Structured Review

Santa Cruz Biotechnology antibodies js k
<t>JS-K</t> increased the activity of <t>PP2A</t> in SMMC7721 and HepG2 cells. a The levels of PP2A were measured by ELISA kit. The cells were treated with different concentrations of JS-K for 24 h and the cell lysates were prepared and assayed by enzyme-linked immunosorbent assay. b The effects of JS-K on protein levels of PP2A complexs. c Effects of JS-K on protein levels of PP2A substrates. d Effects of Carboxy-PTIO on the expression of PP2A-C and PP2A substrates. e Effects of JS-K on protein levels of PP2A substrates through silencing PP2A-C. f Effects of JS-K on protein levels of PP2A substrates through overexpression of PP2A-C. The cells were transfected for 48 h before treatment with JS-K for 24 h. Data are mean ± SD. n = 3 for each concentration. * P
Antibodies Js K, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 2578 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Protein phosphatase 2A activation mechanism contributes to JS-K induced caspase-dependent apoptosis in human hepatocellular carcinoma cells"

Article Title: Protein phosphatase 2A activation mechanism contributes to JS-K induced caspase-dependent apoptosis in human hepatocellular carcinoma cells

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-018-0823-2

JS-K increased the activity of PP2A in SMMC7721 and HepG2 cells. a The levels of PP2A were measured by ELISA kit. The cells were treated with different concentrations of JS-K for 24 h and the cell lysates were prepared and assayed by enzyme-linked immunosorbent assay. b The effects of JS-K on protein levels of PP2A complexs. c Effects of JS-K on protein levels of PP2A substrates. d Effects of Carboxy-PTIO on the expression of PP2A-C and PP2A substrates. e Effects of JS-K on protein levels of PP2A substrates through silencing PP2A-C. f Effects of JS-K on protein levels of PP2A substrates through overexpression of PP2A-C. The cells were transfected for 48 h before treatment with JS-K for 24 h. Data are mean ± SD. n = 3 for each concentration. * P
Figure Legend Snippet: JS-K increased the activity of PP2A in SMMC7721 and HepG2 cells. a The levels of PP2A were measured by ELISA kit. The cells were treated with different concentrations of JS-K for 24 h and the cell lysates were prepared and assayed by enzyme-linked immunosorbent assay. b The effects of JS-K on protein levels of PP2A complexs. c Effects of JS-K on protein levels of PP2A substrates. d Effects of Carboxy-PTIO on the expression of PP2A-C and PP2A substrates. e Effects of JS-K on protein levels of PP2A substrates through silencing PP2A-C. f Effects of JS-K on protein levels of PP2A substrates through overexpression of PP2A-C. The cells were transfected for 48 h before treatment with JS-K for 24 h. Data are mean ± SD. n = 3 for each concentration. * P

Techniques Used: Activity Assay, Enzyme-linked Immunosorbent Assay, Expressing, Over Expression, Transfection, Concentration Assay

JS-K inhibits the growth of rat primary hepatic carcinoma in vivo ( n = 8). a The photographs of livers in control, DEN-treated and JS-K-treated groups. b - c IHC staining of PCNA and PP2A (200 × magnifcation) in control group, DEN group, and DEN+JS-K groups (0.25 mg/kg and 0.5 mg/kg). d The expression levels of PP2A-C, β-catenin, c-Myc and p-Bcl-2 in rat model of primary hepatic carcinoma were analyzed by western blot
Figure Legend Snippet: JS-K inhibits the growth of rat primary hepatic carcinoma in vivo ( n = 8). a The photographs of livers in control, DEN-treated and JS-K-treated groups. b - c IHC staining of PCNA and PP2A (200 × magnifcation) in control group, DEN group, and DEN+JS-K groups (0.25 mg/kg and 0.5 mg/kg). d The expression levels of PP2A-C, β-catenin, c-Myc and p-Bcl-2 in rat model of primary hepatic carcinoma were analyzed by western blot

Techniques Used: In Vivo, Immunohistochemistry, Staining, Expressing, Western Blot

Effects of okadaic acid (OA) on JS-K-induced cell apoptosis and PP2A activation in SMMC7721 and HepG2 cells. a Effect of OA treatment as PP2A inhibitor on cell apoptosis. The cells were pretreated with OA (1 nM) for 1 h, and then stimulated with JS-K (10 μM) for 24 h. The apoptosis was assessed by flow cytometry. b Effect of OA treatment as PP2A inhibitor on the expression of apoptotic-related protein. c Effect of OA treatment as PP2A inhibitor on the expression of PP2A-C and its substrates. The cells were pretreated with OA (1 nM) for 1 h, and then stimulated with JS-K (10 μM) for 24 h. The expressions of protein were assessed by Western blotting analysis. Data are mean ± SD. n = 3 for each concentration. * P
Figure Legend Snippet: Effects of okadaic acid (OA) on JS-K-induced cell apoptosis and PP2A activation in SMMC7721 and HepG2 cells. a Effect of OA treatment as PP2A inhibitor on cell apoptosis. The cells were pretreated with OA (1 nM) for 1 h, and then stimulated with JS-K (10 μM) for 24 h. The apoptosis was assessed by flow cytometry. b Effect of OA treatment as PP2A inhibitor on the expression of apoptotic-related protein. c Effect of OA treatment as PP2A inhibitor on the expression of PP2A-C and its substrates. The cells were pretreated with OA (1 nM) for 1 h, and then stimulated with JS-K (10 μM) for 24 h. The expressions of protein were assessed by Western blotting analysis. Data are mean ± SD. n = 3 for each concentration. * P

