Structured Review

Cell Signaling Technology Inc anti cleaved caspase 3
The Myc 3′ WRE controls proliferation but not apoptosis in the colons of Apc Min/ + mice. (A) Ki67- and cleaved <t>caspase</t> 3 (CASP3)-stained sections of preneoplastic colons and colonic tumors from Apc Min/ + (top) and Apc Min/ + Myc 3′ WRE −/− (bottom) mice. Representative images are shown ( n = 4 mice analyzed per genotype). Arrowheads indicate CASP3 + cells. (B) Quantification of Ki67 + (left) and CASP3 + (right) cells in preneoplastic colons of Apc Min/ + and Apc Min/ + Myc 3′ WRE −/− mice ( n = 4 mice analyzed, with cells counted in a total of 100 crypts per genotype). (C) Same as panel B except that Ki67 + and CASP3 + cells were analyzed in tumor sections prepared from mice with the indicated genotypes ( n = 4 mice, with 40 fields of view examined per genotype). In panels B and C, errors are standard errors of the means (**, P
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1) Product Images from "The Myc 3′ Wnt-Responsive Element Suppresses Colonic Tumorigenesis"

Article Title: The Myc 3′ Wnt-Responsive Element Suppresses Colonic Tumorigenesis

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00969-13

The Myc 3′ WRE controls proliferation but not apoptosis in the colons of Apc Min/ + mice. (A) Ki67- and cleaved caspase 3 (CASP3)-stained sections of preneoplastic colons and colonic tumors from Apc Min/ + (top) and Apc Min/ + Myc 3′ WRE −/− (bottom) mice. Representative images are shown ( n = 4 mice analyzed per genotype). Arrowheads indicate CASP3 + cells. (B) Quantification of Ki67 + (left) and CASP3 + (right) cells in preneoplastic colons of Apc Min/ + and Apc Min/ + Myc 3′ WRE −/− mice ( n = 4 mice analyzed, with cells counted in a total of 100 crypts per genotype). (C) Same as panel B except that Ki67 + and CASP3 + cells were analyzed in tumor sections prepared from mice with the indicated genotypes ( n = 4 mice, with 40 fields of view examined per genotype). In panels B and C, errors are standard errors of the means (**, P
Figure Legend Snippet: The Myc 3′ WRE controls proliferation but not apoptosis in the colons of Apc Min/ + mice. (A) Ki67- and cleaved caspase 3 (CASP3)-stained sections of preneoplastic colons and colonic tumors from Apc Min/ + (top) and Apc Min/ + Myc 3′ WRE −/− (bottom) mice. Representative images are shown ( n = 4 mice analyzed per genotype). Arrowheads indicate CASP3 + cells. (B) Quantification of Ki67 + (left) and CASP3 + (right) cells in preneoplastic colons of Apc Min/ + and Apc Min/ + Myc 3′ WRE −/− mice ( n = 4 mice analyzed, with cells counted in a total of 100 crypts per genotype). (C) Same as panel B except that Ki67 + and CASP3 + cells were analyzed in tumor sections prepared from mice with the indicated genotypes ( n = 4 mice, with 40 fields of view examined per genotype). In panels B and C, errors are standard errors of the means (**, P

Techniques Used: Mouse Assay, Staining

2) Product Images from "Opa1 overexpression protects from early onset Mpv17-/--related mouse kidney disease"

Article Title: Opa1 overexpression protects from early onset Mpv17-/--related mouse kidney disease

Journal: bioRxiv

doi: 10.1101/2020.03.18.996561

Caspase 3 staining in kidney sections and isolated podocytes. A) A Immunohistochemical staining with anti-caspase 3 antibody. Scale bar: 50μm (upper row); 20μm (lower row). B) Confocal micrographs from primary podocytes derived from described genotypes showing cytoplasm staining (GAPDH in green), apoptotic cells (Cleaved CASPASE-3 in red) and nuclei (DAPI in blue). Maximum intensity projection of Z-stacks is shown. Scale bars: 50 μm. OnOn the right panel, qquantification of caspase 3-positive cells. Data are presented as mean ± SD. Symbols * and § represent the significance levels vs. WT and Mpv17 -/- , respectively, calculated by one‐way ANOVA with Tukey’s post hoc multiple comparison test − : **** p
Figure Legend Snippet: Caspase 3 staining in kidney sections and isolated podocytes. A) A Immunohistochemical staining with anti-caspase 3 antibody. Scale bar: 50μm (upper row); 20μm (lower row). B) Confocal micrographs from primary podocytes derived from described genotypes showing cytoplasm staining (GAPDH in green), apoptotic cells (Cleaved CASPASE-3 in red) and nuclei (DAPI in blue). Maximum intensity projection of Z-stacks is shown. Scale bars: 50 μm. OnOn the right panel, qquantification of caspase 3-positive cells. Data are presented as mean ± SD. Symbols * and § represent the significance levels vs. WT and Mpv17 -/- , respectively, calculated by one‐way ANOVA with Tukey’s post hoc multiple comparison test − : **** p

Techniques Used: Staining, Isolation, Immunohistochemistry, Derivative Assay

3) Product Images from "PSMA-homing dsRNA chimeric protein vector kills prostate cancer cells and activates anti-tumor bystander responses"

Article Title: PSMA-homing dsRNA chimeric protein vector kills prostate cancer cells and activates anti-tumor bystander responses

Journal: Oncotarget

doi: 10.18632/oncotarget.15733

dsRB-SCP/polyIC selectively induces apoptosis of PSMA-overexpressing cells A. Cells were seeded in triplicate, grown overnight, and treated with dsRB-SCP/polyIC, polyIC alone or dsRB-SCP alone, as indicated, for 100 h. Viability was quantified using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). Results (mean and standard deviation) are representative of two independent experiments (**** P ≤ 0.0001, treatment with dsRB-SCP/polyIC of LNCaP vs PC3; **** P ≤ 0.0001 treatment of LNCaP with dsRB-SCP/polyIC vs polyIC alone). B. Surviving cells remained permanently arrested. Cells were seeded in triplicate, grown overnight, and treated as indicated. Medium was replaced and viability was quantified after 100/172/344 h using CellTiter-Glo (**** P ≤ 0.0001 dsRB-SCP/polyIC treatment vs UT). Control cells were unable to proliferate beyond 2.5 doublings because they had reached full confluence. C. LNCaP cells were treated for the indicated times with dsRB-SCP/ polyIC or polyIC alone, lysed and subjected to western blot analysis to detect full-length and cleaved Caspase-3 and PARP.
Figure Legend Snippet: dsRB-SCP/polyIC selectively induces apoptosis of PSMA-overexpressing cells A. Cells were seeded in triplicate, grown overnight, and treated with dsRB-SCP/polyIC, polyIC alone or dsRB-SCP alone, as indicated, for 100 h. Viability was quantified using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). Results (mean and standard deviation) are representative of two independent experiments (**** P ≤ 0.0001, treatment with dsRB-SCP/polyIC of LNCaP vs PC3; **** P ≤ 0.0001 treatment of LNCaP with dsRB-SCP/polyIC vs polyIC alone). B. Surviving cells remained permanently arrested. Cells were seeded in triplicate, grown overnight, and treated as indicated. Medium was replaced and viability was quantified after 100/172/344 h using CellTiter-Glo (**** P ≤ 0.0001 dsRB-SCP/polyIC treatment vs UT). Control cells were unable to proliferate beyond 2.5 doublings because they had reached full confluence. C. LNCaP cells were treated for the indicated times with dsRB-SCP/ polyIC or polyIC alone, lysed and subjected to western blot analysis to detect full-length and cleaved Caspase-3 and PARP.

Techniques Used: Cell Viability Assay, Standard Deviation, Western Blot

4) Product Images from "Liraglutide, a glucagon-like peptide-1 analog, inhibits high glucose-induced oxidative stress and apoptosis in neonatal rat cardiomyocytes"

Article Title: Liraglutide, a glucagon-like peptide-1 analog, inhibits high glucose-induced oxidative stress and apoptosis in neonatal rat cardiomyocytes

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2019.7388

Cleaved caspase-3 and full length caspase-3 protein expression was determined via western blotting. Data presented as the mean ± standard deviation of three independent experiments. In the HG group, cleaved caspase-3/full length caspase-3 protein levels were significantly increased. Treatment with liraglutide significantly reduced the HG-induced increase in cleaved caspase-3/full length caspase-3 protein expression. **P
Figure Legend Snippet: Cleaved caspase-3 and full length caspase-3 protein expression was determined via western blotting. Data presented as the mean ± standard deviation of three independent experiments. In the HG group, cleaved caspase-3/full length caspase-3 protein levels were significantly increased. Treatment with liraglutide significantly reduced the HG-induced increase in cleaved caspase-3/full length caspase-3 protein expression. **P

Techniques Used: Expressing, Western Blot, Standard Deviation

5) Product Images from "Adrenomedullin has a cytoprotective role against endoplasmic reticulum stress for pancreatic β‐cells in autocrine and paracrine manners"

Article Title: Adrenomedullin has a cytoprotective role against endoplasmic reticulum stress for pancreatic β‐cells in autocrine and paracrine manners

Journal: Journal of Diabetes Investigation

doi: 10.1111/jdi.13218

Adrenomedullin (ADM) protects MIN6 cells from thapsigargin (TG)‐induced apoptosis. (a) MIN6 cells were cultured for 24 h with rat full‐length ADM peptide or human partial ADM peptide (hum 22–52) at 0.1 or 100 nmol/L in the presence or absence of 1 μmol/L TG. After the culturing, cleaved caspase 3 levels were determined. α‐Tubulin was used as the protein loading control. All data are presented as the mean ± standard error of the mean of four or five independent experiments. Welch’s t ‐test: * P
Figure Legend Snippet: Adrenomedullin (ADM) protects MIN6 cells from thapsigargin (TG)‐induced apoptosis. (a) MIN6 cells were cultured for 24 h with rat full‐length ADM peptide or human partial ADM peptide (hum 22–52) at 0.1 or 100 nmol/L in the presence or absence of 1 μmol/L TG. After the culturing, cleaved caspase 3 levels were determined. α‐Tubulin was used as the protein loading control. All data are presented as the mean ± standard error of the mean of four or five independent experiments. Welch’s t ‐test: * P

Techniques Used: Cell Culture

6) Product Images from "Combination BET Family Protein and HDAC Inhibition Synergistically Elicits Chondrosarcoma Cell Apoptosis Through RAD51-Related DNA Damage Repair"

Article Title: Combination BET Family Protein and HDAC Inhibition Synergistically Elicits Chondrosarcoma Cell Apoptosis Through RAD51-Related DNA Damage Repair

Journal: Cancer Management and Research

doi: 10.2147/CMAR.S254412

Combination treatment with JQ1 and HDACIs leads to apoptosis of chondrosarcoma cells. ( A ) SW 1353 cells were treated with the indicated inhibitors (JQ1: 20µM, SAHA: 1 µM) for 48 h, and then apoptotic cells were assessed by flow cytometry using the Annexin V-FITC/PI kit. ( B ) The percentage of apoptotic cells was calculated from three independent experiments. ( C – D ) After the indicated treatment, SW 1353 cells were incubated with 10 μM DCFH-DA in serum-free medium at 37 °C for 20 min. DCF fluorescence intensity (FI) was detected by flow cytometry, the relative DCF-FI was calculated and presented. ( E – I ) SW 1353 and Hs 819.T cells were treated with JQ1 (20 µM), SAHA (1 µM for SW 1353 and 2 µM for Hs 819.T)/PANO (10 nM for both cell lines) or their combinations for 48 h. The total cell lysate was prepared and the expression levels of Bcl-2, Bcl-XL, and caspase-3 were determined by immunoblotting analysis, and the protein expression was quantified using the Image J software (n = 3). * P
Figure Legend Snippet: Combination treatment with JQ1 and HDACIs leads to apoptosis of chondrosarcoma cells. ( A ) SW 1353 cells were treated with the indicated inhibitors (JQ1: 20µM, SAHA: 1 µM) for 48 h, and then apoptotic cells were assessed by flow cytometry using the Annexin V-FITC/PI kit. ( B ) The percentage of apoptotic cells was calculated from three independent experiments. ( C – D ) After the indicated treatment, SW 1353 cells were incubated with 10 μM DCFH-DA in serum-free medium at 37 °C for 20 min. DCF fluorescence intensity (FI) was detected by flow cytometry, the relative DCF-FI was calculated and presented. ( E – I ) SW 1353 and Hs 819.T cells were treated with JQ1 (20 µM), SAHA (1 µM for SW 1353 and 2 µM for Hs 819.T)/PANO (10 nM for both cell lines) or their combinations for 48 h. The total cell lysate was prepared and the expression levels of Bcl-2, Bcl-XL, and caspase-3 were determined by immunoblotting analysis, and the protein expression was quantified using the Image J software (n = 3). * P

Techniques Used: Flow Cytometry, Incubation, Fluorescence, Expressing, Software

7) Product Images from "MicroRNA-218 Enhances the Radiosensitivity of Human Cervical Cancer via Promoting Radiation Induced Apoptosis"

Article Title: MicroRNA-218 Enhances the Radiosensitivity of Human Cervical Cancer via Promoting Radiation Induced Apoptosis

Journal: International Journal of Medical Sciences

doi: 10.7150/ijms.8880

Upregulation of miR-218 increased radiation induced apoptosis. (A) in HeLa cells, combination of miR-218 mimics transfection and radition induced much more apoptosis than miR-218 mimics transfection alone or radiation alone; (B) both miR-218 mimics transfection and radition increased the expression of cleaved caspase 3 and cleaved PARP, the combination of the two approaches further upregulated the level of cleaved caspase 3 and cleaved PARP; (C) the combining treatment also worked well in the other three human cervical cancer cell lines (NS: not significant; *: P
Figure Legend Snippet: Upregulation of miR-218 increased radiation induced apoptosis. (A) in HeLa cells, combination of miR-218 mimics transfection and radition induced much more apoptosis than miR-218 mimics transfection alone or radiation alone; (B) both miR-218 mimics transfection and radition increased the expression of cleaved caspase 3 and cleaved PARP, the combination of the two approaches further upregulated the level of cleaved caspase 3 and cleaved PARP; (C) the combining treatment also worked well in the other three human cervical cancer cell lines (NS: not significant; *: P

Techniques Used: Transfection, Expressing

8) Product Images from "Downregulation of Bcl-2 Expression by miR-34a Mediates Palmitate-Induced Min6 Cells Apoptosis"

Article Title: Downregulation of Bcl-2 Expression by miR-34a Mediates Palmitate-Induced Min6 Cells Apoptosis

Journal: Journal of Diabetes Research

doi: 10.1155/2014/258695

MiR-34a involves in palmitate-induced Min6 cells apoptosis. (a) miR-34a expression level was elevated in response to palmitate treatment. (b) ectopic expression of miR-34a enhanced Min6 cell apoptosis. Cells transduced or not with miR-34a were treated with palmitate (500 μ M) or not for 2 days. The apoptotic rate of cells was determined by scoring the cells displaying pycnotic nucleus and/or fragmented nucleus. Cleaved caspase-3 expression (c) and caspase-3 activity (d) were carried out to confirm the proapoptotic role of miR-34a in Min6 cells. For (a), (b), (c) and (d), data are shown as means ± SD of three independent experiments. * P
Figure Legend Snippet: MiR-34a involves in palmitate-induced Min6 cells apoptosis. (a) miR-34a expression level was elevated in response to palmitate treatment. (b) ectopic expression of miR-34a enhanced Min6 cell apoptosis. Cells transduced or not with miR-34a were treated with palmitate (500 μ M) or not for 2 days. The apoptotic rate of cells was determined by scoring the cells displaying pycnotic nucleus and/or fragmented nucleus. Cleaved caspase-3 expression (c) and caspase-3 activity (d) were carried out to confirm the proapoptotic role of miR-34a in Min6 cells. For (a), (b), (c) and (d), data are shown as means ± SD of three independent experiments. * P

Techniques Used: Expressing, Activity Assay

9) Product Images from "Basal Cancer Cell Survival Involves JNK2 Suppression of a Novel JNK1/c-Jun/Bcl-3 Apoptotic Network"

Article Title: Basal Cancer Cell Survival Involves JNK2 Suppression of a Novel JNK1/c-Jun/Bcl-3 Apoptotic Network

Journal: PLoS ONE

doi: 10.1371/journal.pone.0007305

JNK2 constitutively suppresses JNK1-mediated apoptosis in a range of human cancer cell lines but not in non-cancer cells. (A) JNK1 mRNA levels, and (B) JNK1, JNK2, and also p53 and p21 protein levels determined 48 h after transfection with JNK1- and JNK2-siRNAs as indicated ( Methods ). (C) Phase contrast images of HCT116 cells 48 h after transfection with JNK1- and JNK2-siRNAs. (D) Apoptosis determined by annexin V labelling of siRNA-treated cells relative to untreated controls in a range of human cell lines ( Methods . Note: apoptosis results are representative of at least three repeat experiments unless error bars are included for repeat analyses of a single experiment). (E) Caspase 8 and its cleavage products, activated caspase 7 and effector caspase 3 following RNAi-mediated silencing of JNK1 and JNK2 as indicated. Actin = loading controls. HCT116 p53+/+ and HCT116 p53−/− are isogenic clones of human HCT116 colorectal cancer cells ( Methods ).
Figure Legend Snippet: JNK2 constitutively suppresses JNK1-mediated apoptosis in a range of human cancer cell lines but not in non-cancer cells. (A) JNK1 mRNA levels, and (B) JNK1, JNK2, and also p53 and p21 protein levels determined 48 h after transfection with JNK1- and JNK2-siRNAs as indicated ( Methods ). (C) Phase contrast images of HCT116 cells 48 h after transfection with JNK1- and JNK2-siRNAs. (D) Apoptosis determined by annexin V labelling of siRNA-treated cells relative to untreated controls in a range of human cell lines ( Methods . Note: apoptosis results are representative of at least three repeat experiments unless error bars are included for repeat analyses of a single experiment). (E) Caspase 8 and its cleavage products, activated caspase 7 and effector caspase 3 following RNAi-mediated silencing of JNK1 and JNK2 as indicated. Actin = loading controls. HCT116 p53+/+ and HCT116 p53−/− are isogenic clones of human HCT116 colorectal cancer cells ( Methods ).

