Structured Review

Becton Dickinson anti cleaved caspase 3
Inhibition of Shh signaling does not affect apoptosis in the germinal zone of the developing ferret cerebral cortex. pCAG-EGFP plus either pCAG-HhipΔC22 or pCAG control vector was electroporated at E33, and the brains were dissected at P1. ( A ) Sections were stained with anti-cleaved <t>caspase</t> 3 (cCasp3) antibody and Hoechst 33342. Scale bars = 100 μm. ( B ) Quantification of cCasp3-positive cells in the ISVZ and the OSVZ. The numbers of immunopositive cells on the electroporated side were divided by those on the non-electroporated side. The numbers of cCasp3-positive cells were not affected by HhipΔC22. n = 3 animals for each condition. Bars present mean ± SD. ns, not significant. Student's t -test.
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Images

1) Product Images from "A discrete subtype of neural progenitor crucial for cortical folding in the gyrencephalic mammalian brain"

Article Title: A discrete subtype of neural progenitor crucial for cortical folding in the gyrencephalic mammalian brain

Journal: eLife

doi: 10.7554/eLife.54873

Inhibition of Shh signaling does not affect apoptosis in the germinal zone of the developing ferret cerebral cortex. pCAG-EGFP plus either pCAG-HhipΔC22 or pCAG control vector was electroporated at E33, and the brains were dissected at P1. ( A ) Sections were stained with anti-cleaved caspase 3 (cCasp3) antibody and Hoechst 33342. Scale bars = 100 μm. ( B ) Quantification of cCasp3-positive cells in the ISVZ and the OSVZ. The numbers of immunopositive cells on the electroporated side were divided by those on the non-electroporated side. The numbers of cCasp3-positive cells were not affected by HhipΔC22. n = 3 animals for each condition. Bars present mean ± SD. ns, not significant. Student's t -test.
Figure Legend Snippet: Inhibition of Shh signaling does not affect apoptosis in the germinal zone of the developing ferret cerebral cortex. pCAG-EGFP plus either pCAG-HhipΔC22 or pCAG control vector was electroporated at E33, and the brains were dissected at P1. ( A ) Sections were stained with anti-cleaved caspase 3 (cCasp3) antibody and Hoechst 33342. Scale bars = 100 μm. ( B ) Quantification of cCasp3-positive cells in the ISVZ and the OSVZ. The numbers of immunopositive cells on the electroporated side were divided by those on the non-electroporated side. The numbers of cCasp3-positive cells were not affected by HhipΔC22. n = 3 animals for each condition. Bars present mean ± SD. ns, not significant. Student's t -test.

Techniques Used: Inhibition, Plasmid Preparation, Staining

2) Product Images from "Integration of Multiple Signaling Pathway Activities Resolves K-RAS / N-RAS Mutation Paradox in Colon Epithelial Cell Response to Inflammatory Cytokine Stimulation"

Article Title: Integration of Multiple Signaling Pathway Activities Resolves K-RAS / N-RAS Mutation Paradox in Colon Epithelial Cell Response to Inflammatory Cytokine Stimulation

Journal: Integrative biology : quantitative biosciences from nano to macro

doi: 10.1039/b925935j

Cells with mutations in both N-RAS and K-RAS are sensitive to TNFα. (A) Overview of the RAS genotypes of the three cell lines. (B) Levels of active (RBD) and total K- and N-RAS for each cell line. (C) The percentage of apoptotic cells was determined by flow cytometry from the percentage of cells that were positive for both cleaved caspase-3 and cleaved PARP. (D) DLD-1 and DLD-N cells demonstrated a similar sensitivity to TNFα, while DKs8-N cells were more resistant.
Figure Legend Snippet: Cells with mutations in both N-RAS and K-RAS are sensitive to TNFα. (A) Overview of the RAS genotypes of the three cell lines. (B) Levels of active (RBD) and total K- and N-RAS for each cell line. (C) The percentage of apoptotic cells was determined by flow cytometry from the percentage of cells that were positive for both cleaved caspase-3 and cleaved PARP. (D) DLD-1 and DLD-N cells demonstrated a similar sensitivity to TNFα, while DKs8-N cells were more resistant.

Techniques Used: Flow Cytometry, Cytometry

A PLSR model built on single-mutant signaling and apoptosis is able to predict dual-mutant apoptosis. (A) The PLSR model accurately predicted the elevated sensitivity of DLD-N cells to TNFα. (B) Scores for the three cell lines in the first and second component. The first component is consistent with a ‘TNFα treatment’ axis. (C) Loadings for the first two components of the single-mutant PLSR model. CP indicates the loadings for the response metric of cleaved caspase-3 and cleaved PARP positive cells.
Figure Legend Snippet: A PLSR model built on single-mutant signaling and apoptosis is able to predict dual-mutant apoptosis. (A) The PLSR model accurately predicted the elevated sensitivity of DLD-N cells to TNFα. (B) Scores for the three cell lines in the first and second component. The first component is consistent with a ‘TNFα treatment’ axis. (C) Loadings for the first two components of the single-mutant PLSR model. CP indicates the loadings for the response metric of cleaved caspase-3 and cleaved PARP positive cells.

Techniques Used: Mutagenesis

3) Product Images from "Multifaceted role of BTLA in the control of CD8+ T cell fate after antigen encounter"

Article Title: Multifaceted role of BTLA in the control of CD8+ T cell fate after antigen encounter

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

doi: 10.1158/1078-0432.CCR-16-1217

BTLA signaling motifs show no effect on tumor killing, while Grb2 motif augments IL-2 production and T cell proliferation ( a ) Schematic diagram depicts the structure of BTLA WT (left), BTLAΔGrb2 (middle), and BTLAΔITSM (right). Signaling motifs with modified Tyrosine to Phenylalanine are indicated by a dotted pattern over a black background. A table summarizing the phenotype observed with the expression of BTLA or of the different BTLA constructs is presented on the right panel. ( b ) B16 OVA (mouse melanoma tumor positive for OVA) or B16F10 (mouse melanoma tumor negative for OVA) were stained with eFluor670® and co-cultured with OT-1 BTLA KO T cells overexpressing WT BTLA or BTLA mutants at the following T cell-to-tumor cell ratios (1:10, 1:3, and 1:1). Tumor cell death is depicted by the percentage of caspase-3 positive cells. N=3 independent experiments ( c ) OT-1 BTLA KO T cells overexpressing WT BTLA or its variants were re-stimulated with dendritic cells pulsed with OVA peptide. TNF-α and IFN-γ production by virally transduced OT-1 BTLA KO T cells was evaluated by intracellular staining. Bar graph depicts the percentage of positive cells (left panel) and mean fluorescence intensity (MFI) (right panel). Each bar represents three independent experiments. (Two-way ANOVA; * P
Figure Legend Snippet: BTLA signaling motifs show no effect on tumor killing, while Grb2 motif augments IL-2 production and T cell proliferation ( a ) Schematic diagram depicts the structure of BTLA WT (left), BTLAΔGrb2 (middle), and BTLAΔITSM (right). Signaling motifs with modified Tyrosine to Phenylalanine are indicated by a dotted pattern over a black background. A table summarizing the phenotype observed with the expression of BTLA or of the different BTLA constructs is presented on the right panel. ( b ) B16 OVA (mouse melanoma tumor positive for OVA) or B16F10 (mouse melanoma tumor negative for OVA) were stained with eFluor670® and co-cultured with OT-1 BTLA KO T cells overexpressing WT BTLA or BTLA mutants at the following T cell-to-tumor cell ratios (1:10, 1:3, and 1:1). Tumor cell death is depicted by the percentage of caspase-3 positive cells. N=3 independent experiments ( c ) OT-1 BTLA KO T cells overexpressing WT BTLA or its variants were re-stimulated with dendritic cells pulsed with OVA peptide. TNF-α and IFN-γ production by virally transduced OT-1 BTLA KO T cells was evaluated by intracellular staining. Bar graph depicts the percentage of positive cells (left panel) and mean fluorescence intensity (MFI) (right panel). Each bar represents three independent experiments. (Two-way ANOVA; * P

Techniques Used: Modification, Expressing, Construct, Staining, Cell Culture, Fluorescence

CD8 + BTLA + TIL demonstrate a superior anti-tumor effect in vivo ( a ) MART1-reactive CD8 + TIL (left and mid panel) and TIL 2549 (right panel) were sorted into BTLA + and BTLA − subsets. MEL 526 (melanoma tumor expressing MART-1 antigen, left and mid graph) or autologous tumor line 2549 (right graph) were stained with eFluor670® and co-cultured with TIL at the following TIL-to-tumor cell ratios (1:10, 1:3, and 1:1). Tumor cell death is measured by the percentage of caspase-3 positive cells. ( b) Ten million sorted CD8 + BTLA + or sorted CD8 + BTLA − TIL were intravenously injected into tumor bearing mice previously subcutaneously implanted with either MEL 526 or autologous melanoma tumor line 2549. Tumor burden was measured using calipers and diameter graphed as mm 2 . ( c ) Bar graph shows the percentage of CD45 + CD8 + in the peripheral blood on days 2, 4, 6, and 8 post-adoptive transfer in the same experiment described in ( b ). N= 5–8 animals per group. * P
Figure Legend Snippet: CD8 + BTLA + TIL demonstrate a superior anti-tumor effect in vivo ( a ) MART1-reactive CD8 + TIL (left and mid panel) and TIL 2549 (right panel) were sorted into BTLA + and BTLA − subsets. MEL 526 (melanoma tumor expressing MART-1 antigen, left and mid graph) or autologous tumor line 2549 (right graph) were stained with eFluor670® and co-cultured with TIL at the following TIL-to-tumor cell ratios (1:10, 1:3, and 1:1). Tumor cell death is measured by the percentage of caspase-3 positive cells. ( b) Ten million sorted CD8 + BTLA + or sorted CD8 + BTLA − TIL were intravenously injected into tumor bearing mice previously subcutaneously implanted with either MEL 526 or autologous melanoma tumor line 2549. Tumor burden was measured using calipers and diameter graphed as mm 2 . ( c ) Bar graph shows the percentage of CD45 + CD8 + in the peripheral blood on days 2, 4, 6, and 8 post-adoptive transfer in the same experiment described in ( b ). N= 5–8 animals per group. * P

Techniques Used: In Vivo, Expressing, Staining, Cell Culture, Injection, Mouse Assay, Adoptive Transfer Assay

4) Product Images from "Targeting invadopodia-mediated breast cancer metastasis by using ABL kinase inhibitors"

Article Title: Targeting invadopodia-mediated breast cancer metastasis by using ABL kinase inhibitors

Journal: Oncotarget

doi: 10.18632/oncotarget.25243

Primary tumor growth is not affected by ABL kinase inhibitors MDA-MB 231/Dendra2 cells were injected into the mammary fat pad of 10-week-old SCID female mice and allowed to grow until the tumor reached the size of 100 mm 3 . At day 56 following injection, mice were treated by oral gavage with vehicle (5% DMSO, 2% hydroxypropyl cellulose, 0.5% Tween-80), 100 mg/kg imatinib, 70 mg/kg nilotinib or 100 mg/kg GNF-5 once a day, 5 days a week, for four weeks. ( A ) Time-dependent tumor growth. Tumor growth was assessed twice a week by measuring two perpendicular diameters and calculating tumor size in mm 3 . Treatment initiation is shown as red dotted line. n = 12 (vehicle), n = 10 (imatinib), n = 12 (nilotinib), n = 10 (GNF-5) mice per group from two independent experiments. ( B ) Primary tumors were dissected at the end of experiment and subjected to immunohistochemistry. Representative images of primary tumor sections stained with anti-PCNA (proliferation), anti-cleaved caspase 3 (apoptosis), and anti-CD31 (angiogenesis). ( C ) Quantification of PCNA positive cells (red/pink) normalized to DAPI positive cells (blue). The average percentage of proliferating cells in total cells per field is shown. ( D ) Quantification of apoptotic cells (cleaved caspase 3 positive cells). Shown is the number of apoptotic cells per field. ( E ) Quantification of CD-31 positive blood vessels. For all quantifications, n = 50 random fields from 5 tumors per condition. Bar, 100 µm.
Figure Legend Snippet: Primary tumor growth is not affected by ABL kinase inhibitors MDA-MB 231/Dendra2 cells were injected into the mammary fat pad of 10-week-old SCID female mice and allowed to grow until the tumor reached the size of 100 mm 3 . At day 56 following injection, mice were treated by oral gavage with vehicle (5% DMSO, 2% hydroxypropyl cellulose, 0.5% Tween-80), 100 mg/kg imatinib, 70 mg/kg nilotinib or 100 mg/kg GNF-5 once a day, 5 days a week, for four weeks. ( A ) Time-dependent tumor growth. Tumor growth was assessed twice a week by measuring two perpendicular diameters and calculating tumor size in mm 3 . Treatment initiation is shown as red dotted line. n = 12 (vehicle), n = 10 (imatinib), n = 12 (nilotinib), n = 10 (GNF-5) mice per group from two independent experiments. ( B ) Primary tumors were dissected at the end of experiment and subjected to immunohistochemistry. Representative images of primary tumor sections stained with anti-PCNA (proliferation), anti-cleaved caspase 3 (apoptosis), and anti-CD31 (angiogenesis). ( C ) Quantification of PCNA positive cells (red/pink) normalized to DAPI positive cells (blue). The average percentage of proliferating cells in total cells per field is shown. ( D ) Quantification of apoptotic cells (cleaved caspase 3 positive cells). Shown is the number of apoptotic cells per field. ( E ) Quantification of CD-31 positive blood vessels. For all quantifications, n = 50 random fields from 5 tumors per condition. Bar, 100 µm.

