

N-cadherin, MMP-7, and apoptosis in human IMA and atherosclerotic plaques. Representative western blot for N-cadherin ( A ) and cleaved caspase-3 ( B ) in IMA and coronary and carotid atherosclerotic plaques, n = 3 per group. β-Tubulin is shown as loading control. Bar chart in ( A ) shows densitometric analysis of N-cadherin western blots. ( C ) MMP activity in IMA and atherosclerotic plaques, n = 15 and 20. * indicates a significant difference from IMA.
Figure Legend Snippet: N-cadherin, MMP-7, and apoptosis in human IMA and atherosclerotic plaques. Representative western blot for N-cadherin ( A ) and cleaved caspase-3 ( B ) in IMA and coronary and carotid atherosclerotic plaques, n = 3 per group. β-Tubulin is shown as loading control. Bar chart in ( A ) shows densitometric analysis of N-cadherin western blots. ( C ) MMP activity in IMA and atherosclerotic plaques, n = 15 and 20. * indicates a significant difference from IMA.

Techniques Used: Western Blot, Activity Assay

44) Product Images from "Survivin-targeting miR-542-3p overcomes HER3 signaling-induced chemoresistance and enhances the antitumor activity of paclitaxel against HER2-overexpressing breast cancer"

Article Title: Survivin-targeting miR-542-3p overcomes HER3 signaling-induced chemoresistance and enhances the antitumor activity of paclitaxel against HER2-overexpressing breast cancer

Journal: Cancer letters

doi: 10.1016/j.canlet.2018.01.065

The combination of miR-542-3p and paclitaxel significantly downregulates Survivin, inhibits proliferation, and induces apoptosis of HCC1954 breast cancer cells in vivo The tumors obtained from the animal experiments were evaluated by IHC analysis of Ki67, Survivin and cleaved caspase-3. A , Data show the representative images of representative tumors with hematoxylin and eosin (H E) staining and immunostaining of Ki67, Survivin and cleaved caspase-3 (Cleaved Casp-3). B , The IHC slides were observed by two independent personnel. The tumor cells with positive staining of Ki67, Survivin, or cleaved caspase-3 were counted from three randomly selected areas in each slide. The three areas were first identified by scanning the entire slide at ×10 magnification, and then the positively stained cells were counted at ×20 magnification using an Olympus B×40 microscope (Tokyo, Japan). A minimum of 500 tumor cells were evaluated. If differences occurred between spot intensities, the most positive spot was considered. The evaluations were recorded as percentages of positively stained target cells in each field. The bar graphs show the mean percentages of positively stained cells in each field. Bars , SD.
Figure Legend Snippet: The combination of miR-542-3p and paclitaxel significantly downregulates Survivin, inhibits proliferation, and induces apoptosis of HCC1954 breast cancer cells in vivo The tumors obtained from the animal experiments were evaluated by IHC analysis of Ki67, Survivin and cleaved caspase-3. A , Data show the representative images of representative tumors with hematoxylin and eosin (H E) staining and immunostaining of Ki67, Survivin and cleaved caspase-3 (Cleaved Casp-3). B , The IHC slides were observed by two independent personnel. The tumor cells with positive staining of Ki67, Survivin, or cleaved caspase-3 were counted from three randomly selected areas in each slide. The three areas were first identified by scanning the entire slide at ×10 magnification, and then the positively stained cells were counted at ×20 magnification using an Olympus B×40 microscope (Tokyo, Japan). A minimum of 500 tumor cells were evaluated. If differences occurred between spot intensities, the most positive spot was considered. The evaluations were recorded as percentages of positively stained target cells in each field. The bar graphs show the mean percentages of positively stained cells in each field. Bars , SD.

Techniques Used: In Vivo, Immunohistochemistry, Staining, Immunostaining, Microscopy

The mimic of miR-542-3p effectively downregulates Survivin and significantly enhances paclitaxel-induced apoptosis in HER2-positive breast cancer cells SKBR3.B3.1 or SKBR3.B3.2 cells were transfected with the negative control miRNA mimic (Control, 40 nmol/L) or the mimic of miR-203 (40 nmol/L) or miR-542-3p (10 nmol/L) for 24 h. A , Cells were collected for western blot analyses of Survivin, Mcl-1, Bcl-xL, or β-actin. B C , The cells were then untreated or treated with paclitaxel (3 nmol/L) for another 24 h and subjected to western blot analyses of PARP, cleaved caspase-3 (C-Casp-3), or β-actin ( B ) or a specific apoptosis ELISA ( C ). Bars , S.D. Data represent the results of three independent experiments.
Figure Legend Snippet: The mimic of miR-542-3p effectively downregulates Survivin and significantly enhances paclitaxel-induced apoptosis in HER2-positive breast cancer cells SKBR3.B3.1 or SKBR3.B3.2 cells were transfected with the negative control miRNA mimic (Control, 40 nmol/L) or the mimic of miR-203 (40 nmol/L) or miR-542-3p (10 nmol/L) for 24 h. A , Cells were collected for western blot analyses of Survivin, Mcl-1, Bcl-xL, or β-actin. B C , The cells were then untreated or treated with paclitaxel (3 nmol/L) for another 24 h and subjected to western blot analyses of PARP, cleaved caspase-3 (C-Casp-3), or β-actin ( B ) or a specific apoptosis ELISA ( C ). Bars , S.D. Data represent the results of three independent experiments.

Techniques Used: Transfection, Negative Control, Western Blot, Enzyme-linked Immunosorbent Assay

45) Product Images from "Increased Apopototic Chondrocytes in Articular Cartilage of Adults Heterozygous SirT1 Mice"

Article Title: Increased Apopototic Chondrocytes in Articular Cartilage of Adults Heterozygous SirT1 Mice

Journal: Annals of the rheumatic diseases

doi: 10.1136/ard.2011.200504

9M SirT1 +/− mice show enhanced cleaved caspase 3 as compared to WT
Figure Legend Snippet: 9M SirT1 +/− mice show enhanced cleaved caspase 3 as compared to WT

Techniques Used: Mouse Assay

46) Product Images from "Identification of C/EBP? Target Genes in ALK+ Anaplastic Large Cell Lymphoma (ALCL) by Gene Expression Profiling and Chromatin Immunoprecipitation"

Article Title: Identification of C/EBP? Target Genes in ALK+ Anaplastic Large Cell Lymphoma (ALCL) by Gene Expression Profiling and Chromatin Immunoprecipitation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0064544

Influence of G0S2 knockdown on cell proliferation, apoptosis and cell cycle. Validation in primary cases. ( A ) Flow cytometric analysis of transduced SUDHL-1 cells and untreated controls four days after infection. The percentage of GFP-positive cells represents the percentage of infected cells. ( B ) RT-qPCR analysis of G0S2 mRNA in the transduced SUDHL-1 cells four days after infection. Values were normalized to TBP and data were analyzed according to the 2 -ΔΔCp method. Results are depicted as mRNA amount relative to untreated SUDHL-1 cells. Error bars indicate SD (n = 3). ( C ) Proliferation curves of the controls and G0S2-shRNA infected SUDHL-1 cells up to 5 days after infection. Error bars indicate SD (n = 3). ( D ) Annexin V/propidium iodide staining of the controls and the G0S2-shRNA transduced SUDHL-1 cells four days after infection. ( E ) Cell cycle distribution of the control and G0S2-shRNA infected SUDHL-1 cells four days after infection. The differences between the uninfected control and the G0S2-shRNA-infected cells are given in percentage in the table. SUDHL-1 = uninfected cells, pG-NS = pGIPZ vector with non-silencing shRNA, pG-G0S2 = virus containing the pGIPZ vector and the G0S2-shRNA sequence, PI = propidium iodide. ( F ) Western Blot analysis of caspase 3 and cleaved caspase 3 in G0S2-shRNA transduced SUDHL-1 cells four days after infection. Each lane contains 30 µg cytoplasmic extract. 15 µg protein extract of Bortezomib-treated SUDHL-1 cells were used as control. α-Tubulin was used as loading control. ( G ) Box plot of G0S2 expression levels obtained by RT-qPCR analysis in 6 ALK+, 8 ALK- and 4 normal lymph node samples.
Figure Legend Snippet: Influence of G0S2 knockdown on cell proliferation, apoptosis and cell cycle. Validation in primary cases. ( A ) Flow cytometric analysis of transduced SUDHL-1 cells and untreated controls four days after infection. The percentage of GFP-positive cells represents the percentage of infected cells. ( B ) RT-qPCR analysis of G0S2 mRNA in the transduced SUDHL-1 cells four days after infection. Values were normalized to TBP and data were analyzed according to the 2 -ΔΔCp method. Results are depicted as mRNA amount relative to untreated SUDHL-1 cells. Error bars indicate SD (n = 3). ( C ) Proliferation curves of the controls and G0S2-shRNA infected SUDHL-1 cells up to 5 days after infection. Error bars indicate SD (n = 3). ( D ) Annexin V/propidium iodide staining of the controls and the G0S2-shRNA transduced SUDHL-1 cells four days after infection. ( E ) Cell cycle distribution of the control and G0S2-shRNA infected SUDHL-1 cells four days after infection. The differences between the uninfected control and the G0S2-shRNA-infected cells are given in percentage in the table. SUDHL-1 = uninfected cells, pG-NS = pGIPZ vector with non-silencing shRNA, pG-G0S2 = virus containing the pGIPZ vector and the G0S2-shRNA sequence, PI = propidium iodide. ( F ) Western Blot analysis of caspase 3 and cleaved caspase 3 in G0S2-shRNA transduced SUDHL-1 cells four days after infection. Each lane contains 30 µg cytoplasmic extract. 15 µg protein extract of Bortezomib-treated SUDHL-1 cells were used as control. α-Tubulin was used as loading control. ( G ) Box plot of G0S2 expression levels obtained by RT-qPCR analysis in 6 ALK+, 8 ALK- and 4 normal lymph node samples.

Techniques Used: Flow Cytometry, Infection, Quantitative RT-PCR, shRNA, Staining, Plasmid Preparation, Sequencing, Western Blot, Expressing

47) Product Images from "The Flavonoid Jaceosidin from Artemisia princeps Induces Apoptotic Cell Death and Inhibits the Akt Pathway in Oral Cancer Cells"

Article Title: The Flavonoid Jaceosidin from Artemisia princeps Induces Apoptotic Cell Death and Inhibits the Akt Pathway in Oral Cancer Cells

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2018/5765047

Jaceosidin triggers cleavage of caspase-9, PARP, and caspase-3 and decreases Akt phosphorylation; OSCC cells were treated with the indicated concentrations of jaceosidin for 48 h and cleaved caspase-9, PARP, and caspase-3 proteins and phosphorylated Akt were examined using western blotting. Paclitaxel (PTX) was used as a positive control for caspase and PARP cleavage; (a) HSC-3 cells; (b) Ca9.22 cells.
Figure Legend Snippet: Jaceosidin triggers cleavage of caspase-9, PARP, and caspase-3 and decreases Akt phosphorylation; OSCC cells were treated with the indicated concentrations of jaceosidin for 48 h and cleaved caspase-9, PARP, and caspase-3 proteins and phosphorylated Akt were examined using western blotting. Paclitaxel (PTX) was used as a positive control for caspase and PARP cleavage; (a) HSC-3 cells; (b) Ca9.22 cells.

Techniques Used: Western Blot, Positive Control

48) Product Images from "Axonal outgrowth is associated with increased ERK 1/2 activation but decreased caspase 3 linked cell death in Schwann cells after immediate nerve repair in rats"

Article Title: Axonal outgrowth is associated with increased ERK 1/2 activation but decreased caspase 3 linked cell death in Schwann cells after immediate nerve repair in rats

Journal: BMC Neuroscience

doi: 10.1186/1471-2202-12-12

Double staining with S-100 and cleaved caspase 3 in a control nerve (A) and in a repaired (B) sciatic nerve from the distal nerve segment . The staining indicated that cleaved caspase 3 stained cell nuclei (green) was associated with S-100 staining (red). DAPI-stained cells (blue) were used to localize cell nuclei. Length of bars 50 μm (A) and 25 μm (B).
Figure Legend Snippet: Double staining with S-100 and cleaved caspase 3 in a control nerve (A) and in a repaired (B) sciatic nerve from the distal nerve segment . The staining indicated that cleaved caspase 3 stained cell nuclei (green) was associated with S-100 staining (red). DAPI-stained cells (blue) were used to localize cell nuclei. Length of bars 50 μm (A) and 25 μm (B).

Techniques Used: Double Staining, Staining

Schematic drawing showing (A) the experimental design with immediate (I), delayed (D) and no nerve repairs (N and DN) and (B) the sites where p-ERK 1/2 and cleaved caspase 3 were analyzed .
Figure Legend Snippet: Schematic drawing showing (A) the experimental design with immediate (I), delayed (D) and no nerve repairs (N and DN) and (B) the sites where p-ERK 1/2 and cleaved caspase 3 were analyzed .

Techniques Used:

Results of immunocytochemical staining for ERK1/2 and cleaved caspase 3 analyzed in the distal nerve segment after immediate, delayed and no nerve repairs . Immunocytochemical staining for p-ERK1/2 (A) in longitudinal sections at the site of the lesion (a)-(d) [(a) group I (immediate repair), (b) group N (no repair), (c) group D (delayed repair), (d) group DN (delayed no repair)], and in the distal nerve segment (e)-(h) [(e) group I, (f) group N, (g) group D, (h) group DN]. Contralateral uninjured nerve is shown in (i). The scale bar represents 100 μm. The graphs show the percentages of immunopositive area for p-ERK1/2 (B) expressed as mean + SD. The stars indicate p-values *
Figure Legend Snippet: Results of immunocytochemical staining for ERK1/2 and cleaved caspase 3 analyzed in the distal nerve segment after immediate, delayed and no nerve repairs . Immunocytochemical staining for p-ERK1/2 (A) in longitudinal sections at the site of the lesion (a)-(d) [(a) group I (immediate repair), (b) group N (no repair), (c) group D (delayed repair), (d) group DN (delayed no repair)], and in the distal nerve segment (e)-(h) [(e) group I, (f) group N, (g) group D, (h) group DN]. Contralateral uninjured nerve is shown in (i). The scale bar represents 100 μm. The graphs show the percentages of immunopositive area for p-ERK1/2 (B) expressed as mean + SD. The stars indicate p-values *

Techniques Used: Staining

Graphs of regression analyses between length of neurofilaments (mm, dependent) and p-ERK 1/2 and cleaved caspase 3 (independent) at site of lesion and in distal nerve segment . For p-values and r 2 see Results.
Figure Legend Snippet: Graphs of regression analyses between length of neurofilaments (mm, dependent) and p-ERK 1/2 and cleaved caspase 3 (independent) at site of lesion and in distal nerve segment . For p-values and r 2 see Results.