Techniques Used: Transfection, Clone Assay

10) Product Images from "Decay accelerating factor (CD55) protects neuronal cells from chemical hypoxia-induced injury"

Article Title: Decay accelerating factor (CD55) protects neuronal cells from chemical hypoxia-induced injury

Journal: Journal of Neuroinflammation

doi: 10.1186/1742-2094-7-24

DAF suppresses the expression of activated caspase in neurons exposed to chemical hypoxia . Primary rat cortical neurons were exposed to glucose free medium containing 1.5 mM NaCN for 1 hr then allowed to recover overnight in normal medium with or without DAF. (a) Cell lysates were analyzed by immunoblot using anti-active caspase-3, anti-caspase-9, and anti-β-actin antibodies. (b) Data was quantified by densitometry (n = 10). (c and d) Cells were marked with anti-active caspase-3 (red), anti-MAC (green) antibodies, and DAPI (blue) (n = 3). Scale bar = 50 μm. (one-way ANOVA followed by Newman-Keuls test. * p
Figure Legend Snippet: DAF suppresses the expression of activated caspase in neurons exposed to chemical hypoxia . Primary rat cortical neurons were exposed to glucose free medium containing 1.5 mM NaCN for 1 hr then allowed to recover overnight in normal medium with or without DAF. (a) Cell lysates were analyzed by immunoblot using anti-active caspase-3, anti-caspase-9, and anti-β-actin antibodies. (b) Data was quantified by densitometry (n = 10). (c and d) Cells were marked with anti-active caspase-3 (red), anti-MAC (green) antibodies, and DAPI (blue) (n = 3). Scale bar = 50 μm. (one-way ANOVA followed by Newman-Keuls test. * p

Techniques Used: Expressing

11) Product Images from "The HMG-CoA reductase inhibitor, simvastatin, exhibits anti-metastatic and anti-tumorigenic effects in ovarian cancer"

Article Title: The HMG-CoA reductase inhibitor, simvastatin, exhibits anti-metastatic and anti-tumorigenic effects in ovarian cancer

Journal: Oncotarget

doi:

Simvastatin reduced tumor growth of orthotropic xenografts of serous ovarian cancer M909 cells were injected into left side of the ovarian bursa of 6–8 week old mice. When the tumors reached ~50 mm 3  (approximately 12 days after injection), the mice were treated with placebo or 3 mg/kg simvastatin once a day for 4 weeks. Tumor volume  A.  and weight  B.  were recorded during or after 4 weeks treatment. Simvastatin reduced serum cholesterol in the mice  C.  VEGF was measured by ELISA assay in mouse serum and tumor tissues  D.  The changes of Ki-67, cleaved caspase 3, HMGCR, phosphorylated-AKT and phosphorylated-p42/44 were assessed by immunohistochemistry in ovarian tumor tissues  E.  (* p
Figure Legend Snippet: Simvastatin reduced tumor growth of orthotropic xenografts of serous ovarian cancer M909 cells were injected into left side of the ovarian bursa of 6–8 week old mice. When the tumors reached ~50 mm 3 (approximately 12 days after injection), the mice were treated with placebo or 3 mg/kg simvastatin once a day for 4 weeks. Tumor volume A. and weight B. were recorded during or after 4 weeks treatment. Simvastatin reduced serum cholesterol in the mice C. VEGF was measured by ELISA assay in mouse serum and tumor tissues D. The changes of Ki-67, cleaved caspase 3, HMGCR, phosphorylated-AKT and phosphorylated-p42/44 were assessed by immunohistochemistry in ovarian tumor tissues E. (* p

Techniques Used: Injection, Mouse Assay, Enzyme-linked Immunosorbent Assay, Immunohistochemistry

Simvastatin increased apoptosis in ovarian cancer cells The Hey A. and SKOV3 B. cell lines were cultured for 24 h and then treated with simvastatin at different doses for 24 h. Apoptosis was examined by Annexin V assay in Cellometer. Caspase-3, Caspase-9 and BCL-2 were determined by Western immunoblotting after exposure to simvastatin for 10 h or 24 h C and D. Each experiment was performed three times. C in graphs refers to control. (* p
Figure Legend Snippet: Simvastatin increased apoptosis in ovarian cancer cells The Hey A. and SKOV3 B. cell lines were cultured for 24 h and then treated with simvastatin at different doses for 24 h. Apoptosis was examined by Annexin V assay in Cellometer. Caspase-3, Caspase-9 and BCL-2 were determined by Western immunoblotting after exposure to simvastatin for 10 h or 24 h C and D. Each experiment was performed three times. C in graphs refers to control. (* p

Techniques Used: Cell Culture, Annexin V Assay, Western Blot

12) Product Images from "EMAPII Monoclonal Antibody Ameliorates Influenza A Virus-Induced Lung Injury"

Article Title: EMAPII Monoclonal Antibody Ameliorates Influenza A Virus-Induced Lung Injury

Journal: Molecular Therapy

doi: 10.1016/j.ymthe.2018.05.017

IAV-Induced Lung Injury Is Accompanied by EMAPII Release and Induction of Pulmonary Apoptosis Mice were administered 750 pfu/mouse IAV to lung and analyzed for (A) BALF level of EMAPII, (B) BALF level of caspase 3/7 activity, (C) lung levels of EMAPII, (D) lung levels of cleaved caspase 3. n = 7 for control group and 3–4 for all other groups; data are presented as mean ± SEM. *p 
Figure Legend Snippet: IAV-Induced Lung Injury Is Accompanied by EMAPII Release and Induction of Pulmonary Apoptosis Mice were administered 750 pfu/mouse IAV to lung and analyzed for (A) BALF level of EMAPII, (B) BALF level of caspase 3/7 activity, (C) lung levels of EMAPII, (D) lung levels of cleaved caspase 3. n = 7 for control group and 3–4 for all other groups; data are presented as mean ± SEM. *p 

Techniques Used: Mouse Assay, Activity Assay

IAV-Induced Apoptosis Is Potentiated by EMAPII and Suppressed by EMAPII mAb HLMVECs (A) and A549 (B) were stimulated with 1 pfu/cell IAV (A) in the presence of 30 μg/mL recombinant EMAPII (E) or (B) 10 μg/mL control IgG or EMAPII mAb, then analyzed for cleaved and total caspase 3 levels. n = 3–5, data are presented as mean ± SEM; *p
Figure Legend Snippet: IAV-Induced Apoptosis Is Potentiated by EMAPII and Suppressed by EMAPII mAb HLMVECs (A) and A549 (B) were stimulated with 1 pfu/cell IAV (A) in the presence of 30 μg/mL recombinant EMAPII (E) or (B) 10 μg/mL control IgG or EMAPII mAb, then analyzed for cleaved and total caspase 3 levels. n = 3–5, data are presented as mean ± SEM; *p

Techniques Used: Recombinant

EMAPII mAb Subcutaneous Injections Reduce Levels of Released EMAPII, Weight Loss, and Indices of Lung Injury in IAV-Infected Mice (A) Mice received 750 pfu/mouse IAV or equal volume of saline (cntr). Half of IAV-infected mice were treated with EMAPII mAb (2.5 mg/kg) on days 4, 6, and 8 post-infection. Mice were analyzed for (B) EMAPII levels in BALF, (C) IAV-induced weight loss, (D) conscious blood oxygenation, (E) lung edema and protein in BALF (day 9), and (F) BALF caspase 3/7 activity (day 9). n = 5 for all groups; data are presented as mean ± SEM. *p 
Figure Legend Snippet: EMAPII mAb Subcutaneous Injections Reduce Levels of Released EMAPII, Weight Loss, and Indices of Lung Injury in IAV-Infected Mice (A) Mice received 750 pfu/mouse IAV or equal volume of saline (cntr). Half of IAV-infected mice were treated with EMAPII mAb (2.5 mg/kg) on days 4, 6, and 8 post-infection. Mice were analyzed for (B) EMAPII levels in BALF, (C) IAV-induced weight loss, (D) conscious blood oxygenation, (E) lung edema and protein in BALF (day 9), and (F) BALF caspase 3/7 activity (day 9). n = 5 for all groups; data are presented as mean ± SEM. *p 

Techniques Used: Infection, Mouse Assay, Activity Assay

IAV Induces Apoptosis and EMAPII Release in Pulmonary Endothelium and Epithelium (A–C and E) Human pulmonary artery endothelial cells (HPAEC), normal human bronchial epithelial cells (NHBEC), human lung microvascular endothelial cells (HLMVEC), and human alveolar epithelial line A549 were stimulated with 1 pfu/cell IAV and then analyzed for (A and B) caspase 3 cleavage, caspase 3 (A) and EMAPII (B) expression, (C) surface annexin V staining, (D) released EMAPII levels, (E) surface EMAPII staining, or (F) concomitant caspase 3 cleavage and surface EMAPII staining. *p
Figure Legend Snippet: IAV Induces Apoptosis and EMAPII Release in Pulmonary Endothelium and Epithelium (A–C and E) Human pulmonary artery endothelial cells (HPAEC), normal human bronchial epithelial cells (NHBEC), human lung microvascular endothelial cells (HLMVEC), and human alveolar epithelial line A549 were stimulated with 1 pfu/cell IAV and then analyzed for (A and B) caspase 3 cleavage, caspase 3 (A) and EMAPII (B) expression, (C) surface annexin V staining, (D) released EMAPII levels, (E) surface EMAPII staining, or (F) concomitant caspase 3 cleavage and surface EMAPII staining. *p

Techniques Used: Expressing, Staining

IAV-Induced Lung Injury Is Accompanied by Surface EMAPII Translocation and Caspase 3 Cleavage in Cells of Hematopoietic and Endothelial Lineage Mice infected with 750 pfu/mouse IAV were sacrificed at day 5 (A and B) or day 7 (C and D). Lungs were digested and subjected to concomitant staining for surface CD31/CD326/CD45 and EMAPII (A and C) or permeabilized and subjected to concomitant staining for CD31/CD326/CD45 and cleaved caspase 3 (B and D). n = 3–5; data are presented as mean ± SEM. *p
Figure Legend Snippet: IAV-Induced Lung Injury Is Accompanied by Surface EMAPII Translocation and Caspase 3 Cleavage in Cells of Hematopoietic and Endothelial Lineage Mice infected with 750 pfu/mouse IAV were sacrificed at day 5 (A and B) or day 7 (C and D). Lungs were digested and subjected to concomitant staining for surface CD31/CD326/CD45 and EMAPII (A and C) or permeabilized and subjected to concomitant staining for CD31/CD326/CD45 and cleaved caspase 3 (B and D). n = 3–5; data are presented as mean ± SEM. *p

Techniques Used: Translocation Assay, Mouse Assay, Infection, Staining

13) Product Images from "Cessation of Renal Morphogenesis in Mice"

Article Title: Cessation of Renal Morphogenesis in Mice

Journal: Developmental biology

doi: 10.1016/j.ydbio.2007.08.021

Capping nephrogenic mesenchyme rapidly converted to nephrons in the immediate postnatal period Whole mount immunostaining with antibody to CITED1 and imaging by confocal microscopy demonstrates large clusters of nephrogenic mesenchyme (red) at birth (P0). The patches of nephrogenic mesenchyme were smaller by P1 and were scarce by P2. (The images are merged images of all Z-stack slices from the surface of the kidney to a depth of 35μ.) In situ hybridization for Six2 demonstrated the same change in capping mesenchyme. There were no apparent changes in patterns of proliferation or apoptosis. Staining with antibody to PHH3 (red) identified proliferating cells within the mesenchyme, nephrons, and ureeric bud at E16.5 and P0. Because the capping mesenchyme was nearly absent by P2, the proliferation that was seen was mostly in developing nephrons rather than in the mesenchyme. Proliferation also continued in the ureteric bud. E-cadherin expression (blue) was used to help identify structures and was seen within ureteric bud derivatives and more mature nephrons. (Images are single slices at a depth of 25μ for E16.5 and P0, and at a depth of 16μ for P2 and P3). Staining of P0 kidneys with antibody to cleaved Caspase 3 (red) showed only sparse apoptotic activity in the surface nephrogenic region (0-25μ) in mesenchymal or ureteric bud cells at all ages examined. In contrast, apoptotic activity was common deeper within the kidney (30-55μ). (Images are merged images of all slices of a Z-stack series between the surface and 25μ and between 30 and 55μ. The magnification of all confocal microscopy images is the same. green bar = 50μ) Whole mount in situ hybridization showed diminished expression of Foxd1 in the postnatal day 3 kidney. During the first 3 postnatal days, WT-1 stained renal vesicles replace the metanephric mesenchyme that had surrounded the ureteric bud branch tips. The staining pattern of the mesenchyme with antibody to WT1 is similar in the embryonic (E16.5) and P0 kidney. By P2 renal vesicles (arrows) replace the nephrogenic mesenchyme near the surface of the kidney. By P3 those vesicles develop further and show polarized expression of WT1 (arrowhead) opposite the ureteric bud tip. (Circles were placed around vesicles or glomerular anlagen to illustrate the manner in which these structures were measured. The images are single optical slices at a depth of 20μ from the surface for E16.5 and 15μ for the rest.) A similar pattern was seen for Pax2 which is expressed in the ureteric bud, capping mesenchyme and early nephron. The mesenchyme staining remained similar in intensity to that in the tips and early nephrons, and demonstrated the replacement of the mesenchyme around the tips by nascent nephrons (15μ from the surface).
Figure Legend Snippet: Capping nephrogenic mesenchyme rapidly converted to nephrons in the immediate postnatal period Whole mount immunostaining with antibody to CITED1 and imaging by confocal microscopy demonstrates large clusters of nephrogenic mesenchyme (red) at birth (P0). The patches of nephrogenic mesenchyme were smaller by P1 and were scarce by P2. (The images are merged images of all Z-stack slices from the surface of the kidney to a depth of 35μ.) In situ hybridization for Six2 demonstrated the same change in capping mesenchyme. There were no apparent changes in patterns of proliferation or apoptosis. Staining with antibody to PHH3 (red) identified proliferating cells within the mesenchyme, nephrons, and ureeric bud at E16.5 and P0. Because the capping mesenchyme was nearly absent by P2, the proliferation that was seen was mostly in developing nephrons rather than in the mesenchyme. Proliferation also continued in the ureteric bud. E-cadherin expression (blue) was used to help identify structures and was seen within ureteric bud derivatives and more mature nephrons. (Images are single slices at a depth of 25μ for E16.5 and P0, and at a depth of 16μ for P2 and P3). Staining of P0 kidneys with antibody to cleaved Caspase 3 (red) showed only sparse apoptotic activity in the surface nephrogenic region (0-25μ) in mesenchymal or ureteric bud cells at all ages examined. In contrast, apoptotic activity was common deeper within the kidney (30-55μ). (Images are merged images of all slices of a Z-stack series between the surface and 25μ and between 30 and 55μ. The magnification of all confocal microscopy images is the same. green bar = 50μ) Whole mount in situ hybridization showed diminished expression of Foxd1 in the postnatal day 3 kidney. During the first 3 postnatal days, WT-1 stained renal vesicles replace the metanephric mesenchyme that had surrounded the ureteric bud branch tips. The staining pattern of the mesenchyme with antibody to WT1 is similar in the embryonic (E16.5) and P0 kidney. By P2 renal vesicles (arrows) replace the nephrogenic mesenchyme near the surface of the kidney. By P3 those vesicles develop further and show polarized expression of WT1 (arrowhead) opposite the ureteric bud tip. (Circles were placed around vesicles or glomerular anlagen to illustrate the manner in which these structures were measured. The images are single optical slices at a depth of 20μ from the surface for E16.5 and 15μ for the rest.) A similar pattern was seen for Pax2 which is expressed in the ureteric bud, capping mesenchyme and early nephron. The mesenchyme staining remained similar in intensity to that in the tips and early nephrons, and demonstrated the replacement of the mesenchyme around the tips by nascent nephrons (15μ from the surface).