Techniques Used: Multiple Displacement Amplification, Injection, Mouse Assay, Immunohistochemistry, Staining

5) Product Images from "Downregulation of CRABP2 Inhibit the Tumorigenesis of Hepatocellular Carcinoma In Vivo and In Vitro"

Article Title: Downregulation of CRABP2 Inhibit the Tumorigenesis of Hepatocellular Carcinoma In Vivo and In Vitro

Journal: BioMed Research International

doi: 10.1155/2020/3098327

Effects of CRABP2 on ERK-VEGF pathway. (a)Western blotting results of ERK-VEGF pathway-related proteins and apoptosis-related proteins of HpeG2 and Hun7 cells. Expression of cleaved caspase-3 (b) and Bax (d) was increased, while Bcl-2 (c), p-ERK/ERK ratio (e), VEGF (f), and p-VEGFR2/VEGFR2 (g) decreased after CRABP2-shRNAs transfection in HpeG2 and Hun7 cells. n = 7. One-way ANOVA was used in multigroup comparison. All data are presented as mean ± SEM. ∗∗ P
Figure Legend Snippet: Effects of CRABP2 on ERK-VEGF pathway. (a)Western blotting results of ERK-VEGF pathway-related proteins and apoptosis-related proteins of HpeG2 and Hun7 cells. Expression of cleaved caspase-3 (b) and Bax (d) was increased, while Bcl-2 (c), p-ERK/ERK ratio (e), VEGF (f), and p-VEGFR2/VEGFR2 (g) decreased after CRABP2-shRNAs transfection in HpeG2 and Hun7 cells. n = 7. One-way ANOVA was used in multigroup comparison. All data are presented as mean ± SEM. ∗∗ P

Techniques Used: Expressing, Transfection

6) Product Images from "Targeting invadopodia-mediated breast cancer metastasis by using ABL kinase inhibitors"

Article Title: Targeting invadopodia-mediated breast cancer metastasis by using ABL kinase inhibitors

Journal: Oncotarget

doi: 10.18632/oncotarget.25243

Primary tumor growth is not affected by ABL kinase inhibitors MDA-MB 231/Dendra2 cells were injected into the mammary fat pad of 10-week-old SCID female mice and allowed to grow until the tumor reached the size of 100 mm 3 . At day 56 following injection, mice were treated by oral gavage with vehicle (5% DMSO, 2% hydroxypropyl cellulose, 0.5% Tween-80), 100 mg/kg imatinib, 70 mg/kg nilotinib or 100 mg/kg GNF-5 once a day, 5 days a week, for four weeks. ( A ) Time-dependent tumor growth. Tumor growth was assessed twice a week by measuring two perpendicular diameters and calculating tumor size in mm 3 . Treatment initiation is shown as red dotted line. n = 12 (vehicle), n = 10 (imatinib), n = 12 (nilotinib), n = 10 (GNF-5) mice per group from two independent experiments. ( B ) Primary tumors were dissected at the end of experiment and subjected to immunohistochemistry. Representative images of primary tumor sections stained with anti-PCNA (proliferation), anti-cleaved caspase 3 (apoptosis), and anti-CD31 (angiogenesis). ( C ) Quantification of PCNA positive cells (red/pink) normalized to DAPI positive cells (blue). The average percentage of proliferating cells in total cells per field is shown. ( D ) Quantification of apoptotic cells (cleaved caspase 3 positive cells). Shown is the number of apoptotic cells per field. ( E ) Quantification of CD-31 positive blood vessels. For all quantifications, n = 50 random fields from 5 tumors per condition. Bar, 100 µm.
Figure Legend Snippet: Primary tumor growth is not affected by ABL kinase inhibitors MDA-MB 231/Dendra2 cells were injected into the mammary fat pad of 10-week-old SCID female mice and allowed to grow until the tumor reached the size of 100 mm 3 . At day 56 following injection, mice were treated by oral gavage with vehicle (5% DMSO, 2% hydroxypropyl cellulose, 0.5% Tween-80), 100 mg/kg imatinib, 70 mg/kg nilotinib or 100 mg/kg GNF-5 once a day, 5 days a week, for four weeks. ( A ) Time-dependent tumor growth. Tumor growth was assessed twice a week by measuring two perpendicular diameters and calculating tumor size in mm 3 . Treatment initiation is shown as red dotted line. n = 12 (vehicle), n = 10 (imatinib), n = 12 (nilotinib), n = 10 (GNF-5) mice per group from two independent experiments. ( B ) Primary tumors were dissected at the end of experiment and subjected to immunohistochemistry. Representative images of primary tumor sections stained with anti-PCNA (proliferation), anti-cleaved caspase 3 (apoptosis), and anti-CD31 (angiogenesis). ( C ) Quantification of PCNA positive cells (red/pink) normalized to DAPI positive cells (blue). The average percentage of proliferating cells in total cells per field is shown. ( D ) Quantification of apoptotic cells (cleaved caspase 3 positive cells). Shown is the number of apoptotic cells per field. ( E ) Quantification of CD-31 positive blood vessels. For all quantifications, n = 50 random fields from 5 tumors per condition. Bar, 100 µm.

Techniques Used: Multiple Displacement Amplification, Injection, Mouse Assay, Immunohistochemistry, Staining

7) Product Images from "The Stimulus-Dependent Gradient of Cyp26B1+ Olfactory Sensory Neurons Is Necessary for the Functional Integrity of the Olfactory Sensory Map"

Article Title: The Stimulus-Dependent Gradient of Cyp26B1+ Olfactory Sensory Neurons Is Necessary for the Functional Integrity of the Olfactory Sensory Map

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2247-15.2015

OMP promoter transgene-driven Cyp26B1 overexpression selectively in OSNs results in increased cell division and caspase-3 activation. A – E , Double OMP (green)/Cyp26B1 (magenta) immunohistochemical analysis of the olfactory epithelia (OE) and olfactory bulbs (OBs) of a 5-week-old control mouse ( B , D ) and a transgenic littermate (OMP-Cyp26B1, A , C , E ). Cyp26B1 expression is increased in OSNs and unaltered in scattered glomerular cells (arrowheads in B , C ). Encircled in B and C are individual glomerular OMP + neuropils. Scale bars, 50 μm. F , G , Quantifications of pH3 + dividing cells at PD3, PD14 and PD45 ( F ) and activated caspase-3 + cells at PD3, PD14, and PD45 ( G ) in the olfactory epithelium of OMP-Cyp26B1 (gray bars) and littermate control mice (open bars). All values represent mean and SD per hemisection ( n = 48), *** p
Figure Legend Snippet: OMP promoter transgene-driven Cyp26B1 overexpression selectively in OSNs results in increased cell division and caspase-3 activation. A – E , Double OMP (green)/Cyp26B1 (magenta) immunohistochemical analysis of the olfactory epithelia (OE) and olfactory bulbs (OBs) of a 5-week-old control mouse ( B , D ) and a transgenic littermate (OMP-Cyp26B1, A , C , E ). Cyp26B1 expression is increased in OSNs and unaltered in scattered glomerular cells (arrowheads in B , C ). Encircled in B and C are individual glomerular OMP + neuropils. Scale bars, 50 μm. F , G , Quantifications of pH3 + dividing cells at PD3, PD14 and PD45 ( F ) and activated caspase-3 + cells at PD3, PD14, and PD45 ( G ) in the olfactory epithelium of OMP-Cyp26B1 (gray bars) and littermate control mice (open bars). All values represent mean and SD per hemisection ( n = 48), *** p

Techniques Used: Over Expression, Activation Assay, Immunohistochemistry, Transgenic Assay, Expressing, Mouse Assay

Cyp26B1 inhibits differentiation of Z4 OSNs at a stage before onset of OR expression A , Double OMP (magenta) and SCG10 (green) immunohistochemical analyses of Z1 and Z4 in the olfactory epithelium of PD5, PD14, and 6-month-old Cyp26B1 transgenic and littermate control mice. B – D , Quantifications of immunohistochemical analysis of Z1 and Z4 at PD14. Shown are OMP + and SCG10 + area sizes in Z1 and Z4 in Cyp26B1 transgenic (gray bars) and littermate control (open bars) mice. Shown are also quantifications, within the same areas, of number of cells that are immunoreactive for pH3 and activated caspase-3 (Casp3). C , Total number Hoechst + nuclei located in the neuronal layer of Z4 at PD5 in Cyp26B1 transgenic (gray bars) and littermate control (open bars) mice. D , Quantification of OSNs immunoreactive for ATF5 in Z1 and Z4 at PD5 in Cyp26B1 transgenic (gray bars) and littermate control (open bars) mice. E , ATF5 (magenta) immunohistochemical analysis of olfactory epithelium of PD5 Cyp26B1 transgenic mice and littermate control mice. All values represent mean and SD; n = 48, ** p
Figure Legend Snippet: Cyp26B1 inhibits differentiation of Z4 OSNs at a stage before onset of OR expression A , Double OMP (magenta) and SCG10 (green) immunohistochemical analyses of Z1 and Z4 in the olfactory epithelium of PD5, PD14, and 6-month-old Cyp26B1 transgenic and littermate control mice. B – D , Quantifications of immunohistochemical analysis of Z1 and Z4 at PD14. Shown are OMP + and SCG10 + area sizes in Z1 and Z4 in Cyp26B1 transgenic (gray bars) and littermate control (open bars) mice. Shown are also quantifications, within the same areas, of number of cells that are immunoreactive for pH3 and activated caspase-3 (Casp3). C , Total number Hoechst + nuclei located in the neuronal layer of Z4 at PD5 in Cyp26B1 transgenic (gray bars) and littermate control (open bars) mice. D , Quantification of OSNs immunoreactive for ATF5 in Z1 and Z4 at PD5 in Cyp26B1 transgenic (gray bars) and littermate control (open bars) mice. E , ATF5 (magenta) immunohistochemical analysis of olfactory epithelium of PD5 Cyp26B1 transgenic mice and littermate control mice. All values represent mean and SD; n = 48, ** p

Techniques Used: Expressing, Immunohistochemistry, Transgenic Assay, Mouse Assay

8) Product Images from "Gyrification of the cerebral cortex requires FGF signaling in the mammalian brain"

Article Title: Gyrification of the cerebral cortex requires FGF signaling in the mammalian brain

Journal: eLife

doi: 10.7554/eLife.29285

FGF signaling is required for cell proliferation in the OSVZ of the developing ferret cortex. pCAG-EGFP plus either pCAG-sFGFR3 or pCAG control vector was electroporated at E33, and the brains were dissected at P6. Sections were stained with anti-Ki-67 ( A and B ), anti-phospho-histone H3 (pHH3) ( C and D ), anti-phosphorylated vimentin (pVim) ( E and F ) and anti-cleaved caspase 3 (cCasp3) ( G and H ) antibodies. Scale bars = 100 μm. ( A, C, E and G ) Immunohistochemical images around the VZ, the ISVZ and the OSVZ. ( B, D, F and H ) Quantification of positive cells in the VZ, the ISVZ and the OSVZ. Note that Ki-67-, pHH3- and pVim-positive cells were significantly reduced by sFGFR3 in the OSVZ selectively. n = 3 animals for each condition. Bars present mean ± SD. *p
Figure Legend Snippet: FGF signaling is required for cell proliferation in the OSVZ of the developing ferret cortex. pCAG-EGFP plus either pCAG-sFGFR3 or pCAG control vector was electroporated at E33, and the brains were dissected at P6. Sections were stained with anti-Ki-67 ( A and B ), anti-phospho-histone H3 (pHH3) ( C and D ), anti-phosphorylated vimentin (pVim) ( E and F ) and anti-cleaved caspase 3 (cCasp3) ( G and H ) antibodies. Scale bars = 100 μm. ( A, C, E and G ) Immunohistochemical images around the VZ, the ISVZ and the OSVZ. ( B, D, F and H ) Quantification of positive cells in the VZ, the ISVZ and the OSVZ. Note that Ki-67-, pHH3- and pVim-positive cells were significantly reduced by sFGFR3 in the OSVZ selectively. n = 3 animals for each condition. Bars present mean ± SD. *p

Techniques Used: Plasmid Preparation, Staining, Immunohistochemistry

9) Product Images from "Combined mTOR and MEK inhibition is an effective therapy in a novel mouse model for angiosarcoma"

Article Title: Combined mTOR and MEK inhibition is an effective therapy in a novel mouse model for angiosarcoma

Journal: Oncotarget

doi: 10.18632/oncotarget.25345

Five-day treatment with trametinib, rapamycin, or a combination of both results in disease regression ( A ) Western blots of signaling molecules on protein lysates prepared from tumors isolated from treated TKO mice comparing vehicle, rapamycin, trametinib or both drugs all administered at a dose of 1 mg/kg. ( B ) IHC demonstrating proliferation (KI67), apoptosis (cleaved caspase-3 and TUNEL), mTOR (pS6), and MAPK (pMAPK) pathway signaling in tumors from the same groups of mice as above. Scale bar in the top right panel is 50 µm and applies to all panels. ( C ) Quantification of KI67 staining from (B). * P ≤ 0.05 applies to all comparisons indicated. ( D ) Quantification of TUNEL staining from (B). ( E ) Comparison of the fold change in tumor volume over the course of the five day treatments. ** P ≤ 0.01 applies to all treatment groups compared to vehicle.
Figure Legend Snippet: Five-day treatment with trametinib, rapamycin, or a combination of both results in disease regression ( A ) Western blots of signaling molecules on protein lysates prepared from tumors isolated from treated TKO mice comparing vehicle, rapamycin, trametinib or both drugs all administered at a dose of 1 mg/kg. ( B ) IHC demonstrating proliferation (KI67), apoptosis (cleaved caspase-3 and TUNEL), mTOR (pS6), and MAPK (pMAPK) pathway signaling in tumors from the same groups of mice as above. Scale bar in the top right panel is 50 µm and applies to all panels. ( C ) Quantification of KI67 staining from (B). * P ≤ 0.05 applies to all comparisons indicated. ( D ) Quantification of TUNEL staining from (B). ( E ) Comparison of the fold change in tumor volume over the course of the five day treatments. ** P ≤ 0.01 applies to all treatment groups compared to vehicle.