Techniques Used: Activation Assay, Flow Cytometry, Cytometry, Expressing, Western Blot, Concentration Assay

Effects of FTY720 on JS-K-induced cell apoptosis and PP2A activation in SMMC7721 and HepG2 cells. a Effect of FTY720 treatment as PP2A agonist on cell apoptosis. The cells were pre-treated with FTY720 (2 μM) for 1 h, and then stimulated with JS-K (10 μM) for 24 h. The apoptosis was assessed by flow cytometry. b Effect of FTY720 treatment as PP2A agonist on the expression of apoptotic-related protein. c Effect of FTY720 treatment as PP2A agonist on the expression of PP2A-C and its substrates. The cells were pretreated with FTY720 (2 μM) for 1 h, and then stimulated with JS-K (10 μM) for 24 h. The expressions of protein were assessed by Western blotting analysis. Data are mean ± SD. n = 3 for each concentration. * P
Figure Legend Snippet: Effects of FTY720 on JS-K-induced cell apoptosis and PP2A activation in SMMC7721 and HepG2 cells. a Effect of FTY720 treatment as PP2A agonist on cell apoptosis. The cells were pre-treated with FTY720 (2 μM) for 1 h, and then stimulated with JS-K (10 μM) for 24 h. The apoptosis was assessed by flow cytometry. b Effect of FTY720 treatment as PP2A agonist on the expression of apoptotic-related protein. c Effect of FTY720 treatment as PP2A agonist on the expression of PP2A-C and its substrates. The cells were pretreated with FTY720 (2 μM) for 1 h, and then stimulated with JS-K (10 μM) for 24 h. The expressions of protein were assessed by Western blotting analysis. Data are mean ± SD. n = 3 for each concentration. * P

Techniques Used: Activation Assay, Flow Cytometry, Cytometry, Expressing, Western Blot, Concentration Assay

Differential effects of JS-K on cell death, apoptosis and PP2A activation in five HHC cells and L02 cells. a Dose-dependent effects of JS-K on cell death in the five HCC cells and L02 cells. The cells were exposed to JS-K at the indicated concentrations for 24, 48, 72 h. b Dose-dependent effects of JS-K on cell apoptosis in the five HCC cells and L02 cells. c - d Effects of JS-K on the expression of PP2A. The cells were treated with JS-K at the indicated concentration for 24 h. Cell lysates were prepared and assayed for PP2A by Western blotting. Data are mean ± SD. n = 3 for each concentration. * P
Figure Legend Snippet: Differential effects of JS-K on cell death, apoptosis and PP2A activation in five HHC cells and L02 cells. a Dose-dependent effects of JS-K on cell death in the five HCC cells and L02 cells. The cells were exposed to JS-K at the indicated concentrations for 24, 48, 72 h. b Dose-dependent effects of JS-K on cell apoptosis in the five HCC cells and L02 cells. c - d Effects of JS-K on the expression of PP2A. The cells were treated with JS-K at the indicated concentration for 24 h. Cell lysates were prepared and assayed for PP2A by Western blotting. Data are mean ± SD. n = 3 for each concentration. * P

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

2) Product Images from "Sodium azide induces mitochondria-mediated apoptosis in PC12 cells through Pgc-1α-associated signaling pathway"

Article Title: Sodium azide induces mitochondria-mediated apoptosis in PC12 cells through Pgc-1α-associated signaling pathway

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2019.9853

NaN 3 induces mitochondria-mediated apoptosis through the expression of Pgc-1α-associated proteins in PC12 cells. (A) Expression levels of Pgc-1α, Nrf-1, Nrf-2, Tfam and Cox IV detected by western blot analysis. (B) Expression levels of procaspase-3, Bax, Bcl-2 and cyt-c detected by western blot analysis. (C) Expression levels of pan-calcineurin A, CaMKII, p-CaMKII, p38 MAPK, p-p38 MAPK, Erk1/2 and p-Erk1/2 detected by western blot analysis. β-actin and GAPDH were used as the internal control. Band intensity ratios for each group are presented as mean ± standard deviation (n=3). *P
Figure Legend Snippet: NaN 3 induces mitochondria-mediated apoptosis through the expression of Pgc-1α-associated proteins in PC12 cells. (A) Expression levels of Pgc-1α, Nrf-1, Nrf-2, Tfam and Cox IV detected by western blot analysis. (B) Expression levels of procaspase-3, Bax, Bcl-2 and cyt-c detected by western blot analysis. (C) Expression levels of pan-calcineurin A, CaMKII, p-CaMKII, p38 MAPK, p-p38 MAPK, Erk1/2 and p-Erk1/2 detected by western blot analysis. β-actin and GAPDH were used as the internal control. Band intensity ratios for each group are presented as mean ± standard deviation (n=3). *P

Techniques Used: Expressing, Pyrolysis Gas Chromatography, Western Blot, Standard Deviation

3) Product Images from "Protective Effects and Mechanisms of N-Phenethyl Caffeamide from UVA-Induced Skin Damage in Human Epidermal Keratinocytes through Nrf2/HO-1 Regulation"

Article Title: Protective Effects and Mechanisms of N-Phenethyl Caffeamide from UVA-Induced Skin Damage in Human Epidermal Keratinocytes through Nrf2/HO-1 Regulation