Techniques Used:

49) Product Images from "Morphology and Function of the Lamb Ileum following Preterm Birth"

Article Title: Morphology and Function of the Lamb Ileum following Preterm Birth

Journal: Frontiers in Pediatrics

doi: 10.3389/fped.2018.00008

Representative images of Ki67 + proliferating cells (A,B) in the crypts and activated caspase 3 + apoptotic cells (C,D) in the villi, as indicated by brown staining (arrows), from term (A,C) and preterm (B,D) lamb ileums. Representative images of periodic acid Schiff-stained villi from term (E) and preterm (F) lambs, with goblet cells indicated by magenta-colored staining of the cytoplasm (arrows). Representative images of villi from term (G) and preterm (H) lambs, showing the apical membranes of enterocytes (arrows) colored blue from alkaline phosphatase staining. Scale bars = 10 µm. Graphs show the mean proportion of proliferating cells in the crypts (I) , and the proportion of apoptotic (J) , goblet (K) , and enterocyte (L) cells in the crypt-villus units of term and preterm lamb ileums.
Figure Legend Snippet: Representative images of Ki67 + proliferating cells (A,B) in the crypts and activated caspase 3 + apoptotic cells (C,D) in the villi, as indicated by brown staining (arrows), from term (A,C) and preterm (B,D) lamb ileums. Representative images of periodic acid Schiff-stained villi from term (E) and preterm (F) lambs, with goblet cells indicated by magenta-colored staining of the cytoplasm (arrows). Representative images of villi from term (G) and preterm (H) lambs, showing the apical membranes of enterocytes (arrows) colored blue from alkaline phosphatase staining. Scale bars = 10 µm. Graphs show the mean proportion of proliferating cells in the crypts (I) , and the proportion of apoptotic (J) , goblet (K) , and enterocyte (L) cells in the crypt-villus units of term and preterm lamb ileums.

Techniques Used: Staining

50) Product Images from "Reovirus Activates Transforming Growth Factor ? and Bone Morphogenetic Protein Signaling Pathways in the Central Nervous System That Contribute to Neuronal Survival following Infection ▿"

Article Title: Reovirus Activates Transforming Growth Factor ? and Bone Morphogenetic Protein Signaling Pathways in the Central Nervous System That Contribute to Neuronal Survival following Infection ▿

Journal: Journal of Virology

doi: 10.1128/JVI.02433-08

Inhibition of reovirus-induced TGF-β signal activation increases apoptosis in vivo. (A) Whole-brain lysates from mock-infected and reovirus-infected (1,000 PFU, i.c. injection) Swiss Webster pups sacrificed at day 8 postinfection following daily intraperitoneal treatment with TGF-βRI inhibitor (TGF-βRI Inh III) or vehicle control were resolved on 10% polyacrylamide gels, transferred to nitrocellulose membranes, and probed with anti-pSMAD3, anti-cleaved caspase 3 (cl-Casp3), anti-cleaved PARP (cl-PARP), and anti-β-actin. Immunoblots are representative of three individual replicates per treatment group from multiple litters. (B to D) Immunohistochemistry staining of the same treatment groups revealed no differences in viral antigen (green) or in the distribution of cleaved caspase 3 (red) in animals treated with vehicle control or TGF-βRI inhibitor. Images shown are of the cingulate cortex. Original magnification, ×400.
Figure Legend Snippet: Inhibition of reovirus-induced TGF-β signal activation increases apoptosis in vivo. (A) Whole-brain lysates from mock-infected and reovirus-infected (1,000 PFU, i.c. injection) Swiss Webster pups sacrificed at day 8 postinfection following daily intraperitoneal treatment with TGF-βRI inhibitor (TGF-βRI Inh III) or vehicle control were resolved on 10% polyacrylamide gels, transferred to nitrocellulose membranes, and probed with anti-pSMAD3, anti-cleaved caspase 3 (cl-Casp3), anti-cleaved PARP (cl-PARP), and anti-β-actin. Immunoblots are representative of three individual replicates per treatment group from multiple litters. (B to D) Immunohistochemistry staining of the same treatment groups revealed no differences in viral antigen (green) or in the distribution of cleaved caspase 3 (red) in animals treated with vehicle control or TGF-βRI inhibitor. Images shown are of the cingulate cortex. Original magnification, ×400.

Techniques Used: Inhibition, Activation Assay, In Vivo, Infection, Injection, Western Blot, Immunohistochemistry, Staining

BMP6 ligand treatment of reovirus-infected MCCs prevents apoptosis. (A to C) Mock-infected (A) or reovirus-infected (B and C) (MOI, 100) primary MCCs were treated daily with vehicle control (B) or BMP6 ligand (C) and analyzed at day 4 postinfection using immunocytochemistry with antibodies to cleaved caspase 3 (green) and microtubule-associated protein 2 (red). Nuclear staining (Hoechst) is shown in blue. (D) Blinded cell counts of anti-cleaved caspase 3 (anti-cl-caspase 3)-positive cells in three replicate experiments show a threefold ( P
Figure Legend Snippet: BMP6 ligand treatment of reovirus-infected MCCs prevents apoptosis. (A to C) Mock-infected (A) or reovirus-infected (B and C) (MOI, 100) primary MCCs were treated daily with vehicle control (B) or BMP6 ligand (C) and analyzed at day 4 postinfection using immunocytochemistry with antibodies to cleaved caspase 3 (green) and microtubule-associated protein 2 (red). Nuclear staining (Hoechst) is shown in blue. (D) Blinded cell counts of anti-cleaved caspase 3 (anti-cl-caspase 3)-positive cells in three replicate experiments show a threefold ( P

Techniques Used: Infection, Immunocytochemistry, Staining

51) Product Images from "H19 induced by oxidative stress confers temozolomide resistance in human glioma cells via activating NF-κB signaling"

Article Title: H19 induced by oxidative stress confers temozolomide resistance in human glioma cells via activating NF-κB signaling

Journal: OncoTargets and therapy

doi: 10.2147/OTT.S173244

Knockdown of H19 decreases chemoresistance of glioma cells to TMZ. Notes:  ( A ) Relative expression of H19 was detected by qRT-PCR in U251 TMZ  and LN229 TMZ  cells after transfected with control siRNAs or two individual H19 siRNAs. ( B ) MTT assay was performed to determine the cell viabilities of transfected U251 TMZ  and LN229 TMZ  cells with TMZ (0, 100, 250, 500, 1,000, 1,500 or 2,000 µM) treatments. ( C ) Transfected U251 TMZ  and LN229 TMZ  cells were treated with 1,750 µM TMZ for 24 hours. The protein level of cleaved caspase 3, cyclin D1, XIAP and Bcl-2 was assessed by immunoblotting. α-Tubulin was used as the internal control.  ** P
Figure Legend Snippet: Knockdown of H19 decreases chemoresistance of glioma cells to TMZ. Notes: ( A ) Relative expression of H19 was detected by qRT-PCR in U251 TMZ and LN229 TMZ cells after transfected with control siRNAs or two individual H19 siRNAs. ( B ) MTT assay was performed to determine the cell viabilities of transfected U251 TMZ and LN229 TMZ cells with TMZ (0, 100, 250, 500, 1,000, 1,500 or 2,000 µM) treatments. ( C ) Transfected U251 TMZ and LN229 TMZ cells were treated with 1,750 µM TMZ for 24 hours. The protein level of cleaved caspase 3, cyclin D1, XIAP and Bcl-2 was assessed by immunoblotting. α-Tubulin was used as the internal control. ** P

Techniques Used: Expressing, Quantitative RT-PCR, Transfection, MTT Assay

H 2 O 2 -induced oxidative stress reversed TMZ sensitivity caused by H19 knockdown. Notes: ( A ) qRT-PCR was performed to determine the H19 expression of transfected U251 TMZ and LN229 TMZ cells with H19 knockdown combined with or without 1 µM H 2 O 2 . ( B ) MTT assay was performed to determine the cell viabilities of transfected U251 TMZ and LN229 TMZ cells with TMZ (0, 100, 250, 500, 1,000, 1,500 or 2,000 µM) treatments combined with or without 1 µM H 2 O 2 . ( C ) The protein level of cleaved caspase 3 was assessed by immunoblotting as described in ( A ). α-Tubulin was used as the internal control. ** P
Figure Legend Snippet: H 2 O 2 -induced oxidative stress reversed TMZ sensitivity caused by H19 knockdown. Notes: ( A ) qRT-PCR was performed to determine the H19 expression of transfected U251 TMZ and LN229 TMZ cells with H19 knockdown combined with or without 1 µM H 2 O 2 . ( B ) MTT assay was performed to determine the cell viabilities of transfected U251 TMZ and LN229 TMZ cells with TMZ (0, 100, 250, 500, 1,000, 1,500 or 2,000 µM) treatments combined with or without 1 µM H 2 O 2 . ( C ) The protein level of cleaved caspase 3 was assessed by immunoblotting as described in ( A ). α-Tubulin was used as the internal control. ** P

Techniques Used: Quantitative RT-PCR, Expressing, Transfection, MTT Assay

Overexpression of H19 increases chemoresistance of glioma cells to TMZ. Notes:  ( A ) Relative expression of H19 was detected by qRT-PCR in U251 and LN229 cells after transfected with control plasmid or H19 overexpression plasmid. ( B ) MTT assay was performed to determine the cell viabilities of transfected U251 and LN229 cells with TMZ (0, 100, 250, 500, 1,000, 1,500 or 2,000 µM) treatments. ( C ) Transfected U251 and LN229 cells were treated with 1,000 µM TMZ for 24 hours. The protein level of cleaved caspase 3, cyclin D1, XIAP and Bcl-2 was assessed by immunoblotting. α-Tubulin was used as the internal control.  ** P
Figure Legend Snippet: Overexpression of H19 increases chemoresistance of glioma cells to TMZ. Notes: ( A ) Relative expression of H19 was detected by qRT-PCR in U251 and LN229 cells after transfected with control plasmid or H19 overexpression plasmid. ( B ) MTT assay was performed to determine the cell viabilities of transfected U251 and LN229 cells with TMZ (0, 100, 250, 500, 1,000, 1,500 or 2,000 µM) treatments. ( C ) Transfected U251 and LN229 cells were treated with 1,000 µM TMZ for 24 hours. The protein level of cleaved caspase 3, cyclin D1, XIAP and Bcl-2 was assessed by immunoblotting. α-Tubulin was used as the internal control. ** P

Techniques Used: Over Expression, Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, MTT Assay

52) Product Images from "Expression and Y435-phosphorylation of Abelson interactor 1 (Abi1) promotes tumour cell adhesion, extracellular matrix degradation and invasion by colorectal carcinoma cells"

Article Title: Expression and Y435-phosphorylation of Abelson interactor 1 (Abi1) promotes tumour cell adhesion, extracellular matrix degradation and invasion by colorectal carcinoma cells

Journal: Molecular Cancer

doi: 10.1186/1476-4598-13-145

Functional role of Abi1 phosphorylation in MMP-9 secretion and CRC cell invasion. A , western immunoblotting of CHD1 cell lysate after control (DMSO) treatment or application of STI571 as indicated confirms inhibition of Abi1 Y435-phosphorylation through STI571, while levels of total Abi1, phospho-Erk1/2 and total Erk1/2 slightly increase upon STI treatment. Levels of cleaved caspase-3 are barely detectable. B , MMP-9 ELISA from cell culture supernatant after control (DMSO) treatment or application of STI571 shows a non-significant decrease in MMP-9 secretion after treatment with 5-50 μM STI571. C , increasing concentrations of STI571 inhibit CHD1 CRC cell invasion through reconstituted basement membrane in a dose-dependent manner. Representative microphotographs (right) show DAPI-counterstained cells that invaded through the sealed pores and were harvested from the lower chamber. DMSO, dimethyl sulfoxide; MMP-9, matrix metalloproteinase 9; n.s., not significant. *p
Figure Legend Snippet: Functional role of Abi1 phosphorylation in MMP-9 secretion and CRC cell invasion. A , western immunoblotting of CHD1 cell lysate after control (DMSO) treatment or application of STI571 as indicated confirms inhibition of Abi1 Y435-phosphorylation through STI571, while levels of total Abi1, phospho-Erk1/2 and total Erk1/2 slightly increase upon STI treatment. Levels of cleaved caspase-3 are barely detectable. B , MMP-9 ELISA from cell culture supernatant after control (DMSO) treatment or application of STI571 shows a non-significant decrease in MMP-9 secretion after treatment with 5-50 μM STI571. C , increasing concentrations of STI571 inhibit CHD1 CRC cell invasion through reconstituted basement membrane in a dose-dependent manner. Representative microphotographs (right) show DAPI-counterstained cells that invaded through the sealed pores and were harvested from the lower chamber. DMSO, dimethyl sulfoxide; MMP-9, matrix metalloproteinase 9; n.s., not significant. *p

Techniques Used: Functional Assay, Western Blot, Inhibition, Enzyme-linked Immunosorbent Assay, Cell Culture

53) Product Images from "Neuroprotective Effect of β-Caryophyllene on Cerebral Ischemia-Reperfusion Injury via Regulation of Necroptotic Neuronal Death and Inflammation: In Vivo and in Vitro"

Article Title: Neuroprotective Effect of β-Caryophyllene on Cerebral Ischemia-Reperfusion Injury via Regulation of Necroptotic Neuronal Death and Inflammation: In Vivo and in Vitro

Journal: Frontiers in Neuroscience

doi: 10.3389/fnins.2017.00583

BCP inhibits a component of necrotic cell death induced by I/R. (A) Representative microphotographs of TUNEL and cleaved caspase-3-positive cells in the cerebral cortex 48 h after MCAO. Scale bar 100 μm. Areas outlined in yellow are enlarged in lower panels. White arrows show necroptotic cells, and yellow arrows show apoptotic cells. Scale bar 20 μm. (B) Representative images of TUNEL-positive cells in the hippocampal CA1 region after I/R in mice treated with or without BCP (72 mg/kg). Scale bar 100 μm. (C,D) Percentage of apoptosis (cells with TUNEL and cleaved caspase-3 double positivity) and necrosis (cells with TUNEL-positive showing no cleaved caspase-3 activation) in the mouse brain in response to I/R treatment. Data are presented as the mean ± SEM ( n = 3). ## p
Figure Legend Snippet: BCP inhibits a component of necrotic cell death induced by I/R. (A) Representative microphotographs of TUNEL and cleaved caspase-3-positive cells in the cerebral cortex 48 h after MCAO. Scale bar 100 μm. Areas outlined in yellow are enlarged in lower panels. White arrows show necroptotic cells, and yellow arrows show apoptotic cells. Scale bar 20 μm. (B) Representative images of TUNEL-positive cells in the hippocampal CA1 region after I/R in mice treated with or without BCP (72 mg/kg). Scale bar 100 μm. (C,D) Percentage of apoptosis (cells with TUNEL and cleaved caspase-3 double positivity) and necrosis (cells with TUNEL-positive showing no cleaved caspase-3 activation) in the mouse brain in response to I/R treatment. Data are presented as the mean ± SEM ( n = 3). ## p