Techniques Used: Immunostaining, Imaging, Confocal Microscopy, In Situ Hybridization, Staining, Expressing, Activity Assay

14) Product Images from "β-catenin nuclear translocation induced by HIF-1α overexpression leads to the radioresistance of prostate cancer"

Article Title: β-catenin nuclear translocation induced by HIF-1α overexpression leads to the radioresistance of prostate cancer

Journal: International Journal of Oncology

doi: 10.3892/ijo.2018.4368

Effect of β-catenin nuclear translocation on apoptosis and non-homologous end joining (NHEJ) repairing potential in the prostate cancer cell lines, LNCaP and C4-2B. (A and B) Flow cytometric analysis of the cell cycle distribution (G0/G1, S and G2/M phase) at 72 h in the negative control, HIF-1α over-expression and β-catenin silenced groups. (C) Western blot analysis of the protein expression of apoptotic regulators (including caspase-3, cleaved-caspase-3, caspase-7, cleaved-caspase-7, cleaved-PARP-1 and Bax), anti-apoptotic proteins (Bcl-2 and Bcl-xL), the DNA double strand breaks (DSB) marker, γH2AX, and NHEJ repair proteins (Ku70, Ku80 and DNA-PKcs) at 72 h in the negative control, HIF-1α overexpression and β-catenin silenced groups.
Figure Legend Snippet: Effect of β-catenin nuclear translocation on apoptosis and non-homologous end joining (NHEJ) repairing potential in the prostate cancer cell lines, LNCaP and C4-2B. (A and B) Flow cytometric analysis of the cell cycle distribution (G0/G1, S and G2/M phase) at 72 h in the negative control, HIF-1α over-expression and β-catenin silenced groups. (C) Western blot analysis of the protein expression of apoptotic regulators (including caspase-3, cleaved-caspase-3, caspase-7, cleaved-caspase-7, cleaved-PARP-1 and Bax), anti-apoptotic proteins (Bcl-2 and Bcl-xL), the DNA double strand breaks (DSB) marker, γH2AX, and NHEJ repair proteins (Ku70, Ku80 and DNA-PKcs) at 72 h in the negative control, HIF-1α overexpression and β-catenin silenced groups.

Techniques Used: Translocation Assay, Non-Homologous End Joining, Flow Cytometry, Negative Control, Over Expression, Western Blot, Expressing, Marker

Effect of β-catenin nuclear translocation on cell cycle distribution, apoptosis and non-homologous end joining (NHEJ) repair following irradiation. (A–D) Results of flow cytometry of the cell cycle distribution and sub-G1 cell ratio at 72 h in the negative control, HIF-1α overexpression and β-catenin silenced groups of LNCaP and C4-2B cells following in vitro irradiation. (E and F) Colony formation assay showing colony-forming capability at 72 h in the negative control, HIF-1α overexpression and β-catenin silenced groups of LNCaP and C4-2B cells following in vitro irradiation. (G and H) ELISA results at 72 h showing cell death (DNA fragmentation) in the negative control, HIF-1α overexpression and β-catenin silenced groups of LNCaP and C4-2B cells following in vitro irradiation. (I) Western blot analysis of the protein expression of cell cycle regulators (p21, CDK1, p-CDK1, Chk1, p-Chk1, Chk2, p-Chk2, Rb and p-Rb), apoptotic proteins (including caspase-3, cleaved-caspase-3, caspase-7, cleaved-caspase-7, cleaved-PARP-1 and Bax), anti-apoptotic proteins (Bcl-2 and Bcl-xL), the DNA double strand breaks (DSB) marker, γH2AX, and non-homologous end joining (NHEJ) repair proteins (Ku70, Ku80 and DNA-PKCs) at 72 h in the negative control, HIF-1α overexpression and β-catenin silenced groups of LNCaP and C4-2B cells following in vitro irradiation.
Figure Legend Snippet: Effect of β-catenin nuclear translocation on cell cycle distribution, apoptosis and non-homologous end joining (NHEJ) repair following irradiation. (A–D) Results of flow cytometry of the cell cycle distribution and sub-G1 cell ratio at 72 h in the negative control, HIF-1α overexpression and β-catenin silenced groups of LNCaP and C4-2B cells following in vitro irradiation. (E and F) Colony formation assay showing colony-forming capability at 72 h in the negative control, HIF-1α overexpression and β-catenin silenced groups of LNCaP and C4-2B cells following in vitro irradiation. (G and H) ELISA results at 72 h showing cell death (DNA fragmentation) in the negative control, HIF-1α overexpression and β-catenin silenced groups of LNCaP and C4-2B cells following in vitro irradiation. (I) Western blot analysis of the protein expression of cell cycle regulators (p21, CDK1, p-CDK1, Chk1, p-Chk1, Chk2, p-Chk2, Rb and p-Rb), apoptotic proteins (including caspase-3, cleaved-caspase-3, caspase-7, cleaved-caspase-7, cleaved-PARP-1 and Bax), anti-apoptotic proteins (Bcl-2 and Bcl-xL), the DNA double strand breaks (DSB) marker, γH2AX, and non-homologous end joining (NHEJ) repair proteins (Ku70, Ku80 and DNA-PKCs) at 72 h in the negative control, HIF-1α overexpression and β-catenin silenced groups of LNCaP and C4-2B cells following in vitro irradiation.

Techniques Used: Translocation Assay, Non-Homologous End Joining, Irradiation, Flow Cytometry, Cytometry, Negative Control, Over Expression, In Vitro, Colony Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Marker

15) Product Images from "Contributions of the RhoA guanine nucleotide exchange factor Net1 to polyoma middle T antigen-mediated mammary gland tumorigenesis and metastasis"

Article Title: Contributions of the RhoA guanine nucleotide exchange factor Net1 to polyoma middle T antigen-mediated mammary gland tumorigenesis and metastasis

Journal: Breast Cancer Research : BCR

doi: 10.1186/s13058-018-0966-2

Net1 deletion inhibits tumor angiogenesis and causes increased tumor cell death. a Representative examples of H E staining of large tumors in genotypes shown. b Quantification of necrotic area in H E sections over range of tumor sizes for genotypes shown. Five animals per genotype, 4–5 tumors per animal, quantified. c Quantification of necrotic area of all tumors per genotype. d Representative examples of cleaved caspase 3 (CC3) staining in large tumors from genotypes shown. e Quantification of number of CC3 hot spots in large tumors. Entire tumor sections analyzed from 8 mice of each genotype. f Examples of cell determinant 31 (CD31) staining (red) in solid tumors from Net1 +/+ ,PyMT and Net1 −/− ,PyMT mice. DNA shown in blue. g Quantification of CD31 staining. Five areas per sample analyzed; tumors from 4 ( Net1 +/+ ,PyMT ) or 5 ( Net1 −/− ,PyMT ) mice assessed. Bars represent median values. * P
Figure Legend Snippet: Net1 deletion inhibits tumor angiogenesis and causes increased tumor cell death. a Representative examples of H E staining of large tumors in genotypes shown. b Quantification of necrotic area in H E sections over range of tumor sizes for genotypes shown. Five animals per genotype, 4–5 tumors per animal, quantified. c Quantification of necrotic area of all tumors per genotype. d Representative examples of cleaved caspase 3 (CC3) staining in large tumors from genotypes shown. e Quantification of number of CC3 hot spots in large tumors. Entire tumor sections analyzed from 8 mice of each genotype. f Examples of cell determinant 31 (CD31) staining (red) in solid tumors from Net1 +/+ ,PyMT and Net1 −/− ,PyMT mice. DNA shown in blue. g Quantification of CD31 staining. Five areas per sample analyzed; tumors from 4 ( Net1 +/+ ,PyMT ) or 5 ( Net1 −/− ,PyMT ) mice assessed. Bars represent median values. * P

Techniques Used: Staining, Mouse Assay

16) Product Images from "DNA crosslink repair safeguards genomic stability during pre-meiotic germ cell development"

Article Title: DNA crosslink repair safeguards genomic stability during pre-meiotic germ cell development

Journal: Nature genetics

doi: 10.1038/s41588-019-0471-2

Damaged PGCs are eliminated by apoptosis. a-b,  Quantification of the frequency of PGCs (GOF18-GFP + ) and surrounding somatic cells (GOF18-GFP - ) with ( a )  >  5 53BP1 or ( b )  >  10 γ–H2A.X foci per nucleus at E11.5. ( P  value calculated by unpaired 2-tailed  t  test; data represent mean and s.e.m.; at least 100 PGCs or somatic cells were scored per embryo, each point represents data from one embryo, 53BP1  n  = 4, 3, 3, 5, 3 and 3, γ–H2A.X  n  = 3, 3, 3, 4, 4 and 5, left to right).  c , Representative images of E11.5 gonads stained for phospho-Ser15-p53 (pSer15-p53) and GFP (GOF18-GFP). Quantification of PGCs (GOF18-GFP + ) and somatic cells (GOF18-GFP - ) that stain positively for phospho-Ser15-p53 ( P  value calculated by unpaired 2-tailed  t  test; data represent mean and s.e.m.; at least 100 PGCs or somatic cells were scored per embryo, each point represents data from one embryo,  n  = 6, 3, 2, 8, 4 and 7, left to right).  d , Representative images of E11.5 gonads stained for the marker of apoptosis cleaved-Caspase 3 and GFP (GOF18-GFP) and the quantification of the frequency of PGCs (GOF18-GFP + ) and somatic cells (GOF18-GFP - ) that stain positively for cleaved-Caspase 3 ( P  value calculated by unpaired 2-tailed  t  test; data represent mean and s.e.m., at least 100 PGCs or somatic cells were scored per embryo, each point represents data from one embryo,  n  = 10, 3, 3, 8, 4 and 4, left to right).
Figure Legend Snippet: Damaged PGCs are eliminated by apoptosis. a-b, Quantification of the frequency of PGCs (GOF18-GFP + ) and surrounding somatic cells (GOF18-GFP - ) with ( a ) > 5 53BP1 or ( b ) > 10 γ–H2A.X foci per nucleus at E11.5. ( P value calculated by unpaired 2-tailed t test; data represent mean and s.e.m.; at least 100 PGCs or somatic cells were scored per embryo, each point represents data from one embryo, 53BP1 n = 4, 3, 3, 5, 3 and 3, γ–H2A.X n = 3, 3, 3, 4, 4 and 5, left to right). c , Representative images of E11.5 gonads stained for phospho-Ser15-p53 (pSer15-p53) and GFP (GOF18-GFP). Quantification of PGCs (GOF18-GFP + ) and somatic cells (GOF18-GFP - ) that stain positively for phospho-Ser15-p53 ( P value calculated by unpaired 2-tailed t test; data represent mean and s.e.m.; at least 100 PGCs or somatic cells were scored per embryo, each point represents data from one embryo, n = 6, 3, 2, 8, 4 and 7, left to right). d , Representative images of E11.5 gonads stained for the marker of apoptosis cleaved-Caspase 3 and GFP (GOF18-GFP) and the quantification of the frequency of PGCs (GOF18-GFP + ) and somatic cells (GOF18-GFP - ) that stain positively for cleaved-Caspase 3 ( P value calculated by unpaired 2-tailed t test; data represent mean and s.e.m., at least 100 PGCs or somatic cells were scored per embryo, each point represents data from one embryo, n = 10, 3, 3, 8, 4 and 4, left to right).

Techniques Used: Staining, Marker

17) Product Images from "Caspase-3 Is Enriched in Postsynaptic Densities and Increased in Alzheimer's Disease"

Article Title: Caspase-3 Is Enriched in Postsynaptic Densities and Increased in Alzheimer's Disease

Journal:

doi: 10.2353/ajpath.2008.080434

Caspase-3 distribution in cytosolic extracts (cyt), nuclear extracts (nucl), and synaptosomes (syn) from normal and AD brain tissue. The caspase-3 antibody recognizes both procaspase-3 (∼32 kDa) and the large subunit of active/cleaved caspase-3
Figure Legend Snippet: Caspase-3 distribution in cytosolic extracts (cyt), nuclear extracts (nucl), and synaptosomes (syn) from normal and AD brain tissue. The caspase-3 antibody recognizes both procaspase-3 (∼32 kDa) and the large subunit of active/cleaved caspase-3

Techniques Used:

Relative distribution of caspase-3 in synaptosomal extracts and subsynaptic fractions of human brain tissue. A control case was used to prepare synaptosomes (S) and all subsynaptic fractions (PSD, postsynaptic density; PrS, presynaptic; SV, synaptic vesicle).
Figure Legend Snippet: Relative distribution of caspase-3 in synaptosomal extracts and subsynaptic fractions of human brain tissue. A control case was used to prepare synaptosomes (S) and all subsynaptic fractions (PSD, postsynaptic density; PrS, presynaptic; SV, synaptic vesicle).

Techniques Used:

Caspase-3 expression in PSD subsynaptic fractions from control and AD brain tissue. The caspase-3 antibody recognizes both procaspase-3 (∼32 kDa) and the large subunit of active/cleaved caspase-3 (∼14 to 21 kDa). β-Actin was used
Figure Legend Snippet: Caspase-3 expression in PSD subsynaptic fractions from control and AD brain tissue. The caspase-3 antibody recognizes both procaspase-3 (∼32 kDa) and the large subunit of active/cleaved caspase-3 (∼14 to 21 kDa). β-Actin was used

Techniques Used: Expressing

Immunohistochemical expression of activated caspase-3. A to D: Hippocampal formation of nonneuropsychiatric control case ( A , 78-year-old female) and AD case ( B , 81-year-old female). Note the especially intense labeling of synaptic terminal fields in the
Figure Legend Snippet: Immunohistochemical expression of activated caspase-3. A to D: Hippocampal formation of nonneuropsychiatric control case ( A , 78-year-old female) and AD case ( B , 81-year-old female). Note the especially intense labeling of synaptic terminal fields in the

Techniques Used: Immunohistochemistry, Expressing, Labeling

A: Caspase-3 expression in synaptosomes from normal and AD brain tissue. β-Actin was used as a loading control for normalization purposes. B: Densitometric analysis of procaspase-3 expression in synaptosomes in control (Ctrl) and AD groups ( t
Figure Legend Snippet: A: Caspase-3 expression in synaptosomes from normal and AD brain tissue. β-Actin was used as a loading control for normalization purposes. B: Densitometric analysis of procaspase-3 expression in synaptosomes in control (Ctrl) and AD groups ( t

Techniques Used: Expressing

18) Product Images from "Disruption of the Microglial ADP Receptor P2Y13 Enhances Adult Hippocampal Neurogenesis"

Article Title: Disruption of the Microglial ADP Receptor P2Y13 Enhances Adult Hippocampal Neurogenesis

Journal: Frontiers in Cellular Neuroscience

doi: 10.3389/fncel.2018.00134

Increased progenitor cell proliferation and apoptosis in P2ry13 KO mice (young adults). (A–D) Increase in number and proliferative capacity of type 2a cells and type 3 cells in P2ry13 KO mice. (A) From left to right: Double labeling for Tbr2 (green) and DCX (white), Tbr2 (green) and Ki67 (red), Ki67 (red) and DCX (white) and triple labeling for Tbr2 (green), Ki67 (red) and DCX (white). Double-labeled cells are indicated by yellow arrows and triple-labeled cells by yellow arrowheads. (B–D) Resulting quantitative evaluation of the total number of type 2a cells (B) , the total number of proliferating type 2a cells (C) , and the total number of proliferating DCX + cells (D) (six corresponding sections per animal, n = 7). (E) Immunolabeling for cleaved caspase-3 (red) in the GCL. DAPI staining is shown in blue. In both genotypes cleaved caspase-3 + cells were exclusively found in the SGZ and in low numbers. (F) Total number of caspase-3 + cells (14 corresponding sections per animal, n = 7). All graphs represent mean ± SEM. * p
Figure Legend Snippet: Increased progenitor cell proliferation and apoptosis in P2ry13 KO mice (young adults). (A–D) Increase in number and proliferative capacity of type 2a cells and type 3 cells in P2ry13 KO mice. (A) From left to right: Double labeling for Tbr2 (green) and DCX (white), Tbr2 (green) and Ki67 (red), Ki67 (red) and DCX (white) and triple labeling for Tbr2 (green), Ki67 (red) and DCX (white). Double-labeled cells are indicated by yellow arrows and triple-labeled cells by yellow arrowheads. (B–D) Resulting quantitative evaluation of the total number of type 2a cells (B) , the total number of proliferating type 2a cells (C) , and the total number of proliferating DCX + cells (D) (six corresponding sections per animal, n = 7). (E) Immunolabeling for cleaved caspase-3 (red) in the GCL. DAPI staining is shown in blue. In both genotypes cleaved caspase-3 + cells were exclusively found in the SGZ and in low numbers. (F) Total number of caspase-3 + cells (14 corresponding sections per animal, n = 7). All graphs represent mean ± SEM. * p