Techniques Used: Western Blot, Isolation, Mouse Assay, Immunohistochemistry, TUNEL Assay, Staining

10) Product Images from "Cyclin E phosphorylation regulates cell proliferation in hematopoietic and epithelial lineages in vivo"

Article Title: Cyclin E phosphorylation regulates cell proliferation in hematopoietic and epithelial lineages in vivo

Journal: Genes & Development

doi: 10.1101/gad.1650208

Delayed mammary involution in cyclin E T74A T393A mice. ( A ) Mammary acini (indicated by arrowheads) were identified in proximity to ducts (D) and numbers of acinar units were counted per 40× field. Representative 40× micrographs are shown from H E stained FFPE sections of cyclin E wt and cyclin E T74A T393A mammary glands (#4) obtained at involution day 16. Five mice per genotype were compared, with approximately equal-sized litters and matched to involution day number. ( B ) Average numbers of acinar units per field were calculated from 40 fields per mammary gland. ( C ) Whole mounts were prepared from involuting #4 mammary glands contralateral to those studied in A , and the size of the terminal duct lobules (TDLs) were compared. The maximal cross-sectional widths of TDLs were measured from 10× images taken of whole mounts using NIH ImageJ software. Thirty to 40 TDLs per mammary gland were measured. ( D ) Tissue sections from FFPE involuting mammary glands from A were stained for Ki67, and positive-staining epithelial cells per 40× field were counted. Average numbers of Ki67-positive cells per field were calculated. ( E ) Shown are 40× micrographs from anti-activated caspase 3 stained, involuting mammary glands day 21. ( F ) Average numbers of caspase-positive cells per field were calculated.
Figure Legend Snippet: Delayed mammary involution in cyclin E T74A T393A mice. ( A ) Mammary acini (indicated by arrowheads) were identified in proximity to ducts (D) and numbers of acinar units were counted per 40× field. Representative 40× micrographs are shown from H E stained FFPE sections of cyclin E wt and cyclin E T74A T393A mammary glands (#4) obtained at involution day 16. Five mice per genotype were compared, with approximately equal-sized litters and matched to involution day number. ( B ) Average numbers of acinar units per field were calculated from 40 fields per mammary gland. ( C ) Whole mounts were prepared from involuting #4 mammary glands contralateral to those studied in A , and the size of the terminal duct lobules (TDLs) were compared. The maximal cross-sectional widths of TDLs were measured from 10× images taken of whole mounts using NIH ImageJ software. Thirty to 40 TDLs per mammary gland were measured. ( D ) Tissue sections from FFPE involuting mammary glands from A were stained for Ki67, and positive-staining epithelial cells per 40× field were counted. Average numbers of Ki67-positive cells per field were calculated. ( E ) Shown are 40× micrographs from anti-activated caspase 3 stained, involuting mammary glands day 21. ( F ) Average numbers of caspase-positive cells per field were calculated.

Techniques Used: Mouse Assay, Staining, Formalin-fixed Paraffin-Embedded, Software

Analysis of cyclin E T74A T393A expression and activity in epithelial tissues. Micrographs at 40× are shown of sections from FFPE colon tissues obtained from age-matched males ( A ) and mammary glands obtained from females sacrificed during timed breedings ( B ) and stained with an affinity purified cyclin E antibody. ( C ) Epithelial cell proliferation was studied in situ in cyclin E wt , cyclin E T393A , and cyclin E T74A T393A mammary glands by staining tissues for Ki67 proliferation antigen expression and BrdU incorporation. Three age- and gestation time-matched mice per genotype were BrdU pulsed, and positive cells were counted per 40× field ( right panel). ( Left panels) Micrographs at 20× are shown for cyclin E wt and cyclin E T74A T393A mammary glands obtained from pregnant mice at D7.5 pc. At least 40 fields were counted per gland. Bars indicate standard error. ( D ) Apoptosis was detected in mammary tissues from age- and gestation time-matched mice by staining with anti-cleaved caspase 3 antibody, and numbers of positive cells per high power field areshown.
Figure Legend Snippet: Analysis of cyclin E T74A T393A expression and activity in epithelial tissues. Micrographs at 40× are shown of sections from FFPE colon tissues obtained from age-matched males ( A ) and mammary glands obtained from females sacrificed during timed breedings ( B ) and stained with an affinity purified cyclin E antibody. ( C ) Epithelial cell proliferation was studied in situ in cyclin E wt , cyclin E T393A , and cyclin E T74A T393A mammary glands by staining tissues for Ki67 proliferation antigen expression and BrdU incorporation. Three age- and gestation time-matched mice per genotype were BrdU pulsed, and positive cells were counted per 40× field ( right panel). ( Left panels) Micrographs at 20× are shown for cyclin E wt and cyclin E T74A T393A mammary glands obtained from pregnant mice at D7.5 pc. At least 40 fields were counted per gland. Bars indicate standard error. ( D ) Apoptosis was detected in mammary tissues from age- and gestation time-matched mice by staining with anti-cleaved caspase 3 antibody, and numbers of positive cells per high power field areshown.

Techniques Used: Expressing, Activity Assay, Formalin-fixed Paraffin-Embedded, Staining, Affinity Purification, In Situ, BrdU Incorporation Assay, Mouse Assay

11) Product Images from "Infection and Functional Modulation of Human Monocytes and Macrophages by Varicella-Zoster Virus"

Article Title: Infection and Functional Modulation of Human Monocytes and Macrophages by Varicella-Zoster Virus

Journal: Journal of Virology

doi: 10.1128/JVI.01887-18

Viability of VZV-infected monocytes. Monocytes without exposure to HFF and mock- and VZV-infected monocytes were stained with live/dead dye (L/D) and antibodies for VZV gE:gI and intracellular cleaved caspase-3 (CC3). (A) VZV-infected monocytes were identified as VZV gE:gI + (red box), and VZV-exposed monocytes were identified as VZV gE:gI – (orange box). (B) Representative plot of VZV-infected monocytes stratified into viable cells (CC3 – , L/D – ) and cells with early apoptotic (CC3 + , L/D – ), late apoptotic (CC3 + , L/D + ), and nonapoptotic (CC3 – , L/D + ) cell death. (C) The proportions of monocytes (±SEM) at 24 hpi (top) and 48 hpi (bottom) are shown. Statistics were performed on the proportions of cells by repeated-measures two-way analysis of variance with Tukey’s multiple-comparison test. *, P
Figure Legend Snippet: Viability of VZV-infected monocytes. Monocytes without exposure to HFF and mock- and VZV-infected monocytes were stained with live/dead dye (L/D) and antibodies for VZV gE:gI and intracellular cleaved caspase-3 (CC3). (A) VZV-infected monocytes were identified as VZV gE:gI + (red box), and VZV-exposed monocytes were identified as VZV gE:gI – (orange box). (B) Representative plot of VZV-infected monocytes stratified into viable cells (CC3 – , L/D – ) and cells with early apoptotic (CC3 + , L/D – ), late apoptotic (CC3 + , L/D + ), and nonapoptotic (CC3 – , L/D + ) cell death. (C) The proportions of monocytes (±SEM) at 24 hpi (top) and 48 hpi (bottom) are shown. Statistics were performed on the proportions of cells by repeated-measures two-way analysis of variance with Tukey’s multiple-comparison test. *, P

Techniques Used: Infection, Staining

12) Product Images from "Discovery of a drug candidate for GLIS3-associated diabetes"

Article Title: Discovery of a drug candidate for GLIS3-associated diabetes

Journal: Nature Communications

doi: 10.1038/s41467-018-04918-x

A high-content chemical screen identifies galunisertib as a drug candidate to rescue cell death induced by loss of GLIS3 both in vitro and in vivo. a Schematic representation of the high-content chemical screen. b Chemical structure of galunisertib. c Inhibitory curve of galunisertib. d Immunocyto-chemistry analysis of GLIS3 −/− PP2-β cells treated with DMSO or 10 µM galunisertib. Scale bar = 100 μm, scale bar of high magnification insets = 40 μm. e , f Quantification of the cell death rate ( e , the percentage of PI + INS + cells in INS + cells, n = 4) and apoptosis rate ( f , the percentage of cleaved caspase-3 + INS + cells in INS + cells, n = 3) of GLIS3 −/− PP2-β cells treated with DMSO or 10 µM galunisertib. Flow cytometry analysis ( g ) and quantification ( h ) of early apoptotic cells (the percentage of Annexin V + /DAPI − cells) in GLIS3 −/− INS-GFP + PP2-β cells treated with DMSO or 10 μM galunisertib ( n = 6). i Relative number of INS + cells in GLIS3 −/− PP2-β cells treated with DMSO or 10 μM galunisertib. Data are normalized to DMSO-treated values ( n = 4). j Schematic representation of the in vivo transplantation and drug treatment experiments. k Immunohistochemistry analysis of INS, cleaved caspase-3, and STEM121 in the grafts isolated from vehicle- or galunisertib-treated mice. Scale bar = 100 μm. l Quantification of immunohistochemistry data in j ( n = 7). m Quantification of the percentage of apoptotic INS + cells (CAS3 + INS + STEM121 + ) in the INS + population within the grafts from vehicle- or galunisertib-treated mice (INS + STEM121 + , n = 6). CAS3: cleaved caspase-3. P values by unpaired two-tailed t -test were * P
Figure Legend Snippet: A high-content chemical screen identifies galunisertib as a drug candidate to rescue cell death induced by loss of GLIS3 both in vitro and in vivo. a Schematic representation of the high-content chemical screen. b Chemical structure of galunisertib. c Inhibitory curve of galunisertib. d Immunocyto-chemistry analysis of GLIS3 −/− PP2-β cells treated with DMSO or 10 µM galunisertib. Scale bar = 100 μm, scale bar of high magnification insets = 40 μm. e , f Quantification of the cell death rate ( e , the percentage of PI + INS + cells in INS + cells, n = 4) and apoptosis rate ( f , the percentage of cleaved caspase-3 + INS + cells in INS + cells, n = 3) of GLIS3 −/− PP2-β cells treated with DMSO or 10 µM galunisertib. Flow cytometry analysis ( g ) and quantification ( h ) of early apoptotic cells (the percentage of Annexin V + /DAPI − cells) in GLIS3 −/− INS-GFP + PP2-β cells treated with DMSO or 10 μM galunisertib ( n = 6). i Relative number of INS + cells in GLIS3 −/− PP2-β cells treated with DMSO or 10 μM galunisertib. Data are normalized to DMSO-treated values ( n = 4). j Schematic representation of the in vivo transplantation and drug treatment experiments. k Immunohistochemistry analysis of INS, cleaved caspase-3, and STEM121 in the grafts isolated from vehicle- or galunisertib-treated mice. Scale bar = 100 μm. l Quantification of immunohistochemistry data in j ( n = 7). m Quantification of the percentage of apoptotic INS + cells (CAS3 + INS + STEM121 + ) in the INS + population within the grafts from vehicle- or galunisertib-treated mice (INS + STEM121 + , n = 6). CAS3: cleaved caspase-3. P values by unpaired two-tailed t -test were * P

Techniques Used: In Vitro, In Vivo, Flow Cytometry, Cytometry, Transplantation Assay, Immunohistochemistry, Isolation, Mouse Assay, Two Tailed Test

Loss of GLIS3 leads to increased cell death in PP2 and PP2-β cells. a Quantification of early apoptotic cells (the percentage of Annexin V + /DAPI − cells) in WT and GLIS3 −/− ES, DE, PP1, and PP2 cells ( n = 3). b Representative flow cytometry analysis plots of Annexin V staining in WT and GLIS3 −/− cells at D23_L. c Immunostaining for PDX1 and cleaved caspase-3 in WT and GLIS3 −/− cells at D23_L. Scale bar = 40 μm. Annexin V staining ( d ) and quantification ( e ) of early apoptotic cells in the INS-GFP + cells at D30_L ( n = 6). Histogram showing fluorescence intensity ( f ) and quantification of median fluorescence values ( g ) of Annexin V staining of WT and GLIS3 −/− INS + DAPI − PP2-β cells (WT n = 4, GLIS3 −/− n = 6). h PI, cleaved caspase-3 and INS staining of WT and GLIS3 −/− cells at D30_L. Scale bar = 40 μm. i Quantification of cell death rate (the percentage of PI + INS + cells in INS + cells) and apoptosis rate (the percentage of cleaved caspase-3 + INS + cells in INS + cells) of WT and GLIS3 −/− INS + PP2-β cells ( n = 3). j Schematic representation of the in vivo transplantation experiment. k Immunostaining for INS, cleaved caspase-3 and STEM121 in the grafts of mice transplanted with WT or GLIS3 −/− cells. Scale bar = 100 μm. l Quantification of the apoptosis rate (the percentage of cleaved caspase-3 + / STEM121 + cells in STEM121 + cells) within WT and GLIS3 −/− grafts ( n = 7 for WT, n = 4 for GLIS3 −/− ). m Quantification of the percentage of apoptotic INS + cells (CAS3 + PDX1 + STEM121 + ) in the INS + population within the WT and GLIS3 −/− grafts (INS + STEM121 + , WT n = 7, GLIS3 −/− n = 4). CAS3: cleaved caspase-3. P values by unpaired two-tailed Student’s t -test were * P
Figure Legend Snippet: Loss of GLIS3 leads to increased cell death in PP2 and PP2-β cells. a Quantification of early apoptotic cells (the percentage of Annexin V + /DAPI − cells) in WT and GLIS3 −/− ES, DE, PP1, and PP2 cells ( n = 3). b Representative flow cytometry analysis plots of Annexin V staining in WT and GLIS3 −/− cells at D23_L. c Immunostaining for PDX1 and cleaved caspase-3 in WT and GLIS3 −/− cells at D23_L. Scale bar = 40 μm. Annexin V staining ( d ) and quantification ( e ) of early apoptotic cells in the INS-GFP + cells at D30_L ( n = 6). Histogram showing fluorescence intensity ( f ) and quantification of median fluorescence values ( g ) of Annexin V staining of WT and GLIS3 −/− INS + DAPI − PP2-β cells (WT n = 4, GLIS3 −/− n = 6). h PI, cleaved caspase-3 and INS staining of WT and GLIS3 −/− cells at D30_L. Scale bar = 40 μm. i Quantification of cell death rate (the percentage of PI + INS + cells in INS + cells) and apoptosis rate (the percentage of cleaved caspase-3 + INS + cells in INS + cells) of WT and GLIS3 −/− INS + PP2-β cells ( n = 3). j Schematic representation of the in vivo transplantation experiment. k Immunostaining for INS, cleaved caspase-3 and STEM121 in the grafts of mice transplanted with WT or GLIS3 −/− cells. Scale bar = 100 μm. l Quantification of the apoptosis rate (the percentage of cleaved caspase-3 + / STEM121 + cells in STEM121 + cells) within WT and GLIS3 −/− grafts ( n = 7 for WT, n = 4 for GLIS3 −/− ). m Quantification of the percentage of apoptotic INS + cells (CAS3 + PDX1 + STEM121 + ) in the INS + population within the WT and GLIS3 −/− grafts (INS + STEM121 + , WT n = 7, GLIS3 −/− n = 4). CAS3: cleaved caspase-3. P values by unpaired two-tailed Student’s t -test were * P