Techniques Used: TUNEL Assay, Mouse Assay, Activation Assay

54) Product Images from "Potential role of insulin receptor isoforms and IGF receptors in plaque instability of human and experimental atherosclerosis"

Article Title: Potential role of insulin receptor isoforms and IGF receptors in plaque instability of human and experimental atherosclerosis

Journal: Cardiovascular Diabetology

doi: 10.1186/s12933-018-0675-2

Antiapoptotic effect of IGF-IR on VSMCs. a Effect of IGF-IR inhibition by PPP (1 µmol/L) on cleaved caspase 3 levels analyzed by Western blot in IRLoxP +/+ and IR −/− VSMCs stimulated with IGF-I (10 nmol/L) or IGF-II (10 nmol/L) for 24 h. b Dose–response effect of thapsigargin (0-100 nmol/L, 18 h) on cleaved caspase 3 levels in IRLoxP +/+ and IR −/− VSMCs. c Effect of IGF-I or IGF-I pretreatment (10 nmol/L, 1 h) in IRLoxP +/+ and IR −/− VSMCs treated with thapsigargin (100 nmol/L) for 18 h. Experiments were performed 4 or 5 times. *p
Figure Legend Snippet: Antiapoptotic effect of IGF-IR on VSMCs. a Effect of IGF-IR inhibition by PPP (1 µmol/L) on cleaved caspase 3 levels analyzed by Western blot in IRLoxP +/+ and IR −/− VSMCs stimulated with IGF-I (10 nmol/L) or IGF-II (10 nmol/L) for 24 h. b Dose–response effect of thapsigargin (0-100 nmol/L, 18 h) on cleaved caspase 3 levels in IRLoxP +/+ and IR −/− VSMCs. c Effect of IGF-I or IGF-I pretreatment (10 nmol/L, 1 h) in IRLoxP +/+ and IR −/− VSMCs treated with thapsigargin (100 nmol/L) for 18 h. Experiments were performed 4 or 5 times. *p

Techniques Used: Inhibition, Western Blot

Differential contribution of IR isoforms to VSMCs apoptosis. a Effect of thapsigargin (100 nmol/L, 18 h) on cleaved caspase 3 levels analyzed by Western blot in IRA and IRB VSMCs. b Effect of IGF-IR inhibition by PPP (1 µmol/L) on cleaved caspase 3 levels in IRB and IRLoxP +/+ VSMCs treated with thapsigargin (100 nmol/L) for 18 h. Experiments were performed 4 times. *p
Figure Legend Snippet: Differential contribution of IR isoforms to VSMCs apoptosis. a Effect of thapsigargin (100 nmol/L, 18 h) on cleaved caspase 3 levels analyzed by Western blot in IRA and IRB VSMCs. b Effect of IGF-IR inhibition by PPP (1 µmol/L) on cleaved caspase 3 levels in IRB and IRLoxP +/+ VSMCs treated with thapsigargin (100 nmol/L) for 18 h. Experiments were performed 4 times. *p

Techniques Used: Western Blot, Inhibition

55) Product Images from "An image cytometric technique is a concise method to detect adenoviruses and host cell proteins and to monitor the infection and cellular responses induced"

Article Title: An image cytometric technique is a concise method to detect adenoviruses and host cell proteins and to monitor the infection and cellular responses induced

Journal: Virology Journal

doi: 10.1186/s12985-017-0888-0

Image cytometric analysis of E1A and cleaved caspase-3. a MSTO-211H cells were uninfected or infected with AdF35/Sur (10 3 or 10 4 vp/cell) for a period as indicated, stained with Ab against E1A and cleaved caspase-3, and analyzed with Tali image-based cytometer. Representative data are shown. The red lines are tentatively drawn to distinguish stained and unstained cells. A number in image data shows a percentage of the corresponding fraction. b Quantitative analysis of E1A single positive and E1A/cleaved caspase-3 double positive cells (10 4 vp/cell). Averages and SE bars are shown. c Western blot analysis to detect E1A expression in MSTO-211H cells uninfected or infected with AdF35/Sur at a vp/cell dose as indicated for 24 h. (−): uninfected. d E1A intensity per cell analyzed with image cytometry. The E1A expression per cell, corresponding each dot in ( a ), was plotted and the dot numbers are shown in the abscissa axis. Cells showing less than 1800 (arbitrary unit) at the E1A fluorescent intensity were gated out. A red line shows E1A intensity at 2000 and the E1A intensity of positively stained cells ( > 2000 units) was calculated. An average unit of the intensity of the positive cells and SE are shown with statistical analysis (ANOVA)
Figure Legend Snippet: Image cytometric analysis of E1A and cleaved caspase-3. a MSTO-211H cells were uninfected or infected with AdF35/Sur (10 3 or 10 4 vp/cell) for a period as indicated, stained with Ab against E1A and cleaved caspase-3, and analyzed with Tali image-based cytometer. Representative data are shown. The red lines are tentatively drawn to distinguish stained and unstained cells. A number in image data shows a percentage of the corresponding fraction. b Quantitative analysis of E1A single positive and E1A/cleaved caspase-3 double positive cells (10 4 vp/cell). Averages and SE bars are shown. c Western blot analysis to detect E1A expression in MSTO-211H cells uninfected or infected with AdF35/Sur at a vp/cell dose as indicated for 24 h. (−): uninfected. d E1A intensity per cell analyzed with image cytometry. The E1A expression per cell, corresponding each dot in ( a ), was plotted and the dot numbers are shown in the abscissa axis. Cells showing less than 1800 (arbitrary unit) at the E1A fluorescent intensity were gated out. A red line shows E1A intensity at 2000 and the E1A intensity of positively stained cells ( > 2000 units) was calculated. An average unit of the intensity of the positive cells and SE are shown with statistical analysis (ANOVA)

Techniques Used: Infection, Staining, Cytometry, Western Blot, Expressing

Image cytometric analysis of viral and cellular proteins. a NCI-H2452 cells were uninfected or infected with AdF35/Sur (10 3 or 10 4 vp/cell) for a period as indicated, stained with Ab against E1A and cleaved caspase-3, and analyzed with Tali image-based cytometer. Representative data are shown. The red lines are tentatively drawn to distinguish stained and unstained cells. A number in image data shows a percentage of the corresponding fraction. (−): uninfected. Bar graphs shows quantitative analysis of E1A single positive and E1A/cleaved caspase-3 double positive cells in ( a ) (10 4 vp/cell). Averages and SE bars are shown. b NCI-H2452 cells were infected as shown in ( a ) and stained with Ab against hexon and cleaved caspase-3. Bar graph shows quantitative analysis of hexon single positive and hexon/cleaved caspase-3 double positive cells in ( b ) (10 4 vp/cell). c MSTO-211H cells were uninfected or infected with AdF35/MK (10 3 or 10 4 vp/cell) for a period as indicated, stained with Ab against E1A and cleaved caspase-3, and analyzed with Tali image-based cytometer. Bar graph shows quantitative analysis of E1A single positive and E1A/cleaved caspase-3 double positive cells in ( c ) (10 4 vp/cell)
Figure Legend Snippet: Image cytometric analysis of viral and cellular proteins. a NCI-H2452 cells were uninfected or infected with AdF35/Sur (10 3 or 10 4 vp/cell) for a period as indicated, stained with Ab against E1A and cleaved caspase-3, and analyzed with Tali image-based cytometer. Representative data are shown. The red lines are tentatively drawn to distinguish stained and unstained cells. A number in image data shows a percentage of the corresponding fraction. (−): uninfected. Bar graphs shows quantitative analysis of E1A single positive and E1A/cleaved caspase-3 double positive cells in ( a ) (10 4 vp/cell). Averages and SE bars are shown. b NCI-H2452 cells were infected as shown in ( a ) and stained with Ab against hexon and cleaved caspase-3. Bar graph shows quantitative analysis of hexon single positive and hexon/cleaved caspase-3 double positive cells in ( b ) (10 4 vp/cell). c MSTO-211H cells were uninfected or infected with AdF35/MK (10 3 or 10 4 vp/cell) for a period as indicated, stained with Ab against E1A and cleaved caspase-3, and analyzed with Tali image-based cytometer. Bar graph shows quantitative analysis of E1A single positive and E1A/cleaved caspase-3 double positive cells in ( c ) (10 4 vp/cell)

Techniques Used: Infection, Staining, Cytometry

Image cytometric analysis of hexon and cleaved caspase-3. a MSTO-211H cells were uninfected or infected with AdF35/Sur (10 3 or 10 4 vp/cell) for a period as indicated, stained with Ab against hexon and cleaved caspase-3, and analyzed with Tali image-based cytometer. Representative data are shown. The red lines are tentatively drawn to distinguish stained and unstained cells. A number in image data shows a percentage of the corresponding fraction. b Quantitative analysis of hexon single positive and hexon/cleaved caspase-3 double positive cells (10 4 vp/cell). Averages and SE bars are shown. c Hexon intensity per cell analyzed with image cytometry. The hexon expression level per cell, corresponding each dot in ( a ), was plotted and the cell numbers are shown in the abscissa axis. Cells showing less than 1800 (arbitrary unit) at the hexon fluorescent intensity were gated out. A red line shows hexon intensity at 2000 and the hexon intensity of positively stained cells ( > 2000 units) was calculated. An average unit of the intensity of the positive cells and SE are shown with statistical analysis (ANOVA)
Figure Legend Snippet: Image cytometric analysis of hexon and cleaved caspase-3. a MSTO-211H cells were uninfected or infected with AdF35/Sur (10 3 or 10 4 vp/cell) for a period as indicated, stained with Ab against hexon and cleaved caspase-3, and analyzed with Tali image-based cytometer. Representative data are shown. The red lines are tentatively drawn to distinguish stained and unstained cells. A number in image data shows a percentage of the corresponding fraction. b Quantitative analysis of hexon single positive and hexon/cleaved caspase-3 double positive cells (10 4 vp/cell). Averages and SE bars are shown. c Hexon intensity per cell analyzed with image cytometry. The hexon expression level per cell, corresponding each dot in ( a ), was plotted and the cell numbers are shown in the abscissa axis. Cells showing less than 1800 (arbitrary unit) at the hexon fluorescent intensity were gated out. A red line shows hexon intensity at 2000 and the hexon intensity of positively stained cells ( > 2000 units) was calculated. An average unit of the intensity of the positive cells and SE are shown with statistical analysis (ANOVA)

Techniques Used: Infection, Staining, Cytometry, Expressing

56) Product Images from "Erythropoietin attenuates motor neuron programmed cell death in a burn animal model"

Article Title: Erythropoietin attenuates motor neuron programmed cell death in a burn animal model

Journal: PLoS ONE

doi: 10.1371/journal.pone.0190039

EPO attenuates burn-induced motor neruon apoptosis. (A) Expression levels of BCL-2, BAX, and cleaved caspase-3 in the spinal cord ventral horn, measured through Western blotting. (B) Significant reduction in BCL-2/BAX expression was observed in Group B compared with Group A (100%). However, BCL-2/BAX expression was upregulated after EPO treatment. (C) Cleaved caspase-3 expression increased in the burn-induced group compared with the sham-control group. Similar to the notable reduction in cleaved caspase-3 expression following EPO treatment, a decrease in cleaved caspase-3 was observed. *: p
Figure Legend Snippet: EPO attenuates burn-induced motor neruon apoptosis. (A) Expression levels of BCL-2, BAX, and cleaved caspase-3 in the spinal cord ventral horn, measured through Western blotting. (B) Significant reduction in BCL-2/BAX expression was observed in Group B compared with Group A (100%). However, BCL-2/BAX expression was upregulated after EPO treatment. (C) Cleaved caspase-3 expression increased in the burn-induced group compared with the sham-control group. Similar to the notable reduction in cleaved caspase-3 expression following EPO treatment, a decrease in cleaved caspase-3 was observed. *: p

Techniques Used: Expressing, Western Blot

EPO alleviated burn-induced muscle cells apoptosis by Western blotting. Burn injury significantly increased the expression of cleaved caspase-3 in muscle cells vs Group A (p
Figure Legend Snippet: EPO alleviated burn-induced muscle cells apoptosis by Western blotting. Burn injury significantly increased the expression of cleaved caspase-3 in muscle cells vs Group A (p

Techniques Used: Western Blot, Expressing

57) Product Images from "Hypoxia-inducible factor 1? is a new target of microphthalmia-associated transcription factor (MITF) in melanoma cells"

Article Title: Hypoxia-inducible factor 1? is a new target of microphthalmia-associated transcription factor (MITF) in melanoma cells

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200501067

HIF1α plays a pro-survival role in B16 melanoma cells. (A) Immunofluorescence study to detect the presence of cleaved caspase-3 in B16 cells nonstimulated (NS) or stimulated for 24 with forskolin (FK), and nontreated (−St) or treated with staurosporin (+St) for 6 h. Bar, 40 μM. (B) Immunofluorescence analysis of B16 cells transfected with a nonrelevant siRNA (siRNA-Ctrol) or an siRNA to invalidate HIF1α. Cleaved caspase-3 was labeled with a secondary Texas red–conjugated antibody (red) and HIF1α protein was detected by an mAb and a secondary FITC-conjugated secondary one (green). Nuclei were labeled with bisbenzidine (blue). Bar, 20 μM.
Figure Legend Snippet: HIF1α plays a pro-survival role in B16 melanoma cells. (A) Immunofluorescence study to detect the presence of cleaved caspase-3 in B16 cells nonstimulated (NS) or stimulated for 24 with forskolin (FK), and nontreated (−St) or treated with staurosporin (+St) for 6 h. Bar, 40 μM. (B) Immunofluorescence analysis of B16 cells transfected with a nonrelevant siRNA (siRNA-Ctrol) or an siRNA to invalidate HIF1α. Cleaved caspase-3 was labeled with a secondary Texas red–conjugated antibody (red) and HIF1α protein was detected by an mAb and a secondary FITC-conjugated secondary one (green). Nuclei were labeled with bisbenzidine (blue). Bar, 20 μM.