Techniques Used: Mouse Assay, Labeling, Immunolabeling, Staining

19) Product Images from "Resveratrol provides neuroprotection by regulating the JAK2/STAT3/PI3K/AKT/mTOR pathway after stroke in rats"

Article Title: Resveratrol provides neuroprotection by regulating the JAK2/STAT3/PI3K/AKT/mTOR pathway after stroke in rats

Journal: Genes & Diseases

doi: 10.1016/j.gendis.2018.06.001

Effects of resveratrol on mRNA and protein expression of BCL-2, BAX, and cleaved caspase-3 after 24 h of cerebral I/R. (A) RT-qPCR analysis showed significantly higher BLC-2 and lower BAX and cleaved caspase-3 in the resveratrol group than in the vehicle group. These effects were partially reversed by LY294002 and AG490. (B) Typical immunohistochemical photographs of BCL-2, BAX, and caspase-3. (C) Immunohistochemical staining showed more BLC-2-positive and fewer BAX- and cleaved caspase-3-positive cells in the resveratrol group compared to the vehicle group. These effects were partially reversed by LY294002 and AG490. Data are presented as the mean ± SEM . (* P
Figure Legend Snippet: Effects of resveratrol on mRNA and protein expression of BCL-2, BAX, and cleaved caspase-3 after 24 h of cerebral I/R. (A) RT-qPCR analysis showed significantly higher BLC-2 and lower BAX and cleaved caspase-3 in the resveratrol group than in the vehicle group. These effects were partially reversed by LY294002 and AG490. (B) Typical immunohistochemical photographs of BCL-2, BAX, and caspase-3. (C) Immunohistochemical staining showed more BLC-2-positive and fewer BAX- and cleaved caspase-3-positive cells in the resveratrol group compared to the vehicle group. These effects were partially reversed by LY294002 and AG490. Data are presented as the mean ± SEM . (* P

Techniques Used: Expressing, Quantitative RT-PCR, Immunohistochemistry, Staining

Effects of resveratrol on protein expression of JAK2, STAT3, AKT, mTOR, p-JAK2, p-STAT3, p-AKT, p-mTOR, BCL-2, BAX, and cleaved caspase-3 at 24 h after reperfusion. (A) The protein expression of p-AKT was increased 1.66-fold in the resveratrol group compared to the vehicle group, which was partially reversed by LY294002 (PI3K inhibitor). (B) The protein expression of p-mTOR was increased 1.66-fold in the resveratrol group compared to the vehicle group, which was also partially reversed by LY294002. (C) JAK2 inhibitor AG490 significantly decreased the protein expression of p-AKT compared with the resveratrol group. (D) The effect of PI3K inhibitor LY294002 on p-JAK2 after resveratrol treatment was not significant. (E) JAK2 inhibitor AG490 significantly decreased the protein expression of p-STAT3 compared with the resveratrol group. (F) Western blot indicated significantly higher BLC-2 and lower BAX and cleaved caspase-3 in the resveratrol group than in the vehicle group. These effects were partially reversed by LY294002 and AG490. Data are presented as mean ± SEM . (* P
Figure Legend Snippet: Effects of resveratrol on protein expression of JAK2, STAT3, AKT, mTOR, p-JAK2, p-STAT3, p-AKT, p-mTOR, BCL-2, BAX, and cleaved caspase-3 at 24 h after reperfusion. (A) The protein expression of p-AKT was increased 1.66-fold in the resveratrol group compared to the vehicle group, which was partially reversed by LY294002 (PI3K inhibitor). (B) The protein expression of p-mTOR was increased 1.66-fold in the resveratrol group compared to the vehicle group, which was also partially reversed by LY294002. (C) JAK2 inhibitor AG490 significantly decreased the protein expression of p-AKT compared with the resveratrol group. (D) The effect of PI3K inhibitor LY294002 on p-JAK2 after resveratrol treatment was not significant. (E) JAK2 inhibitor AG490 significantly decreased the protein expression of p-STAT3 compared with the resveratrol group. (F) Western blot indicated significantly higher BLC-2 and lower BAX and cleaved caspase-3 in the resveratrol group than in the vehicle group. These effects were partially reversed by LY294002 and AG490. Data are presented as mean ± SEM . (* P

Techniques Used: Expressing, Western Blot

20) Product Images from "Diosmetin suppresses human prostate cancer cell proliferation through the induction of apoptosis and cell cycle arrest"

Article Title: Diosmetin suppresses human prostate cancer cell proliferation through the induction of apoptosis and cell cycle arrest

Journal: International Journal of Oncology

doi: 10.3892/ijo.2018.4407

Schematic diagram of the proposed cell growth arrest signaling pathways mediated by diosmetin in human prostate cancer cells. Overexpression of c-Myc, Cdk4 and cyclin D1 in prostate cancer cells drives cell growth. Diosmetin treatment inhibits the expression of c-Myc and its downstream molecules, cyclin D1 and Cdk4, conversely increasing FOXO3a and p27 Kip1 expression levels to arrest cell growth. Moreover, diosmetin treatment potentiates the apoptotic machinery by modulating cleaved caspase-3 and cleaved PARP in prostate cancer cells.
Figure Legend Snippet: Schematic diagram of the proposed cell growth arrest signaling pathways mediated by diosmetin in human prostate cancer cells. Overexpression of c-Myc, Cdk4 and cyclin D1 in prostate cancer cells drives cell growth. Diosmetin treatment inhibits the expression of c-Myc and its downstream molecules, cyclin D1 and Cdk4, conversely increasing FOXO3a and p27 Kip1 expression levels to arrest cell growth. Moreover, diosmetin treatment potentiates the apoptotic machinery by modulating cleaved caspase-3 and cleaved PARP in prostate cancer cells.

Techniques Used: Over Expression, Expressing

Dose-response effects of diosmetin on apoptotic and anti-apoptotic molecules. (A) LNCaP and PC-3 cells were exposed to two concentrations of diosmetin (10 and 20 µ M) for 24 h. Total cell lysates were prepared and using SDS-PAGE, lysates proteins were resolved and then subjected to western blot analysis for the evaluation of XIAP, Bax, Bcl-2, cleaved PARP and cleaved caspase-3. Lanes marked ‘0’ are the DMSO control (0.2%)-treated cells. For protein band density, densitometric analysis was performed and protein expressions levels were correlated to express relative controls (B and C). For loading control blots were stripped and reprobed with GAPDH. * P
Figure Legend Snippet: Dose-response effects of diosmetin on apoptotic and anti-apoptotic molecules. (A) LNCaP and PC-3 cells were exposed to two concentrations of diosmetin (10 and 20 µ M) for 24 h. Total cell lysates were prepared and using SDS-PAGE, lysates proteins were resolved and then subjected to western blot analysis for the evaluation of XIAP, Bax, Bcl-2, cleaved PARP and cleaved caspase-3. Lanes marked ‘0’ are the DMSO control (0.2%)-treated cells. For protein band density, densitometric analysis was performed and protein expressions levels were correlated to express relative controls (B and C). For loading control blots were stripped and reprobed with GAPDH. * P

Techniques Used: SDS Page, Western Blot

21) Product Images from "Novel compounds protect auditory hair cells against gentamycin-induced apoptosis by maintaining the expression level of H3K4me2"

Article Title: Novel compounds protect auditory hair cells against gentamycin-induced apoptosis by maintaining the expression level of H3K4me2

Journal: Drug Delivery

doi: 10.1080/10717544.2018.1461277

The new compounds reduce apoptosis in the organ of Corti after gentamycin exposure. (A) The schematic diagram of the experiment. (B) The level of cleaved caspase-3 in the organ of Corti decreased in the presence of compound A (c) and compound B (d) when compared with the gentamycin-only group (b). The normal group with no treatment was used as controls (a). (C) The quantification of HCs (a) and myosin VIIa/cleaved caspase-3 double-positive cells (b) in the different groups. Confocal images were taken from the middle turn of each group. The red spots represent the cleaved caspase-3 signal, and anti-myosin VIIa antibody (green) is used as the HC marker. Scale bar =25 μm. Six cochleae were used for each group. Data are expressed as the mean ± S.E. * p
Figure Legend Snippet: The new compounds reduce apoptosis in the organ of Corti after gentamycin exposure. (A) The schematic diagram of the experiment. (B) The level of cleaved caspase-3 in the organ of Corti decreased in the presence of compound A (c) and compound B (d) when compared with the gentamycin-only group (b). The normal group with no treatment was used as controls (a). (C) The quantification of HCs (a) and myosin VIIa/cleaved caspase-3 double-positive cells (b) in the different groups. Confocal images were taken from the middle turn of each group. The red spots represent the cleaved caspase-3 signal, and anti-myosin VIIa antibody (green) is used as the HC marker. Scale bar =25 μm. Six cochleae were used for each group. Data are expressed as the mean ± S.E. * p

Techniques Used: Marker

22) Product Images from "Studies on the non-invasive anticancer remedy of the triple combination of epigallocatechin gallate, pulsed electric field, and ultrasound"

Article Title: Studies on the non-invasive anticancer remedy of the triple combination of epigallocatechin gallate, pulsed electric field, and ultrasound

Journal: PLoS ONE

doi: 10.1371/journal.pone.0201920

Effects of the combined triple treatment on the viability of the HepG2 cells. The cell viability (24 h) was determined using MTT assay. (A) The HepG2 cells were triple treated with the EGCG (0 to 200 μM), 0.3 W/cm 2 US exposure (60 min), and consecutive 60 V/cm PEF for 24 h. The result revealed that the triple treatment could overcome the tolerance of cancer cells to EGCG and cause the death of HepG2 cells. (B) The triple treatment decreased the phosphorylation of Akt protein and increased the conversion of LC3-I to LC3-II in the HepG2 cells. The band intensities of LC3-II and LC3-I were quantified to calculate the LC3-II/LC3-I ratio. (C) The triple treatment triggered the activations of caspase-3 and PARP in the HepG2 cells. The cleaved PARP was quantified relative to the full-length PARP. β-actin was used as a loading control for each Western blot assay. (*** is used for P
Figure Legend Snippet: Effects of the combined triple treatment on the viability of the HepG2 cells. The cell viability (24 h) was determined using MTT assay. (A) The HepG2 cells were triple treated with the EGCG (0 to 200 μM), 0.3 W/cm 2 US exposure (60 min), and consecutive 60 V/cm PEF for 24 h. The result revealed that the triple treatment could overcome the tolerance of cancer cells to EGCG and cause the death of HepG2 cells. (B) The triple treatment decreased the phosphorylation of Akt protein and increased the conversion of LC3-I to LC3-II in the HepG2 cells. The band intensities of LC3-II and LC3-I were quantified to calculate the LC3-II/LC3-I ratio. (C) The triple treatment triggered the activations of caspase-3 and PARP in the HepG2 cells. The cleaved PARP was quantified relative to the full-length PARP. β-actin was used as a loading control for each Western blot assay. (*** is used for P

Techniques Used: MTT Assay, Western Blot

Effects of the combined triple treatment on the intracellular proteins in the PANC-1 cells. The protein expression levels of p-Akt, LC3-II, PARP, cleaved caspase-9, and cleaved caspase-3 were measured using Western blot analysis. β-actin was used as a loading control. (A) The triple treatment decreased the phosphorylation of Akt protein and increased the conversion of LC3-I to LC3-II. The band intensities of LC3-II and LC3-I were quantified to calculate the LC3-II/LC3-I ratio. (B) Apoptotic proteins were activated after the cells were treated with the combined triple treatment. The cleaved PARP was quantified relative to the full-length PARP. These results suggested that both the apoptosis and the autophagy were triggered in the PANC-1 cells by the triple combined treatment. (*** is used for P
Figure Legend Snippet: Effects of the combined triple treatment on the intracellular proteins in the PANC-1 cells. The protein expression levels of p-Akt, LC3-II, PARP, cleaved caspase-9, and cleaved caspase-3 were measured using Western blot analysis. β-actin was used as a loading control. (A) The triple treatment decreased the phosphorylation of Akt protein and increased the conversion of LC3-I to LC3-II. The band intensities of LC3-II and LC3-I were quantified to calculate the LC3-II/LC3-I ratio. (B) Apoptotic proteins were activated after the cells were treated with the combined triple treatment. The cleaved PARP was quantified relative to the full-length PARP. These results suggested that both the apoptosis and the autophagy were triggered in the PANC-1 cells by the triple combined treatment. (*** is used for P

Techniques Used: Expressing, Western Blot

Effects of the autophagy and apoptosis inhibitors on the triple treatment-induced growth inhibition in the PANC-1 cells. The 3-MA and Z-VAD-FMK were used for the autophagy and apoptosis inhibitor analysis, respectively. (A) The PANC-1 cells were pretreated with the autophagy inhibitor 3-MA and the caspase inhibitor Z-VAD-FMK before the triple treatment for 24 h. The result showed that both 3-MA and Z-VAD-FMK could partially enervate the triple treatment-induced growth inhibition in the PANC-1 cells. (B) The pretreatment of Z-VAD-FMK significantly reduced the levels of cleaved caspase-3 and cleaved PARP. The cleaved PARP was quantified relative to the full-length PARP. (C) The pretreatment of 3-MA blocked the conversion of LC3-I to LC3-II. The band intensities of LC3-II and LC3-I were quantified to calculate the LC3-II/LC3-I ratio. β-actin was used as a loading control for each Western blot assay. (D) To better understand the role of autophagy in the triple treatment, 3-MA pretreatment was applied to the combination treatments using US stimulation. (* is used for P
Figure Legend Snippet: Effects of the autophagy and apoptosis inhibitors on the triple treatment-induced growth inhibition in the PANC-1 cells. The 3-MA and Z-VAD-FMK were used for the autophagy and apoptosis inhibitor analysis, respectively. (A) The PANC-1 cells were pretreated with the autophagy inhibitor 3-MA and the caspase inhibitor Z-VAD-FMK before the triple treatment for 24 h. The result showed that both 3-MA and Z-VAD-FMK could partially enervate the triple treatment-induced growth inhibition in the PANC-1 cells. (B) The pretreatment of Z-VAD-FMK significantly reduced the levels of cleaved caspase-3 and cleaved PARP. The cleaved PARP was quantified relative to the full-length PARP. (C) The pretreatment of 3-MA blocked the conversion of LC3-I to LC3-II. The band intensities of LC3-II and LC3-I were quantified to calculate the LC3-II/LC3-I ratio. β-actin was used as a loading control for each Western blot assay. (D) To better understand the role of autophagy in the triple treatment, 3-MA pretreatment was applied to the combination treatments using US stimulation. (* is used for P

Techniques Used: Inhibition, Western Blot

23) Product Images from "An adjustment in BMP4 function represents a treatment for diabetic nephropathy and podocyte injury"

Article Title: An adjustment in BMP4 function represents a treatment for diabetic nephropathy and podocyte injury

Journal: Scientific Reports

doi: 10.1038/s41598-018-31464-9

BMP4 induced apoptotic signaling in cultured podocytes. (A) Cultured podocytes were treated with BMP4 (20 ng/ml) at the indicated time points. BMP4 treatment of podocytes increased levels of phosphorylated p38 in a time-dependent manner. (B) BMP4 decreased nephrin expression and increased cleaved caspase-3 levels after 12 days of stimulation. (C) Cultured podocytes were untreated or incubated with dorsomorphin (Dorso), SB203580, SB242235 or SB202190 for 24 hr and then stimulated with BMP4. Each inhibitor was added to the culture medium at a concentration of 500 nM. The pp38 levels were decreased in cells treated with dorsomorphin, SB242235 and SB202190. The pSmad1 levels were decreased in response to dorsomorphin treatments. The cleaved caspase-3 levels were decreased in cells treated with SB242235 and SB202190. Total cell lysates were immunoblotted with anti-pp38, anti-nephrin, anti-cleaved caspase-3, anti-pSmad1, anti-GAPDH, anti-β-actin and anti-α-tubulin antibodies.
Figure Legend Snippet: BMP4 induced apoptotic signaling in cultured podocytes. (A) Cultured podocytes were treated with BMP4 (20 ng/ml) at the indicated time points. BMP4 treatment of podocytes increased levels of phosphorylated p38 in a time-dependent manner. (B) BMP4 decreased nephrin expression and increased cleaved caspase-3 levels after 12 days of stimulation. (C) Cultured podocytes were untreated or incubated with dorsomorphin (Dorso), SB203580, SB242235 or SB202190 for 24 hr and then stimulated with BMP4. Each inhibitor was added to the culture medium at a concentration of 500 nM. The pp38 levels were decreased in cells treated with dorsomorphin, SB242235 and SB202190. The pSmad1 levels were decreased in response to dorsomorphin treatments. The cleaved caspase-3 levels were decreased in cells treated with SB242235 and SB202190. Total cell lysates were immunoblotted with anti-pp38, anti-nephrin, anti-cleaved caspase-3, anti-pSmad1, anti-GAPDH, anti-β-actin and anti-α-tubulin antibodies.