Techniques Used: Flow Cytometry, Cytometry, Staining, Immunostaining, Fluorescence, In Vivo, Transplantation Assay, Mouse Assay, Two Tailed Test

13) Product Images from "Retinoic acid receptor and CNGA2 channel signaling are part of a regulatory feedback loop controlling axonal convergence and survival of olfactory sensory neurons"

Article Title: Retinoic acid receptor and CNGA2 channel signaling are part of a regulatory feedback loop controlling axonal convergence and survival of olfactory sensory neurons

Journal: The FASEB Journal

doi: 10.1096/fj.11-192450

Nrp1, Kirrel-2, and activated caspase-3 expression in the glomerular layer of the olfactory bulb. A ) Immunohistochemical analysis of the glomerular layer in PD14 mice with antibodies specific for OMP, Nrp1, and Kirrel-2. Axonal OMP is unaltered, whereas Nrp1 is higher, in glomeruli of dnRAR transgenics. Kirrel-2 immunoreactivity is reduced in OSN axons of dnRAR transgenics. Arrow indicates a glomerulus in a dnRAR transgenic mouse that contains a neuropil with mixed high and low Kirrel-2 regions. B ) Double-activated caspase-3 (Casp-3; red) and Kirrel-2 (green) immunohistochemical analysis of glomeruli of PD14 dnRAR transgenic mice. a–c ) Two glomeruli are shown; one contains OSN axons with high levels of Kirrel-2 and no activated caspase-3 (arrows), whereas the other glomerulus contains axons that express low levels of Kirrel-2 and high levels of activated caspase-3 (arrowheads). d–f ) Two glomeruli are shown; one contains axons with high levels of Kirrel-2 and no activated caspase-3 (arrows), whereas the other is a heterogeneous glomerulus with regions that show an inverse relationship between axonal Kirrel-2 expression and caspase-3 activation (arrowhead). Dashed outlines delineate individual glomeruli.
Figure Legend Snippet: Nrp1, Kirrel-2, and activated caspase-3 expression in the glomerular layer of the olfactory bulb. A ) Immunohistochemical analysis of the glomerular layer in PD14 mice with antibodies specific for OMP, Nrp1, and Kirrel-2. Axonal OMP is unaltered, whereas Nrp1 is higher, in glomeruli of dnRAR transgenics. Kirrel-2 immunoreactivity is reduced in OSN axons of dnRAR transgenics. Arrow indicates a glomerulus in a dnRAR transgenic mouse that contains a neuropil with mixed high and low Kirrel-2 regions. B ) Double-activated caspase-3 (Casp-3; red) and Kirrel-2 (green) immunohistochemical analysis of glomeruli of PD14 dnRAR transgenic mice. a–c ) Two glomeruli are shown; one contains OSN axons with high levels of Kirrel-2 and no activated caspase-3 (arrows), whereas the other glomerulus contains axons that express low levels of Kirrel-2 and high levels of activated caspase-3 (arrowheads). d–f ) Two glomeruli are shown; one contains axons with high levels of Kirrel-2 and no activated caspase-3 (arrows), whereas the other is a heterogeneous glomerulus with regions that show an inverse relationship between axonal Kirrel-2 expression and caspase-3 activation (arrowhead). Dashed outlines delineate individual glomeruli.

Techniques Used: Expressing, Immunohistochemistry, Mouse Assay, Transgenic Assay, Activation Assay

Correlations among neuronal activity, CNGA2 deficiency, Kirrel-2, caspase-3 activation, and cell death. A ) Representative images of activated caspase-3 immunohistochemical analyses of control (cont) and heterozygous female CNGA2-null mice (CNGA2 +/− ) at PD10. More cells were positive for activated caspase-3 (red signal) in the olfactory epithelium of CNGA2 +/− compared with control. Dashed lines indicate basal lamina. B ) Representative images showing double activated caspase-3 (Casp-3; red) and Kirrel-2 (green) immunohistochemical analysis of glomeruli of PD10 CNGA2 +/− female mice. Three glomeruli contain OSN axons with high Kirrel-2 levels and no activated caspase-3 (arrows), whereas the 2 glomeruli contain axons that express low levels of Kirrel-2 and high levels of activated caspase-3 (arrowheads). C ) Quantifications of total numbers of OSNs expressing specific ORs (MOL2.3, P2, MOR256-17, and K21) in 1-mo-old controls (solid bars), hemizygous dnRAR transgenic mice (shaded bars), and homozygous dnRAR transgenic mice (open bars). Numbers of OR-positive OSNs are given as percentage of controls (%OR+). D ) Quantification of activated caspase-3-positive OSNs in the olfactory epithelium of PD10 mice subjected to unilateral naris closure at PD1. Bars show the number of activated caspase-3-positive cells (Casp+) per section in epithelia ipsilateral (closed) or contralateral (open) to the closed nostril. Naris closure does not inhibit dnRAR-dependent caspase-3 activation. Instead, the fold inductions of dnRAR-dependent caspase-3 activation for open and closed nostrils were 1.6 ± 0.3 and 2.4 ± 0.3, respectively. E ) Immunohistochemical analyses of activated caspase-3 and tyrosine hydroxylase (TH) immunoreactivity in the glomerular layer of PD10 dnRAR transgenic mice subjected to unilateral naris closure at PD1. Closure reduced postsynaptic TH expression in both control and dnRAR transgenic mice, without an apparent change in axonal caspase-3 activation. Dashed outlines delineate individual glomeruli. Scale bars = 40 μm ( A , B ); 50 μm ( C , D ). All values are given as means ± sd ; n = 3. * P
Figure Legend Snippet: Correlations among neuronal activity, CNGA2 deficiency, Kirrel-2, caspase-3 activation, and cell death. A ) Representative images of activated caspase-3 immunohistochemical analyses of control (cont) and heterozygous female CNGA2-null mice (CNGA2 +/− ) at PD10. More cells were positive for activated caspase-3 (red signal) in the olfactory epithelium of CNGA2 +/− compared with control. Dashed lines indicate basal lamina. B ) Representative images showing double activated caspase-3 (Casp-3; red) and Kirrel-2 (green) immunohistochemical analysis of glomeruli of PD10 CNGA2 +/− female mice. Three glomeruli contain OSN axons with high Kirrel-2 levels and no activated caspase-3 (arrows), whereas the 2 glomeruli contain axons that express low levels of Kirrel-2 and high levels of activated caspase-3 (arrowheads). C ) Quantifications of total numbers of OSNs expressing specific ORs (MOL2.3, P2, MOR256-17, and K21) in 1-mo-old controls (solid bars), hemizygous dnRAR transgenic mice (shaded bars), and homozygous dnRAR transgenic mice (open bars). Numbers of OR-positive OSNs are given as percentage of controls (%OR+). D ) Quantification of activated caspase-3-positive OSNs in the olfactory epithelium of PD10 mice subjected to unilateral naris closure at PD1. Bars show the number of activated caspase-3-positive cells (Casp+) per section in epithelia ipsilateral (closed) or contralateral (open) to the closed nostril. Naris closure does not inhibit dnRAR-dependent caspase-3 activation. Instead, the fold inductions of dnRAR-dependent caspase-3 activation for open and closed nostrils were 1.6 ± 0.3 and 2.4 ± 0.3, respectively. E ) Immunohistochemical analyses of activated caspase-3 and tyrosine hydroxylase (TH) immunoreactivity in the glomerular layer of PD10 dnRAR transgenic mice subjected to unilateral naris closure at PD1. Closure reduced postsynaptic TH expression in both control and dnRAR transgenic mice, without an apparent change in axonal caspase-3 activation. Dashed outlines delineate individual glomeruli. Scale bars = 40 μm ( A , B ); 50 μm ( C , D ). All values are given as means ± sd ; n = 3. * P

Techniques Used: Activity Assay, Activation Assay, Immunohistochemistry, Mouse Assay, Expressing, Transgenic Assay

14) Product Images from "Discovery of a drug candidate for GLIS3-associated diabetes"

Article Title: Discovery of a drug candidate for GLIS3-associated diabetes

Journal: Nature Communications

doi: 10.1038/s41467-018-04918-x

A high-content chemical screen identifies galunisertib as a drug candidate to rescue cell death induced by loss of GLIS3 both in vitro and in vivo. a Schematic representation of the high-content chemical screen. b Chemical structure of galunisertib. c Inhibitory curve of galunisertib. d Immunocyto-chemistry analysis of GLIS3 −/− PP2-β cells treated with DMSO or 10 µM galunisertib. Scale bar = 100 μm, scale bar of high magnification insets = 40 μm. e , f Quantification of the cell death rate ( e , the percentage of PI + INS + cells in INS + cells, n = 4) and apoptosis rate ( f , the percentage of cleaved caspase-3 + INS + cells in INS + cells, n = 3) of GLIS3 −/− PP2-β cells treated with DMSO or 10 µM galunisertib. Flow cytometry analysis ( g ) and quantification ( h ) of early apoptotic cells (the percentage of Annexin V + /DAPI − cells) in GLIS3 −/− INS-GFP + PP2-β cells treated with DMSO or 10 μM galunisertib ( n = 6). i Relative number of INS + cells in GLIS3 −/− PP2-β cells treated with DMSO or 10 μM galunisertib. Data are normalized to DMSO-treated values ( n = 4). j Schematic representation of the in vivo transplantation and drug treatment experiments. k Immunohistochemistry analysis of INS, cleaved caspase-3, and STEM121 in the grafts isolated from vehicle- or galunisertib-treated mice. Scale bar = 100 μm. l Quantification of immunohistochemistry data in j ( n = 7). m Quantification of the percentage of apoptotic INS + cells (CAS3 + INS + STEM121 + ) in the INS + population within the grafts from vehicle- or galunisertib-treated mice (INS + STEM121 + , n = 6). CAS3: cleaved caspase-3. P values by unpaired two-tailed t -test were * P
Figure Legend Snippet: A high-content chemical screen identifies galunisertib as a drug candidate to rescue cell death induced by loss of GLIS3 both in vitro and in vivo. a Schematic representation of the high-content chemical screen. b Chemical structure of galunisertib. c Inhibitory curve of galunisertib. d Immunocyto-chemistry analysis of GLIS3 −/− PP2-β cells treated with DMSO or 10 µM galunisertib. Scale bar = 100 μm, scale bar of high magnification insets = 40 μm. e , f Quantification of the cell death rate ( e , the percentage of PI + INS + cells in INS + cells, n = 4) and apoptosis rate ( f , the percentage of cleaved caspase-3 + INS + cells in INS + cells, n = 3) of GLIS3 −/− PP2-β cells treated with DMSO or 10 µM galunisertib. Flow cytometry analysis ( g ) and quantification ( h ) of early apoptotic cells (the percentage of Annexin V + /DAPI − cells) in GLIS3 −/− INS-GFP + PP2-β cells treated with DMSO or 10 μM galunisertib ( n = 6). i Relative number of INS + cells in GLIS3 −/− PP2-β cells treated with DMSO or 10 μM galunisertib. Data are normalized to DMSO-treated values ( n = 4). j Schematic representation of the in vivo transplantation and drug treatment experiments. k Immunohistochemistry analysis of INS, cleaved caspase-3, and STEM121 in the grafts isolated from vehicle- or galunisertib-treated mice. Scale bar = 100 μm. l Quantification of immunohistochemistry data in j ( n = 7). m Quantification of the percentage of apoptotic INS + cells (CAS3 + INS + STEM121 + ) in the INS + population within the grafts from vehicle- or galunisertib-treated mice (INS + STEM121 + , n = 6). CAS3: cleaved caspase-3. P values by unpaired two-tailed t -test were * P

Techniques Used: In Vitro, In Vivo, Flow Cytometry, Cytometry, Transplantation Assay, Immunohistochemistry, Isolation, Mouse Assay, Two Tailed Test