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms20010164

Effect of K36 on UVA-induced Bcl-2 expression in human epidermal keratinocytes. The representative image of the western blot ( a ) and the average value of the triplicate experiment ( b ); significant difference versus nonirradiated group: ###, p
Figure Legend Snippet: Effect of K36 on UVA-induced Bcl-2 expression in human epidermal keratinocytes. The representative image of the western blot ( a ) and the average value of the triplicate experiment ( b ); significant difference versus nonirradiated group: ###, p

Techniques Used: Expressing, Western Blot

4) Product Images from "MHY440, a Novel Topoisomerase Ι Inhibitor, Induces Cell Cycle Arrest and Apoptosis via a ROS-Dependent DNA Damage Signaling Pathway in AGS Human Gastric Cancer Cells"

Article Title: MHY440, a Novel Topoisomerase Ι Inhibitor, Induces Cell Cycle Arrest and Apoptosis via a ROS-Dependent DNA Damage Signaling Pathway in AGS Human Gastric Cancer Cells

Journal: Molecules

doi: 10.3390/molecules24010096

The effect of caspases on MHY440-induced apoptosis in AGS cells. ( A ) MHY440-treated cell lysates were assayed for caspase-3, -8, and -9 activities using DEVD-pNA, IETD-pNA and LEHD-pNA substrates, respectively. The emitted fluorescent products were measured. Data are expressed as the means ± SD of triplicate samples. The results represent one of three independent experiments. ( B ) Cells were pretreated with 40 μM Z-VAD-FMK for 30 min and then treated with 2.5 μM MHY440 for 24 h. Cells were stained with PI and analyzed using flow cytometry. The results are expressed as means ± SD of three individual experiments. Significance was determined using Student’s t -test (* p
Figure Legend Snippet: The effect of caspases on MHY440-induced apoptosis in AGS cells. ( A ) MHY440-treated cell lysates were assayed for caspase-3, -8, and -9 activities using DEVD-pNA, IETD-pNA and LEHD-pNA substrates, respectively. The emitted fluorescent products were measured. Data are expressed as the means ± SD of triplicate samples. The results represent one of three independent experiments. ( B ) Cells were pretreated with 40 μM Z-VAD-FMK for 30 min and then treated with 2.5 μM MHY440 for 24 h. Cells were stained with PI and analyzed using flow cytometry. The results are expressed as means ± SD of three individual experiments. Significance was determined using Student’s t -test (* p

Techniques Used: Staining, Flow Cytometry, Cytometry

5) Product Images from "Protective Effect of Hesperidin Against Sepsis-Induced Lung Injury by Inducing the Heat-Stable Protein 70 (Hsp70)/Toll-Like Receptor 4 (TLR4)/ Myeloid Differentiation Primary Response 88 (MyD88) Pathway"

Article Title: Protective Effect of Hesperidin Against Sepsis-Induced Lung Injury by Inducing the Heat-Stable Protein 70 (Hsp70)/Toll-Like Receptor 4 (TLR4)/ Myeloid Differentiation Primary Response 88 (MyD88) Pathway

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

doi: 10.12659/MSM.912490

Effect of hesperidin on the expression of Bcl-2, caspase-3, TLR-4, and HSP70 protein in the lung tissue of CLP-induced lung injury mice. Mean ±SEM (n=6). ## p
Figure Legend Snippet: Effect of hesperidin on the expression of Bcl-2, caspase-3, TLR-4, and HSP70 protein in the lung tissue of CLP-induced lung injury mice. Mean ±SEM (n=6). ## p

Techniques Used: Expressing, Mouse Assay

6) Product Images from "Yap1 safeguards mouse embryonic stem cells from excessive apoptosis during differentiation"

Article Title: Yap1 safeguards mouse embryonic stem cells from excessive apoptosis during differentiation

Journal: eLife

doi: 10.7554/eLife.40167

( A ) Signal tracks indicating Yap1 (red) and p300 (blue) ChIP-seq performed in ESCs (+LIF for Yap1, 2i for p300) and dESCs (-LIF for Yap1, EpiLC for p300).
Figure Legend Snippet: ( A ) Signal tracks indicating Yap1 (red) and p300 (blue) ChIP-seq performed in ESCs (+LIF for Yap1, 2i for p300) and dESCs (-LIF for Yap1, EpiLC for p300).