Techniques Used: Cell Culture, Expressing, Incubation, Concentration Assay

Diabetic mice and BMP4 induced mice exhibited apoptotic damage in glomeruli. (A) Images of immunohistochemistry for cleaved caspase-3 in the DM model, Bmp4 tgm and CAG-B4xPod-Cre mice. The WT DM mice (c) exhibited an increase in the positively stained area compared with the WT control mice (a). Yellow represents the area in which cleaved caspase-3 and nephrin expression overlap (c, arrowhead). Bmp4 +/− DM mice (d) exhibited a reduced positively stained area compared with WT DM mice. Bmp4 tgm (+) (f) and CAG-B4xPod-Cre (h) mice displayed increased positively stained areas after Cre-mediated recombination. Cleaved caspase-3-stained areas are shown in red in Bmp4 tgm (+) (f, arrowhead) and green in CAG-B4xPod-Cre mice (h, arrowhead). (B) Percentage of apoptotic cells in the glomeruli, as determined by a TUNEL assay. Apoptosis was frequently observed in the glomeruli of WT DM (c), Bmp4 tgm (+) (j, red color) and CAG-B4xPod-Cre mice (l). Bmp4 +/− DM mice (d) exhibited reduced apoptosis. TUNEL staining is shown in green and red; blue represents the counterstain.
Figure Legend Snippet: Diabetic mice and BMP4 induced mice exhibited apoptotic damage in glomeruli. (A) Images of immunohistochemistry for cleaved caspase-3 in the DM model, Bmp4 tgm and CAG-B4xPod-Cre mice. The WT DM mice (c) exhibited an increase in the positively stained area compared with the WT control mice (a). Yellow represents the area in which cleaved caspase-3 and nephrin expression overlap (c, arrowhead). Bmp4 +/− DM mice (d) exhibited a reduced positively stained area compared with WT DM mice. Bmp4 tgm (+) (f) and CAG-B4xPod-Cre (h) mice displayed increased positively stained areas after Cre-mediated recombination. Cleaved caspase-3-stained areas are shown in red in Bmp4 tgm (+) (f, arrowhead) and green in CAG-B4xPod-Cre mice (h, arrowhead). (B) Percentage of apoptotic cells in the glomeruli, as determined by a TUNEL assay. Apoptosis was frequently observed in the glomeruli of WT DM (c), Bmp4 tgm (+) (j, red color) and CAG-B4xPod-Cre mice (l). Bmp4 +/− DM mice (d) exhibited reduced apoptosis. TUNEL staining is shown in green and red; blue represents the counterstain.

Techniques Used: Mouse Assay, Immunohistochemistry, Staining, Expressing, TUNEL Assay

24) Product Images from "Efficacy of Prednisolone in Generated Myotubes Derived From Fibroblasts of Duchenne Muscular Dystrophy Patients"

Article Title: Efficacy of Prednisolone in Generated Myotubes Derived From Fibroblasts of Duchenne Muscular Dystrophy Patients

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2018.01402

Efficacy of prednisolone in reversing DMD pathology. (A) Immunostaining using skeletal muscle marker (myosin heavy chain, MHC), and apoptosis marker (cleaved caspase-3, Cas). Prednisolone (PDN) restored decreased MHC area and increased apoptosis cells. Scale bars = 100 μm. (B) Quantification of the MHC area of myotubes from control individual, non-treated myotubes from DMD (NT), and prednisolone-treated myotubes from DMD (PDN). Data are shown as means ± SEM ( n = 4). (C) Western blot analysis of cleaved caspase-3 in control individual (Ctrl), NT, and PDN. Data are shown as means ± SEM ( n = 5). # p
Figure Legend Snippet: Efficacy of prednisolone in reversing DMD pathology. (A) Immunostaining using skeletal muscle marker (myosin heavy chain, MHC), and apoptosis marker (cleaved caspase-3, Cas). Prednisolone (PDN) restored decreased MHC area and increased apoptosis cells. Scale bars = 100 μm. (B) Quantification of the MHC area of myotubes from control individual, non-treated myotubes from DMD (NT), and prednisolone-treated myotubes from DMD (PDN). Data are shown as means ± SEM ( n = 4). (C) Western blot analysis of cleaved caspase-3 in control individual (Ctrl), NT, and PDN. Data are shown as means ± SEM ( n = 5). # p

Techniques Used: Immunostaining, Marker, Western Blot

25) Product Images from "Identification and Characterization of NTB451 as a Potential Inhibitor of Necroptosis"

Article Title: Identification and Characterization of NTB451 as a Potential Inhibitor of Necroptosis

Journal: Molecules

doi: 10.3390/molecules23112884

Effect of NTB451 on TNF-α-induced NF-κB activation and on the apoptotic cell death induced by TNF-α and FasL. ( A ) L929 cells were treated with TNF-α (400 units/mL) for the indicated times in the presence or absence of NTB451 (20 µM). Cells were lysed and immunoblotted with the antibodies against phospho-IκB, IκB, and β-actin. ( B ) L929 cells were treated with TNF-α (400 units/mL) plus CHX (10 µg/mL) for 3 h in the presence or absence of NTB451 (20 µM) or Nec-1 (10 µM), and cell apoptosis was analyzed by immunoblotting with anti-cleaved PARP and anti-cleaved caspase-3 in cell lysates. * indicates a nonspecific band. ( C ) Apoptotic cell death in Jurkat cells was induced with 5% FasL for 3 h in the presence or absence of NTB451 (20 µM), Nec-1 (10 µM) or zVAD (20 µM). Then, the cells were stained with PE-conjugated Annexin V and TO-PRO-3 (10 nM), followed by FACS analysis.
Figure Legend Snippet: Effect of NTB451 on TNF-α-induced NF-κB activation and on the apoptotic cell death induced by TNF-α and FasL. ( A ) L929 cells were treated with TNF-α (400 units/mL) for the indicated times in the presence or absence of NTB451 (20 µM). Cells were lysed and immunoblotted with the antibodies against phospho-IκB, IκB, and β-actin. ( B ) L929 cells were treated with TNF-α (400 units/mL) plus CHX (10 µg/mL) for 3 h in the presence or absence of NTB451 (20 µM) or Nec-1 (10 µM), and cell apoptosis was analyzed by immunoblotting with anti-cleaved PARP and anti-cleaved caspase-3 in cell lysates. * indicates a nonspecific band. ( C ) Apoptotic cell death in Jurkat cells was induced with 5% FasL for 3 h in the presence or absence of NTB451 (20 µM), Nec-1 (10 µM) or zVAD (20 µM). Then, the cells were stained with PE-conjugated Annexin V and TO-PRO-3 (10 nM), followed by FACS analysis.

Techniques Used: Activation Assay, Staining, FACS

26) Product Images from "DIAPH1 Is Upregulated and Inhibits Cell Apoptosis through ATR/p53/Caspase-3 Signaling Pathway in Laryngeal Squamous Cell Carcinoma"

Article Title: DIAPH1 Is Upregulated and Inhibits Cell Apoptosis through ATR/p53/Caspase-3 Signaling Pathway in Laryngeal Squamous Cell Carcinoma

Journal: Disease Markers

doi: 10.1155/2019/6716472

DIAPH1 knockdown promoted apoptosis via ATR/p53/caspase-3 signaling pathway. (a) Western blot analysis of ATR, p-p53, Bax, and cleaved caspase-3, -8, and -9 in DIAPH1 knockdown (SH1) and control (NC) of AMC-HN-8 and FD-LSC-1 cells. Numbers labelled under the bands were the relative expression and the relative expression of NC cells was set as 1. (b) The schematic graph of apoptotic signaling pathway mediated by DIAPH1.
Figure Legend Snippet: DIAPH1 knockdown promoted apoptosis via ATR/p53/caspase-3 signaling pathway. (a) Western blot analysis of ATR, p-p53, Bax, and cleaved caspase-3, -8, and -9 in DIAPH1 knockdown (SH1) and control (NC) of AMC-HN-8 and FD-LSC-1 cells. Numbers labelled under the bands were the relative expression and the relative expression of NC cells was set as 1. (b) The schematic graph of apoptotic signaling pathway mediated by DIAPH1.

Techniques Used: Western Blot, Expressing

27) Product Images from "Formation of high molecular weight p62 by CORM-3"

Article Title: Formation of high molecular weight p62 by CORM-3

Journal: PLoS ONE

doi: 10.1371/journal.pone.0210474

Generation of HMW-FN by CORM-3 in A549 and H9c2 cells. A549 cells (A-E) or H9c2 cells (G-K) treated with verteporfin (10 μM, 6 hours) or CORM-3 (0–1 mM, 24 or 72 hours) were subjected to immunoblot analysis using anti-p62 and anti-cleaved caspase 3 (c-cas3) antibodies. #, uncharacterized fragment. Percentages of HMW-p62 in total (HMW+mono) p62 and relative levels of c-cas3 (p17) to actin are also shown. (D and J) Percentages of LDH released from A549 (D) and H9c2 (J) cells into the medium 72 hours after treatment with the indicated concentrations of CORM-3. Graphs show means and S.E. (n = 4). **, p
Figure Legend Snippet: Generation of HMW-FN by CORM-3 in A549 and H9c2 cells. A549 cells (A-E) or H9c2 cells (G-K) treated with verteporfin (10 μM, 6 hours) or CORM-3 (0–1 mM, 24 or 72 hours) were subjected to immunoblot analysis using anti-p62 and anti-cleaved caspase 3 (c-cas3) antibodies. #, uncharacterized fragment. Percentages of HMW-p62 in total (HMW+mono) p62 and relative levels of c-cas3 (p17) to actin are also shown. (D and J) Percentages of LDH released from A549 (D) and H9c2 (J) cells into the medium 72 hours after treatment with the indicated concentrations of CORM-3. Graphs show means and S.E. (n = 4). **, p

Techniques Used:

CORM-3 induces the death of MEFs. (A and B) MEFs were treated with the indicated concentrations (0, 0.1, or 1 mM) of CORM-3 for 24 or 48 hours (A), and the relative levels of p17 fragment of cleaved-caspase-3 to actin (B and C) as well as LDH release into the medium (D and E) are shown. #, uncharacterized fragment. Data represent the means and S.E. *, p
Figure Legend Snippet: CORM-3 induces the death of MEFs. (A and B) MEFs were treated with the indicated concentrations (0, 0.1, or 1 mM) of CORM-3 for 24 or 48 hours (A), and the relative levels of p17 fragment of cleaved-caspase-3 to actin (B and C) as well as LDH release into the medium (D and E) are shown. #, uncharacterized fragment. Data represent the means and S.E. *, p

Techniques Used:

28) Product Images from "miR-573 regulates cell proliferation and apoptosis by targeting Bax in nucleus pulposus cells"

Article Title: miR-573 regulates cell proliferation and apoptosis by targeting Bax in nucleus pulposus cells

Journal: Cellular & Molecular Biology Letters

doi: 10.1186/s11658-018-0132-y

Overexpression of miR-573 markedly increased the expression of Bcl-2 and decreased the expression of cleaved caspase-3 and cleaved caspase-9 in degenerative NP cells. Protein expression levels of Bcl-2, cleaved caspase-3, cleaved caspase-9, caspase-3 and caspase-9 in degenerative NP cells were evaluated using Western blotting assay. *** P
Figure Legend Snippet: Overexpression of miR-573 markedly increased the expression of Bcl-2 and decreased the expression of cleaved caspase-3 and cleaved caspase-9 in degenerative NP cells. Protein expression levels of Bcl-2, cleaved caspase-3, cleaved caspase-9, caspase-3 and caspase-9 in degenerative NP cells were evaluated using Western blotting assay. *** P

Techniques Used: Over Expression, Expressing, Western Blot

29) Product Images from "Cilostazol protects hepatocytes against alcohol-induced apoptosis via activation of AMPK pathway"

Article Title: Cilostazol protects hepatocytes against alcohol-induced apoptosis via activation of AMPK pathway

Journal: PLoS ONE

doi: 10.1371/journal.pone.0211415

The role of AMPK in the effects of cilostazol on ethanol-induced apoptosis of hepatocytes. (A) Cells were treated with cilostazol (0 ~ 100 μM) in the presence or absence of compound C (10 μM) for 10 min. Phosphorylated- and total-AMPK and ACC were detected by Western blotting. (B—D) Cells were treated with ethanol (100 and 200 mM) in the presence of AICAR (100 μM), cilostazol (100 μM), compound C (10 μM) or compound C (10 μM) plus cilostazol (100 μM) for 24 h. Cleaved caspase-3 (C-caspase-3) or cleaved PARP (C-PARP) were detected by Western blotting (B, C). The caspase-3 activity was measured (D). (E) Cells were treated with AMPK siRNA or scramble siRNA for 48 h and then treated with cilostazol (100 μM) for 10 min. Phosphorylated- and total-AMPK and ACC were detected by Western blotting. (F, G) Cells were treated with AMPK siRNA or scramble siRNA for 24 h and then stimulated with ethanol (100 mM) in the presence or absence of cilostazol (100 μM) for 24 h. Caspase-3 activity (F) and cell viability (G) were measured. Representative microscopic images from three independent experiments were presented (magnification, x 400). The blots are representative of three independent experiments. Data represented as fold increases is mean±S.E.M. of three independent experiments. * P
Figure Legend Snippet: The role of AMPK in the effects of cilostazol on ethanol-induced apoptosis of hepatocytes. (A) Cells were treated with cilostazol (0 ~ 100 μM) in the presence or absence of compound C (10 μM) for 10 min. Phosphorylated- and total-AMPK and ACC were detected by Western blotting. (B—D) Cells were treated with ethanol (100 and 200 mM) in the presence of AICAR (100 μM), cilostazol (100 μM), compound C (10 μM) or compound C (10 μM) plus cilostazol (100 μM) for 24 h. Cleaved caspase-3 (C-caspase-3) or cleaved PARP (C-PARP) were detected by Western blotting (B, C). The caspase-3 activity was measured (D). (E) Cells were treated with AMPK siRNA or scramble siRNA for 48 h and then treated with cilostazol (100 μM) for 10 min. Phosphorylated- and total-AMPK and ACC were detected by Western blotting. (F, G) Cells were treated with AMPK siRNA or scramble siRNA for 24 h and then stimulated with ethanol (100 mM) in the presence or absence of cilostazol (100 μM) for 24 h. Caspase-3 activity (F) and cell viability (G) were measured. Representative microscopic images from three independent experiments were presented (magnification, x 400). The blots are representative of three independent experiments. Data represented as fold increases is mean±S.E.M. of three independent experiments. * P

Techniques Used: Western Blot, Activity Assay

The regulation of AMPK by LKB1 and CaMKK2 in cilostazol treated hepatocytes. (A) Cells were treated with LKB1- and CaMKK2- siRNA or scramble siRNA for 48 h and then treated with cilostazol (100 μM) for 10 min. Phosphorylated- and total-LKB1, CaMKK2, AMPK and ACC were detected by Western blotting. (B, C) Cells were treated with LKB1- and CaMKK2- siRNA or scramble siRNA for 24 h and then stimulated with ethanol (100 mM) in the presence or absence of cilostazol (100 μM) for 24 h. Caspase-3 activity (B) and cell viability (C) were measured. (D) Cells were treated with cilostazol (100 μM) for 10 min in the presence or absence of KT5720 (1 μM), SQ22536 (400 μM) or STO-609 (5 μM). Phosphorylated- and total- AMPK and ACC were detected by Western blotting. (E) Cells were treated with ethanol (100 mM) in the presence or absence of cilostazol (100 μM), STO-609 (5 μM) or KT5720 (1 μM) for 24 h. Then, cell viability was measured by MTS assay. The blots are representative of three independent experiments. Data represented as fold increases is mean±S.E.M. of three independent experiments. *** P
Figure Legend Snippet: The regulation of AMPK by LKB1 and CaMKK2 in cilostazol treated hepatocytes. (A) Cells were treated with LKB1- and CaMKK2- siRNA or scramble siRNA for 48 h and then treated with cilostazol (100 μM) for 10 min. Phosphorylated- and total-LKB1, CaMKK2, AMPK and ACC were detected by Western blotting. (B, C) Cells were treated with LKB1- and CaMKK2- siRNA or scramble siRNA for 24 h and then stimulated with ethanol (100 mM) in the presence or absence of cilostazol (100 μM) for 24 h. Caspase-3 activity (B) and cell viability (C) were measured. (D) Cells were treated with cilostazol (100 μM) for 10 min in the presence or absence of KT5720 (1 μM), SQ22536 (400 μM) or STO-609 (5 μM). Phosphorylated- and total- AMPK and ACC were detected by Western blotting. (E) Cells were treated with ethanol (100 mM) in the presence or absence of cilostazol (100 μM), STO-609 (5 μM) or KT5720 (1 μM) for 24 h. Then, cell viability was measured by MTS assay. The blots are representative of three independent experiments. Data represented as fold increases is mean±S.E.M. of three independent experiments. *** P