Loss of GLIS3 leads to increased cell death in PP2 and PP2-β cells. a Quantification of early apoptotic cells (the percentage of Annexin V + /DAPI − cells) in WT and GLIS3 −/− ES, DE, PP1, and PP2 cells ( n = 3). b Representative flow cytometry analysis plots of Annexin V staining in WT and GLIS3 −/− cells at D23_L. c Immunostaining for PDX1 and cleaved caspase-3 in WT and GLIS3 −/− cells at D23_L. Scale bar = 40 μm. Annexin V staining ( d ) and quantification ( e ) of early apoptotic cells in the INS-GFP + cells at D30_L ( n = 6). Histogram showing fluorescence intensity ( f ) and quantification of median fluorescence values ( g ) of Annexin V staining of WT and GLIS3 −/− INS + DAPI − PP2-β cells (WT n = 4, GLIS3 −/− n = 6). h PI, cleaved caspase-3 and INS staining of WT and GLIS3 −/− cells at D30_L. Scale bar = 40 μm. i Quantification of cell death rate (the percentage of PI + INS + cells in INS + cells) and apoptosis rate (the percentage of cleaved caspase-3 + INS + cells in INS + cells) of WT and GLIS3 −/− INS + PP2-β cells ( n = 3). j Schematic representation of the in vivo transplantation experiment. k Immunostaining for INS, cleaved caspase-3 and STEM121 in the grafts of mice transplanted with WT or GLIS3 −/− cells. Scale bar = 100 μm. l Quantification of the apoptosis rate (the percentage of cleaved caspase-3 + / STEM121 + cells in STEM121 + cells) within WT and GLIS3 −/− grafts ( n = 7 for WT, n = 4 for GLIS3 −/− ). m Quantification of the percentage of apoptotic INS + cells (CAS3 + PDX1 + STEM121 + ) in the INS + population within the WT and GLIS3 −/− grafts (INS + STEM121 + , WT n = 7, GLIS3 −/− n = 4). CAS3: cleaved caspase-3. P values by unpaired two-tailed Student’s t -test were * P
Figure Legend Snippet: Loss of GLIS3 leads to increased cell death in PP2 and PP2-β cells. a Quantification of early apoptotic cells (the percentage of Annexin V + /DAPI − cells) in WT and GLIS3 −/− ES, DE, PP1, and PP2 cells ( n = 3). b Representative flow cytometry analysis plots of Annexin V staining in WT and GLIS3 −/− cells at D23_L. c Immunostaining for PDX1 and cleaved caspase-3 in WT and GLIS3 −/− cells at D23_L. Scale bar = 40 μm. Annexin V staining ( d ) and quantification ( e ) of early apoptotic cells in the INS-GFP + cells at D30_L ( n = 6). Histogram showing fluorescence intensity ( f ) and quantification of median fluorescence values ( g ) of Annexin V staining of WT and GLIS3 −/− INS + DAPI − PP2-β cells (WT n = 4, GLIS3 −/− n = 6). h PI, cleaved caspase-3 and INS staining of WT and GLIS3 −/− cells at D30_L. Scale bar = 40 μm. i Quantification of cell death rate (the percentage of PI + INS + cells in INS + cells) and apoptosis rate (the percentage of cleaved caspase-3 + INS + cells in INS + cells) of WT and GLIS3 −/− INS + PP2-β cells ( n = 3). j Schematic representation of the in vivo transplantation experiment. k Immunostaining for INS, cleaved caspase-3 and STEM121 in the grafts of mice transplanted with WT or GLIS3 −/− cells. Scale bar = 100 μm. l Quantification of the apoptosis rate (the percentage of cleaved caspase-3 + / STEM121 + cells in STEM121 + cells) within WT and GLIS3 −/− grafts ( n = 7 for WT, n = 4 for GLIS3 −/− ). m Quantification of the percentage of apoptotic INS + cells (CAS3 + PDX1 + STEM121 + ) in the INS + population within the WT and GLIS3 −/− grafts (INS + STEM121 + , WT n = 7, GLIS3 −/− n = 4). CAS3: cleaved caspase-3. P values by unpaired two-tailed Student’s t -test were * P

Techniques Used: Flow Cytometry, Cytometry, Staining, Immunostaining, Fluorescence, In Vivo, Transplantation Assay, Mouse Assay, Two Tailed Test

15) Product Images from "Combined mTOR and MEK inhibition is an effective therapy in a novel mouse model for angiosarcoma"

Article Title: Combined mTOR and MEK inhibition is an effective therapy in a novel mouse model for angiosarcoma

Journal: Oncotarget

doi: 10.18632/oncotarget.25345

Five-day treatment with trametinib, rapamycin, or a combination of both results in disease regression ( A ) Western blots of signaling molecules on protein lysates prepared from tumors isolated from treated TKO mice comparing vehicle, rapamycin, trametinib or both drugs all administered at a dose of 1 mg/kg. ( B ) IHC demonstrating proliferation (KI67), apoptosis (cleaved caspase-3 and TUNEL), mTOR (pS6), and MAPK (pMAPK) pathway signaling in tumors from the same groups of mice as above. Scale bar in the top right panel is 50 µm and applies to all panels. ( C ) Quantification of KI67 staining from (B). * P ≤ 0.05 applies to all comparisons indicated. ( D ) Quantification of TUNEL staining from (B). ( E ) Comparison of the fold change in tumor volume over the course of the five day treatments. ** P ≤ 0.01 applies to all treatment groups compared to vehicle.
Figure Legend Snippet: Five-day treatment with trametinib, rapamycin, or a combination of both results in disease regression ( A ) Western blots of signaling molecules on protein lysates prepared from tumors isolated from treated TKO mice comparing vehicle, rapamycin, trametinib or both drugs all administered at a dose of 1 mg/kg. ( B ) IHC demonstrating proliferation (KI67), apoptosis (cleaved caspase-3 and TUNEL), mTOR (pS6), and MAPK (pMAPK) pathway signaling in tumors from the same groups of mice as above. Scale bar in the top right panel is 50 µm and applies to all panels. ( C ) Quantification of KI67 staining from (B). * P ≤ 0.05 applies to all comparisons indicated. ( D ) Quantification of TUNEL staining from (B). ( E ) Comparison of the fold change in tumor volume over the course of the five day treatments. ** P ≤ 0.01 applies to all treatment groups compared to vehicle.

Techniques Used: Western Blot, Isolation, Mouse Assay, Immunohistochemistry, TUNEL Assay, Staining

Long-Term Treatment using both rapamycin and trametinib results in sustained disease regression ( A ) Comparison of change in tumor volume in long-term treatments of tumor-bearing TKO mice. The red arrow represents when mice were taken off treatment at 140 days. Black arrows with lower-case letters represent the time points at which samples were analyzed in ( D ). The table below indicates the number of mice remaining from each cohort at the indicated time points. ( B ) Disease-related mortality analyzed by Kaplan–Meier. Non-tumor related deaths are indicated by the black tick marks. Statistical significance beside each curve is compared with the dual drug treatment. ** P ≤ 0.01, **** P ≤ 0.0001. ( C ) Waterfall plot showing the best responses in drug-treated tumors and maximum growth in vehicle-treated tumors. ( D ) Immunohistochemistry comparing mTOR and MAPK signaling, proliferation and apoptosis at various timepoints. Letters in the lefthand panels correspond to the timepoints when tissue was harvested as delineated in (A). Scale bar in vehicle cleaved caspase-3 panel represents 50 µm and applies to all panels. ( E ) Quantification of TUNEL staining shown in (D). ( F ) Quantification of KI67 staining shown in (D).
Figure Legend Snippet: Long-Term Treatment using both rapamycin and trametinib results in sustained disease regression ( A ) Comparison of change in tumor volume in long-term treatments of tumor-bearing TKO mice. The red arrow represents when mice were taken off treatment at 140 days. Black arrows with lower-case letters represent the time points at which samples were analyzed in ( D ). The table below indicates the number of mice remaining from each cohort at the indicated time points. ( B ) Disease-related mortality analyzed by Kaplan–Meier. Non-tumor related deaths are indicated by the black tick marks. Statistical significance beside each curve is compared with the dual drug treatment. ** P ≤ 0.01, **** P ≤ 0.0001. ( C ) Waterfall plot showing the best responses in drug-treated tumors and maximum growth in vehicle-treated tumors. ( D ) Immunohistochemistry comparing mTOR and MAPK signaling, proliferation and apoptosis at various timepoints. Letters in the lefthand panels correspond to the timepoints when tissue was harvested as delineated in (A). Scale bar in vehicle cleaved caspase-3 panel represents 50 µm and applies to all panels. ( E ) Quantification of TUNEL staining shown in (D). ( F ) Quantification of KI67 staining shown in (D).

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

16) Product Images from "Protective effect of Xuebijing injection on D-galactosamine- and lipopolysaccharide-induced acute liver injury in rats through the regulation of p38 MAPK, MMP-9 and HO-1 expression by increasing TIPE2 expression"

Article Title: Protective effect of Xuebijing injection on D-galactosamine- and lipopolysaccharide-induced acute liver injury in rats through the regulation of p38 MAPK, MMP-9 and HO-1 expression by increasing TIPE2 expression

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2016.2749

Effect of the administration of Xuebijing injection (XBJ) on the positive expression of cleaved caspase-3 in the liver tissue of lipopolysaccharide (LPS) plus D-galactosamine (D-Gal)-exposed rats. The groups of rats were challenged with LPS, 10 µ g/kg plus D-Gal, 500 mg/kg and treated with XBJ 24 h later. Immunostaining was performed on the liver sections following antigen retrieval using Retrievagen A. Representative images of immunostaining show the positive expression levels of cleaved caspase-3 in the five groups of rats (immunofluorescence staining; magnification, ×200):(A) control group, (B) model group and (C–E) treatment groups (XBJ, at doses of 5, 10 and 15 ml/kg, respectively).
Figure Legend Snippet: Effect of the administration of Xuebijing injection (XBJ) on the positive expression of cleaved caspase-3 in the liver tissue of lipopolysaccharide (LPS) plus D-galactosamine (D-Gal)-exposed rats. The groups of rats were challenged with LPS, 10 µ g/kg plus D-Gal, 500 mg/kg and treated with XBJ 24 h later. Immunostaining was performed on the liver sections following antigen retrieval using Retrievagen A. Representative images of immunostaining show the positive expression levels of cleaved caspase-3 in the five groups of rats (immunofluorescence staining; magnification, ×200):(A) control group, (B) model group and (C–E) treatment groups (XBJ, at doses of 5, 10 and 15 ml/kg, respectively).

Techniques Used: Injection, Expressing, Immunostaining, Immunofluorescence, Staining

Administration of Xuebijing injection (XBJ) decreases the positive expression of cleaved caspase-3 in the liver tissues of lipopolysaccharide (LPS) plus D-galactosamine (D-Gal)-exposed rats. The groups of rats were challenged with LPS, 10 µ g/kg plus D-Gal, 500 mg/kg and treated with XBJ 24 h later. Immunostaining was performed on the liver sections following antigen retrieval using Retrievagen A. Using Image-Pro Plus image analysis software, the cleaved caspase-3 positive expression levels in the liver tissue were calculated. Data are presented as the means ± SD of one experiment consisting of three replicates. The experiments were performed in triplicate. ** P
Figure Legend Snippet: Administration of Xuebijing injection (XBJ) decreases the positive expression of cleaved caspase-3 in the liver tissues of lipopolysaccharide (LPS) plus D-galactosamine (D-Gal)-exposed rats. The groups of rats were challenged with LPS, 10 µ g/kg plus D-Gal, 500 mg/kg and treated with XBJ 24 h later. Immunostaining was performed on the liver sections following antigen retrieval using Retrievagen A. Using Image-Pro Plus image analysis software, the cleaved caspase-3 positive expression levels in the liver tissue were calculated. Data are presented as the means ± SD of one experiment consisting of three replicates. The experiments were performed in triplicate. ** P

Techniques Used: Injection, Expressing, Immunostaining, Software

17) Product Images from "Discovery of a drug candidate for GLIS3-associated diabetes"

Article Title: Discovery of a drug candidate for GLIS3-associated diabetes

Journal: Nature Communications

doi: 10.1038/s41467-018-04918-x

A high-content chemical screen identifies galunisertib as a drug candidate to rescue cell death induced by loss of GLIS3 both in vitro and in vivo. a Schematic representation of the high-content chemical screen. b Chemical structure of galunisertib. c Inhibitory curve of galunisertib. d Immunocyto-chemistry analysis of GLIS3 −/− PP2-β cells treated with DMSO or 10 µM galunisertib. Scale bar = 100 μm, scale bar of high magnification insets = 40 μm. e , f Quantification of the cell death rate ( e , the percentage of PI + INS + cells in INS + cells, n = 4) and apoptosis rate ( f , the percentage of cleaved caspase-3 + INS + cells in INS + cells, n = 3) of GLIS3 −/− PP2-β cells treated with DMSO or 10 µM galunisertib. Flow cytometry analysis ( g ) and quantification ( h ) of early apoptotic cells (the percentage of Annexin V + /DAPI − cells) in GLIS3 −/− INS-GFP + PP2-β cells treated with DMSO or 10 μM galunisertib ( n = 6). i Relative number of INS + cells in GLIS3 −/− PP2-β cells treated with DMSO or 10 μM galunisertib. Data are normalized to DMSO-treated values ( n = 4). j Schematic representation of the in vivo transplantation and drug treatment experiments. k Immunohistochemistry analysis of INS, cleaved caspase-3, and STEM121 in the grafts isolated from vehicle- or galunisertib-treated mice. Scale bar = 100 μm. l Quantification of immunohistochemistry data in j ( n = 7). m Quantification of the percentage of apoptotic INS + cells (CAS3 + INS + STEM121 + ) in the INS + population within the grafts from vehicle- or galunisertib-treated mice (INS + STEM121 + , n = 6). CAS3: cleaved caspase-3. P values by unpaired two-tailed t -test were * P
Figure Legend Snippet: A high-content chemical screen identifies galunisertib as a drug candidate to rescue cell death induced by loss of GLIS3 both in vitro and in vivo. a Schematic representation of the high-content chemical screen. b Chemical structure of galunisertib. c Inhibitory curve of galunisertib. d Immunocyto-chemistry analysis of GLIS3 −/− PP2-β cells treated with DMSO or 10 µM galunisertib. Scale bar = 100 μm, scale bar of high magnification insets = 40 μm. e , f Quantification of the cell death rate ( e , the percentage of PI + INS + cells in INS + cells, n = 4) and apoptosis rate ( f , the percentage of cleaved caspase-3 + INS + cells in INS + cells, n = 3) of GLIS3 −/− PP2-β cells treated with DMSO or 10 µM galunisertib. Flow cytometry analysis ( g ) and quantification ( h ) of early apoptotic cells (the percentage of Annexin V + /DAPI − cells) in GLIS3 −/− INS-GFP + PP2-β cells treated with DMSO or 10 μM galunisertib ( n = 6). i Relative number of INS + cells in GLIS3 −/− PP2-β cells treated with DMSO or 10 μM galunisertib. Data are normalized to DMSO-treated values ( n = 4). j Schematic representation of the in vivo transplantation and drug treatment experiments. k Immunohistochemistry analysis of INS, cleaved caspase-3, and STEM121 in the grafts isolated from vehicle- or galunisertib-treated mice. Scale bar = 100 μm. l Quantification of immunohistochemistry data in j ( n = 7). m Quantification of the percentage of apoptotic INS + cells (CAS3 + INS + STEM121 + ) in the INS + population within the grafts from vehicle- or galunisertib-treated mice (INS + STEM121 + , n = 6). CAS3: cleaved caspase-3. P values by unpaired two-tailed t -test were * P