Techniques Used: Immunofluorescence, Transfection, Labeling

58) Product Images from "Honokiol Ameliorates Myocardial Ischemia/Reperfusion Injury in Type 1 Diabetic Rats by Reducing Oxidative Stress and Apoptosis through Activating the SIRT1-Nrf2 Signaling Pathway"

Article Title: Honokiol Ameliorates Myocardial Ischemia/Reperfusion Injury in Type 1 Diabetic Rats by Reducing Oxidative Stress and Apoptosis through Activating the SIRT1-Nrf2 Signaling Pathway

Journal: Oxidative Medicine and Cellular Longevity

doi: 10.1155/2018/3159801

EX527 abolished HKL-induced suppression on myocardial apoptosis in MI/R-injured diabetic myocardium. (a) Representative images of TUNEL staining (300x, bar = 100 μ m). The apoptotic cells were detected by TUNEL (green), and the nuclei were detected by DAPI (blue). (b) Myocardial apoptotic index. (c) Representative blots. (d) Bcl-2/Bax ratio. (e) Cleaved caspase 3 expression. (f) Cytosolic cytochrome c expression. (g) Representative blots. (h) SIRT1 expression. (i) Relative SIRT1 activity. (j) Nrf2 nuclear translocation. Data are presented as the mean ± SEM ( n = 6 in each group). #/## P
Figure Legend Snippet: EX527 abolished HKL-induced suppression on myocardial apoptosis in MI/R-injured diabetic myocardium. (a) Representative images of TUNEL staining (300x, bar = 100 μ m). The apoptotic cells were detected by TUNEL (green), and the nuclei were detected by DAPI (blue). (b) Myocardial apoptotic index. (c) Representative blots. (d) Bcl-2/Bax ratio. (e) Cleaved caspase 3 expression. (f) Cytosolic cytochrome c expression. (g) Representative blots. (h) SIRT1 expression. (i) Relative SIRT1 activity. (j) Nrf2 nuclear translocation. Data are presented as the mean ± SEM ( n = 6 in each group). #/## P

Techniques Used: TUNEL Assay, Staining, Expressing, Activity Assay, Translocation Assay

Type 1 diabetic rats subjected to MI/R surgery showed further weakened cardiac function, increased myocardial oxidative stress and apoptosis, and reduced myocardial SIRT1 signaling. (a) Representative M-mode images by echocardiography. Echocardiographic assessment was performed after 72 h of reperfusion. (b) Left ventricular ejection fraction (LVEF). (c) Left ventricular fractional shortening (LVFS). (d) Representative blots. (e) SIRT1 expression. (f) Relative SIRT1 activity. (g) Bcl-2/Bax ratio. (h) Cleaved caspase 3 expression. (i) gp91 phox expression. (j) Representative images of DHE staining (300x, bar = 100 μ m). (k) DHE intensity. Data are presented as the mean ± SEM ( n = 6 in each group). $$ P
Figure Legend Snippet: Type 1 diabetic rats subjected to MI/R surgery showed further weakened cardiac function, increased myocardial oxidative stress and apoptosis, and reduced myocardial SIRT1 signaling. (a) Representative M-mode images by echocardiography. Echocardiographic assessment was performed after 72 h of reperfusion. (b) Left ventricular ejection fraction (LVEF). (c) Left ventricular fractional shortening (LVFS). (d) Representative blots. (e) SIRT1 expression. (f) Relative SIRT1 activity. (g) Bcl-2/Bax ratio. (h) Cleaved caspase 3 expression. (i) gp91 phox expression. (j) Representative images of DHE staining (300x, bar = 100 μ m). (k) DHE intensity. Data are presented as the mean ± SEM ( n = 6 in each group). $$ P

Techniques Used: Expressing, Activity Assay, Staining

SIRT1 siRNA and Nrf2 siRNA transfection blunted HKL-induced antiapoptotic effects against SIR injury in HG-treated H9c2 cells. (a) Cellular morphology (100x). (b) Cellular viability. (c) Representative images of TUNEL staining (200x, bar = 200 μ m). The apoptotic cells were detected by TUNEL (green), and the nuclei were detected by DAPI (blue). (d) Cellular apoptotic index. (e) Representative blots. (f) Bcl-2/Bax ratio. (g) Cleaved caspase 3 expression. (h) Cytosolic cytochrome c expression. Data are presented as the mean ± SEM ( n = 6 in each group). a/aa P
Figure Legend Snippet: SIRT1 siRNA and Nrf2 siRNA transfection blunted HKL-induced antiapoptotic effects against SIR injury in HG-treated H9c2 cells. (a) Cellular morphology (100x). (b) Cellular viability. (c) Representative images of TUNEL staining (200x, bar = 200 μ m). The apoptotic cells were detected by TUNEL (green), and the nuclei were detected by DAPI (blue). (d) Cellular apoptotic index. (e) Representative blots. (f) Bcl-2/Bax ratio. (g) Cleaved caspase 3 expression. (h) Cytosolic cytochrome c expression. Data are presented as the mean ± SEM ( n = 6 in each group). a/aa P

Techniques Used: Transfection, TUNEL Assay, Staining, Expressing

59) Product Images from "Role of PPAR-β/δ/miR-17/TXNIP pathway in neuronal apoptosis after neonatal hypoxic-ischemic injury in rats"

Article Title: Role of PPAR-β/δ/miR-17/TXNIP pathway in neuronal apoptosis after neonatal hypoxic-ischemic injury in rats

Journal: Neuropharmacology

doi: 10.1016/j.neuropharm.2018.08.003

Effects of GW0742 on apoptosis via the PPAR-β/δ/TXNIP/Trx-1/p-ASK1/p-p38 signaling pathway at 72 hours post hypoxia-ischemia (HI). (A) Representative picture of western blot data showing bands of the expression levels of TXNIP, thioredoxin-1 (Trx-1), p-ASK1, p-p38, Bcl-2, Bax and cleaved caspase-3 either with GW0742 treatment alone or GW0742+DMSO in corn oil, GW0742+GSK3787, GW0742+control CRISPR, GW0742+TXNIP CRISPR, GW0742+control LNA antimir, GW0742+antimir-17-5p groups. (B–G) Western blot data quantification of bands showed that GW0742 significantly increased Trx-1 and Bcl-2/Bax ratio and significantly decreased TXNIP, p-ASK1, p-p38 and caspase-3 cleavage when compared with HI+vehicle. GSK3787, TXNIP CRISPR activation plasmid and antimir-17-5p showed to significantly increase TXNIP (B), p-ASK1 (D), p-p38 (E) and cleavage of caspase-3 (G) expression, but decrease Trx-1 (C), Bcl-2/Bax (F) expression when compared with GW0742+DMSO in corn oil, GW0742+control CRISPR and GW0742+control LNA groups, respectively. * p
Figure Legend Snippet: Effects of GW0742 on apoptosis via the PPAR-β/δ/TXNIP/Trx-1/p-ASK1/p-p38 signaling pathway at 72 hours post hypoxia-ischemia (HI). (A) Representative picture of western blot data showing bands of the expression levels of TXNIP, thioredoxin-1 (Trx-1), p-ASK1, p-p38, Bcl-2, Bax and cleaved caspase-3 either with GW0742 treatment alone or GW0742+DMSO in corn oil, GW0742+GSK3787, GW0742+control CRISPR, GW0742+TXNIP CRISPR, GW0742+control LNA antimir, GW0742+antimir-17-5p groups. (B–G) Western blot data quantification of bands showed that GW0742 significantly increased Trx-1 and Bcl-2/Bax ratio and significantly decreased TXNIP, p-ASK1, p-p38 and caspase-3 cleavage when compared with HI+vehicle. GSK3787, TXNIP CRISPR activation plasmid and antimir-17-5p showed to significantly increase TXNIP (B), p-ASK1 (D), p-p38 (E) and cleavage of caspase-3 (G) expression, but decrease Trx-1 (C), Bcl-2/Bax (F) expression when compared with GW0742+DMSO in corn oil, GW0742+control CRISPR and GW0742+control LNA groups, respectively. * p

Techniques Used: Western Blot, Expressing, CRISPR, Activation Assay, Plasmid Preparation

60) Product Images from "Pioglitazone Attenuates Palmitate-Induced Inflammation and Endoplasmic Reticulum Stress in Pancreatic β-Cells"

Article Title: Pioglitazone Attenuates Palmitate-Induced Inflammation and Endoplasmic Reticulum Stress in Pancreatic β-Cells

Journal: Endocrinology and Metabolism

doi: 10.3803/EnM.2018.33.1.105

Pioglitazone (Pio) improves palmitate (PA)-induced β-cell impairment via repression of the inflammatory response and endoplasmic reticulum (ER) stress. (A) Mouse insulinoma 6 (MIN6) cells were incubated with 0.5 mM PA in the presence or absence of 10 µM Pio for 24 hours, and the glucose-stimulated (1 or 4.5 g/L) insulin secretion of MIN6 cells was evaluated. Secreted insulin was measured by a mouse insulin enzyme-linked immunosorbent assay (ELISA) kit. The values are representative of 6 independent experiments. (B) Expression of cleaved caspase-3 (c-casp3), an apoptotic protein, was measured by Western blot analysis. (C) Poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) activity is represented as the percentage of relative absorbance compared to the vehicle group. (D) Transcription of tumor necrosis factor α ( TNFA ), interleukin 6 ( IL6 ), and IL1B was measured by real time reverse-transcription polymerase chain reaction, and normalized with β-actin. (E) The ER stress proteins, including phosphor-eukaryotic translation initiation factor 2α (p-eIF2α), glucose-regulated protein 78 (GRP78), cleaved-activating transcription factor 6 (c-ATF6), and C/EBP homologous protein (CHOP), were measured by Western blot analysis and (F) the ratio of p-eIF2α, GRP78, and CHOP to β-actin was described. Each value represents the mean of three experiments. t-casp3, total caspase-3; Veh, vehicle; PA, palmitate. a P
Figure Legend Snippet: Pioglitazone (Pio) improves palmitate (PA)-induced β-cell impairment via repression of the inflammatory response and endoplasmic reticulum (ER) stress. (A) Mouse insulinoma 6 (MIN6) cells were incubated with 0.5 mM PA in the presence or absence of 10 µM Pio for 24 hours, and the glucose-stimulated (1 or 4.5 g/L) insulin secretion of MIN6 cells was evaluated. Secreted insulin was measured by a mouse insulin enzyme-linked immunosorbent assay (ELISA) kit. The values are representative of 6 independent experiments. (B) Expression of cleaved caspase-3 (c-casp3), an apoptotic protein, was measured by Western blot analysis. (C) Poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) activity is represented as the percentage of relative absorbance compared to the vehicle group. (D) Transcription of tumor necrosis factor α ( TNFA ), interleukin 6 ( IL6 ), and IL1B was measured by real time reverse-transcription polymerase chain reaction, and normalized with β-actin. (E) The ER stress proteins, including phosphor-eukaryotic translation initiation factor 2α (p-eIF2α), glucose-regulated protein 78 (GRP78), cleaved-activating transcription factor 6 (c-ATF6), and C/EBP homologous protein (CHOP), were measured by Western blot analysis and (F) the ratio of p-eIF2α, GRP78, and CHOP to β-actin was described. Each value represents the mean of three experiments. t-casp3, total caspase-3; Veh, vehicle; PA, palmitate. a P

Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Activity Assay, Reverse Transcription Polymerase Chain Reaction

61) Product Images from "Triptolide induces Sertoli cell apoptosis in mice via ROS/JNK-dependent activation of the mitochondrial pathway and inhibition of Nrf2-mediated antioxidant response"

Article Title: Triptolide induces Sertoli cell apoptosis in mice via ROS/JNK-dependent activation of the mitochondrial pathway and inhibition of Nrf2-mediated antioxidant response

Journal: Acta Pharmacologica Sinica

doi: 10.1038/aps.2017.95

TP induced testicular injury in mice. (A) The testis indexes of mice after the treatment of TP for 14 days; (B) The MDA levels in testicular tissue; (C) The SDH activations in testicular tissue; (D) The SOD activations in testicular tissue; (E) Activations of caspase-3 in testicular tissue were determined by classical colorimetric method by commercial kits; (F) The mRNA expression of FSHR was detected by real-time PCR in testicular tissues. Data represent the mean±SD of eight independent experiments. * P
Figure Legend Snippet: TP induced testicular injury in mice. (A) The testis indexes of mice after the treatment of TP for 14 days; (B) The MDA levels in testicular tissue; (C) The SDH activations in testicular tissue; (D) The SOD activations in testicular tissue; (E) Activations of caspase-3 in testicular tissue were determined by classical colorimetric method by commercial kits; (F) The mRNA expression of FSHR was detected by real-time PCR in testicular tissues. Data represent the mean±SD of eight independent experiments. * P

Techniques Used: Mouse Assay, Multiple Displacement Amplification, Expressing, Real-time Polymerase Chain Reaction

62) Product Images from "Plectin protects podocytes from adriamycin‐induced apoptosis and F‐actin cytoskeletal disruption through the integrin α6β4/ FAK/p38 MAPK pathway, et al. Plectin protects podocytes from adriamycin‐induced apoptosis and F‐actin cytoskeletal disruption through the integrin α6β4/FAK/p38 MAPK pathway"

Article Title: Plectin protects podocytes from adriamycin‐induced apoptosis and F‐actin cytoskeletal disruption through the integrin α6β4/ FAK/p38 MAPK pathway, et al. Plectin protects podocytes from adriamycin‐induced apoptosis and F‐actin cytoskeletal disruption through the integrin α6β4/FAK/p38 MAPK pathway

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.13816

ADR contributed to the development of proteinuria and renal dysfunction by inhibiting plectin expression and activating the integrin α6β4/ FAK /p38 pathway. ADR induced nephropathy was introduced by a single injection of 7.5 mg/kg ADR via the tail vein. All rats were killed at the end of the 4th week after ADR injection. A, Light microscopic examination revealed glomerular atrophy and disappearance as well as renal tubular swelling after ADR treatment. B, TEM examination of the NC group revealed the presence of normal glomerular ultrastructure. TEM examination of the ADR group revealed the presence of diffuse foot process effacement, GBM thickening, slit diaphragm loss and mesangial sclerosis. C‐D, Western blot showed that WT 1 and synaptopodin protein expression levels were decreased and that desmin protein expression levels were increased in the ADR group compared with the NC group. E, Western blot showed that plectin expression was suppressed in the ADR ‐treated group (n = 5) compared with the NC group (n = 5). F‐G, Immunohistochemical analysis showed that plectin expression in glomeruli was decreased in ADR treated kidney tissues (n = 5) compared with normal kidney tissues (n = 5). H‐I, Western blot showed that integrin α6β4, FAK and p38 were activated by ADR treatment. J‐K, Western blot showed that cleaved caspase‐3 and Bax protein expression was increased in the ADR group (n = 5) compared with the NC group (n = 5). ** P
Figure Legend Snippet: ADR contributed to the development of proteinuria and renal dysfunction by inhibiting plectin expression and activating the integrin α6β4/ FAK /p38 pathway. ADR induced nephropathy was introduced by a single injection of 7.5 mg/kg ADR via the tail vein. All rats were killed at the end of the 4th week after ADR injection. A, Light microscopic examination revealed glomerular atrophy and disappearance as well as renal tubular swelling after ADR treatment. B, TEM examination of the NC group revealed the presence of normal glomerular ultrastructure. TEM examination of the ADR group revealed the presence of diffuse foot process effacement, GBM thickening, slit diaphragm loss and mesangial sclerosis. C‐D, Western blot showed that WT 1 and synaptopodin protein expression levels were decreased and that desmin protein expression levels were increased in the ADR group compared with the NC group. E, Western blot showed that plectin expression was suppressed in the ADR ‐treated group (n = 5) compared with the NC group (n = 5). F‐G, Immunohistochemical analysis showed that plectin expression in glomeruli was decreased in ADR treated kidney tissues (n = 5) compared with normal kidney tissues (n = 5). H‐I, Western blot showed that integrin α6β4, FAK and p38 were activated by ADR treatment. J‐K, Western blot showed that cleaved caspase‐3 and Bax protein expression was increased in the ADR group (n = 5) compared with the NC group (n = 5). ** P