Techniques Used: Western Blot, Activity Assay, MTS Assay

The role of cAMP in the effects of cilostazol on ethanol-induced apoptosis of hepatocytes. (A) Cells were treated with cilostazol (100 μM), forskolin (10 μM) for 30 min in the presence or absence of SQ22536 (400 μM). Intracellular cAMP concentration was measured. (B) Cells were treated with ethanol (100 mM) for 24 h in the presence of forskolin (1 ~ 10 μM), cilostazol (100 μM), or SQ22536 (400 μM). Caspase-3 activity was measured. Data represented as fold increases are mean±S.E.M. of three independent experiments. ** P
Figure Legend Snippet: The role of cAMP in the effects of cilostazol on ethanol-induced apoptosis of hepatocytes. (A) Cells were treated with cilostazol (100 μM), forskolin (10 μM) for 30 min in the presence or absence of SQ22536 (400 μM). Intracellular cAMP concentration was measured. (B) Cells were treated with ethanol (100 mM) for 24 h in the presence of forskolin (1 ~ 10 μM), cilostazol (100 μM), or SQ22536 (400 μM). Caspase-3 activity was measured. Data represented as fold increases are mean±S.E.M. of three independent experiments. ** P

Techniques Used: Concentration Assay, Activity Assay

Effects of cilostazol on ethanol-induced apoptosis of hepatocytes. (A, B) Cells were treated with 100 mM of ethanol for 24 h in the presence or absence of different concentration of cilostazol (0 ~ 100 μM). Cell viability (A) and caspase-3 activity (B) were measured by MTS assay and activity assay as described in “methods”. (C—E) Cells were treated with 100 mM or 200 mM ethanol for 24 h in the presence or absence of cilostazol (100 μM) alone or together with compound C (10 μM). The cleaved caspase-3 (C-caspase-3) and cleaved PARP (C-PARP) were detected by Western blotting. The blots are representative of four independent experiments. Protein levels were normalized to GAPDH. The relative band intensities are presented (C). Nuclear morphology was monitored by fluorescence microscopy after staining cells with Hoechst 33342. Arrows indicate nuclear condensation or nuclear fragmentation (D). TUNEL positive cells were monitored by fluorescence microscopy after staining cells with propidium iodide (red) and TUNEL (green) (E). Representative microscopic images from three independent experiments were presented (magnification, x 400). Data represented as fold increases are mean±S.E.M. of three independent experiments. * P
Figure Legend Snippet: Effects of cilostazol on ethanol-induced apoptosis of hepatocytes. (A, B) Cells were treated with 100 mM of ethanol for 24 h in the presence or absence of different concentration of cilostazol (0 ~ 100 μM). Cell viability (A) and caspase-3 activity (B) were measured by MTS assay and activity assay as described in “methods”. (C—E) Cells were treated with 100 mM or 200 mM ethanol for 24 h in the presence or absence of cilostazol (100 μM) alone or together with compound C (10 μM). The cleaved caspase-3 (C-caspase-3) and cleaved PARP (C-PARP) were detected by Western blotting. The blots are representative of four independent experiments. Protein levels were normalized to GAPDH. The relative band intensities are presented (C). Nuclear morphology was monitored by fluorescence microscopy after staining cells with Hoechst 33342. Arrows indicate nuclear condensation or nuclear fragmentation (D). TUNEL positive cells were monitored by fluorescence microscopy after staining cells with propidium iodide (red) and TUNEL (green) (E). Representative microscopic images from three independent experiments were presented (magnification, x 400). Data represented as fold increases are mean±S.E.M. of three independent experiments. * P

Techniques Used: Concentration Assay, Activity Assay, MTS Assay, Western Blot, Fluorescence, Microscopy, Staining, TUNEL Assay

Effects of cilostazol on apoptosis and AMPK signaling in liver from binge alcohol treated mice. (A) After ethanol binge drinking (5 g/kg body weight) by oral gavage, caspase-3 activity was measured at different times. (B—D) Mice were subjected to intraperitoneal injection of cilostazol (10 mg/kg/day) for 4 days following a single dose of ethanol (5 g/kg) intake. Liver samples were taken at 6 h after ethanol intake. (C) Liver sections were stained with H E (a-c) or anti-cleaved caspase-3 antibody (d-f). Cleaved caspase-3 positive cells showed cytoplasmic tan staining (magnification, x 400). Caspase-3 activity and AMPK signaling were measured by activity assay (B) and Western blotting (D), respectively. Three representative lanes of five different samples are shown in each group. Data represented as fold increases (A, B) or % of relative band intensity (D) are mean±S.E.M. (n = 5 mice per group). * P
Figure Legend Snippet: Effects of cilostazol on apoptosis and AMPK signaling in liver from binge alcohol treated mice. (A) After ethanol binge drinking (5 g/kg body weight) by oral gavage, caspase-3 activity was measured at different times. (B—D) Mice were subjected to intraperitoneal injection of cilostazol (10 mg/kg/day) for 4 days following a single dose of ethanol (5 g/kg) intake. Liver samples were taken at 6 h after ethanol intake. (C) Liver sections were stained with H E (a-c) or anti-cleaved caspase-3 antibody (d-f). Cleaved caspase-3 positive cells showed cytoplasmic tan staining (magnification, x 400). Caspase-3 activity and AMPK signaling were measured by activity assay (B) and Western blotting (D), respectively. Three representative lanes of five different samples are shown in each group. Data represented as fold increases (A, B) or % of relative band intensity (D) are mean±S.E.M. (n = 5 mice per group). * P

Techniques Used: Mouse Assay, Activity Assay, Injection, Staining, Western Blot

30) Product Images from "miR-16-2-3p inhibits cell proliferation and migration and induces apoptosis by targeting PDPK1 in maxillary primordium mesenchymal cells"

Article Title: miR-16-2-3p inhibits cell proliferation and migration and induces apoptosis by targeting PDPK1 in maxillary primordium mesenchymal cells

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2019.4070

miR-16-2-3p inhibition decreases MPMC apoptosis. (A) Flow cytometry analysis was performed to measure cell apoptosis in siNC and si16-2-3-p MPMC groups. (B) Quantification of the flow cytometry data. (C) Western blot analysis of cleaved caspase-3 protein expression. Independent experiments were repeated at least 3 times, and the values are expressed as the mean ± standard deviation. * P
Figure Legend Snippet: miR-16-2-3p inhibition decreases MPMC apoptosis. (A) Flow cytometry analysis was performed to measure cell apoptosis in siNC and si16-2-3-p MPMC groups. (B) Quantification of the flow cytometry data. (C) Western blot analysis of cleaved caspase-3 protein expression. Independent experiments were repeated at least 3 times, and the values are expressed as the mean ± standard deviation. * P

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

Effect of miR-16-2-3p on MPMC apoptosis. (A) Apoptosis in preNC and pre16-2-3p MPMC groups was assessed by flow cytometry analysis. (B) Quantification of the level of apoptosis in preNC and pre16-2-3p groups. (C) Western blot analysis of cleaved caspase-3 protein expression. Independent experiments were repeated at least 3 times, and the values are expressed as the mean ± standard deviation. ** P
Figure Legend Snippet: Effect of miR-16-2-3p on MPMC apoptosis. (A) Apoptosis in preNC and pre16-2-3p MPMC groups was assessed by flow cytometry analysis. (B) Quantification of the level of apoptosis in preNC and pre16-2-3p groups. (C) Western blot analysis of cleaved caspase-3 protein expression. Independent experiments were repeated at least 3 times, and the values are expressed as the mean ± standard deviation. ** P

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

31) Product Images from "Phosphatase Actin Regulator-1 (PHACTR-1) Knockdown Suppresses Cell Proliferation and Migration and Promotes Cell Apoptosis in the bEnd.3 Mouse Brain Capillary Endothelial Cell Line"

Article Title: Phosphatase Actin Regulator-1 (PHACTR-1) Knockdown Suppresses Cell Proliferation and Migration and Promotes Cell Apoptosis in the bEnd.3 Mouse Brain Capillary Endothelial Cell Line

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

doi: 10.12659/MSM.912586

The expression of MMP-2, MMP-9, Bax, Bcl-2, cleaved caspase-3, and caspase-3 in the bEnd.3 mouse brain capillary endothelial cells. ( A, C, E ) Western blot s for protein expressions of MMP-2, MMP-9, Bax, Bcl-2, cleaved caspase-3, and caspase-3 in the NC and KD cell groups. β-tubulin and GAPDH were used as the housekeeping proteins. ( B, D, F ) Bar graphs showed the quantification of MMP-2, MMP-9 protein expression and the Bax/Bcl-2 ratio and cleaved caspase-3/caspase-3 ratio in the NC and KD cell groups. Data are presented as the mean ±SD. ** P
Figure Legend Snippet: The expression of MMP-2, MMP-9, Bax, Bcl-2, cleaved caspase-3, and caspase-3 in the bEnd.3 mouse brain capillary endothelial cells. ( A, C, E ) Western blot s for protein expressions of MMP-2, MMP-9, Bax, Bcl-2, cleaved caspase-3, and caspase-3 in the NC and KD cell groups. β-tubulin and GAPDH were used as the housekeeping proteins. ( B, D, F ) Bar graphs showed the quantification of MMP-2, MMP-9 protein expression and the Bax/Bcl-2 ratio and cleaved caspase-3/caspase-3 ratio in the NC and KD cell groups. Data are presented as the mean ±SD. ** P

Techniques Used: Expressing, Western Blot

32) Product Images from "Celecoxib-induced apoptosis is enhanced by ABT-737 and by inhibition of autophagy in human colorectal cancer cells"

Article Title: Celecoxib-induced apoptosis is enhanced by ABT-737 and by inhibition of autophagy in human colorectal cancer cells

Journal: Autophagy

doi:

Prosurvival Bcl-2 proteins negatively regulate celecoxib (coxib)-induced apoptosis. (A) SW480 colon cancer cells with/without ectopic Bcl-2 expression were incubated with celecoxib and/or ABT-737 at indicated doses for 48 h at 37°C. Cytotoxicity was determined by the MTS assay with absorbance measured at 492 nm. Cells were treated with celecoxib at the given doses (48 h) and lysates were analyzed for caspase-3 cleavage. Columns , mean of triplicate experiments; bars , SD. *p
Figure Legend Snippet: Prosurvival Bcl-2 proteins negatively regulate celecoxib (coxib)-induced apoptosis. (A) SW480 colon cancer cells with/without ectopic Bcl-2 expression were incubated with celecoxib and/or ABT-737 at indicated doses for 48 h at 37°C. Cytotoxicity was determined by the MTS assay with absorbance measured at 492 nm. Cells were treated with celecoxib at the given doses (48 h) and lysates were analyzed for caspase-3 cleavage. Columns , mean of triplicate experiments; bars , SD. *p

Techniques Used: Expressing, Incubation, MTS Assay

33) Product Images from "Niaspan Treatment Induces Neuroprotection After Stroke"

Article Title: Niaspan Treatment Induces Neuroprotection After Stroke

Journal: Neurobiology of disease

doi: 10.1016/j.nbd.2010.05.034

Western Blot Assay: Niaspan Treatment Of Stroke Reduces TNF-Alpha And Cleaved Caspase-3 Expression And Increases P-Akt Activity In The Ischemic Brain
Figure Legend Snippet: Western Blot Assay: Niaspan Treatment Of Stroke Reduces TNF-Alpha And Cleaved Caspase-3 Expression And Increases P-Akt Activity In The Ischemic Brain

Techniques Used: Western Blot, Expressing, Activity Assay

34) Product Images from "Loss of PTPN12 Stimulates Progression of ErbB2-Dependent Breast Cancer by Enhancing Cell Survival, Migration, and Epithelial-to-Mesenchymal Transition"

Article Title: Loss of PTPN12 Stimulates Progression of ErbB2-Dependent Breast Cancer by Enhancing Cell Survival, Migration, and Epithelial-to-Mesenchymal Transition

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00741-15

Impact of PTPN12 deficiency on breast cancer cell proliferation and survival. (A) Expression of Ki67 in primary tumors (top) or tumor-derived cell lines (bottom) was analyzed by immunofluorescence. Staining of representative samples with DAPI, which marks nuclei, and a merged image are shown. Bars, 25 μm. The graphs show mean fluorescence intensity in 10 different fields from 3 tumor samples per genotype, with standard deviations. ns, not significant (for tumor tissues,  P  = 0.4986; for cell lines,  P  = 0.7062). WT, wild-type mice (MMTV-NIC  Ptpn12 +/+ ); PTPN12 KO, PTPN12-deficient mice (MMTV-NIC  Ptpn12 fl/fl ).  ( B) Quantitative real-time PCR analysis of Ki67 expression in tumor-derived cell lines or primary tumor tissues. RNA samples from four epithelial cell lines or tumor tissues of each genotype were analyzed for quantitative PCR. Ratios of relative expression in KO compared to WT are depicted. Average values with standard deviations are shown. (C) Proliferation of multiple tumor-derived cell lines over 6 days was analyzed using the cell proliferation dye WST1. Absorbance at 450 nm was measured. Average values from triplicates with standard deviations are shown. (D) Activated (cleaved) caspase 3 was measured in primary tumors using immunohistochemistry. Representative samples are shown on the left. Bars, 60 μm. Percentages of cells positive for activated caspase 3 in 25 fields of six tumor samples per genotype are depicted on the right. Average values with standard deviations are shown. **,  P  = 0.0032. (E )  Susceptibility of tumor-derived cell lines to anoikis was determined by staining with annexin V and propidium iodide (PI) and flow cytometry. Representative samples are shown on the left. Annexin V- and PI-positive cells (circled, with percentages) correspond to apoptotic cells. The bar graph shows data for four independent cell lines of each genotype. Values are averages from 3 independent experiments, with standard deviations. *,  P  = 0.0161.
Figure Legend Snippet: Impact of PTPN12 deficiency on breast cancer cell proliferation and survival. (A) Expression of Ki67 in primary tumors (top) or tumor-derived cell lines (bottom) was analyzed by immunofluorescence. Staining of representative samples with DAPI, which marks nuclei, and a merged image are shown. Bars, 25 μm. The graphs show mean fluorescence intensity in 10 different fields from 3 tumor samples per genotype, with standard deviations. ns, not significant (for tumor tissues, P = 0.4986; for cell lines, P = 0.7062). WT, wild-type mice (MMTV-NIC Ptpn12 +/+ ); PTPN12 KO, PTPN12-deficient mice (MMTV-NIC Ptpn12 fl/fl ). ( B) Quantitative real-time PCR analysis of Ki67 expression in tumor-derived cell lines or primary tumor tissues. RNA samples from four epithelial cell lines or tumor tissues of each genotype were analyzed for quantitative PCR. Ratios of relative expression in KO compared to WT are depicted. Average values with standard deviations are shown. (C) Proliferation of multiple tumor-derived cell lines over 6 days was analyzed using the cell proliferation dye WST1. Absorbance at 450 nm was measured. Average values from triplicates with standard deviations are shown. (D) Activated (cleaved) caspase 3 was measured in primary tumors using immunohistochemistry. Representative samples are shown on the left. Bars, 60 μm. Percentages of cells positive for activated caspase 3 in 25 fields of six tumor samples per genotype are depicted on the right. Average values with standard deviations are shown. **, P = 0.0032. (E ) Susceptibility of tumor-derived cell lines to anoikis was determined by staining with annexin V and propidium iodide (PI) and flow cytometry. Representative samples are shown on the left. Annexin V- and PI-positive cells (circled, with percentages) correspond to apoptotic cells. The bar graph shows data for four independent cell lines of each genotype. Values are averages from 3 independent experiments, with standard deviations. *, P = 0.0161.