Techniques Used: In Vitro, In Vivo, Flow Cytometry, Cytometry, Transplantation Assay, Immunohistochemistry, Isolation, Mouse Assay, Two Tailed Test

Loss of GLIS3 leads to increased cell death in PP2 and PP2-β cells. a Quantification of early apoptotic cells (the percentage of Annexin V + /DAPI − cells) in WT and GLIS3 −/− ES, DE, PP1, and PP2 cells ( n = 3). b Representative flow cytometry analysis plots of Annexin V staining in WT and GLIS3 −/− cells at D23_L. c Immunostaining for PDX1 and cleaved caspase-3 in WT and GLIS3 −/− cells at D23_L. Scale bar = 40 μm. Annexin V staining ( d ) and quantification ( e ) of early apoptotic cells in the INS-GFP + cells at D30_L ( n = 6). Histogram showing fluorescence intensity ( f ) and quantification of median fluorescence values ( g ) of Annexin V staining of WT and GLIS3 −/− INS + DAPI − PP2-β cells (WT n = 4, GLIS3 −/− n = 6). h PI, cleaved caspase-3 and INS staining of WT and GLIS3 −/− cells at D30_L. Scale bar = 40 μm. i Quantification of cell death rate (the percentage of PI + INS + cells in INS + cells) and apoptosis rate (the percentage of cleaved caspase-3 + INS + cells in INS + cells) of WT and GLIS3 −/− INS + PP2-β cells ( n = 3). j Schematic representation of the in vivo transplantation experiment. k Immunostaining for INS, cleaved caspase-3 and STEM121 in the grafts of mice transplanted with WT or GLIS3 −/− cells. Scale bar = 100 μm. l Quantification of the apoptosis rate (the percentage of cleaved caspase-3 + / STEM121 + cells in STEM121 + cells) within WT and GLIS3 −/− grafts ( n = 7 for WT, n = 4 for GLIS3 −/− ). m Quantification of the percentage of apoptotic INS + cells (CAS3 + PDX1 + STEM121 + ) in the INS + population within the WT and GLIS3 −/− grafts (INS + STEM121 + , WT n = 7, GLIS3 −/− n = 4). CAS3: cleaved caspase-3. P values by unpaired two-tailed Student’s t -test were * P
Figure Legend Snippet: Loss of GLIS3 leads to increased cell death in PP2 and PP2-β cells. a Quantification of early apoptotic cells (the percentage of Annexin V + /DAPI − cells) in WT and GLIS3 −/− ES, DE, PP1, and PP2 cells ( n = 3). b Representative flow cytometry analysis plots of Annexin V staining in WT and GLIS3 −/− cells at D23_L. c Immunostaining for PDX1 and cleaved caspase-3 in WT and GLIS3 −/− cells at D23_L. Scale bar = 40 μm. Annexin V staining ( d ) and quantification ( e ) of early apoptotic cells in the INS-GFP + cells at D30_L ( n = 6). Histogram showing fluorescence intensity ( f ) and quantification of median fluorescence values ( g ) of Annexin V staining of WT and GLIS3 −/− INS + DAPI − PP2-β cells (WT n = 4, GLIS3 −/− n = 6). h PI, cleaved caspase-3 and INS staining of WT and GLIS3 −/− cells at D30_L. Scale bar = 40 μm. i Quantification of cell death rate (the percentage of PI + INS + cells in INS + cells) and apoptosis rate (the percentage of cleaved caspase-3 + INS + cells in INS + cells) of WT and GLIS3 −/− INS + PP2-β cells ( n = 3). j Schematic representation of the in vivo transplantation experiment. k Immunostaining for INS, cleaved caspase-3 and STEM121 in the grafts of mice transplanted with WT or GLIS3 −/− cells. Scale bar = 100 μm. l Quantification of the apoptosis rate (the percentage of cleaved caspase-3 + / STEM121 + cells in STEM121 + cells) within WT and GLIS3 −/− grafts ( n = 7 for WT, n = 4 for GLIS3 −/− ). m Quantification of the percentage of apoptotic INS + cells (CAS3 + PDX1 + STEM121 + ) in the INS + population within the WT and GLIS3 −/− grafts (INS + STEM121 + , WT n = 7, GLIS3 −/− n = 4). CAS3: cleaved caspase-3. P values by unpaired two-tailed Student’s t -test were * P

Techniques Used: Flow Cytometry, Cytometry, Staining, Immunostaining, Fluorescence, In Vivo, Transplantation Assay, Mouse Assay, Two Tailed Test

18) Product Images from "MicroRNA 22 Regulates Cell Cycle Length in Cerebellar Granular Neuron Precursors"

Article Title: MicroRNA 22 Regulates Cell Cycle Length in Cerebellar Granular Neuron Precursors

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00338-13

Expression of miR-22 increases cell cycle length but does not induce neuronal differentiation of CGNPs. Freshly isolated CGNPs were transfected with the indicated constructs and cultured for 48 h or longer in Shh (3 μg/ml)-containing medium to study different aspects of cell cycle progression. The percentage of cells labeled with each specific marker (X) among the GFP-positive cells (X/GFP ×100) was calculated for each marker. The bar graphs show the means ± SD of at least 3 independent experiments. The total number of GFP-positive cells counted for each data point is indicated in each bar. Constructs: pMICRO (empty vector), Scr22 (scrambled miR-22), miR-22, dec22 (miR-22 decoy). (A) Progression through the G 1 restriction point was studied by anti-retinoblastoma PS807 labeling. (B) G 1 -to-S and G 2 -to-M progression was studied with anti-Cdc2 PY15 antibody. (C) Anti-phospho-histone 3 was used to measure the percentage of cells in M phase. (D) Representative FACS analysis of GFP/DAPI-stained cells. The percentage of cells in each phase is indicated above each bar. A minimum of 2,500 GFP-positive cells were evaluated in each transfection. (E) Apoptosis was studied by cleaved caspase 3 staining. A B27-deprived culture (Scr-B27) was used as a positive control for apoptosis. (F to H) Neuronal differentiation of CGNPs was studied at 48 and 72 h by staining with Tuj1 and HuC/D neural markers. BMP2 treatment at 100 ng/ml was included as a positive control for neuronal differentiation. (I) The effect of miR-22 expression on cell cycle duration was studied by cumulative BrdU staining. Tendency lines for miR-22 or Scr22 were fitted with the GraphPad Prism statistical program. The growth fraction is given by the intersection between the plateau lines and the y axis (arrowheads). Total cycle duration (Tc) minus the duration of the S phase (Ts) is given by the intersection between the tendency lines and the x axis (arrows). The S-phase duration is calculated from the intersection between the tendency lines and the y axis. The equation formula and the R 2 coefficient of determination are shown. The number of cells counted for each data point ranged from 483 to 2,019 with a mean of 972.
Figure Legend Snippet: Expression of miR-22 increases cell cycle length but does not induce neuronal differentiation of CGNPs. Freshly isolated CGNPs were transfected with the indicated constructs and cultured for 48 h or longer in Shh (3 μg/ml)-containing medium to study different aspects of cell cycle progression. The percentage of cells labeled with each specific marker (X) among the GFP-positive cells (X/GFP ×100) was calculated for each marker. The bar graphs show the means ± SD of at least 3 independent experiments. The total number of GFP-positive cells counted for each data point is indicated in each bar. Constructs: pMICRO (empty vector), Scr22 (scrambled miR-22), miR-22, dec22 (miR-22 decoy). (A) Progression through the G 1 restriction point was studied by anti-retinoblastoma PS807 labeling. (B) G 1 -to-S and G 2 -to-M progression was studied with anti-Cdc2 PY15 antibody. (C) Anti-phospho-histone 3 was used to measure the percentage of cells in M phase. (D) Representative FACS analysis of GFP/DAPI-stained cells. The percentage of cells in each phase is indicated above each bar. A minimum of 2,500 GFP-positive cells were evaluated in each transfection. (E) Apoptosis was studied by cleaved caspase 3 staining. A B27-deprived culture (Scr-B27) was used as a positive control for apoptosis. (F to H) Neuronal differentiation of CGNPs was studied at 48 and 72 h by staining with Tuj1 and HuC/D neural markers. BMP2 treatment at 100 ng/ml was included as a positive control for neuronal differentiation. (I) The effect of miR-22 expression on cell cycle duration was studied by cumulative BrdU staining. Tendency lines for miR-22 or Scr22 were fitted with the GraphPad Prism statistical program. The growth fraction is given by the intersection between the plateau lines and the y axis (arrowheads). Total cycle duration (Tc) minus the duration of the S phase (Ts) is given by the intersection between the tendency lines and the x axis (arrows). The S-phase duration is calculated from the intersection between the tendency lines and the y axis. The equation formula and the R 2 coefficient of determination are shown. The number of cells counted for each data point ranged from 483 to 2,019 with a mean of 972.

Techniques Used: Expressing, Isolation, Transfection, Construct, Cell Culture, Labeling, Marker, Plasmid Preparation, FACS, Staining, Positive Control, BrdU Staining

19) Product Images from "HSP90 inhibition enhances cancer immunotherapy by upregulating interferon response genes"

Article Title: HSP90 inhibition enhances cancer immunotherapy by upregulating interferon response genes

Journal: Nature Communications

doi: 10.1038/s41467-017-00449-z

HSP90 inhibitor effect on T-cell-mediated killing requires IFIT genes. a Gene expression analysis of cell lines treated with ganetespib. b , c Quantitative real-time PCR (qRT-PCR) and western blot analysis showing upregulation of IFIT1, IFIT2 and IFIT3 following ganetespib treatment in human melanoma cell line 2400. D = DMSO and G = ganetespib. Interferon alpha (IFN-α) used as a positive control. An increase in cleaved PARP indicates efficacy of the HSP90 inhibition by ganetespib. d qRT-PCR to verify silencing of IFIT1, IFIT2 and IFIT3 in 2400. Control = cell line transduced with scrambled shRNA and IFITall = cell line transduced simultaneously with IFIT1, IFIT2 and IFIT3 shRNAs. e 2400 Control and IFITall cell lines treated with ganetespib at 250 nM, co-cultured with autologous T cells and assayed for cleaved caspase 3 by flow cytometry. f qRT-PCR verifying overexpression of IFIT1, IFIT2 and IFIT3 over GFP control in 2400. g 2400 GFP and IFIT overexpressing cell lines co-cultured with autologous T cells and assayed for cleaved caspase 3. h Western blots showing a decrease in BCL2 protein after overexpression of IFIT1, IFIT2 and IFIT3. The data represented as mean ± s.e.m. * P
Figure Legend Snippet: HSP90 inhibitor effect on T-cell-mediated killing requires IFIT genes. a Gene expression analysis of cell lines treated with ganetespib. b , c Quantitative real-time PCR (qRT-PCR) and western blot analysis showing upregulation of IFIT1, IFIT2 and IFIT3 following ganetespib treatment in human melanoma cell line 2400. D = DMSO and G = ganetespib. Interferon alpha (IFN-α) used as a positive control. An increase in cleaved PARP indicates efficacy of the HSP90 inhibition by ganetespib. d qRT-PCR to verify silencing of IFIT1, IFIT2 and IFIT3 in 2400. Control = cell line transduced with scrambled shRNA and IFITall = cell line transduced simultaneously with IFIT1, IFIT2 and IFIT3 shRNAs. e 2400 Control and IFITall cell lines treated with ganetespib at 250 nM, co-cultured with autologous T cells and assayed for cleaved caspase 3 by flow cytometry. f qRT-PCR verifying overexpression of IFIT1, IFIT2 and IFIT3 over GFP control in 2400. g 2400 GFP and IFIT overexpressing cell lines co-cultured with autologous T cells and assayed for cleaved caspase 3. h Western blots showing a decrease in BCL2 protein after overexpression of IFIT1, IFIT2 and IFIT3. The data represented as mean ± s.e.m. * P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Positive Control, Inhibition, Transduction, shRNA, Cell Culture, Flow Cytometry, Cytometry, Over Expression