Techniques Used: Chromatin Immunoprecipitation

Modulation of the expression of individual anti- or pro-apoptotic genes influences cell death during differentiation. ( A ) Immunoblot of Yap1 and Bcl-xL after OE in Yap1 KO cells relative to WT or empty vector Yap1 KO in +LIF. β-actin was used as a loading control. ( B ) Representative brightfield and fluorescent microscopy images of Yap1 KO cells showing ZsGreen fluorescence ±Dox. Scale bar, 400 μm. ( C ) RT-qPCR measuring the expression of Bcl-2 in WT and Yap1 KO cells during differentiation (72 hr)±Dox (48 hr, 500 ng/mL). ( D ) Immunoblot of Bcl-2 in Yap1 KO cells ± Dox (48 hr, 500 ng/mL) in +LIF. ( E ) Immunoblot of Taz in WT and Yap1 KO cells ± Dox (48 hr, 500 ng/mL) in +LIF. ( F ) Immunoblot of Bcl-xL and Mcl-1 after siRNA KD in WT cells in -LIF (48 hr). ( G ) Immunoblot of Bcl-2 after shRNA KD in -LIF (72 hr). ( H ) Quantification of fold increase in cell death from Figure 6E and F observed upon KD of Bcl-xL, Mcl-1, or Bcl-2, relative to control KD, in -LIF (72 hr). ( I ) RT-qPCR measuring the expression of lineage markers in WT cells transfected with siRNA against Bcl-xL or Mcl-1 in -LIF (72 hr). Expression is indicated as a fold change compared to control siRNA. ( J ) RT-qPCR measur ing the induction of lineage markers in WT cells transduced with shRNA against Bcl-2 in -LIF relative to +LIF (72 hr). All data are expressed as mean ±standard deviation (n = 3 independent samples). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.
Figure Legend Snippet: Modulation of the expression of individual anti- or pro-apoptotic genes influences cell death during differentiation. ( A ) Immunoblot of Yap1 and Bcl-xL after OE in Yap1 KO cells relative to WT or empty vector Yap1 KO in +LIF. β-actin was used as a loading control. ( B ) Representative brightfield and fluorescent microscopy images of Yap1 KO cells showing ZsGreen fluorescence ±Dox. Scale bar, 400 μm. ( C ) RT-qPCR measuring the expression of Bcl-2 in WT and Yap1 KO cells during differentiation (72 hr)±Dox (48 hr, 500 ng/mL). ( D ) Immunoblot of Bcl-2 in Yap1 KO cells ± Dox (48 hr, 500 ng/mL) in +LIF. ( E ) Immunoblot of Taz in WT and Yap1 KO cells ± Dox (48 hr, 500 ng/mL) in +LIF. ( F ) Immunoblot of Bcl-xL and Mcl-1 after siRNA KD in WT cells in -LIF (48 hr). ( G ) Immunoblot of Bcl-2 after shRNA KD in -LIF (72 hr). ( H ) Quantification of fold increase in cell death from Figure 6E and F observed upon KD of Bcl-xL, Mcl-1, or Bcl-2, relative to control KD, in -LIF (72 hr). ( I ) RT-qPCR measuring the expression of lineage markers in WT cells transfected with siRNA against Bcl-xL or Mcl-1 in -LIF (72 hr). Expression is indicated as a fold change compared to control siRNA. ( J ) RT-qPCR measur ing the induction of lineage markers in WT cells transduced with shRNA against Bcl-2 in -LIF relative to +LIF (72 hr). All data are expressed as mean ±standard deviation (n = 3 independent samples). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

Techniques Used: Expressing, Plasmid Preparation, Microscopy, Fluorescence, Quantitative RT-PCR, shRNA, Transfection, Transduction, Standard Deviation, Two Tailed Test

Yap1 expression in KO/KD/OE cell lines, STS sensitivity, and caspase expression during ES cell differentiation. ( A ) Immunoblot of Yap1 to verify knockout in Yap1 KO cells. β-actin was used as a loading control. ( B ) Representative brightfield microscopy images of WT and Yap1 KO ES cells in ±LIF. Scale bar, 200 μm. ( C ) Immunoblot of Yap1 to verify knockout of Yap1 in three different ESC lines (J1, E14, and CJ7). β-actin was used as a loading control. J1 clone #5 was used as a positive control for knockout. ( D ) RT-qPCR measuring the expression of Yap1 after lentiviral shRNA-mediated Yap1 KD in differentiating WT ESCs (-LIF 72 hr). ( E ) LDH assay measuring cell death of Yap1 KD vs. control KD cells during differentiation (-LIF 72 hr). ( F ) Immunoblot of Yap1 to verify stable overexpression (OE) of FLAG-Bio-Yap1 in three different clones compared to WT ESCs. β-actin was used as a loading control. ( G ) Immunoblot of cleaved Casp3 and cleaved Parp1 in WT and Yap1 KO cells that had been treated with 1 μM STS for the indicated number of hours during differentiation (treatment started 43–48 hr after withdrawal of LIF depending on the length of STS treatment). ( H ) RT-qPCR measuring the expression of Casp9 upon shRNA-mediated lentiviral KD in WT and Yap1 KO cells during differentiation (72 hr) relative to empty vector KD. ( I ) RT-qPCR measuring the expression of Casp2, Casp3, Casp6, Casp7, Casp8, and Casp9 in Yap1 KO cells compared to WT cells in ±LIF. All data are expressed as mean ±standard deviation (n = 3 independent samples). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.
Figure Legend Snippet: Yap1 expression in KO/KD/OE cell lines, STS sensitivity, and caspase expression during ES cell differentiation. ( A ) Immunoblot of Yap1 to verify knockout in Yap1 KO cells. β-actin was used as a loading control. ( B ) Representative brightfield microscopy images of WT and Yap1 KO ES cells in ±LIF. Scale bar, 200 μm. ( C ) Immunoblot of Yap1 to verify knockout of Yap1 in three different ESC lines (J1, E14, and CJ7). β-actin was used as a loading control. J1 clone #5 was used as a positive control for knockout. ( D ) RT-qPCR measuring the expression of Yap1 after lentiviral shRNA-mediated Yap1 KD in differentiating WT ESCs (-LIF 72 hr). ( E ) LDH assay measuring cell death of Yap1 KD vs. control KD cells during differentiation (-LIF 72 hr). ( F ) Immunoblot of Yap1 to verify stable overexpression (OE) of FLAG-Bio-Yap1 in three different clones compared to WT ESCs. β-actin was used as a loading control. ( G ) Immunoblot of cleaved Casp3 and cleaved Parp1 in WT and Yap1 KO cells that had been treated with 1 μM STS for the indicated number of hours during differentiation (treatment started 43–48 hr after withdrawal of LIF depending on the length of STS treatment). ( H ) RT-qPCR measuring the expression of Casp9 upon shRNA-mediated lentiviral KD in WT and Yap1 KO cells during differentiation (72 hr) relative to empty vector KD. ( I ) RT-qPCR measuring the expression of Casp2, Casp3, Casp6, Casp7, Casp8, and Casp9 in Yap1 KO cells compared to WT cells in ±LIF. All data are expressed as mean ±standard deviation (n = 3 independent samples). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