Techniques Used: Expressing, Derivative Assay, Immunofluorescence, Staining, Fluorescence, Mouse Assay, Real-time Polymerase Chain Reaction, Immunohistochemistry, Flow Cytometry, Cytometry

35) Product Images from "Chronic cardiotoxicity of doxorubicin involves activation of myocardial and circulating matrix metalloproteinases in rats"

Article Title: Chronic cardiotoxicity of doxorubicin involves activation of myocardial and circulating matrix metalloproteinases in rats

Journal: Acta Pharmacologica Sinica

doi: 10.1038/aps.2011.194

Effect of chronic DOX treatment on protein levels iNOS and cleaved caspase-3. For the analysis protein fractions isolated from the left ventricular tissue of control (C) and doxorubicin-treated (DOX) rat hearts obtained 4 weeks (C-4, DOX-4) or 8 weeks
Figure Legend Snippet: Effect of chronic DOX treatment on protein levels iNOS and cleaved caspase-3. For the analysis protein fractions isolated from the left ventricular tissue of control (C) and doxorubicin-treated (DOX) rat hearts obtained 4 weeks (C-4, DOX-4) or 8 weeks

Techniques Used: Isolation

36) Product Images from "Fanconi DNA repair pathway is required for survival and long-term maintenance of neural progenitors"

Article Title: Fanconi DNA repair pathway is required for survival and long-term maintenance of neural progenitors

Journal: The EMBO Journal

doi: 10.1038/emboj.2008.14

Apoptosis is increased in FA VZ. ( A ) DAPI and cleaved-caspase-3 immunostaining are shown in a fanca −/− E14.5 neocortex section. Arrows pointed out the apoptotic cells in the VZ. Nonspecific staining was observed on blood vessels (stars) and on meninges (dotted lines). Colocalization of pycnotic nuclei and cleaved-caspase-3 staining is shown in the enlargement of the white box ( A′ ). White arrows pointed out the isolated apoptotic cells and red arrows pointed out a doublet of apoptotic cells. ( B ) Quantifications of pycnotic nuclei and ( C ) cleaved-caspase-3-positive cells in the VZ of E13.5 and E14.5 embryonic brains show a significant increase of apoptosis in fanca −/− and fancg −/− compared with fanca +/+ and fancg +/+ controls. ( D ) Apoptotic cells in the fancg −/− neocortex express nestin, an NSPC marker. The number of mice is indicated within the bars. V, ventricle. Scale bars, 10 μm. * P
Figure Legend Snippet: Apoptosis is increased in FA VZ. ( A ) DAPI and cleaved-caspase-3 immunostaining are shown in a fanca −/− E14.5 neocortex section. Arrows pointed out the apoptotic cells in the VZ. Nonspecific staining was observed on blood vessels (stars) and on meninges (dotted lines). Colocalization of pycnotic nuclei and cleaved-caspase-3 staining is shown in the enlargement of the white box ( A′ ). White arrows pointed out the isolated apoptotic cells and red arrows pointed out a doublet of apoptotic cells. ( B ) Quantifications of pycnotic nuclei and ( C ) cleaved-caspase-3-positive cells in the VZ of E13.5 and E14.5 embryonic brains show a significant increase of apoptosis in fanca −/− and fancg −/− compared with fanca +/+ and fancg +/+ controls. ( D ) Apoptotic cells in the fancg −/− neocortex express nestin, an NSPC marker. The number of mice is indicated within the bars. V, ventricle. Scale bars, 10 μm. * P

Techniques Used: Immunostaining, Staining, Isolation, Marker, Mouse Assay

Apoptosis is increased in the adult FA SVZ. ( A ) Pycnotic nuclei and cleaved-caspase-3 staining (white arrows) are shown in the SVZ of adult fancg −/− mice. Colocalization of pycnotic nuclei and cleaved-caspase-3 staining is showed in an enlargement (lower panel). ( B ) Quantifications of cleaved-caspase-3-positive cells per SVZ cross section showed significant elevated apoptosis in the SVZ of fanca −/− and fancg −/− compared with fanca +/+ and fancg +/+ controls. The number of mice is indicated within the bars. V, ventricle. *** P
Figure Legend Snippet: Apoptosis is increased in the adult FA SVZ. ( A ) Pycnotic nuclei and cleaved-caspase-3 staining (white arrows) are shown in the SVZ of adult fancg −/− mice. Colocalization of pycnotic nuclei and cleaved-caspase-3 staining is showed in an enlargement (lower panel). ( B ) Quantifications of cleaved-caspase-3-positive cells per SVZ cross section showed significant elevated apoptosis in the SVZ of fanca −/− and fancg −/− compared with fanca +/+ and fancg +/+ controls. The number of mice is indicated within the bars. V, ventricle. *** P

Techniques Used: Staining, Mouse Assay

NSPC self-renewal in vitro is altered in FA mice. Growth, proliferation, and apoptosis were examined in neurosphere cultures from embryonic ( A – C ) and adult ( D – F ) NSPCs. (A) Population doublings of fanca −/− embryonic NSPCs were reduced in comparison with fanca +/+ controls. ( B , E ) Proliferating NSPCs were determined by flow cytometry after BrdU incorporation (30 min or 1 h for embryonic or adult NSPCs, respectively). ( C , F ) Analysis of cleaved-caspase-3 by flow cytometry revealed increased level of apoptosis in fanca −/− and fancg −/− embryonic and adult NSPCs in comparison with WT controls. ( D ) Population doublings of adult NSPCs were drastically reduced compared with WT controls. The number of mice is indicated within the bars. ** P
Figure Legend Snippet: NSPC self-renewal in vitro is altered in FA mice. Growth, proliferation, and apoptosis were examined in neurosphere cultures from embryonic ( A – C ) and adult ( D – F ) NSPCs. (A) Population doublings of fanca −/− embryonic NSPCs were reduced in comparison with fanca +/+ controls. ( B , E ) Proliferating NSPCs were determined by flow cytometry after BrdU incorporation (30 min or 1 h for embryonic or adult NSPCs, respectively). ( C , F ) Analysis of cleaved-caspase-3 by flow cytometry revealed increased level of apoptosis in fanca −/− and fancg −/− embryonic and adult NSPCs in comparison with WT controls. ( D ) Population doublings of adult NSPCs were drastically reduced compared with WT controls. The number of mice is indicated within the bars. ** P

Techniques Used: In Vitro, Mouse Assay, Flow Cytometry, Cytometry, BrdU Incorporation Assay

37) Product Images from "Effects of axon degeneration on oligodendrocyte lineage cells: Dorsal rhizotomy evokes a repair response while axon degeneration rostral to spinal contusion induces both repair and apoptosis"

Article Title: Effects of axon degeneration on oligodendrocyte lineage cells: Dorsal rhizotomy evokes a repair response while axon degeneration rostral to spinal contusion induces both repair and apoptosis

Journal: Glia

doi: 10.1002/glia.21009

RNA oxidation preceded the presence of cleaved caspase-3 after SCI. Double labeling in the dorsal funiculus from control, and spinal injured cases surviving for 1 and 8 days. 15A3-IR (A, D G) and cleaved caspase-3 IR (B, E H; Merged in C, F I) are shown. Scale bar in A=20um and applies to all.
Figure Legend Snippet: RNA oxidation preceded the presence of cleaved caspase-3 after SCI. Double labeling in the dorsal funiculus from control, and spinal injured cases surviving for 1 and 8 days. 15A3-IR (A, D G) and cleaved caspase-3 IR (B, E H; Merged in C, F I) are shown. Scale bar in A=20um and applies to all.

Techniques Used: Labeling

38) Product Images from "Inhibition of pluripotent stem cell-derived teratoma formation by small molecules"

Article Title: Inhibition of pluripotent stem cell-derived teratoma formation by small molecules

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1303669110

QC treatment blocks teratoma formation from undifferentiated hESCs without affecting lineage-specific differentiation. ( A ) OCT-4 (red), active caspase-3 (green), and DAPI (blue) IFC images of differentiated hESCs (spontaneous differentiation for 5 d)
Figure Legend Snippet: QC treatment blocks teratoma formation from undifferentiated hESCs without affecting lineage-specific differentiation. ( A ) OCT-4 (red), active caspase-3 (green), and DAPI (blue) IFC images of differentiated hESCs (spontaneous differentiation for 5 d)

Techniques Used:

39) Product Images from "Deletion of the de novo DNA methyltransferase Dnmt3a promotes lung tumor progression"

Article Title: Deletion of the de novo DNA methyltransferase Dnmt3a promotes lung tumor progression

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1114946108

Dnmt3a-deficient tumors have a higher proliferation index. ( A and B ) Representative photomicrographs of immunohistochemical staining for the proliferation marker K i -67 in Dnmt3a WT ( A ) and KO mice ( B ). The positive signal localizes to the nuclei. ( C ) Plotting of proliferation index (positive nuclei/mm 2 ) against tumor size (mm 2 ). Dnmt3a-deficient tumors had a significantly higher proliferation index compared with WT tumors ( P = 0.0006; the proliferation index was also influenced by tumor size, P = 0.032). ( D ) Representative photomicrograph of immunohistochemical staining for the apoptosis marker cleaved caspase-3 shows that positive cells are rare in Dnmt3a-deficient and WT tumors. ( Inset ) Details of two positive cells with cytoplasmic staining. (Scale bars: A and B , 50 μm; C , 200 μm.)
Figure Legend Snippet: Dnmt3a-deficient tumors have a higher proliferation index. ( A and B ) Representative photomicrographs of immunohistochemical staining for the proliferation marker K i -67 in Dnmt3a WT ( A ) and KO mice ( B ). The positive signal localizes to the nuclei. ( C ) Plotting of proliferation index (positive nuclei/mm 2 ) against tumor size (mm 2 ). Dnmt3a-deficient tumors had a significantly higher proliferation index compared with WT tumors ( P = 0.0006; the proliferation index was also influenced by tumor size, P = 0.032). ( D ) Representative photomicrograph of immunohistochemical staining for the apoptosis marker cleaved caspase-3 shows that positive cells are rare in Dnmt3a-deficient and WT tumors. ( Inset ) Details of two positive cells with cytoplasmic staining. (Scale bars: A and B , 50 μm; C , 200 μm.)

Techniques Used: Immunohistochemistry, Staining, Marker, Mouse Assay

40) Product Images from "Bergamot Polyphenols Improve Dyslipidemia and Pathophysiological Features in a Mouse Model of Non-Alcoholic Fatty Liver Disease"

Article Title: Bergamot Polyphenols Improve Dyslipidemia and Pathophysiological Features in a Mouse Model of Non-Alcoholic Fatty Liver Disease

Journal: Scientific Reports

doi: 10.1038/s41598-020-59485-3

Effects of BPF99 on inflammatory, metabolic and stress-related markers associated with NASH. Whole cell lysates were prepared from liver tissue of mice fed a normal chow diet and tap water (NC NW) or high fructose/glucose, high fat Western Diet, treated with vehicle (WD SW Vehicle) or 50 mg/kg/day BPF99 (WD SW BPF99). ( A ) Immunoblot analysis were performed for NF-kB; phosphorylated and total JNK (p54, p46); phosphorylated and total p38; phosphorylated and total AMPK; phosphorylated and total ACC, Caspase-3; COL1A1; PIIINP; PARP; cleaved Caspase-3 and cleaved PARP product p89 were not visualized despite long exposure times. GAPDH and Tubulin were used as a control for protein loading. Western blot analysis showed that ( B ) pACC/ACC and ( C ) PhosphoAMPK/AMPK ratio levels were up-regulated in WD SW-fed mice treated with vehicle or BPF99. The activation of ( D ) JNK and ( E ) p38 (shown as phosphorylated/total protein ratio) was markedly increased in NASH mice treated with vehicle compared to nonsteatotic controls. Treatment with 50 mg/kg/d BPF99 significantly reduced the phosphorylation levels of the MAP kinases effectors in the livers of WD SW-fed mice compared to the vehicle group compared to NC NW group. ( F ) NF-kB was increased in WD SW-fed mice treated with vehicle or BPF99. ( G ) Caspase-3 levels were similar in NASH animals treated with vehicle or BPF99 and higher compared to control animals. ( H ) PARP protein levels were higher in NASH animals treated with vehicle, whereas BPF99 reduced PARP expression to control levels. ( I ) Procollagen-1 and ( L ) procollagen-3 levels were higher in WD SW-fed mice treated with vehicle compared to control animals. BPF99 was able to reduce Procollagen I and III levels. NC NW (n = 4, red bar); WD SW Vehicle (n = 10, orange bar); WD SW BPF99 (n = 10, green bar). Data are expressed as the mean ± SEM. *p
Figure Legend Snippet: Effects of BPF99 on inflammatory, metabolic and stress-related markers associated with NASH. Whole cell lysates were prepared from liver tissue of mice fed a normal chow diet and tap water (NC NW) or high fructose/glucose, high fat Western Diet, treated with vehicle (WD SW Vehicle) or 50 mg/kg/day BPF99 (WD SW BPF99). ( A ) Immunoblot analysis were performed for NF-kB; phosphorylated and total JNK (p54, p46); phosphorylated and total p38; phosphorylated and total AMPK; phosphorylated and total ACC, Caspase-3; COL1A1; PIIINP; PARP; cleaved Caspase-3 and cleaved PARP product p89 were not visualized despite long exposure times. GAPDH and Tubulin were used as a control for protein loading. Western blot analysis showed that ( B ) pACC/ACC and ( C ) PhosphoAMPK/AMPK ratio levels were up-regulated in WD SW-fed mice treated with vehicle or BPF99. The activation of ( D ) JNK and ( E ) p38 (shown as phosphorylated/total protein ratio) was markedly increased in NASH mice treated with vehicle compared to nonsteatotic controls. Treatment with 50 mg/kg/d BPF99 significantly reduced the phosphorylation levels of the MAP kinases effectors in the livers of WD SW-fed mice compared to the vehicle group compared to NC NW group. ( F ) NF-kB was increased in WD SW-fed mice treated with vehicle or BPF99. ( G ) Caspase-3 levels were similar in NASH animals treated with vehicle or BPF99 and higher compared to control animals. ( H ) PARP protein levels were higher in NASH animals treated with vehicle, whereas BPF99 reduced PARP expression to control levels. ( I ) Procollagen-1 and ( L ) procollagen-3 levels were higher in WD SW-fed mice treated with vehicle compared to control animals. BPF99 was able to reduce Procollagen I and III levels. NC NW (n = 4, red bar); WD SW Vehicle (n = 10, orange bar); WD SW BPF99 (n = 10, green bar). Data are expressed as the mean ± SEM. *p

Techniques Used: Mouse Assay, Western Blot, Activation Assay, Expressing

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Western Blot:

Article Title: EMAPII Monoclonal Antibody Ameliorates Influenza A Virus-Induced Lung Injury
Article Snippet: .. Alternatively, cells were digested with 1% SDS-containing PBS and analyzed by western blot with anti-cleaved caspase 3, anti-caspase 3 (Cell Signaling, Danvers, MA, #49662 and #96625, respectively), rabbit anti-EMAPII and anti-β-actin antibodies (Sigma, St. Louis, MO, #A5441). .. Measurement of Transendothelial Permeability Transendothelial electrical resistance (TER) was measured using the highly sensitive biophysical assay with an electrical cell-substrate impedance sensor as described previously.

Incubation:

Article Title: A Novel Galectin-1 Inhibitor Discovered through One-Bead-Two-Compounds Library Potentiates the Anti-tumor Effects of Paclitaxel in vivo
Article Snippet: .. The tissue sections were then incubated with the specific antibody against cleaved-caspase 3 and Ki-67(Cell signaling) overnight, rinsed extensively three times with PBS. ..

Article Title: MicroRNA-218 Enhances the Radiosensitivity of Human Cervical Cancer via Promoting Radiation Induced Apoptosis
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other:

Article Title: Decay accelerating factor (CD55) protects neuronal cells from chemical hypoxia-induced injury
Article Snippet: Anti-Caspase 9, Anti-cleaved Caspase 3 (activated Caspase 3) and anti-Tyr416-Src antibodies (activated Src) were purchased from Cell Signaling (Danvers, MA).

Immunoprecipitation:

Article Title: Inhibition of mitochondrial fusion is an early and critical event in breast cancer cell apoptosis by dietary chemopreventative benzyl isothiocyanate
Article Snippet: .. Sources of the antibodies were as follows: anti-phospho (S616) dynamin-1-like protein (Drp1), anti-total Drp1, anti-mitofusin (Mfn) 2, and anti-cleaved caspase 3 antibodies were from Cell Signaling Technology (Beverley, MA); anti-Mfn1, anti-mitochondrial fission 1 protein (Fis1), anti-Bak and anti-Bax antibody was from Santa Cruz Biotechnology (Dallas, TX); anti-optic atrophy 1 (Opa1) antibody was from Abcam (Cambridge, MA); anti-active Bax (for immunoprecipitation) antibodies were from BD Biosciences (San Jose, CA); anti-actin antibody was from Sigma Aldrich (St. Louis, MO); anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody was from GeneTex (Irvine, CA); and anti-active Bak (for immunoprecipitation) antibody was from Calbiochem (Billerica, MA). .. MitoTracker green and Hoechst 33342 were purchased from Invitrogen-Life Technologies whereas 4′,6-diamidino-2-phenylindole (DAPI) was from Sigma-Aldrich.