HSP90 inhibition enhances T-cell mediated killing of melanoma cells. a Tableau depiction of screen results from patient-derived cell line 2549. HSP90 inhibitors 1—17-DMAG, 2—BIIB021 and 3—17-AAG are highlighted. b Cleaved caspase 3 percentage following treatment with varying concentrations of ganetespib and autologous TILs in human melanoma cell lines 2549 and 2400. c Isobolograms depicting the synergism between ganetespib and T-cell killing. Points > 1 indicate antagonism, points = 1 indicate additivity and points
Figure Legend Snippet: HSP90 inhibition enhances T-cell mediated killing of melanoma cells. a Tableau depiction of screen results from patient-derived cell line 2549. HSP90 inhibitors 1—17-DMAG, 2—BIIB021 and 3—17-AAG are highlighted. b Cleaved caspase 3 percentage following treatment with varying concentrations of ganetespib and autologous TILs in human melanoma cell lines 2549 and 2400. c Isobolograms depicting the synergism between ganetespib and T-cell killing. Points > 1 indicate antagonism, points = 1 indicate additivity and points

Techniques Used: Inhibition, Derivative Assay

20) Product Images from "Discovery of a drug candidate for GLIS3-associated diabetes"

Article Title: Discovery of a drug candidate for GLIS3-associated diabetes

Journal: Nature Communications

doi: 10.1038/s41467-018-04918-x

A high-content chemical screen identifies galunisertib as a drug candidate to rescue cell death induced by loss of GLIS3 both in vitro and in vivo. a Schematic representation of the high-content chemical screen. b Chemical structure of galunisertib. c Inhibitory curve of galunisertib. d Immunocyto-chemistry analysis of GLIS3 −/− PP2-β cells treated with DMSO or 10 µM galunisertib. Scale bar = 100 μm, scale bar of high magnification insets = 40 μm. e , f Quantification of the cell death rate ( e , the percentage of PI + INS + cells in INS + cells, n = 4) and apoptosis rate ( f , the percentage of cleaved caspase-3 + INS + cells in INS + cells, n = 3) of GLIS3 −/− PP2-β cells treated with DMSO or 10 µM galunisertib. Flow cytometry analysis ( g ) and quantification ( h ) of early apoptotic cells (the percentage of Annexin V + /DAPI − cells) in GLIS3 −/− INS-GFP + PP2-β cells treated with DMSO or 10 μM galunisertib ( n = 6). i Relative number of INS + cells in GLIS3 −/− PP2-β cells treated with DMSO or 10 μM galunisertib. Data are normalized to DMSO-treated values ( n = 4). j Schematic representation of the in vivo transplantation and drug treatment experiments. k Immunohistochemistry analysis of INS, cleaved caspase-3, and STEM121 in the grafts isolated from vehicle- or galunisertib-treated mice. Scale bar = 100 μm. l Quantification of immunohistochemistry data in j ( n = 7). m Quantification of the percentage of apoptotic INS + cells (CAS3 + INS + STEM121 + ) in the INS + population within the grafts from vehicle- or galunisertib-treated mice (INS + STEM121 + , n = 6). CAS3: cleaved caspase-3. P values by unpaired two-tailed t -test were * P
Figure Legend Snippet: A high-content chemical screen identifies galunisertib as a drug candidate to rescue cell death induced by loss of GLIS3 both in vitro and in vivo. a Schematic representation of the high-content chemical screen. b Chemical structure of galunisertib. c Inhibitory curve of galunisertib. d Immunocyto-chemistry analysis of GLIS3 −/− PP2-β cells treated with DMSO or 10 µM galunisertib. Scale bar = 100 μm, scale bar of high magnification insets = 40 μm. e , f Quantification of the cell death rate ( e , the percentage of PI + INS + cells in INS + cells, n = 4) and apoptosis rate ( f , the percentage of cleaved caspase-3 + INS + cells in INS + cells, n = 3) of GLIS3 −/− PP2-β cells treated with DMSO or 10 µM galunisertib. Flow cytometry analysis ( g ) and quantification ( h ) of early apoptotic cells (the percentage of Annexin V + /DAPI − cells) in GLIS3 −/− INS-GFP + PP2-β cells treated with DMSO or 10 μM galunisertib ( n = 6). i Relative number of INS + cells in GLIS3 −/− PP2-β cells treated with DMSO or 10 μM galunisertib. Data are normalized to DMSO-treated values ( n = 4). j Schematic representation of the in vivo transplantation and drug treatment experiments. k Immunohistochemistry analysis of INS, cleaved caspase-3, and STEM121 in the grafts isolated from vehicle- or galunisertib-treated mice. Scale bar = 100 μm. l Quantification of immunohistochemistry data in j ( n = 7). m Quantification of the percentage of apoptotic INS + cells (CAS3 + INS + STEM121 + ) in the INS + population within the grafts from vehicle- or galunisertib-treated mice (INS + STEM121 + , n = 6). CAS3: cleaved caspase-3. P values by unpaired two-tailed t -test were * P

Techniques Used: In Vitro, In Vivo, Flow Cytometry, Cytometry, Transplantation Assay, Immunohistochemistry, Isolation, Mouse Assay, Two Tailed Test

Loss of GLIS3 leads to increased cell death in PP2 and PP2-β cells. a Quantification of early apoptotic cells (the percentage of Annexin V + /DAPI − cells) in WT and GLIS3 −/− ES, DE, PP1, and PP2 cells ( n = 3). b Representative flow cytometry analysis plots of Annexin V staining in WT and GLIS3 −/− cells at D23_L. c Immunostaining for PDX1 and cleaved caspase-3 in WT and GLIS3 −/− cells at D23_L. Scale bar = 40 μm. Annexin V staining ( d ) and quantification ( e ) of early apoptotic cells in the INS-GFP + cells at D30_L ( n = 6). Histogram showing fluorescence intensity ( f ) and quantification of median fluorescence values ( g ) of Annexin V staining of WT and GLIS3 −/− INS + DAPI − PP2-β cells (WT n = 4, GLIS3 −/− n = 6). h PI, cleaved caspase-3 and INS staining of WT and GLIS3 −/− cells at D30_L. Scale bar = 40 μm. i Quantification of cell death rate (the percentage of PI + INS + cells in INS + cells) and apoptosis rate (the percentage of cleaved caspase-3 + INS + cells in INS + cells) of WT and GLIS3 −/− INS + PP2-β cells ( n = 3). j Schematic representation of the in vivo transplantation experiment. k Immunostaining for INS, cleaved caspase-3 and STEM121 in the grafts of mice transplanted with WT or GLIS3 −/− cells. Scale bar = 100 μm. l Quantification of the apoptosis rate (the percentage of cleaved caspase-3 + / STEM121 + cells in STEM121 + cells) within WT and GLIS3 −/− grafts ( n = 7 for WT, n = 4 for GLIS3 −/− ). m Quantification of the percentage of apoptotic INS + cells (CAS3 + PDX1 + STEM121 + ) in the INS + population within the WT and GLIS3 −/− grafts (INS + STEM121 + , WT n = 7, GLIS3 −/− n = 4). CAS3: cleaved caspase-3. P values by unpaired two-tailed Student’s t -test were * P
Figure Legend Snippet: Loss of GLIS3 leads to increased cell death in PP2 and PP2-β cells. a Quantification of early apoptotic cells (the percentage of Annexin V + /DAPI − cells) in WT and GLIS3 −/− ES, DE, PP1, and PP2 cells ( n = 3). b Representative flow cytometry analysis plots of Annexin V staining in WT and GLIS3 −/− cells at D23_L. c Immunostaining for PDX1 and cleaved caspase-3 in WT and GLIS3 −/− cells at D23_L. Scale bar = 40 μm. Annexin V staining ( d ) and quantification ( e ) of early apoptotic cells in the INS-GFP + cells at D30_L ( n = 6). Histogram showing fluorescence intensity ( f ) and quantification of median fluorescence values ( g ) of Annexin V staining of WT and GLIS3 −/− INS + DAPI − PP2-β cells (WT n = 4, GLIS3 −/− n = 6). h PI, cleaved caspase-3 and INS staining of WT and GLIS3 −/− cells at D30_L. Scale bar = 40 μm. i Quantification of cell death rate (the percentage of PI + INS + cells in INS + cells) and apoptosis rate (the percentage of cleaved caspase-3 + INS + cells in INS + cells) of WT and GLIS3 −/− INS + PP2-β cells ( n = 3). j Schematic representation of the in vivo transplantation experiment. k Immunostaining for INS, cleaved caspase-3 and STEM121 in the grafts of mice transplanted with WT or GLIS3 −/− cells. Scale bar = 100 μm. l Quantification of the apoptosis rate (the percentage of cleaved caspase-3 + / STEM121 + cells in STEM121 + cells) within WT and GLIS3 −/− grafts ( n = 7 for WT, n = 4 for GLIS3 −/− ). m Quantification of the percentage of apoptotic INS + cells (CAS3 + PDX1 + STEM121 + ) in the INS + population within the WT and GLIS3 −/− grafts (INS + STEM121 + , WT n = 7, GLIS3 −/− n = 4). CAS3: cleaved caspase-3. P values by unpaired two-tailed Student’s t -test were * P

Techniques Used: Flow Cytometry, Cytometry, Staining, Immunostaining, Fluorescence, In Vivo, Transplantation Assay, Mouse Assay, Two Tailed Test

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Article Snippet: .. Membranes were blocked with 5% skimmed milk for one hour and then incubated with the following primary antibodies: anti-Bcl-2 (BD Biosciences), anti-Bax (BD Biosciences), anti-cleaved-caspase-3 (BD Biosciences), anti-CRABP2 (Cell Signaling Technology), anti-ERK (Abcam), anti-p-ERK (Abcam), anti-VGEF (Cell Signaling Technology), anti-VEGFR2 (Cell Signaling Technology), anti-p-VEGFR2 (Cell Signaling Technology), and anti-GAPDH (Cell Signaling Technology) at 4°C overnight. .. The next day, the membranes were incubated with Horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, Danvers, USA) for 2 h. ECL reagents were used to detect the immunoreactivity, and the protein bands were accessed by ImageJ software (National Institutes of Health, MD, USA).

Article Title: The Stimulus-Dependent Gradient of Cyp26B1+ Olfactory Sensory Neurons Is Necessary for the Functional Integrity of the Olfactory Sensory Map
Article Snippet: .. Background was blocked by incubation for 1 h in 3% normal donkey serum in PBS with 0.1% Triton X-100, followed by overnight incubation at 4°C in blocking solution containing anti-NQO1 (dilution 1:500; catalog #ab2346; Abcam), anti-NCAM2 (1:200; catalog #AS05 077; Agrisera), anti-Cyp26B1 (1:200; catalog #21555-1-AP; ProteinTech), anti-β-galactosidase (1:1000; catalog #559762, MP-Cappel), anti-AC3 (1:500; catalog #C-20; Santa Cruz Biotechnology), anti-cleaved caspase 3 (1:500; catalog #559565; BD Biosciences), anti-phospho-histone 3 (pH3, 1:500; catalog #06-570; Millipore), anti-OMP (1:1000; catalog #544-10001; Wako Chemicals), and anti-SCG10 (Stathmin-1; 1:500, catalog #AS02024; Agrisera). .. After washes, specific fluorescent immunostaining was visualized after incubation 1 h at room temperature with Alexa Fluor 488/546-conjugated anti-goat IgG or Alexa Fluor 488/546-conjugated anti-rabbit IgG (1:1000; Invitrogen).

Fluorescence In Situ Hybridization:

Article Title: Targeting invadopodia-mediated breast cancer metastasis by using ABL kinase inhibitors
Article Snippet: .. Antibodies and reagents For immunofluorescence, anti-cortactin (ab-33333) and anti-PCNA were obtained from Abcam; anti-Arp2 (H-84) (SC-15389) and anti-Tks5 (FISH M-300) (SC-30122) were obtained from Santa Cruz Biotechnology; anti-pY421-cortactin (C0739) was obtained from Sigma-Aldrich; anti-CD31 was obtained from BD Biosciences; anti-cleaved caspase 3 was obtained from Cell Signaling Technology. .. Rhodamine-labeled phalloidin and Alexa Fluor-conjugated secondary antibodies were obtained from Molecular Probes (Thermo Fisher Scientific).

Article Title: Targeting invadopodia-mediated breast cancer metastasis by using ABL kinase inhibitors
Article Snippet: .. For immunofluorescence, anti-cortactin (ab-33333) and anti-PCNA were obtained from Abcam; anti-Arp2 (H-84) (SC-15389) and anti-Tks5 (FISH M-300) (SC-30122) were obtained from Santa Cruz Biotechnology; anti-pY421-cortactin (C0739) was obtained from Sigma-Aldrich; anti-CD31 was obtained from BD Biosciences; anti-cleaved caspase 3 was obtained from Cell Signaling Technology. .. Rhodamine-labeled phalloidin and Alexa Fluor-conjugated secondary antibodies were obtained from Molecular Probes (Thermo Fisher Scientific).

Immunofluorescence:

Article Title: Targeting invadopodia-mediated breast cancer metastasis by using ABL kinase inhibitors
Article Snippet: .. Antibodies and reagents For immunofluorescence, anti-cortactin (ab-33333) and anti-PCNA were obtained from Abcam; anti-Arp2 (H-84) (SC-15389) and anti-Tks5 (FISH M-300) (SC-30122) were obtained from Santa Cruz Biotechnology; anti-pY421-cortactin (C0739) was obtained from Sigma-Aldrich; anti-CD31 was obtained from BD Biosciences; anti-cleaved caspase 3 was obtained from Cell Signaling Technology. .. Rhodamine-labeled phalloidin and Alexa Fluor-conjugated secondary antibodies were obtained from Molecular Probes (Thermo Fisher Scientific).