Techniques Used: Expressing, Cell Differentiation, Knock-Out, Microscopy, Positive Control, Quantitative RT-PCR, shRNA, Lactate Dehydrogenase Assay, Over Expression, Clone Assay, Plasmid Preparation, Standard Deviation, Two Tailed Test

Depletion or loss of Yap1 leads to dysregulation of apoptosis-related genes. ( A ) Representative confocal images (63X oil objective) of immunocytochemistry of WT and Yap1 KO ESCs in -LIF (72 hr). Blue represents the nucleus, red represents Bcl-2 (top 6) or Mcl-1 (bottom 6), and yellow represents mitochondria. White squares indicate location of zoom images. Scale bar, 20 μm. ( B ) Quantification of fluorescence corresponding to Bcl-2, Mcl-1, and mitochondria using ImageJ, normalized to the number of nuclei in each view as stained by NucBlue. ( C ) RT-qPCR of WT ESCs with transient OE of Yap1 (48 hr) in -LIF (72 hr) normalized to empty vector. Blue indicates anti-apoptotic genes, red indicates pro-apoptotic genes. ( D ) Boxplots of expression of pro-apoptotic genes and anti-apoptotic genes in Yap1 OE cells versus BirA cells (log 2 ) in +LIF. Plus symbols represent the average and middle red bands represent the median. Outliers are represented by hollow circles. Significance stars indicate p-values from paired t-test. ( E ) Boxplots of expression of pro- and anti-apoptotic genes in Yap1 KD and empty KD in -LIF relative to +LIF (log2). Plus symbols represent the average and middle red bands represent the median. Outliers are represented by hollow circles. Significance stars indicate p-values from paired t-test. All data are expressed as mean ±standard deviation (n = 3 independent samples unless otherwise stated). Two sample two-tailed t-test (unless otherwise specified) compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.
Figure Legend Snippet: Depletion or loss of Yap1 leads to dysregulation of apoptosis-related genes. ( A ) Representative confocal images (63X oil objective) of immunocytochemistry of WT and Yap1 KO ESCs in -LIF (72 hr). Blue represents the nucleus, red represents Bcl-2 (top 6) or Mcl-1 (bottom 6), and yellow represents mitochondria. White squares indicate location of zoom images. Scale bar, 20 μm. ( B ) Quantification of fluorescence corresponding to Bcl-2, Mcl-1, and mitochondria using ImageJ, normalized to the number of nuclei in each view as stained by NucBlue. ( C ) RT-qPCR of WT ESCs with transient OE of Yap1 (48 hr) in -LIF (72 hr) normalized to empty vector. Blue indicates anti-apoptotic genes, red indicates pro-apoptotic genes. ( D ) Boxplots of expression of pro-apoptotic genes and anti-apoptotic genes in Yap1 OE cells versus BirA cells (log 2 ) in +LIF. Plus symbols represent the average and middle red bands represent the median. Outliers are represented by hollow circles. Significance stars indicate p-values from paired t-test. ( E ) Boxplots of expression of pro- and anti-apoptotic genes in Yap1 KD and empty KD in -LIF relative to +LIF (log2). Plus symbols represent the average and middle red bands represent the median. Outliers are represented by hollow circles. Significance stars indicate p-values from paired t-test. All data are expressed as mean ±standard deviation (n = 3 independent samples unless otherwise stated). Two sample two-tailed t-test (unless otherwise specified) compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

Techniques Used: Immunocytochemistry, Fluorescence, Staining, Quantitative RT-PCR, Plasmid Preparation, Expressing, Standard Deviation, Two Tailed Test

Verification of KD and OE of pro-apoptotic factors Bmf and Puma. ( A ) RT-qPCR measuring the expression of Bmf and Puma in Yap1 KO cells during differentiation (48 hr) relative to empty vector KD (n = 3). ( B ) Immunoblot of Bmf and Puma after KD in Yap1 KO cells during differentiation (48 hr) relative to empty vector KD. β-actin was used as a loading control. ( C ) RT-qPCR measuring the expression of Bmf and Puma in WT and Yap1 KO ESCs ± Dox (24 hr) in +LIF (n = 2). ( D ) Immunoblot of Bmf and Puma after OE in WT ESCs in +LIF. β-actin was used as a loading control.
Figure Legend Snippet: Verification of KD and OE of pro-apoptotic factors Bmf and Puma. ( A ) RT-qPCR measuring the expression of Bmf and Puma in Yap1 KO cells during differentiation (48 hr) relative to empty vector KD (n = 3). ( B ) Immunoblot of Bmf and Puma after KD in Yap1 KO cells during differentiation (48 hr) relative to empty vector KD. β-actin was used as a loading control. ( C ) RT-qPCR measuring the expression of Bmf and Puma in WT and Yap1 KO ESCs ± Dox (24 hr) in +LIF (n = 2). ( D ) Immunoblot of Bmf and Puma after OE in WT ESCs in +LIF. β-actin was used as a loading control.