Techniques Used: Expressing, Injection, Transmission Electron Microscopy, Western Blot, Immunohistochemistry

p38 induced podocyte apoptosis by activating Bax and caspase‐3. For the ADR group, podocyte was treated with 0.5 μg/ mL ADR for 12 h. For the ADR + plectin group or ADR + MOCK group, podocyte was incubated with pEGFP ‐N1‐plectin plasmids or empty vectors for 6 h and then cultured normally for 42 h, 0.5 μg/ mL ADR was added 12 h prior to cell harvest. For the NC + siPlectin group and NC + Scramble group, podocyte was transfected with siPlectin or scramble RNA respectively at a final concentration of 20 nmol/L for 4 h and then cultured normally for 68 h. For p38‐ MAPK inhibitor study, podocyte was preincubated with the p38 inhibitor SB 203580 (5 μmol/L) or DMSO vehicle for 1 h before siPlectin transfection and then incubated for an additional 72 h until podocyte collection. A‐B, Western blot showed that Bax and cleaved caspase‐3 protein expression levels were elevated in ADR ‐treated podocyte compared with NC podocyte and that recovering plectin expression inhibited the activation of these pro‐apoptotic proteins. C‐D, Western blot showed that siPlectin transfection increased Bax and cleaved caspase‐3 protein expression in the NC + siPlectin group compared with the NC + Scramble group, whereas p38 inhibition blocked the siPlectin‐induced activation of these proteins. Data shown are representative of three independent experiments (n = 3). * P
Figure Legend Snippet: p38 induced podocyte apoptosis by activating Bax and caspase‐3. For the ADR group, podocyte was treated with 0.5 μg/ mL ADR for 12 h. For the ADR + plectin group or ADR + MOCK group, podocyte was incubated with pEGFP ‐N1‐plectin plasmids or empty vectors for 6 h and then cultured normally for 42 h, 0.5 μg/ mL ADR was added 12 h prior to cell harvest. For the NC + siPlectin group and NC + Scramble group, podocyte was transfected with siPlectin or scramble RNA respectively at a final concentration of 20 nmol/L for 4 h and then cultured normally for 68 h. For p38‐ MAPK inhibitor study, podocyte was preincubated with the p38 inhibitor SB 203580 (5 μmol/L) or DMSO vehicle for 1 h before siPlectin transfection and then incubated for an additional 72 h until podocyte collection. A‐B, Western blot showed that Bax and cleaved caspase‐3 protein expression levels were elevated in ADR ‐treated podocyte compared with NC podocyte and that recovering plectin expression inhibited the activation of these pro‐apoptotic proteins. C‐D, Western blot showed that siPlectin transfection increased Bax and cleaved caspase‐3 protein expression in the NC + siPlectin group compared with the NC + Scramble group, whereas p38 inhibition blocked the siPlectin‐induced activation of these proteins. Data shown are representative of three independent experiments (n = 3). * P

Techniques Used: Incubation, Cell Culture, Transfection, Concentration Assay, Western Blot, Expressing, Activation Assay, Inhibition

63) Product Images from "Requirement of JNK1 for endothelial cell injury in atherogenesis"

Article Title: Requirement of JNK1 for endothelial cell injury in atherogenesis

Journal: Atherosclerosis

doi: 10.1016/j.atherosclerosis.2014.05.950

JNK1 regulates endothelial injury in hypercholesterolemia. LDLR −/− , LDLR −/− /JNK1 −/− or LDLR −/− /MKP-1 −/− mice were exposed to a high fat diet for 6 weeks. (A) Caspase-3 activation was measured in EC by en face staining of susceptible (S) or protected (P) regions of the aorta (red; n = 6 per group). Endothelial marker is CD31 (green) and nuclei were stained using ToPro-3 (purple). (B) En face TUNEL staining was carried out at susceptible (S) or protected (P) regions of the aorta (green; n = 6 per group). Endothelial marker is lectin (red). Representative images (positive cells indicated by arrows) and quantitation of active caspase-3 and TUNEL-positive cells (mean ± SD) are shown. * p
Figure Legend Snippet: JNK1 regulates endothelial injury in hypercholesterolemia. LDLR −/− , LDLR −/− /JNK1 −/− or LDLR −/− /MKP-1 −/− mice were exposed to a high fat diet for 6 weeks. (A) Caspase-3 activation was measured in EC by en face staining of susceptible (S) or protected (P) regions of the aorta (red; n = 6 per group). Endothelial marker is CD31 (green) and nuclei were stained using ToPro-3 (purple). (B) En face TUNEL staining was carried out at susceptible (S) or protected (P) regions of the aorta (green; n = 6 per group). Endothelial marker is lectin (red). Representative images (positive cells indicated by arrows) and quantitation of active caspase-3 and TUNEL-positive cells (mean ± SD) are shown. * p

Techniques Used: Mouse Assay, Activation Assay, Staining, Marker, TUNEL Assay, Quantitation Assay

64) Product Images from "Icotinib inhibits the proliferation of hepatocellular carcinoma cells in vitro and in vivo dependently on EGFR activation and PDL1 expression"

Article Title: Icotinib inhibits the proliferation of hepatocellular carcinoma cells in vitro and in vivo dependently on EGFR activation and PDL1 expression

Journal: OncoTargets and therapy

doi: 10.2147/OTT.S179844

Effect of icotinib on expression of C-caspase 3, BCL2, p-EGFR, and PDL1 in SMMC7721 and Huh7 cells. Notes:  ( A ) Representative Western blot images; ( B ) quantification analysis of protein expression by ImageJ. ** P
Figure Legend Snippet: Effect of icotinib on expression of C-caspase 3, BCL2, p-EGFR, and PDL1 in SMMC7721 and Huh7 cells. Notes: ( A ) Representative Western blot images; ( B ) quantification analysis of protein expression by ImageJ. ** P

Techniques Used: Expressing, Western Blot

65) Product Images from "Valproic acid enhances the viability of random pattern skin flaps: involvement of enhancing angiogenesis and inhibiting oxidative stress and apoptosis"

Article Title: Valproic acid enhances the viability of random pattern skin flaps: involvement of enhancing angiogenesis and inhibiting oxidative stress and apoptosis

Journal: Drug Design, Development and Therapy

doi: 10.2147/DDDT.S186222

VPA reduced apoptosis in random skin flaps. Notes: ( A ) Cleaved CASP3 expression in each group as assessed by IHC (original magnification ×200). ( B ) Protein expression of cleaved CASP3 in each group as assessed by Western blot analysis. The gels have been run under the same experimental conditions, and cropped blots are used here. ( C ) The optical density values of cleaved CASP3. ( D ) Densitometry result of cleaved CASP3 protein expression in the two groups. Values are expressed as mean ± SEM, n=6 per group. * P
Figure Legend Snippet: VPA reduced apoptosis in random skin flaps. Notes: ( A ) Cleaved CASP3 expression in each group as assessed by IHC (original magnification ×200). ( B ) Protein expression of cleaved CASP3 in each group as assessed by Western blot analysis. The gels have been run under the same experimental conditions, and cropped blots are used here. ( C ) The optical density values of cleaved CASP3. ( D ) Densitometry result of cleaved CASP3 protein expression in the two groups. Values are expressed as mean ± SEM, n=6 per group. * P

Techniques Used: Expressing, Immunohistochemistry, Western Blot

66) Product Images from "Ischemia-induced Neuronal Cell Death Is Mediated by Chemokine Receptor CX3CR1"

Article Title: Ischemia-induced Neuronal Cell Death Is Mediated by Chemokine Receptor CX3CR1

Journal: Scientific Reports

doi: 10.1038/s41598-017-18774-0

Coupling of CX3CR1 to apoptotic neuronal cell death in pMCAO mice. ( A ) Triple staining with cleaved Caspase-3 (apoptotic marker), CX3CR1, and NeuN (neuron marker) was performed and demonstrated that CX3CR1 was coupled to apoptotic neurons in ischemic damage 24 hours following pMCAO. Scale bars = 100 µm, n = 4/group. ( B ) Semi-quantification analysis showed that the number of Caspase-3/CX3CR1/NeuN positive cells was significantly increased in the ipsilateral hemisphere (**p
Figure Legend Snippet: Coupling of CX3CR1 to apoptotic neuronal cell death in pMCAO mice. ( A ) Triple staining with cleaved Caspase-3 (apoptotic marker), CX3CR1, and NeuN (neuron marker) was performed and demonstrated that CX3CR1 was coupled to apoptotic neurons in ischemic damage 24 hours following pMCAO. Scale bars = 100 µm, n = 4/group. ( B ) Semi-quantification analysis showed that the number of Caspase-3/CX3CR1/NeuN positive cells was significantly increased in the ipsilateral hemisphere (**p

Techniques Used: Mouse Assay, Staining, Marker

CX3CR1 deficiency decreased Caspase-3 positive neuronal cells after pMCAO. ( A ) Immunohistochemical staining of Caspase-3 antibody in the ipsilateral (1) peri-infarct region, (2) hippocampus, and (3) striatum 24 hours after pMCAO. ( B ) Semi-quantification analysis of the number of Caspase-3 positive cells demonstrated that CX3CR1 deficiency decreased the number of apoptotic cells in the peri-infarction region, hippocampus, and striatum (*p
Figure Legend Snippet: CX3CR1 deficiency decreased Caspase-3 positive neuronal cells after pMCAO. ( A ) Immunohistochemical staining of Caspase-3 antibody in the ipsilateral (1) peri-infarct region, (2) hippocampus, and (3) striatum 24 hours after pMCAO. ( B ) Semi-quantification analysis of the number of Caspase-3 positive cells demonstrated that CX3CR1 deficiency decreased the number of apoptotic cells in the peri-infarction region, hippocampus, and striatum (*p

Techniques Used: Immunohistochemistry, Staining

67) Product Images from "Potent anti-tumor activity of a syringolin analog in multiple myeloma: a dual inhibitor of proteasome activity targeting β2 and β5 subunits"

Article Title: Potent anti-tumor activity of a syringolin analog in multiple myeloma: a dual inhibitor of proteasome activity targeting β2 and β5 subunits

Journal: Oncotarget

doi: 10.18632/oncotarget.24160

Evaluation of syringolog-1 activity in primary MM cells from patients ( A – B ) Cytotoxicity of syringolog-1 (Sy) or bortezomib (Btz) was tested on 8 primary MM cells. IC 50 values of Sy on each primary MM cell were calculated from the triplicate experiments. A. 5 cases clinically sensitive to Btz-based therapy. (B) 3 cases clinically refractory to Btz-based therapy. RR: relapsed and or refractory, ND: newly diagnosed. ( C ) 20S proteasome activities in 8 primary MM cells were evaluated after incubation with or without 10 nM of Sy or Btz for 6 h. ( D ) Four peripheral blood mononuclear cells from healthy individuals were analyzed for cytotoxic effect induced by Sy treatment at the indicated dose for 48 h. ( E ) Accumulation of poly-ubiquitinated proteins and expression of cleaved caspase-3 were evaluated in 3 primary MM cell lines after incubation with 10 nM of syringolog-1 or Btz for 16 h. * represents statistically significant ( p
Figure Legend Snippet: Evaluation of syringolog-1 activity in primary MM cells from patients ( A – B ) Cytotoxicity of syringolog-1 (Sy) or bortezomib (Btz) was tested on 8 primary MM cells. IC 50 values of Sy on each primary MM cell were calculated from the triplicate experiments. A. 5 cases clinically sensitive to Btz-based therapy. (B) 3 cases clinically refractory to Btz-based therapy. RR: relapsed and or refractory, ND: newly diagnosed. ( C ) 20S proteasome activities in 8 primary MM cells were evaluated after incubation with or without 10 nM of Sy or Btz for 6 h. ( D ) Four peripheral blood mononuclear cells from healthy individuals were analyzed for cytotoxic effect induced by Sy treatment at the indicated dose for 48 h. ( E ) Accumulation of poly-ubiquitinated proteins and expression of cleaved caspase-3 were evaluated in 3 primary MM cell lines after incubation with 10 nM of syringolog-1 or Btz for 16 h. * represents statistically significant ( p

Techniques Used: Activity Assay, Incubation, Expressing

68) Product Images from "Kaempferol induces autophagic cell death via IRE1-JNK-CHOP pathway and inhibition of G9a in gastric cancer cells"

Article Title: Kaempferol induces autophagic cell death via IRE1-JNK-CHOP pathway and inhibition of G9a in gastric cancer cells

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-0930-1

Inhibition of AMPKα/ULK1 attenuates cell death in kaempferol-treated GC cells. a AGS and SNU-638 cells were treated with kaempferol (50 μM) in a time-dependent manner (0, 8, 16, and 24 h). Cell lysates were loaded used in the western blot assay and detected antibodies targeting p-AMPKα (Thr172), AMPKα, p-ULK1 (Ser555), ULK1, p-mTOR (Ser2448), mTOR, p-p70S6K (Thr389), and p70S6K involved in autophagy induction. β-actin was used as a protein loading control. b , c The effect of cell viability, LDH release, and Western blot analysis for p-AMPKα, p-ULK1, p-mTOR, cleaved caspase-3, and LC3B in kaempferol (50 μM, 24 h)-treated AGS and SNU-638 cells in the presence or absence of compound C (2 μM, 24 h); * p
Figure Legend Snippet: Inhibition of AMPKα/ULK1 attenuates cell death in kaempferol-treated GC cells. a AGS and SNU-638 cells were treated with kaempferol (50 μM) in a time-dependent manner (0, 8, 16, and 24 h). Cell lysates were loaded used in the western blot assay and detected antibodies targeting p-AMPKα (Thr172), AMPKα, p-ULK1 (Ser555), ULK1, p-mTOR (Ser2448), mTOR, p-p70S6K (Thr389), and p70S6K involved in autophagy induction. β-actin was used as a protein loading control. b , c The effect of cell viability, LDH release, and Western blot analysis for p-AMPKα, p-ULK1, p-mTOR, cleaved caspase-3, and LC3B in kaempferol (50 μM, 24 h)-treated AGS and SNU-638 cells in the presence or absence of compound C (2 μM, 24 h); * p

Techniques Used: Inhibition, Western Blot

69) Product Images from "Cyclovirobuxine D Inhibits Cell Proliferation and Induces Mitochondria-Mediated Apoptosis in Human Gastric Cancer Cells"

Article Title: Cyclovirobuxine D Inhibits Cell Proliferation and Induces Mitochondria-Mediated Apoptosis in Human Gastric Cancer Cells

Journal: Molecules

doi: 10.3390/molecules201119729

CVB-D induces expression of apoptosis-related proteins in MGC-803 and MKN28 cells. The protein bands of cleaved Caspase-3, Bax and Bcl-2 were detected using western blotting in CVB-D (0, 30, 60 and 120 µmol/L) treated MGC-803 ( left ); and MKN28 cells ( right ). β-actin was used as the control protein.
Figure Legend Snippet: CVB-D induces expression of apoptosis-related proteins in MGC-803 and MKN28 cells. The protein bands of cleaved Caspase-3, Bax and Bcl-2 were detected using western blotting in CVB-D (0, 30, 60 and 120 µmol/L) treated MGC-803 ( left ); and MKN28 cells ( right ). β-actin was used as the control protein.