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    Cell Signaling Technology Inc rabbit anti casp3
    Premature apoptosis occured in C9ORF72 spinal MNs over ageing (a) Apoptosis in MAP2-positive neurons (in red) was revealed as cytosolic rim staining around Hoechst-positive nuclei (in blue) for cleaved caspase 3 <t>(Casp3,</t> in green) by confocal IF microscopy at D80 endpoints (fig. 3). Dotted boxed areas in image galleries are shown magnified on the right. Note the striking accumulation of Casp3 in parental C9 and C9-KO that was phenocopied by WT-KO. Conversely, apoptosis hardly occurred in C9-GC and parental Ctrl1. Scale bars = 10µm. (b) Quantification of (a), percentage of apoptotic cells in MAP2-positive population. Asterisks: highly significant increase in any pairwise comparison with unlabeled conditions, one-way ANOVA with Bonferroni post test, *P≤0.05, **P≤0.01, ***P≤0.001, N=60 images from 3 independent experiments, error bars=SD. All unlabeled conditions (i.e. with no asterisk) were not significantly different amongst themselves in any pairwise comparison. Remaining comparisons were: WT-KO vs C9: ***, WT-KO vs C9-KO: ***, C9 vs C9-KO: ***.
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    TRAP1 C501S mutant exerts protective role towards staurosporine-induced cell death A ) Western blot analyses of HeLa cell lysates were performed to check the expression levels of WT and C501S mutant forms of GFP-fused TRAP1 (TRAP1-GFP) which can be discriminated by the endogenous variant (TRAP1) by the use of an anti-TRAP1 antibody. Western blot shown is representative of at least n = 3 independent experiments giving similar results.  B ) The number of dead cells was evaluated by Trypan blue exclusion test in control cells transfected with an empty vector (Mock), as well as in cell expressing TRAP1 WT  (WT) and TRAP1 C501S  (C501S) treated for 4 h with 200 nM staurosporine (STS), in the presence or absence of the NO donor DETA-NONOate (250 μM, maintained for 22-24 h before STS treatment). Data are shown as fold change of dead cells with respect to untreated cells, and represent the mean ± SEM of n =5 independent experiments done in duplicate. * p  = 0.05; ** p  = 0.01.  C ) Western blot analyses of HeLa cells treated as in (B) were performed to evaluate cleaved PARP1 and pro-caspase 3 (Casp3) protein levels. Vinculin and α-actin were used as loading controls. Densitometric analyses (denso) of PARP1/Vinculin or Casp3/actin are shown below each lane. Western blots shown are representative of at least n = 3 independent experiments giving similar results.
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    <t>Caspase</t> 3 staining in kidney sections and isolated podocytes. A) A Immunohistochemical staining with anti-caspase 3 antibody. Scale bar: 50μm (upper row); 20μm (lower row). B) Confocal micrographs from primary podocytes derived from described genotypes showing cytoplasm staining (GAPDH in green), apoptotic cells (Cleaved CASPASE-3 in red) and nuclei (DAPI in blue). Maximum intensity projection of Z-stacks is shown. Scale bars: 50 μm. OnOn the right panel, qquantification of caspase 3-positive cells. Data are presented as mean ± SD. Symbols * and § represent the significance levels vs. WT and Mpv17 -/- , respectively, calculated by one‐way ANOVA with Tukey’s post hoc multiple comparison test − : **** p
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    SARS-CoV-2 internalizes and replicates within hiPSC-CMs in vitro , eliciting cytopathic effect and contractility alterations. A) Human iPSC-CMs exhibit standard sarcomeric markers including cardiac troponin T (cTnT) and α-actinin with DAPI as nuclear counterstain. B) Immunofluorescence for cTnT and SARS-CoV-2 “spike” protein demonstrates that hiPSC-CMs can be infected by SARS-CoV-2. SARS-CoV-2 spike protein is not present in mock infected cultures. C) HiPSC-CMs after SARS-CoV-2 infection, but not mock infection, exhibit signs of cellular apoptosis, indicated by morphological changes seen in bright field (BF) and cleaved <t>caspase-3</t> production. A second SARS-CoV-2 antibody marks a viral-specific double-stranded intermediate RNA (dsRNA). D) Magnified inset from panel B shows a merged immunofluorescence image for SARS-CoV-2 spike protein and DAPI. Arrows indicate perinuclear accumulation of viral particles, and suggests active viral protein translation and genome replication at perinuclear ribosomes and membranous compartments. E) Magnified inset from panel C shows a merged immunofluorescence image for SARS-CoV-2 dsRNA and DAPI. Arrows indicate perinuclear viral replication sites. F) Quantification of immunofluorescence indicates percentage of total DAPI-positive cells that are positive for spike protein, viral dsRNA, cleaved caspase-3 (CC3), and dsRNA+CC3 in hiPSC-CMs infected with SARS-CoV-2, compared to mock infection. N=5-7 images quantified for each stain for mock and infected conditions. G) Quantification of beats per minute in wells containing hiPSC-CMs with mock infection versus wells containing hiPSC-CMs infected with SARS-CoV-2. N=6 videos recorded for each condition. See Supplemental Movie 1 for representative video clips. * indicates p
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    Premature apoptosis occured in C9ORF72 spinal MNs over ageing (a) Apoptosis in MAP2-positive neurons (in red) was revealed as cytosolic rim staining around Hoechst-positive nuclei (in blue) for cleaved caspase 3 (Casp3, in green) by confocal IF microscopy at D80 endpoints (fig. 3). Dotted boxed areas in image galleries are shown magnified on the right. Note the striking accumulation of Casp3 in parental C9 and C9-KO that was phenocopied by WT-KO. Conversely, apoptosis hardly occurred in C9-GC and parental Ctrl1. Scale bars = 10µm. (b) Quantification of (a), percentage of apoptotic cells in MAP2-positive population. Asterisks: highly significant increase in any pairwise comparison with unlabeled conditions, one-way ANOVA with Bonferroni post test, *P≤0.05, **P≤0.01, ***P≤0.001, N=60 images from 3 independent experiments, error bars=SD. All unlabeled conditions (i.e. with no asterisk) were not significantly different amongst themselves in any pairwise comparison. Remaining comparisons were: WT-KO vs C9: ***, WT-KO vs C9-KO: ***, C9 vs C9-KO: ***.

    Journal: bioRxiv

    Article Title: High content live profiling reveals concomitant gain and loss of function pathomechanisms in C9ORF72 amyotrophic lateral sclerosis

    doi: 10.1101/2020.04.15.040394

    Figure Lengend Snippet: Premature apoptosis occured in C9ORF72 spinal MNs over ageing (a) Apoptosis in MAP2-positive neurons (in red) was revealed as cytosolic rim staining around Hoechst-positive nuclei (in blue) for cleaved caspase 3 (Casp3, in green) by confocal IF microscopy at D80 endpoints (fig. 3). Dotted boxed areas in image galleries are shown magnified on the right. Note the striking accumulation of Casp3 in parental C9 and C9-KO that was phenocopied by WT-KO. Conversely, apoptosis hardly occurred in C9-GC and parental Ctrl1. Scale bars = 10µm. (b) Quantification of (a), percentage of apoptotic cells in MAP2-positive population. Asterisks: highly significant increase in any pairwise comparison with unlabeled conditions, one-way ANOVA with Bonferroni post test, *P≤0.05, **P≤0.01, ***P≤0.001, N=60 images from 3 independent experiments, error bars=SD. All unlabeled conditions (i.e. with no asterisk) were not significantly different amongst themselves in any pairwise comparison. Remaining comparisons were: WT-KO vs C9: ***, WT-KO vs C9-KO: ***, C9 vs C9-KO: ***.

    Article Snippet: The following primary antibodies were used: mouse anti-yH2A.X (1:500, Millipore #05- 636), rabbit anti-53BP1 (1:1000, Novusbio NB100-304), chicken anti-MAP2 (1:1000, Abcam ab5392), rabbit anti-Casp3 (1:1000, Cell Signaling #9661), rat anti-GP (1:500, clone 18H8) and mouse anti GA (1:500, clone IAI2) were both generously provided from Dieter Edbauer ( ).

    Techniques: Staining, Microscopy

    TRAP1 C501S mutant exerts protective role towards staurosporine-induced cell death A ) Western blot analyses of HeLa cell lysates were performed to check the expression levels of WT and C501S mutant forms of GFP-fused TRAP1 (TRAP1-GFP) which can be discriminated by the endogenous variant (TRAP1) by the use of an anti-TRAP1 antibody. Western blot shown is representative of at least n = 3 independent experiments giving similar results.  B ) The number of dead cells was evaluated by Trypan blue exclusion test in control cells transfected with an empty vector (Mock), as well as in cell expressing TRAP1 WT  (WT) and TRAP1 C501S  (C501S) treated for 4 h with 200 nM staurosporine (STS), in the presence or absence of the NO donor DETA-NONOate (250 μM, maintained for 22-24 h before STS treatment). Data are shown as fold change of dead cells with respect to untreated cells, and represent the mean ± SEM of n =5 independent experiments done in duplicate. * p  = 0.05; ** p  = 0.01.  C ) Western blot analyses of HeLa cells treated as in (B) were performed to evaluate cleaved PARP1 and pro-caspase 3 (Casp3) protein levels. Vinculin and α-actin were used as loading controls. Densitometric analyses (denso) of PARP1/Vinculin or Casp3/actin are shown below each lane. Western blots shown are representative of at least n = 3 independent experiments giving similar results.

    Journal: bioRxiv

    Article Title: S-nitrosylation affects TRAP1 structure and ATPase activity and modulates cell response to apoptotic stimuli

    doi: 10.1101/859629

    Figure Lengend Snippet: TRAP1 C501S mutant exerts protective role towards staurosporine-induced cell death A ) Western blot analyses of HeLa cell lysates were performed to check the expression levels of WT and C501S mutant forms of GFP-fused TRAP1 (TRAP1-GFP) which can be discriminated by the endogenous variant (TRAP1) by the use of an anti-TRAP1 antibody. Western blot shown is representative of at least n = 3 independent experiments giving similar results. B ) The number of dead cells was evaluated by Trypan blue exclusion test in control cells transfected with an empty vector (Mock), as well as in cell expressing TRAP1 WT (WT) and TRAP1 C501S (C501S) treated for 4 h with 200 nM staurosporine (STS), in the presence or absence of the NO donor DETA-NONOate (250 μM, maintained for 22-24 h before STS treatment). Data are shown as fold change of dead cells with respect to untreated cells, and represent the mean ± SEM of n =5 independent experiments done in duplicate. * p = 0.05; ** p = 0.01. C ) Western blot analyses of HeLa cells treated as in (B) were performed to evaluate cleaved PARP1 and pro-caspase 3 (Casp3) protein levels. Vinculin and α-actin were used as loading controls. Densitometric analyses (denso) of PARP1/Vinculin or Casp3/actin are shown below each lane. Western blots shown are representative of at least n = 3 independent experiments giving similar results.

    Article Snippet: We used the following primary antibodies: anti-HSP75 (TRAP1) and anti-Actin from Santa Cruz Biotechnology; anti-Vinculin from Sigma-Aldrich; anti-PARP1 from Enzo Life Sciences; anti-Caspase 3 from Cell Signaling.

    Techniques: Mutagenesis, Western Blot, Expressing, Variant Assay, Transfection, Plasmid Preparation

    Caspase 3 staining in kidney sections and isolated podocytes. A) A Immunohistochemical staining with anti-caspase 3 antibody. Scale bar: 50μm (upper row); 20μm (lower row). B) Confocal micrographs from primary podocytes derived from described genotypes showing cytoplasm staining (GAPDH in green), apoptotic cells (Cleaved CASPASE-3 in red) and nuclei (DAPI in blue). Maximum intensity projection of Z-stacks is shown. Scale bars: 50 μm. OnOn the right panel, qquantification of caspase 3-positive cells. Data are presented as mean ± SD. Symbols * and § represent the significance levels vs. WT and Mpv17 -/- , respectively, calculated by one‐way ANOVA with Tukey’s post hoc multiple comparison test − : **** p

    Journal: bioRxiv

    Article Title: Opa1 overexpression protects from early onset Mpv17-/--related mouse kidney disease

    doi: 10.1101/2020.03.18.996561

    Figure Lengend Snippet: Caspase 3 staining in kidney sections and isolated podocytes. A) A Immunohistochemical staining with anti-caspase 3 antibody. Scale bar: 50μm (upper row); 20μm (lower row). B) Confocal micrographs from primary podocytes derived from described genotypes showing cytoplasm staining (GAPDH in green), apoptotic cells (Cleaved CASPASE-3 in red) and nuclei (DAPI in blue). Maximum intensity projection of Z-stacks is shown. Scale bars: 50 μm. OnOn the right panel, qquantification of caspase 3-positive cells. Data are presented as mean ± SD. Symbols * and § represent the significance levels vs. WT and Mpv17 -/- , respectively, calculated by one‐way ANOVA with Tukey’s post hoc multiple comparison test − : **** p

    Article Snippet: Antibodies Mouse monoclonal anti-GAPDH (1:1,000; #ab8245; Abcam), rabbit monoclonal anti-TOM20 Alexa Fluor 647 (1:500; #ab209606; Abcam), Alexa Fluor 488 Goat anti-mouse (1:300; #A11004; Invitrogen), Alexa Fluor 647 Goat anti-rabbit (1:300; #A27040; Invitrogen), rabbit polyclonal anti-NPHS2 (1:300; #ab50339; Abcam), mouse monoclonal anti-CYTOKERATIN (1:300; #ab86734; Abcam), anti-cleaved caspase 3 (1:200; Cell Signalling, #9661).

    Techniques: Staining, Isolation, Immunohistochemistry, Derivative Assay

    SARS-CoV-2 internalizes and replicates within hiPSC-CMs in vitro , eliciting cytopathic effect and contractility alterations. A) Human iPSC-CMs exhibit standard sarcomeric markers including cardiac troponin T (cTnT) and α-actinin with DAPI as nuclear counterstain. B) Immunofluorescence for cTnT and SARS-CoV-2 “spike” protein demonstrates that hiPSC-CMs can be infected by SARS-CoV-2. SARS-CoV-2 spike protein is not present in mock infected cultures. C) HiPSC-CMs after SARS-CoV-2 infection, but not mock infection, exhibit signs of cellular apoptosis, indicated by morphological changes seen in bright field (BF) and cleaved caspase-3 production. A second SARS-CoV-2 antibody marks a viral-specific double-stranded intermediate RNA (dsRNA). D) Magnified inset from panel B shows a merged immunofluorescence image for SARS-CoV-2 spike protein and DAPI. Arrows indicate perinuclear accumulation of viral particles, and suggests active viral protein translation and genome replication at perinuclear ribosomes and membranous compartments. E) Magnified inset from panel C shows a merged immunofluorescence image for SARS-CoV-2 dsRNA and DAPI. Arrows indicate perinuclear viral replication sites. F) Quantification of immunofluorescence indicates percentage of total DAPI-positive cells that are positive for spike protein, viral dsRNA, cleaved caspase-3 (CC3), and dsRNA+CC3 in hiPSC-CMs infected with SARS-CoV-2, compared to mock infection. N=5-7 images quantified for each stain for mock and infected conditions. G) Quantification of beats per minute in wells containing hiPSC-CMs with mock infection versus wells containing hiPSC-CMs infected with SARS-CoV-2. N=6 videos recorded for each condition. See Supplemental Movie 1 for representative video clips. * indicates p

    Journal: bioRxiv

    Article Title: Human iPSC-Derived Cardiomyocytes are Susceptible to SARS-CoV-2 Infection

    doi: 10.1101/2020.04.21.051912

    Figure Lengend Snippet: SARS-CoV-2 internalizes and replicates within hiPSC-CMs in vitro , eliciting cytopathic effect and contractility alterations. A) Human iPSC-CMs exhibit standard sarcomeric markers including cardiac troponin T (cTnT) and α-actinin with DAPI as nuclear counterstain. B) Immunofluorescence for cTnT and SARS-CoV-2 “spike” protein demonstrates that hiPSC-CMs can be infected by SARS-CoV-2. SARS-CoV-2 spike protein is not present in mock infected cultures. C) HiPSC-CMs after SARS-CoV-2 infection, but not mock infection, exhibit signs of cellular apoptosis, indicated by morphological changes seen in bright field (BF) and cleaved caspase-3 production. A second SARS-CoV-2 antibody marks a viral-specific double-stranded intermediate RNA (dsRNA). D) Magnified inset from panel B shows a merged immunofluorescence image for SARS-CoV-2 spike protein and DAPI. Arrows indicate perinuclear accumulation of viral particles, and suggests active viral protein translation and genome replication at perinuclear ribosomes and membranous compartments. E) Magnified inset from panel C shows a merged immunofluorescence image for SARS-CoV-2 dsRNA and DAPI. Arrows indicate perinuclear viral replication sites. F) Quantification of immunofluorescence indicates percentage of total DAPI-positive cells that are positive for spike protein, viral dsRNA, cleaved caspase-3 (CC3), and dsRNA+CC3 in hiPSC-CMs infected with SARS-CoV-2, compared to mock infection. N=5-7 images quantified for each stain for mock and infected conditions. G) Quantification of beats per minute in wells containing hiPSC-CMs with mock infection versus wells containing hiPSC-CMs infected with SARS-CoV-2. N=6 videos recorded for each condition. See Supplemental Movie 1 for representative video clips. * indicates p

    Article Snippet: The following antibodies and dilutions were used: α-actinin (1:100, Sigma-Aldrich Cat# A7811, RRID: AB_476766); cardiac troponin T (cTnT, 1:100, Abcam Cat# ab45932, RRID: AB_956386); SARS-CoV-2 spike (S) protein (1:100, BEI Resources NR-616 Monoclonal Anti-SARS-CoV S Protein (Similar to 240C) SARS coronavirus); SARS-CoV-2 double stranded RNA (1:100, J2 clone; Absolute Antibody Inc.); cleaved caspase-3 (1:200, Cell Signaling Technology Cat# 9661, RRID: AB_2341188).

    Techniques: In Vitro, Immunofluorescence, Infection, Staining