Techniques Used: Quantitative RT-PCR, Expressing, Plasmid Preparation

Yap1 binds to distal regulatory elements, primarily through Tead factors. ( A ) Gene feature analysis of Yap1 ChIP-seq quantifying the proportion of Yap1 peaks in promoter, intergenic, upstream, intron, or exon regions. ( B ) Co-IP followed by immunoblot of Tead4 and p300 after pull-down using magnetic streptavidin beads in Yap1 FB cells during differentiation (72 hr). ( C ) Known motif analysis of Yap1 ChIP-seq peaks using BirA ES cells as a background control. Top five motifs with the lowest p-values corresponding to known factors are presented. ( D ) Peak-centered histogram of Yap1 ChIP-seq peaks indicating the presence of the motifs of Tead4, Zic3, and AP-1 complex members JunB and Fra1 (Fosl1). Esrrb is presented as a negative control, as known motif analysis in ( C ) showed no significant enrichment of the Esrrb motif. Input represents 0.03% of total protein lysate. ( E ) Peak-centered histogram of Yap1 ChIP-seq peaks indicating p300 occupancy and H3K27ac presence in ESCs maintained in 2i and EpiLCs, which represent dESCs. ( F ) Gene ontology (GO) analysis of genes bound by Yap1 that are also upregulated (white) or downregulated (black) after Yap1 KD in -LIF (96 hr). ( G ) Schematic of dual luciferase essay using putative Yap1-responsive cis- regulatory elements. ( H ) Schematic of tandem Bcl-2 enhancer creation showing the location of both Yap1-occupied elements that were combined into the same construct. ( I ) Correlation heatmap of YAP1 occupancy on apoptosis-related genes in SF268 glioblastoma cells, NCI-H2052 lung mesothelioma cells, IMR90 lung fibroblasts, and MDA-MB-231 triple negative breast cancer cells. Genes that were not occupied by any factor were removed from the analysis to reduce noise. ( J ) Signal tracks of YAP1 occupancy on MCL1 , BCL2 , and BCL2L1 (BCL-XL) in the cell types described in ( I ). ( K ) Correlation heatmap of occupancy of Yap1 in mouse dES cells (from this study), Tead1 in pre-B progenitor cells, Tead2 in Py2T breast cancer cells, and Tead4 in hemogenic epithelium, all on apoptosis-related genes. Genes that were not occupied by any factor were removed from the analysis to reduce noise. ( L ) Signal tracks of Yap1 (red), Tead1, Tead2, and Tead4 (all in blue) on Mcl1 , Bcl2 , and Bcl2l1 (Bcl-xL) in the cell types described in ( I ).
Figure Legend Snippet: Yap1 binds to distal regulatory elements, primarily through Tead factors. ( A ) Gene feature analysis of Yap1 ChIP-seq quantifying the proportion of Yap1 peaks in promoter, intergenic, upstream, intron, or exon regions. ( B ) Co-IP followed by immunoblot of Tead4 and p300 after pull-down using magnetic streptavidin beads in Yap1 FB cells during differentiation (72 hr). ( C ) Known motif analysis of Yap1 ChIP-seq peaks using BirA ES cells as a background control. Top five motifs with the lowest p-values corresponding to known factors are presented. ( D ) Peak-centered histogram of Yap1 ChIP-seq peaks indicating the presence of the motifs of Tead4, Zic3, and AP-1 complex members JunB and Fra1 (Fosl1). Esrrb is presented as a negative control, as known motif analysis in ( C ) showed no significant enrichment of the Esrrb motif. Input represents 0.03% of total protein lysate. ( E ) Peak-centered histogram of Yap1 ChIP-seq peaks indicating p300 occupancy and H3K27ac presence in ESCs maintained in 2i and EpiLCs, which represent dESCs. ( F ) Gene ontology (GO) analysis of genes bound by Yap1 that are also upregulated (white) or downregulated (black) after Yap1 KD in -LIF (96 hr). ( G ) Schematic of dual luciferase essay using putative Yap1-responsive cis- regulatory elements. ( H ) Schematic of tandem Bcl-2 enhancer creation showing the location of both Yap1-occupied elements that were combined into the same construct. ( I ) Correlation heatmap of YAP1 occupancy on apoptosis-related genes in SF268 glioblastoma cells, NCI-H2052 lung mesothelioma cells, IMR90 lung fibroblasts, and MDA-MB-231 triple negative breast cancer cells. Genes that were not occupied by any factor were removed from the analysis to reduce noise. ( J ) Signal tracks of YAP1 occupancy on MCL1 , BCL2 , and BCL2L1 (BCL-XL) in the cell types described in ( I ). ( K ) Correlation heatmap of occupancy of Yap1 in mouse dES cells (from this study), Tead1 in pre-B progenitor cells, Tead2 in Py2T breast cancer cells, and Tead4 in hemogenic epithelium, all on apoptosis-related genes. Genes that were not occupied by any factor were removed from the analysis to reduce noise. ( L ) Signal tracks of Yap1 (red), Tead1, Tead2, and Tead4 (all in blue) on Mcl1 , Bcl2 , and Bcl2l1 (Bcl-xL) in the cell types described in ( I ).