Techniques Used: Expressing, Western Blot

70) Product Images from "The HDAC Inhibitor, SAHA, Combined with Cisplatin Synergistically Induces Apoptosis in Alpha-fetoprotein-producing Hepatoid Adenocarcinoma Cells"

Article Title: The HDAC Inhibitor, SAHA, Combined with Cisplatin Synergistically Induces Apoptosis in Alpha-fetoprotein-producing Hepatoid Adenocarcinoma Cells

Journal: Acta Histochemica et Cytochemica

doi: 10.1267/ahc.18044

Effect of cisplatin and SAHA on induction of apoptotic cell death in VAT-39 cells. ( A ) Immunohistochemical localization of cleaved caspase-3 in 5 μM cisplatin and 2 μM SAHA-treated VAT-39 cells. ( B ) Counts of cleaved caspase-3-positive cells are shown in the bar graph. ( C ) Western blot analysis detected double bands of cleaved caspase-3 (17 kDa and 19 kDa). Isolated proteins (20 μg) were subjected to SDS-PAGE. β-actin (42 kDa) was used as a loading control. ( D ) Cell death was examined by TUNEL assay using the Mebstain apoptosis TUNEL kit. Arrows indicate TUNEL-positive cells among cisplatin and SAHA-treated cells. *** P
Figure Legend Snippet: Effect of cisplatin and SAHA on induction of apoptotic cell death in VAT-39 cells. ( A ) Immunohistochemical localization of cleaved caspase-3 in 5 μM cisplatin and 2 μM SAHA-treated VAT-39 cells. ( B ) Counts of cleaved caspase-3-positive cells are shown in the bar graph. ( C ) Western blot analysis detected double bands of cleaved caspase-3 (17 kDa and 19 kDa). Isolated proteins (20 μg) were subjected to SDS-PAGE. β-actin (42 kDa) was used as a loading control. ( D ) Cell death was examined by TUNEL assay using the Mebstain apoptosis TUNEL kit. Arrows indicate TUNEL-positive cells among cisplatin and SAHA-treated cells. *** P

Techniques Used: Immunohistochemistry, Western Blot, Isolation, SDS Page, TUNEL Assay

71) Product Images from "Inducible HSP70 antagonizes cisplatin-induced cell apoptosis through inhibition of the MAPK signaling pathway in HGC-27 cells"

Article Title: Inducible HSP70 antagonizes cisplatin-induced cell apoptosis through inhibition of the MAPK signaling pathway in HGC-27 cells

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2018.3789

HSP70 downregulation enhances cisplatin-induced HGC-27 cell apoptosis. HGC-27 cells were transfected with HSP70 shRNA plasmid and control plasmid, and at 48 h post-transfection, cells were stimulated with 5 µ g/ml cisplatin for the indicated times. (A) The morphology of apoptotic cell nuclei was detected by DAPI staining (magnification, ×100). (B) Expression levels of apoptosis-related proteins PARP, pro-caspase-3 and cleaved caspase-3 were detected by western blotting. (C) Flow cytometry was used to determine apoptosis rates (representative plots and quantification is shown). ** P
Figure Legend Snippet: HSP70 downregulation enhances cisplatin-induced HGC-27 cell apoptosis. HGC-27 cells were transfected with HSP70 shRNA plasmid and control plasmid, and at 48 h post-transfection, cells were stimulated with 5 µ g/ml cisplatin for the indicated times. (A) The morphology of apoptotic cell nuclei was detected by DAPI staining (magnification, ×100). (B) Expression levels of apoptosis-related proteins PARP, pro-caspase-3 and cleaved caspase-3 were detected by western blotting. (C) Flow cytometry was used to determine apoptosis rates (representative plots and quantification is shown). ** P

Techniques Used: Transfection, shRNA, Plasmid Preparation, Staining, Expressing, Western Blot, Flow Cytometry, Cytometry

HSP70 overexpression antagonizes cisplatin-induced HGC-27 cell apoptosis. HGC-27 cells were transfected with GFP-HSP70 plasmid or control plasmid. After 24 h, cells were stimulated with 5 µ g/ml cisplatin for the indicated times. (A) Nuclear morphology of apoptotic cells was detected by DAPI staining (magnification, ×100). (B) Expression levels of apoptosis-related proteins PARP, pro-caspase-3 and cleaved caspase-3 were detected by western blotting. (C) Apoptosis rate was determined by flow cytometry (representative plots and quantification is shown). ** P
Figure Legend Snippet: HSP70 overexpression antagonizes cisplatin-induced HGC-27 cell apoptosis. HGC-27 cells were transfected with GFP-HSP70 plasmid or control plasmid. After 24 h, cells were stimulated with 5 µ g/ml cisplatin for the indicated times. (A) Nuclear morphology of apoptotic cells was detected by DAPI staining (magnification, ×100). (B) Expression levels of apoptosis-related proteins PARP, pro-caspase-3 and cleaved caspase-3 were detected by western blotting. (C) Apoptosis rate was determined by flow cytometry (representative plots and quantification is shown). ** P

Techniques Used: Over Expression, Transfection, Plasmid Preparation, Staining, Expressing, Western Blot, Flow Cytometry, Cytometry

MAPK pathway inhibition enhances cisplatin-induced HGC-27 cell apoptosis. (A) HGC-27 cells were pretreated with specific inhibitors for p38, ERK or JNK for 2 h and then treated with cisplatin for 24 h. Expression levels of PARP, cleaved caspase-3 and pro-caspase-3 were detected by western blotting. (B) Apoptotic rate was determined by flow cytometry (representative plots and quantification is shown). (C) HGC-27 cells were pretreated with specific inhibitors for p38, ERK or JNK for 2 h and then treated with cisplatin for 6 h. Phosphorylation of p38, ERK, JNK and the levels of HSP70 were detected by western blotting. * P
Figure Legend Snippet: MAPK pathway inhibition enhances cisplatin-induced HGC-27 cell apoptosis. (A) HGC-27 cells were pretreated with specific inhibitors for p38, ERK or JNK for 2 h and then treated with cisplatin for 24 h. Expression levels of PARP, cleaved caspase-3 and pro-caspase-3 were detected by western blotting. (B) Apoptotic rate was determined by flow cytometry (representative plots and quantification is shown). (C) HGC-27 cells were pretreated with specific inhibitors for p38, ERK or JNK for 2 h and then treated with cisplatin for 6 h. Phosphorylation of p38, ERK, JNK and the levels of HSP70 were detected by western blotting. * P

Techniques Used: Inhibition, Expressing, Western Blot, Flow Cytometry, Cytometry

72) Product Images from "Desmoglein 2 mutant mice develop cardiac fibrosis and dilation"

Article Title: Desmoglein 2 mutant mice develop cardiac fibrosis and dilation

Journal: Basic Research in Cardiology

doi: 10.1007/s00395-011-0175-y

Two-week-old DSG2 mt/mt mice present variable cardiac phenotypes. In situ images of hearts ( a – c ), Kossa stains of histological sections ( d – f ) and cleaved caspase 3-immunohistology ( g – i ) are shown for heterozygous DSG2 mt/wt mice ( a , d , g ) and for homozygous DSG2 mt/mt mice either without visible fibrotic foci ( b , e , f ) or with extensive fibrotic lesions (+fib.; c , f , i ). Note the positive Kossa reaction in the large fibrotic area in ( f ) and the increase of cleaved caspase-3 positive cells ( arrowheads ) within a fibrotic lesion (le; i ) of a DSG2 mt/mt mouse with fibrosis. Scale bars : 5 mm in a (same magnification in b , c ), 200 μm in ( d ) (same magnification in e , f ), 100 μm in ( g ) (same magnification in h , i )
Figure Legend Snippet: Two-week-old DSG2 mt/mt mice present variable cardiac phenotypes. In situ images of hearts ( a – c ), Kossa stains of histological sections ( d – f ) and cleaved caspase 3-immunohistology ( g – i ) are shown for heterozygous DSG2 mt/wt mice ( a , d , g ) and for homozygous DSG2 mt/mt mice either without visible fibrotic foci ( b , e , f ) or with extensive fibrotic lesions (+fib.; c , f , i ). Note the positive Kossa reaction in the large fibrotic area in ( f ) and the increase of cleaved caspase-3 positive cells ( arrowheads ) within a fibrotic lesion (le; i ) of a DSG2 mt/mt mouse with fibrosis. Scale bars : 5 mm in a (same magnification in b , c ), 200 μm in ( d ) (same magnification in e , f ), 100 μm in ( g ) (same magnification in h , i )

Techniques Used: Mouse Assay, In Situ

73) Product Images from "Idelalisib promotes Bim-dependent apoptosis through AKT/FoxO3a in hepatocellular carcinoma"

Article Title: Idelalisib promotes Bim-dependent apoptosis through AKT/FoxO3a in hepatocellular carcinoma

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-0960-8

Idelalisib synergizes with sorafenib or doxorubicin to induce apoptosis via Bim in HCC. a  HepG2 cells were treated with 2.5 μmol/L idelalisib, 5 μmol/L sorafenib, or their combination for 24 h. Bim and Cleaved-caspase 3 expression were analyzed by western blotting and normalized to β-actin. The data represent the mean ± SD of three independent experiments. ** P
Figure Legend Snippet: Idelalisib synergizes with sorafenib or doxorubicin to induce apoptosis via Bim in HCC. a HepG2 cells were treated with 2.5 μmol/L idelalisib, 5 μmol/L sorafenib, or their combination for 24 h. Bim and Cleaved-caspase 3 expression were analyzed by western blotting and normalized to β-actin. The data represent the mean ± SD of three independent experiments. ** P

Techniques Used: Expressing, Western Blot

74) Product Images from "Resveratrol prevents embryonic oxidative stress and apoptosis associated with diabetic embryopathy, and improves glucose and lipid profile of diabetic dam"

Article Title: Resveratrol prevents embryonic oxidative stress and apoptosis associated with diabetic embryopathy, and improves glucose and lipid profile of diabetic dam

Journal: Molecular nutrition & food research

doi: 10.1002/mnfr.201000457

Western blot analysis to assess the activation of caspases. Total embryonic protein was prepared using 1× RIPA lysis buffer and immunoblotted with caspase-9, caspase-8, caspase-7 and cleaved caspase-3 antibodies and finally reprobed and blotted with β-actin antibody to determine the loading differences (A). Diabetes increases the active forms of caspase-9 and caspase-8 as well as increases the level of cleaved caspase-3 for execution of apoptotic processes. RSV treatment significantly suppresses the activation of caspases in embryos of diabetic dams. Caspase-7 expression remained unchanged. Cleaved caspase-3 expression in cranial neural tube region was further confirmed by immunohistochemistry (B). It was detected with Rhodamine labelled secondary antibody (immunofluorescent photomicrographs at 10× and 40× respectively). Further dual immunostaining was performed to compare the activated caspase-3 with TUNEL positive cells and counter stained with DAPI to visualize the nuclear DNA (C). Both were co-localized in most of the cells of neural tube and neural crest. RSV significantly reduced the level of cleaved caspase-3. RSV alone had no effect as evident from control treated with RSV. Red stained cells represent activated caspase-3, green color represents TUNEL positive cells, and yellow color represents cells positive for both cleaved caspase-3 and TUNEL.
Figure Legend Snippet: Western blot analysis to assess the activation of caspases. Total embryonic protein was prepared using 1× RIPA lysis buffer and immunoblotted with caspase-9, caspase-8, caspase-7 and cleaved caspase-3 antibodies and finally reprobed and blotted with β-actin antibody to determine the loading differences (A). Diabetes increases the active forms of caspase-9 and caspase-8 as well as increases the level of cleaved caspase-3 for execution of apoptotic processes. RSV treatment significantly suppresses the activation of caspases in embryos of diabetic dams. Caspase-7 expression remained unchanged. Cleaved caspase-3 expression in cranial neural tube region was further confirmed by immunohistochemistry (B). It was detected with Rhodamine labelled secondary antibody (immunofluorescent photomicrographs at 10× and 40× respectively). Further dual immunostaining was performed to compare the activated caspase-3 with TUNEL positive cells and counter stained with DAPI to visualize the nuclear DNA (C). Both were co-localized in most of the cells of neural tube and neural crest. RSV significantly reduced the level of cleaved caspase-3. RSV alone had no effect as evident from control treated with RSV. Red stained cells represent activated caspase-3, green color represents TUNEL positive cells, and yellow color represents cells positive for both cleaved caspase-3 and TUNEL.

Techniques Used: Western Blot, Activation Assay, Lysis, Expressing, Immunohistochemistry, Immunostaining, TUNEL Assay, Staining

Diabetes-induced oxidative stress analysis and anti-oxidative effect of resveratrol. Total embryonic proteins were extracted and assayed for lipid peroxidation (LPO), total thiol and reduced glutathione (GSH). To assess LPO, thiobarbituric acid reactive substance formation in tissue homogenates was evaluated by the amount of malondialdehyde (MDA) formed (A). Total thiol and reduced GSH levels in tissue homogenates were assessed with DTNB reagent (B, C). Superoxide dismutase expression was analysed by Western blot but no change in the expression was observed (D). RSV significantly suppressed LPO and total thiol level in the embryo of diabetic dams. GSH level was decreased in the embryos of diabetic dam but normalized after RSV treatment. All the parameters in the control treated with RSV group were unchanged. Error bars represent ± 2 std. errors for each mean. Further, consequences of oxidative stress were analysed by immunohistochemistry for 4-Hydroxy-2-Nonenal (HNE) adduct formation (E) and HNE co-localization pattern was also assessed in reference to cleaved caspase 3 by dual immunostaining and counter stained with DAPI (F). RSV significantly suppressed HNE adduct formation in embryos of diabetic dams. Red color represents cells with HNE adduct formation, green color represents activated caspase-3, and yellow color represents cells for both HNE adduct and cleaved caspase-3.
Figure Legend Snippet: Diabetes-induced oxidative stress analysis and anti-oxidative effect of resveratrol. Total embryonic proteins were extracted and assayed for lipid peroxidation (LPO), total thiol and reduced glutathione (GSH). To assess LPO, thiobarbituric acid reactive substance formation in tissue homogenates was evaluated by the amount of malondialdehyde (MDA) formed (A). Total thiol and reduced GSH levels in tissue homogenates were assessed with DTNB reagent (B, C). Superoxide dismutase expression was analysed by Western blot but no change in the expression was observed (D). RSV significantly suppressed LPO and total thiol level in the embryo of diabetic dams. GSH level was decreased in the embryos of diabetic dam but normalized after RSV treatment. All the parameters in the control treated with RSV group were unchanged. Error bars represent ± 2 std. errors for each mean. Further, consequences of oxidative stress were analysed by immunohistochemistry for 4-Hydroxy-2-Nonenal (HNE) adduct formation (E) and HNE co-localization pattern was also assessed in reference to cleaved caspase 3 by dual immunostaining and counter stained with DAPI (F). RSV significantly suppressed HNE adduct formation in embryos of diabetic dams. Red color represents cells with HNE adduct formation, green color represents activated caspase-3, and yellow color represents cells for both HNE adduct and cleaved caspase-3.