Article Title: Targeting invadopodia-mediated breast cancer metastasis by using ABL kinase inhibitors
Article Snippet: .. For immunofluorescence, anti-cortactin (ab-33333) and anti-PCNA were obtained from Abcam; anti-Arp2 (H-84) (SC-15389) and anti-Tks5 (FISH M-300) (SC-30122) were obtained from Santa Cruz Biotechnology; anti-pY421-cortactin (C0739) was obtained from Sigma-Aldrich; anti-CD31 was obtained from BD Biosciences; anti-cleaved caspase 3 was obtained from Cell Signaling Technology. .. Rhodamine-labeled phalloidin and Alexa Fluor-conjugated secondary antibodies were obtained from Molecular Probes (Thermo Fisher Scientific).

Blocking Assay:

Article Title: The Stimulus-Dependent Gradient of Cyp26B1+ Olfactory Sensory Neurons Is Necessary for the Functional Integrity of the Olfactory Sensory Map
Article Snippet: .. Background was blocked by incubation for 1 h in 3% normal donkey serum in PBS with 0.1% Triton X-100, followed by overnight incubation at 4°C in blocking solution containing anti-NQO1 (dilution 1:500; catalog #ab2346; Abcam), anti-NCAM2 (1:200; catalog #AS05 077; Agrisera), anti-Cyp26B1 (1:200; catalog #21555-1-AP; ProteinTech), anti-β-galactosidase (1:1000; catalog #559762, MP-Cappel), anti-AC3 (1:500; catalog #C-20; Santa Cruz Biotechnology), anti-cleaved caspase 3 (1:500; catalog #559565; BD Biosciences), anti-phospho-histone 3 (pH3, 1:500; catalog #06-570; Millipore), anti-OMP (1:1000; catalog #544-10001; Wako Chemicals), and anti-SCG10 (Stathmin-1; 1:500, catalog #AS02024; Agrisera). .. After washes, specific fluorescent immunostaining was visualized after incubation 1 h at room temperature with Alexa Fluor 488/546-conjugated anti-goat IgG or Alexa Fluor 488/546-conjugated anti-rabbit IgG (1:1000; Invitrogen).

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  • 89
    Becton Dickinson pe anti caspase 3 antibodies
    Effect of TAT-BID on the activation of procaspase-3 induced in HeLa cells by doxorubicin (DOX). Cells were treated with 40 μ g/ml TAT-BID or 1 μ M DOX (or both) for 15 h. (A) Western blot analysis of procaspase-3 (31 kDa) cleavage into the active <t>caspase-3</t> form (large fragment, 17 kDa). Caspase-3 and GAPDH were identified using specific antibodies in whole cell extracts. (B) Microscopic images of the cells. Cells were stained with PE-anti-caspase-3 antibodies (active caspase-3, green) and DRAQ5 (nuclei, red) and observed using confocal microscopy.
    Pe Anti Caspase 3 Antibodies, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 89/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Becton Dickinson monoclonal anti caspase 3
    Apoptosis in  Par4 −/−  embryonic fibroblasts. ( A ) Wild-type (WT) and  Par4  null (KO) embryonic fibroblasts were incubated with 10 ng ml −1  of tumour necrosis factor-α (TNF-α), 10 μg ml −1  cycloheximide (CHX) or both for 72 h, after which the percentage of apoptotic cells was determined by TUNEL (TdT-mediated dUTP nick end-labelling) analysis. Results are the mean ± s.d. of three independent experiments with incubations in duplicate. ( B ) Cell extractions were carried out from cultures incubated for 12 h as above and the processing of caspase 3 was analysed by immunoblotting. The data shown are representative of three independent experiments.
    Monoclonal Anti Caspase 3, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Becton Dickinson fitc labeled anti active caspase 3 antibody
    Induction of apoptosis of H1299 cells by peptide 49 . (a) Flow cytometry analysis of H1299 cells after treatment with 10 μM peptide 49 for varying periods of time (0–24 h) and stained with <t>FITC-annexin</t> V and propidium iodide (PI). The percentage of cells in each quartile is indicated. (b) Activation of <t>caspase-3</t> activity in H1299 cells by peptide 49 (20 μM) as monitored by anticaspase-3 immunostaining and flow cytometry.
    Fitc Labeled Anti Active Caspase 3 Antibody, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fitc labeled anti active caspase 3 antibody/product/Becton Dickinson
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    85
    Becton Dickinson pe conjugated anti active caspase 3 abs
    HIV-infected iDCs are resistant to NK-mediated killing. ( A ) iDCs were either uninfected or infected for 24 hours with R5-HIV-1 BaL (1 ng/ml of p24), and further incubated for 24 hours with aNK cells at NK∶DC ratio of 5∶1. Viability of iDCs was assessed using the 7-AAD assay. Living DCs are identified as CD56 neg 7-AAD neg FSC high . Inserts: the maturation stage of DCs was determined via the dual staining of gated living DCs with anti-CD86 and -HLA-DR specific antibodies. Dot plots show one representative out of more than ten independent experiments. Histograms show the mean ± sd of experiments performed with cells from 10 healthy donors. * p = 0.0001. ( B ) Intracellular active <t>caspase-3</t> detection by flow cytometry in iDC and DC HIV cultured alone, or cocultured with aNK cells for 24 hours. Data represent the mean ± sd of three independent experiments. * p
    Pe Conjugated Anti Active Caspase 3 Abs, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of TAT-BID on the activation of procaspase-3 induced in HeLa cells by doxorubicin (DOX). Cells were treated with 40 μ g/ml TAT-BID or 1 μ M DOX (or both) for 15 h. (A) Western blot analysis of procaspase-3 (31 kDa) cleavage into the active caspase-3 form (large fragment, 17 kDa). Caspase-3 and GAPDH were identified using specific antibodies in whole cell extracts. (B) Microscopic images of the cells. Cells were stained with PE-anti-caspase-3 antibodies (active caspase-3, green) and DRAQ5 (nuclei, red) and observed using confocal microscopy.

    Journal: Oncology Reports

    Article Title: Synergy of BID with doxorubicin in the killing of cancer cells

    doi: 10.3892/or.2015.3841

    Figure Lengend Snippet: Effect of TAT-BID on the activation of procaspase-3 induced in HeLa cells by doxorubicin (DOX). Cells were treated with 40 μ g/ml TAT-BID or 1 μ M DOX (or both) for 15 h. (A) Western blot analysis of procaspase-3 (31 kDa) cleavage into the active caspase-3 form (large fragment, 17 kDa). Caspase-3 and GAPDH were identified using specific antibodies in whole cell extracts. (B) Microscopic images of the cells. Cells were stained with PE-anti-caspase-3 antibodies (active caspase-3, green) and DRAQ5 (nuclei, red) and observed using confocal microscopy.

    Article Snippet: Materials and methods Reagents Doxorubicin (DOX), RPMI-1640 medium, Dulbecco’s modified Eagle’s medium (DMEM), trypsin inhibitor from soybean, fetal bovine serum (FBS), penicillin and streptomycin, D-glucose, sodium pyruvate, MTT reagent and anti-GAPDH antibodies were obtained from Sigma-Aldrich (St. Louis, MO, USA); F12K medium from the American Type Culture Collection (ATCC; Manassas, VA, USA); anti-HA antibodies from Santa Cruz Biotechnology (Dallas, TX, USA); anti-caspase-3 antibodies from Cell Signaling Technology (Danvers, MA, USA); anti-PARP1 and PE-anti-caspase-3 antibodies from Beckton-Dickinson (San Jose, CA, USA); DRAQ5 from BioStatus Ltd. (Shepshed, UK); Dako fluorescent mounting medium from Dako North America (Carpinteria, CA, USA); and Giemsa’s azur and May-Grünwald dyes were from Merck Millipore (Billerica, MA, USA).

    Techniques: Activation Assay, Western Blot, Staining, Confocal Microscopy

    Apoptosis in  Par4 −/−  embryonic fibroblasts. ( A ) Wild-type (WT) and  Par4  null (KO) embryonic fibroblasts were incubated with 10 ng ml −1  of tumour necrosis factor-α (TNF-α), 10 μg ml −1  cycloheximide (CHX) or both for 72 h, after which the percentage of apoptotic cells was determined by TUNEL (TdT-mediated dUTP nick end-labelling) analysis. Results are the mean ± s.d. of three independent experiments with incubations in duplicate. ( B ) Cell extractions were carried out from cultures incubated for 12 h as above and the processing of caspase 3 was analysed by immunoblotting. The data shown are representative of three independent experiments.

    Journal: EMBO Reports

    Article Title: Genetic inactivation of Par4 results in hyperactivation of NF-?B and impairment of JNK and p38

    doi: 10.1038/sj.embor.embor769

    Figure Lengend Snippet: Apoptosis in Par4 −/− embryonic fibroblasts. ( A ) Wild-type (WT) and Par4 null (KO) embryonic fibroblasts were incubated with 10 ng ml −1 of tumour necrosis factor-α (TNF-α), 10 μg ml −1 cycloheximide (CHX) or both for 72 h, after which the percentage of apoptotic cells was determined by TUNEL (TdT-mediated dUTP nick end-labelling) analysis. Results are the mean ± s.d. of three independent experiments with incubations in duplicate. ( B ) Cell extractions were carried out from cultures incubated for 12 h as above and the processing of caspase 3 was analysed by immunoblotting. The data shown are representative of three independent experiments.

    Article Snippet: Monoclonal anti-caspase-3 and anti-XIAP antibodies, and the polyclonal anti-JNK antibody were purchased from Becton Dickinson.

    Techniques: Incubation, TUNEL Assay

    Induction of apoptosis of H1299 cells by peptide 49 . (a) Flow cytometry analysis of H1299 cells after treatment with 10 μM peptide 49 for varying periods of time (0–24 h) and stained with FITC-annexin V and propidium iodide (PI). The percentage of cells in each quartile is indicated. (b) Activation of caspase-3 activity in H1299 cells by peptide 49 (20 μM) as monitored by anticaspase-3 immunostaining and flow cytometry.

    Journal: ACS Combinatorial Science

    Article Title: Discovery of a Direct Ras Inhibitor by Screening a Combinatorial Library of Cell-Permeable Bicyclic Peptides

    doi: 10.1021/acscombsci.5b00164

    Figure Lengend Snippet: Induction of apoptosis of H1299 cells by peptide 49 . (a) Flow cytometry analysis of H1299 cells after treatment with 10 μM peptide 49 for varying periods of time (0–24 h) and stained with FITC-annexin V and propidium iodide (PI). The percentage of cells in each quartile is indicated. (b) Activation of caspase-3 activity in H1299 cells by peptide 49 (20 μM) as monitored by anticaspase-3 immunostaining and flow cytometry.

    Article Snippet: The cells were pelleted, washed with BD Perm/Wash buffer (BD biosciences) twice, and incubated with 20 μL of FITC-labeled anti-Active Caspase-3 antibody in 100 μL of BD Perm/Wash buffer for 30 min at room temperature.

    Techniques: Flow Cytometry, Cytometry, Staining, Activation Assay, Activity Assay, Immunostaining

    HIV-infected iDCs are resistant to NK-mediated killing. ( A ) iDCs were either uninfected or infected for 24 hours with R5-HIV-1 BaL (1 ng/ml of p24), and further incubated for 24 hours with aNK cells at NK∶DC ratio of 5∶1. Viability of iDCs was assessed using the 7-AAD assay. Living DCs are identified as CD56 neg 7-AAD neg FSC high . Inserts: the maturation stage of DCs was determined via the dual staining of gated living DCs with anti-CD86 and -HLA-DR specific antibodies. Dot plots show one representative out of more than ten independent experiments. Histograms show the mean ± sd of experiments performed with cells from 10 healthy donors. * p = 0.0001. ( B ) Intracellular active caspase-3 detection by flow cytometry in iDC and DC HIV cultured alone, or cocultured with aNK cells for 24 hours. Data represent the mean ± sd of three independent experiments. * p

    Journal: PLoS Pathogens

    Article Title: Escape of HIV-1-Infected Dendritic Cells from TRAIL-Mediated NK Cell Cytotoxicity during NK-DC Cross-Talk--A Pivotal Role of HMGB1

    doi: 10.1371/journal.ppat.1000862

    Figure Lengend Snippet: HIV-infected iDCs are resistant to NK-mediated killing. ( A ) iDCs were either uninfected or infected for 24 hours with R5-HIV-1 BaL (1 ng/ml of p24), and further incubated for 24 hours with aNK cells at NK∶DC ratio of 5∶1. Viability of iDCs was assessed using the 7-AAD assay. Living DCs are identified as CD56 neg 7-AAD neg FSC high . Inserts: the maturation stage of DCs was determined via the dual staining of gated living DCs with anti-CD86 and -HLA-DR specific antibodies. Dot plots show one representative out of more than ten independent experiments. Histograms show the mean ± sd of experiments performed with cells from 10 healthy donors. * p = 0.0001. ( B ) Intracellular active caspase-3 detection by flow cytometry in iDC and DC HIV cultured alone, or cocultured with aNK cells for 24 hours. Data represent the mean ± sd of three independent experiments. * p

    Article Snippet: For intracellular staining, cells were fixed with 4% PFA, permeabilized using 0.5% BSA, 0.01% NaN3 , 0.5% Saponin buffer, stained for 20 minutes at room temperature with FITC-labeled anti-perforin mAbs (BD), PE-conjugated anti-p24 mAbs (Beckman Coulter), PE-conjugated anti-active caspase-3 Abs (BD), unconjugated rabbit anti-human c-IAP2 polyclonal antibody (clone H-85, Santa Cruz Biotechnology) or c-FLIP antibodies (Santa Cruz biotechnology).

    Techniques: Infection, Incubation, Staining, Flow Cytometry, Cytometry, Cell Culture