Techniques Used: Chromatin Immunoprecipitation, Co-Immunoprecipitation Assay, Negative Control, Luciferase, Construct, Multiple Displacement Amplification

7) Product Images from "LncRNA SNHG20 predicts a poor prognosis and promotes cell progression in epithelial ovarian cancer"

Article Title: LncRNA SNHG20 predicts a poor prognosis and promotes cell progression in epithelial ovarian cancer

Journal: Bioscience Reports

doi: 10.1042/BSR20182186

SNHG20 knockdown regulated several genes related to proliferation and metastasis ( A , B ) The mRNA level of P21, Cyclin D1, Vimentin, and E-cadherin was detected by qRT-PCR in A2780 and CAOV-3 cells transfecting with si-SNHG20-1 or -2 (* P
Figure Legend Snippet: SNHG20 knockdown regulated several genes related to proliferation and metastasis ( A , B ) The mRNA level of P21, Cyclin D1, Vimentin, and E-cadherin was detected by qRT-PCR in A2780 and CAOV-3 cells transfecting with si-SNHG20-1 or -2 (* P

Techniques Used: Quantitative RT-PCR

8) Product Images from "A Triterpenoid from Thalictrum fortunei Induces Apoptosis in BEL-7402 Cells Through the P53-Induced Apoptosis Pathway"

Article Title: A Triterpenoid from Thalictrum fortunei Induces Apoptosis in BEL-7402 Cells Through the P53-Induced Apoptosis Pathway

Journal: Molecules

doi: 10.3390/molecules16119505

Bel-7402 cells were incubated with compound 1 at the concentrations of 20, 40 and 80 μM for 48 h, respectively, then, the different proteins levels were measured by westblot method. ( A ) Effects of compound 1 on the expression levels of P53, Bcl-2 and Bax proteins; ( B ) The ratio of Bcl-2/Bax protein expression. ** p
Figure Legend Snippet: Bel-7402 cells were incubated with compound 1 at the concentrations of 20, 40 and 80 μM for 48 h, respectively, then, the different proteins levels were measured by westblot method. ( A ) Effects of compound 1 on the expression levels of P53, Bcl-2 and Bax proteins; ( B ) The ratio of Bcl-2/Bax protein expression. ** p

Techniques Used: Incubation, Expressing

9) Product Images from "Fisetin Induces Apoptosis Through p53-Mediated Up-Regulation of DR5 Expression in Human Renal Carcinoma Caki Cells"

Article Title: Fisetin Induces Apoptosis Through p53-Mediated Up-Regulation of DR5 Expression in Human Renal Carcinoma Caki Cells

Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

doi: 10.3390/molecules22081285

Fisetin induced apoptosis in a caspase-dependent manner. ( A ) Caki cells were treated with the indicated concentrations of fisetin for 24 h. Caspase activities were determined with colorimetric assays using caspase-3 (DEVDase) assay kits; ( B ) Caki cells were treated with 200 μM fisetin in the presence or absence of 20 μM z-VAD-fmk (z-VAD). The sub-G1 fraction was measured by flow cytometry. The protein expression levels of PARP and actin were determined by Western blotting. The level of actin was used as a loading control; ( C ) Caki cells were treated with the indicated concentrations of fisetin for 24 h. The protein expression levels of DR5, DR4, Fas, c-FLIP, FADD, Bcl-2, Bcl-xL, PUMA and actin were determined by western blotting. The level of actin was used as a loading control; the values in ( A , B ) represent the mean ± SD from three independent samples. * p
Figure Legend Snippet: Fisetin induced apoptosis in a caspase-dependent manner. ( A ) Caki cells were treated with the indicated concentrations of fisetin for 24 h. Caspase activities were determined with colorimetric assays using caspase-3 (DEVDase) assay kits; ( B ) Caki cells were treated with 200 μM fisetin in the presence or absence of 20 μM z-VAD-fmk (z-VAD). The sub-G1 fraction was measured by flow cytometry. The protein expression levels of PARP and actin were determined by Western blotting. The level of actin was used as a loading control; ( C ) Caki cells were treated with the indicated concentrations of fisetin for 24 h. The protein expression levels of DR5, DR4, Fas, c-FLIP, FADD, Bcl-2, Bcl-xL, PUMA and actin were determined by western blotting. The level of actin was used as a loading control; the values in ( A , B ) represent the mean ± SD from three independent samples. * p

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

10) Product Images from "Anticancer activity of bergenin against cervical cancer cells involves apoptosis, cell cycle arrest, inhibition of cell migration and the STAT3 signalling pathway"

Article Title: Anticancer activity of bergenin against cervical cancer cells involves apoptosis, cell cycle arrest, inhibition of cell migration and the STAT3 signalling pathway

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2019.7380

Effect of bergenin at indicated concentrations on the expression of Bax and Bcl-2 proteins in HeLa cells as shown in the western blot. The experiments were carried out in triplicates. The values were considered significant at *P
Figure Legend Snippet: Effect of bergenin at indicated concentrations on the expression of Bax and Bcl-2 proteins in HeLa cells as shown in the western blot. The experiments were carried out in triplicates. The values were considered significant at *P

Techniques Used: Expressing, Western Blot

11) Product Images from "NF-κB inhibition compromises cardiac fibroblast viability under hypoxia"

Article Title: NF-κB inhibition compromises cardiac fibroblast viability under hypoxia