Techniques Used: Multiple Displacement Amplification, Expressing, Western Blot, Immunohistochemistry, Immunostaining, Staining

75) Product Images from "Adipocytes fuel gastric cancer omental metastasis via PITPNC1-mediated fatty acid metabolic reprogramming"

Article Title: Adipocytes fuel gastric cancer omental metastasis via PITPNC1-mediated fatty acid metabolic reprogramming

Journal: Theranostics

doi: 10.7150/thno.28219

PITPNC1-mediated anoikis resistance is dependent on FAO metabolism. (A-E) BGC823 or AGS stably transfected with sh-PITPNC1 or ox-PITPNC1 were seeded and treated with 50 μM etomoxir for 48 h, and the metabolic state of the cells were detected as follows. (A) The basal respiration, maximal respiration, ATP production, and spare respiration capacity was measured after co-culture using Seahorse XF96 extracellular flux analyzer. (B) Fatty acid oxidation rate was measured by Fatty Acid β-Oxidation Kit. (C) ATP level was measured by firefly luciferase ATP Assay Kit. (D) ROS content was detected by measuring the fluorescence intensity of DCF-DA through flow cytometry. (E) Cell adhesion was detected by crystal violet staining after co-culture with adipocytes for 48 h. (F) Vector or PITPNC1 overexpressing BGC823 and AGS was seeded and treated with 50 μM etomoxir, and anoikis was detected by Calcein AM/EthD-1 staining method. (G) PITPNC1-silencing or overexpressing BGC823 was cultured in collagen I 3D culture systems and then treated with 50 μM etomoxir for 48 h. The sphere formation was photographed, and cleaved caspase 3 was examined as an indicator of anoikis by immunofluorescence. (H) The relative change in BAX, BCL2, SREBP1, PPARγ, CD36, CPT1B and PITPNC1 expression was analyzed by Western blot after PITPNC1 overexpression or silencing. Vector or PITPNC1 overexpressing BGC823 and AGS were seeded and treated with etomoxir (I) or ranolazine (J) , and the relative change in BAX, BCL2, CPT1B and PITPNC1 expression was analyzed by Western blot. (K) The nuclear-cytoplasmic distribution of PPARγ after PITPNC1 overexpression or silencing through immunofluorescence. (L) PITPNC1 overexpressing BGC823 and AGS were co-transfected with PPARγ silencing sequence, and the relative change in PPARγ, CPT1B and PITPNC1 expression was analyzed by Western blot. Error bars, SD. * P
Figure Legend Snippet: PITPNC1-mediated anoikis resistance is dependent on FAO metabolism. (A-E) BGC823 or AGS stably transfected with sh-PITPNC1 or ox-PITPNC1 were seeded and treated with 50 μM etomoxir for 48 h, and the metabolic state of the cells were detected as follows. (A) The basal respiration, maximal respiration, ATP production, and spare respiration capacity was measured after co-culture using Seahorse XF96 extracellular flux analyzer. (B) Fatty acid oxidation rate was measured by Fatty Acid β-Oxidation Kit. (C) ATP level was measured by firefly luciferase ATP Assay Kit. (D) ROS content was detected by measuring the fluorescence intensity of DCF-DA through flow cytometry. (E) Cell adhesion was detected by crystal violet staining after co-culture with adipocytes for 48 h. (F) Vector or PITPNC1 overexpressing BGC823 and AGS was seeded and treated with 50 μM etomoxir, and anoikis was detected by Calcein AM/EthD-1 staining method. (G) PITPNC1-silencing or overexpressing BGC823 was cultured in collagen I 3D culture systems and then treated with 50 μM etomoxir for 48 h. The sphere formation was photographed, and cleaved caspase 3 was examined as an indicator of anoikis by immunofluorescence. (H) The relative change in BAX, BCL2, SREBP1, PPARγ, CD36, CPT1B and PITPNC1 expression was analyzed by Western blot after PITPNC1 overexpression or silencing. Vector or PITPNC1 overexpressing BGC823 and AGS were seeded and treated with etomoxir (I) or ranolazine (J) , and the relative change in BAX, BCL2, CPT1B and PITPNC1 expression was analyzed by Western blot. (K) The nuclear-cytoplasmic distribution of PPARγ after PITPNC1 overexpression or silencing through immunofluorescence. (L) PITPNC1 overexpressing BGC823 and AGS were co-transfected with PPARγ silencing sequence, and the relative change in PPARγ, CPT1B and PITPNC1 expression was analyzed by Western blot. Error bars, SD. * P

Techniques Used: Stable Transfection, Transfection, Co-Culture Assay, Luciferase, ATP Assay, Fluorescence, Flow Cytometry, Cytometry, Staining, Plasmid Preparation, Ethidium Homodimer Assay, Cell Culture, Immunofluorescence, Expressing, Western Blot, Over Expression, Sequencing

PITPNC1 promotes GC omental metastasis via fatty acid oxidation. (A-B) GFP-labeled vector or PITPNC1 overexpression GC cells were injected intraperitoneally in nude mice. After 3 days, mice were sacrificed and omentum was extracted and observed under in vivo imaging systems. The fluorescence indicated the intensity of omental metastasis (A). Representative image of omental tumor deposit after HE staining of the omentum tissues (B). (C-D) Vector or PITPNC1-overexpressing BGC823 cells with luciferase were injected intraperitoneally, and in vivo imaging (C) was performed at 1, 4 and 7 weeks after the injection. The results showed PITPNC1 overexpression group exhibited substantially larger omental metastasis compared to vector group, while shPITPNC1 group exhibited opposite results. At 8 weeks after injection, nude mice were sacrificed, and the abdomen was opened to macroscopic observation (D). (E) The omental tumor nodules counted from the in vivo imaging at indicated 1, 4 and 7 weeks after injection showed that silencing PITPNC1 or etomoxir treatment significantly reduced the tumor nodules, while PITPNC1 overexpression reversed the inhibition effect. (F) The weight of nude mice was monitored daily after intraperitoneal injection. Overexpression of PITPNC1 resulted in greater weight loss compared to the vector group or the PITPNC1-silencing group, and additional treatment with etomoxir did not influence the weight. (G) The omental tissues were embedded with paraffin to perform IHC staining. The results showed that silencing PITPNC1 or treatment with etomoxir exhibited decreased expression of Ki-67 but elevated apoptosis marker cleaved caspase-3 expression, while overexpression of PITPNC1 substantially reversed this effect. (H) Representative illustration of the metabolic crosstalk between omental adipocytes and gastric cancer cells in the process of omental metastasis. Adipocyte-secreted IL6 and TNF-α promote the expression of PITPNC1 in GC cancer cells through AKT signaling. PITPNC1 promotes the PPARγ and SREBP1 nuclear translocation, leading to elevated expression of CPT1B and CD36 expression, thereby promoting fatty acid absorption and oxidation, inducing anoikis resistance and omental metastasis.
Figure Legend Snippet: PITPNC1 promotes GC omental metastasis via fatty acid oxidation. (A-B) GFP-labeled vector or PITPNC1 overexpression GC cells were injected intraperitoneally in nude mice. After 3 days, mice were sacrificed and omentum was extracted and observed under in vivo imaging systems. The fluorescence indicated the intensity of omental metastasis (A). Representative image of omental tumor deposit after HE staining of the omentum tissues (B). (C-D) Vector or PITPNC1-overexpressing BGC823 cells with luciferase were injected intraperitoneally, and in vivo imaging (C) was performed at 1, 4 and 7 weeks after the injection. The results showed PITPNC1 overexpression group exhibited substantially larger omental metastasis compared to vector group, while shPITPNC1 group exhibited opposite results. At 8 weeks after injection, nude mice were sacrificed, and the abdomen was opened to macroscopic observation (D). (E) The omental tumor nodules counted from the in vivo imaging at indicated 1, 4 and 7 weeks after injection showed that silencing PITPNC1 or etomoxir treatment significantly reduced the tumor nodules, while PITPNC1 overexpression reversed the inhibition effect. (F) The weight of nude mice was monitored daily after intraperitoneal injection. Overexpression of PITPNC1 resulted in greater weight loss compared to the vector group or the PITPNC1-silencing group, and additional treatment with etomoxir did not influence the weight. (G) The omental tissues were embedded with paraffin to perform IHC staining. The results showed that silencing PITPNC1 or treatment with etomoxir exhibited decreased expression of Ki-67 but elevated apoptosis marker cleaved caspase-3 expression, while overexpression of PITPNC1 substantially reversed this effect. (H) Representative illustration of the metabolic crosstalk between omental adipocytes and gastric cancer cells in the process of omental metastasis. Adipocyte-secreted IL6 and TNF-α promote the expression of PITPNC1 in GC cancer cells through AKT signaling. PITPNC1 promotes the PPARγ and SREBP1 nuclear translocation, leading to elevated expression of CPT1B and CD36 expression, thereby promoting fatty acid absorption and oxidation, inducing anoikis resistance and omental metastasis.

Techniques Used: Labeling, Plasmid Preparation, Over Expression, Injection, Mouse Assay, In Vivo Imaging, Fluorescence, Staining, Luciferase, Inhibition, Immunohistochemistry, Expressing, Marker, Translocation Assay

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Ex Vivo:

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Incubation:

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Cell Culture:

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Expressing:

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BIA-KA:

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Modification:

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Western Blot:

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Immunohistochemistry:

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Protease Inhibitor:

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Infection:

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Imaging:

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Protein Concentration:

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Binding Assay:

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Immunofluorescence:

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Multiple Displacement Amplification:

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Isolation:

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Microscopy:

Article Title: Nucleolar Targeting by Platinum: p53-Independent Apoptosis Follows rRNA Inhibition, Cell-Cycle Arrest, and DNA Compaction
Article Snippet: Then 1:50 dilution NPM/B23 (Santa Cruz, #sc-5564), 1:100 dilution alpha-Tubulin (Cell Signaling, #3873), or 1:100 dilution of cleaved caspase-3 (Cell signaling, # 9662) was added overnight at 4 °C. .. All slides were mounted in VectaShield with DAPI (Vector Laboratories) and viewed using a Zeiss LSM 510 confocal microscope.

Article Title: Development of Aortic Valve Disease in Familial Hypercholesterolemic Swine: Implications for Elucidating Disease Etiology
Article Snippet: Detection of cleaved caspase 3 (2 μg/mL, polyclonal, rabbit; Cell Signaling Technology, Inc., Danvers, MA), CD107a (5 μg/mL, polyclonal, mouse; AbdSerotec, Raleigh, NC), monocyte chemoattractant protein 1 (MCP‐1; 5 μg/mL, polyclonal, rabbit; PeproTech, Rocky Hill, NJ), oxidatively modified apolipoprotein‐B100 (oxApoB; 10 μg/mL, polyclonal, mouse), and malondialdehyde (MDA; 10 μg/mL, polyclonal, mouse) was performed using immunohistochemical methods following the VECTASTAIN Universal Elite ABC Kit protocol (Vector Laboratories). .. Brightfield and fluorescent images were captured using an Olympus IX51 microscope (Olympus, Tokyo, Japan).

Polyacrylamide Gel Electrophoresis:

Article Title: Infiltrating macrophages in diabetic nephropathy promote podocytes apoptosis via TNF-α-ROS-p38MAPK pathway
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Lysis:

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Article Snippet: Heart homogenates and cultured rat neonatal cardiomyocytes were lysed with ice-cold radioimmuneprecipitation assay buffer (New England Biolabs) and cell lysis buffer (Cell Signaling), respectively, with protease inhibitor mix (Amersham Biosciences). .. Membranes were blocked with 5% nonfat dry milk in PBS containing 0.1% Tween 20 for 1 h at room temperature and then probed overnight at 4 °C for LC3 (1:1000; Cell Signaling), p27 (1:1500; Cell Signaling), p62 (1:1000; Cell Signaling), ATG5 (1:1000; Cell Signaling), cleaved caspase 3 (1:1500; Cell Signaling), β-Gal (1:5000; Millipore).

Article Title: Farnesol, a Fungal Quorum-Sensing Molecule Triggers Apoptosis in Human Oral Squamous Carcinoma Cells
Article Snippet: Following the same treatments as described above, cells were incubated and washed twice with ice-cold PBS, followed by lysis using radioimmunoprecipitation assay buffer [50 µM Tris (pH 7.4), 150 µM NaCl, 1% Triton X-100, 1% deoxycholic acid, 150 µM sodium salt, 0.1% SDS, 100 µg/ml phenylmethysulfonyl flouride, 1 µg/ml aprotinin, 1 mM dithiothreitol (DTT), 1 mM sodium orthovanadate] for 10 minutes at 4°C. .. Western blot analysis was performed using a 1:1000 dilution of monoclonal primary antibodies for survivin (Abcam, Cambridge, UK), cleaved-caspase 3 (Cell Signaling, Beverly, MA), and cleaved-caspase 9 (Cell Signaling), with β-actin (Sigma) used as a loading control.

SDS Page:

Article Title: p27 Protein Protects Metabolically Stressed Cardiomyocytes from Apoptosis by Promoting Autophagy *
Article Snippet: Protein extracts were subjected to 4–20% gradient SDS-PAGE (Bio-Rad) and then transferred to PVDF membrane. .. Membranes were blocked with 5% nonfat dry milk in PBS containing 0.1% Tween 20 for 1 h at room temperature and then probed overnight at 4 °C for LC3 (1:1000; Cell Signaling), p27 (1:1500; Cell Signaling), p62 (1:1000; Cell Signaling), ATG5 (1:1000; Cell Signaling), cleaved caspase 3 (1:1500; Cell Signaling), β-Gal (1:5000; Millipore).

Article Title: Reperfusion Differentially Induces Caspase-3 Activation in Ischemic Core and Penumbra After Stroke in Immature Brain
Article Snippet: Samples were boiled for 5 minutes, subjected to SDS-PAGE (40 µg of protein per lane), and transferred to nitrocellulose (Amersham). .. Blots were rinsed with 1×Tris-buffered saline (TBS) and 0.1% Tween (TTBS), blocked with 5% milk/TTBS for 1 hour, and probed with primary antibody against cleaved caspase-3 (1:1000, overnight, 4°C; Cell Signaling, Inc).

Article Title: Infiltrating macrophages in diabetic nephropathy promote podocytes apoptosis via TNF-α-ROS-p38MAPK pathway
Article Snippet: Western blot The total proteins extracted from the renal cortex and cells were separated by sodium dodecyl sulfate -polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. .. The membranes were incubated overnight with primary antibodies against iNOS (Santa, USA), MR (Abcam, UK), cleaved caspase-3 (Cell Signaling Technology, USA), p-p38MAPK, p38MAPK (Cell Signaling Technology, USA) at 4°C followed by incubation with horse reddish peroxidase (HRP) conjugated secondary antibodies for 1h.

Plasmid Preparation:

Article Title: Nucleolar Targeting by Platinum: p53-Independent Apoptosis Follows rRNA Inhibition, Cell-Cycle Arrest, and DNA Compaction
Article Snippet: Then 1:50 dilution NPM/B23 (Santa Cruz, #sc-5564), 1:100 dilution alpha-Tubulin (Cell Signaling, #3873), or 1:100 dilution of cleaved caspase-3 (Cell signaling, # 9662) was added overnight at 4 °C. .. All slides were mounted in VectaShield with DAPI (Vector Laboratories) and viewed using a Zeiss LSM 510 confocal microscope.

Software:

Article Title: Development of Aortic Valve Disease in Familial Hypercholesterolemic Swine: Implications for Elucidating Disease Etiology
Article Snippet: After histological staining, leaflet thickness was measured using ImageJ software (NIH, Bethesda, MD). .. Detection of cleaved caspase 3 (2 μg/mL, polyclonal, rabbit; Cell Signaling Technology, Inc., Danvers, MA), CD107a (5 μg/mL, polyclonal, mouse; AbdSerotec, Raleigh, NC), monocyte chemoattractant protein 1 (MCP‐1; 5 μg/mL, polyclonal, rabbit; PeproTech, Rocky Hill, NJ), oxidatively modified apolipoprotein‐B100 (oxApoB; 10 μg/mL, polyclonal, mouse), and malondialdehyde (MDA; 10 μg/mL, polyclonal, mouse) was performed using immunohistochemical methods following the VECTASTAIN Universal Elite ABC Kit protocol (Vector Laboratories).

Article Title: Differential induction of apoptosis in human breast cancer cell lines by phenethyl isothiocyanate, a glutathione depleting agent
Article Snippet: Immunoblots were performed as previously described (Brimmell et al. ) using equal amounts of protein lysates (quantified using the BioRad assay) and antibodies specific for caspase 9, caspase 7, cleaved caspase 9, cleaved caspase 3, (all Cell Signalling Technology, Beverly, MA, USA), BAX, BCL2, NRF2 (all Santa Cruz Biotechnology, Santa Cruz, CA, USA) and a rabbit anti-β-actin antibody (Sigma Chemicals). .. Immunoblot signals were quantified using Quantity One image analysis software (BioRad, Hemel Hempstead, UK).

Radio Immunoprecipitation:

Article Title: Farnesol, a Fungal Quorum-Sensing Molecule Triggers Apoptosis in Human Oral Squamous Carcinoma Cells
Article Snippet: Following the same treatments as described above, cells were incubated and washed twice with ice-cold PBS, followed by lysis using radioimmunoprecipitation assay buffer [50 µM Tris (pH 7.4), 150 µM NaCl, 1% Triton X-100, 1% deoxycholic acid, 150 µM sodium salt, 0.1% SDS, 100 µg/ml phenylmethysulfonyl flouride, 1 µg/ml aprotinin, 1 mM dithiothreitol (DTT), 1 mM sodium orthovanadate] for 10 minutes at 4°C. .. Western blot analysis was performed using a 1:1000 dilution of monoclonal primary antibodies for survivin (Abcam, Cambridge, UK), cleaved-caspase 3 (Cell Signaling, Beverly, MA), and cleaved-caspase 9 (Cell Signaling), with β-actin (Sigma) used as a loading control.

Marker:

Article Title: Tropism of and Innate Immune Responses to the Novel Human Betacoronavirus Lineage C Virus in Human Ex Vivo Respiratory Organ Cultures
Article Snippet: .. By immunohistochemistry, we found extensive expression of cleaved caspase 3, an apoptosis marker, in ex vivo lung tissue infected with HCoV-EMC ( ) and SARS-CoV ( ) but not in mock-infected ( ) human lung tissue. .. In order to investigate if the apoptosis was induced directly by coronavirus infection in the human lung, we performed costaining by immunohistochemistry of HCoV-EMC antigen (stained in pink) with cleaved caspase 3 (stained in reddish brown) using HCoV-EMC-infected ( ) and SARS-CoV-infected ( ) human lung tissues in ex vivo culture.

Staining:

Article Title: A Novel Chimeric Oncolytic Virus Vector for Improved Safety and Efficacy as a Platform for the Treatment of Hepatocellular Carcinoma
Article Snippet: .. Slices (3 µm in thickness) were stained with hematoxylin-eosin or subjected to immunohistochemical staining using a rabbit monoclonal antibody against cleaved caspase-3 (Cell Signaling Technology, Danvers, MA) on a Bond RX automated staining instrument (Leica, Wetzlar, Germany). .. Analysis of pathological changes and confirmation of positive immunohistochemical reaction were performed by a certified pathologist who was blind to the treatment groups of the specimens.

Article Title: Development of Aortic Valve Disease in Familial Hypercholesterolemic Swine: Implications for Elucidating Disease Etiology
Article Snippet: After histological staining, leaflet thickness was measured using ImageJ software (NIH, Bethesda, MD). .. Detection of cleaved caspase 3 (2 μg/mL, polyclonal, rabbit; Cell Signaling Technology, Inc., Danvers, MA), CD107a (5 μg/mL, polyclonal, mouse; AbdSerotec, Raleigh, NC), monocyte chemoattractant protein 1 (MCP‐1; 5 μg/mL, polyclonal, rabbit; PeproTech, Rocky Hill, NJ), oxidatively modified apolipoprotein‐B100 (oxApoB; 10 μg/mL, polyclonal, mouse), and malondialdehyde (MDA; 10 μg/mL, polyclonal, mouse) was performed using immunohistochemical methods following the VECTASTAIN Universal Elite ABC Kit protocol (Vector Laboratories).

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    Cell Signaling Technology Inc cleaved caspase 3
    DEX improved the apoptosis of rat renal tubular cells induced by acute stress. (a) The apoptosis of renal tissue was measured by the TUNEL Apoptosis kit, in which green cells represent TUNEL-positive apoptotic cells. TUNEL (green) detects apoptosis, DAPI (blue) locates the nucleus, and MERGE is the combination of TUNEL and DAPI. All fluorescence images were observed with a fluorescence microscope at a magnification of 200. Quantitative results of TUNEL analysis. Immunohistochemistry of cleaved <t>caspase</t> 3 protein: (b) C group, (c) AS group, and (d) D + AS group; bars = 50 μ m. Data are expressed as mean ± SEM ( n = 6). ∗∗ p
    Cleaved Caspase 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 298 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    DEX improved the apoptosis of rat renal tubular cells induced by acute stress. (a) The apoptosis of renal tissue was measured by the TUNEL Apoptosis kit, in which green cells represent TUNEL-positive apoptotic cells. TUNEL (green) detects apoptosis, DAPI (blue) locates the nucleus, and MERGE is the combination of TUNEL and DAPI. All fluorescence images were observed with a fluorescence microscope at a magnification of 200. Quantitative results of TUNEL analysis. Immunohistochemistry of cleaved caspase 3 protein: (b) C group, (c) AS group, and (d) D + AS group; bars = 50 μ m. Data are expressed as mean ± SEM ( n = 6). ∗∗ p

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Dexmedetomidine Ameliorates Acute Stress-Induced Kidney Injury by Attenuating Oxidative Stress and Apoptosis through Inhibition of the ROS/JNK Signaling Pathway

    doi: 10.1155/2018/4035310

    Figure Lengend Snippet: DEX improved the apoptosis of rat renal tubular cells induced by acute stress. (a) The apoptosis of renal tissue was measured by the TUNEL Apoptosis kit, in which green cells represent TUNEL-positive apoptotic cells. TUNEL (green) detects apoptosis, DAPI (blue) locates the nucleus, and MERGE is the combination of TUNEL and DAPI. All fluorescence images were observed with a fluorescence microscope at a magnification of 200. Quantitative results of TUNEL analysis. Immunohistochemistry of cleaved caspase 3 protein: (b) C group, (c) AS group, and (d) D + AS group; bars = 50 μ m. Data are expressed as mean ± SEM ( n = 6). ∗∗ p

    Article Snippet: Therefore, the site of kidney injury was determined by measuring localization of cleaved caspase 3 in kidney tissue.

    Techniques: TUNEL Assay, Fluorescence, Microscopy, Immunohistochemistry

    ( A, B ) Western blot analysis was performed to detect the effect of melatonin on factors related to cell apoptosis. Melatonin administration upregulated the level of Bcl-2 but reduced the levels of Bax and cleavage caspase-3 following the onset of SAH (P

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Melatonin Upregulates Nuclear Factor Erythroid-2 Related Factor 2 (Nrf2) and Mediates Mitophagy to Protect Against Early Brain Injury After Subarachnoid Hemorrhage

    doi: 10.12659/MSM.909221

    Figure Lengend Snippet: ( A, B ) Western blot analysis was performed to detect the effect of melatonin on factors related to cell apoptosis. Melatonin administration upregulated the level of Bcl-2 but reduced the levels of Bax and cleavage caspase-3 following the onset of SAH (P

    Article Snippet: In addition, high protein levels of Bax and cleaved caspase-3 were observed in the SAH group, while the melatonin administration decreased the protein levels of Bax and cleaved caspase-3, indicating that melatonin prevented the apoptosis of neurons following the onset of SAH.

    Techniques: Western Blot

    Correlation of the serum levels of 24S-HC with the expression of cleaved caspase-3 at 6hr and 24hr after neonatal HI. A. Protein expression of cleaved caspase-3 in the cortices was measured by western blotting at the indicated time points and presented as the OD ratio to β-actin and normalized to an internal control (IC). Sham vs. HI, *p=0.0112 at 6hr; *p=0.0027 at 24hr; *p=0.0228 at 72hr (n=5–6 for sham animals, n=7–10 for HI animals from 6–72hr). B. Correlation of the serum levels of 24S-HC with the expression of cleaved caspase-3 at 6hr (top) and at 24hr (bottom). Sample number, R 2 and p values are shown in the graphs.

    Journal: Pediatric research

    Article Title: Up-regulation of cholesterol 24-hydroxylase following hypoxia-ischemia in neonatal mouse brain

    doi: 10.1038/pr.2018.49

    Figure Lengend Snippet: Correlation of the serum levels of 24S-HC with the expression of cleaved caspase-3 at 6hr and 24hr after neonatal HI. A. Protein expression of cleaved caspase-3 in the cortices was measured by western blotting at the indicated time points and presented as the OD ratio to β-actin and normalized to an internal control (IC). Sham vs. HI, *p=0.0112 at 6hr; *p=0.0027 at 24hr; *p=0.0228 at 72hr (n=5–6 for sham animals, n=7–10 for HI animals from 6–72hr). B. Correlation of the serum levels of 24S-HC with the expression of cleaved caspase-3 at 6hr (top) and at 24hr (bottom). Sample number, R 2 and p values are shown in the graphs.

    Article Snippet: It is interesting that the expression of CYP46A1 in the ipsilateral cortex was largely localized to the cells undergoing apoptotic cell death as visualized by the expression of cleaved caspase-3 ( ).

    Techniques: Expressing, Western Blot

    Up-regulation of CYP46A1 with a concomitant increase of 24S-HC in the ipsilateral cortex after neonatal HI. A. Protein expression of CYP46A1 was measured by western blotting at the indicated time points and presented as the OD ratio to β-actin and normalized to the values of sham 0hr (graph on the right, sham vs. HI: * p= 0.0339 at 6hr; * p=0.0026 at 24hr; n=5–6 for sham animals, n=6–12 for HI animals at 6–72hr). B. Immunofluorescent staining with anti-CYP46A1 antibody at 24hr after HI showed enhanced expression in the ipsilateral cortex than that in the contralateral side and in the sham animals (left panel s). CYP46A1 staining in the ipsi-cortex was largely co-localized with the expression of cleaved caspase-3 (middle panels). C. Increased production of 24S-HC (ng/mg tissue wet weight) in the ipsilateral cortex at 6hr and 24hr after HI (sham vs. HI: *p= 0.0167 at 6hr; *p=0.0085 at 24hr; n=3–6 for sham animals, n=5–7 for HI animals at 6–72hr). The time course of the changes in 24S-HC in the sham-, contra- and ipsilateral cortex is shown on the right. At 6hr, 24S-HC in the ipsilateral cortex was higher than that in the contralateral side (ipsi- vs. contra-: *p=0.0493). #: Significant difference between the HI ipsi- and the sham animals only, but not between the HI ipsi- and HI contralateral hemisphere.

    Journal: Pediatric research

    Article Title: Up-regulation of cholesterol 24-hydroxylase following hypoxia-ischemia in neonatal mouse brain

    doi: 10.1038/pr.2018.49

    Figure Lengend Snippet: Up-regulation of CYP46A1 with a concomitant increase of 24S-HC in the ipsilateral cortex after neonatal HI. A. Protein expression of CYP46A1 was measured by western blotting at the indicated time points and presented as the OD ratio to β-actin and normalized to the values of sham 0hr (graph on the right, sham vs. HI: * p= 0.0339 at 6hr; * p=0.0026 at 24hr; n=5–6 for sham animals, n=6–12 for HI animals at 6–72hr). B. Immunofluorescent staining with anti-CYP46A1 antibody at 24hr after HI showed enhanced expression in the ipsilateral cortex than that in the contralateral side and in the sham animals (left panel s). CYP46A1 staining in the ipsi-cortex was largely co-localized with the expression of cleaved caspase-3 (middle panels). C. Increased production of 24S-HC (ng/mg tissue wet weight) in the ipsilateral cortex at 6hr and 24hr after HI (sham vs. HI: *p= 0.0167 at 6hr; *p=0.0085 at 24hr; n=3–6 for sham animals, n=5–7 for HI animals at 6–72hr). The time course of the changes in 24S-HC in the sham-, contra- and ipsilateral cortex is shown on the right. At 6hr, 24S-HC in the ipsilateral cortex was higher than that in the contralateral side (ipsi- vs. contra-: *p=0.0493). #: Significant difference between the HI ipsi- and the sham animals only, but not between the HI ipsi- and HI contralateral hemisphere.

    Article Snippet: It is interesting that the expression of CYP46A1 in the ipsilateral cortex was largely localized to the cells undergoing apoptotic cell death as visualized by the expression of cleaved caspase-3 ( ).

    Techniques: Expressing, Western Blot, Staining