Journal: Experimental cell research

doi: 10.1016/j.yexcr.2010.12.024

Effect of hypoxia and Bay 11-7085 on cIAP-2 expression in cardiac fibroblasts Confluent cultures of cardiac fibroblasts in M199 with 10% FBS were exposed to normoxia (N) or hypoxia (H) for 12 hours in the presence or absence of 4μmol/L Bay-11. Western blot analysis was performed as described under Methods, using polyclonal anti-cIAP-2 antibody. Significant induction of cIAP-2 expression was observed in hypoxic cells (H), which was attenuated in NF-κB-inhibited (4μmol/L Bay-11-7085) hypoxic cells (HBay). A representative profile from one of three experiments is shown.
Figure Legend Snippet: Effect of hypoxia and Bay 11-7085 on cIAP-2 expression in cardiac fibroblasts Confluent cultures of cardiac fibroblasts in M199 with 10% FBS were exposed to normoxia (N) or hypoxia (H) for 12 hours in the presence or absence of 4μmol/L Bay-11. Western blot analysis was performed as described under Methods, using polyclonal anti-cIAP-2 antibody. Significant induction of cIAP-2 expression was observed in hypoxic cells (H), which was attenuated in NF-κB-inhibited (4μmol/L Bay-11-7085) hypoxic cells (HBay). A representative profile from one of three experiments is shown.

Techniques Used: Expressing, Western Blot

12) Product Images from "A novel curcumin analogue is a potent chemotherapy candidate for human hepatocellular carcinoma"

Article Title: A novel curcumin analogue is a potent chemotherapy candidate for human hepatocellular carcinoma

Journal: Oncology Letters

doi: 10.3892/ol.2016.5126

Apoptosis rates of SK-HEP-1 cells subjected to GL63 and CUR treatment, as detected by flow cytometry and protein expression analysis. SK-HEP-1 cells were treated with GL63 and CUR (0, 10, 20 and 40 µM) for 24 h. (A) Representative diagrams of annexin V-fluorescein isothiocyanate/propidium iodide staining upon treatment with different concentrations of GL63 and CUR. (B) Bar diagram of apoptotic cell rate from three FACS analyses from three separate treatments. (C) Caspase-9, caspase-3, Cyt-c and PARP levels were determined in SK-HEP-1 cells treated with GL63 and CUR at various concentrations for 24 h. β-actin was used as a protein loading control. (D) Bar diagram represents the expression levels of caspase-9, caspase-3, Cyt-c and PARP, which displayed a concentration-dependent increase upon treatment with GL63 or CUR. (E) Z-DEVD-FMK inhibits GL63-induced apoptosis. SK-HEP-1 cells were incubated with Z-DEVD-FMK (20 µM) or vehicle for 1 h, followed by treatment with or without GL63 (40 µM) for another 24 h, and then apoptosis was evaluated by FACS (**P
Figure Legend Snippet: Apoptosis rates of SK-HEP-1 cells subjected to GL63 and CUR treatment, as detected by flow cytometry and protein expression analysis. SK-HEP-1 cells were treated with GL63 and CUR (0, 10, 20 and 40 µM) for 24 h. (A) Representative diagrams of annexin V-fluorescein isothiocyanate/propidium iodide staining upon treatment with different concentrations of GL63 and CUR. (B) Bar diagram of apoptotic cell rate from three FACS analyses from three separate treatments. (C) Caspase-9, caspase-3, Cyt-c and PARP levels were determined in SK-HEP-1 cells treated with GL63 and CUR at various concentrations for 24 h. β-actin was used as a protein loading control. (D) Bar diagram represents the expression levels of caspase-9, caspase-3, Cyt-c and PARP, which displayed a concentration-dependent increase upon treatment with GL63 or CUR. (E) Z-DEVD-FMK inhibits GL63-induced apoptosis. SK-HEP-1 cells were incubated with Z-DEVD-FMK (20 µM) or vehicle for 1 h, followed by treatment with or without GL63 (40 µM) for another 24 h, and then apoptosis was evaluated by FACS (**P

Techniques Used: Flow Cytometry, Cytometry, Expressing, Staining, FACS, Concentration Assay, Incubation

13) Product Images from "β2-microglobulin has a different regulatory molecular mechanism between ER+ and ER− breast cancer with HER2−"

Article Title: β2-microglobulin has a different regulatory molecular mechanism between ER+ and ER− breast cancer with HER2−

Journal: BMC Cancer

doi: 10.1186/s12885-019-5410-1

Effects of β2M overexpression in two types of HER2 − breast cancer cell lines. pEGFP-C1 (control) or pEGFP-C1-β2M (β2M overexpression group) plasmids were transfected into cells (MCF-7 ( a ), T47D ( b ), MDA-MB-231 ( c ), and Hs578T ( d )) for 36 h. Total RNAs were extracted, and the mRNA levels mentioned above were evaluated using real-time PCR. The relative mRNA levels were normalized to that of GAPDH and shown as a histogram (left of panels a - d ). Meanwhile, whole cell lysates were prepared from cells and analyzed by western blotting for the indicated proteins. Representative immunoblots are shown in the middle of panels a - d . β-actin was used as loading control. Relative protein signal intensities were quantitatively analyzed using Image Lab software and shown as histograms (right of panels a - d ). Values are presented as the mean ± SD; * p
Figure Legend Snippet: Effects of β2M overexpression in two types of HER2 − breast cancer cell lines. pEGFP-C1 (control) or pEGFP-C1-β2M (β2M overexpression group) plasmids were transfected into cells (MCF-7 ( a ), T47D ( b ), MDA-MB-231 ( c ), and Hs578T ( d )) for 36 h. Total RNAs were extracted, and the mRNA levels mentioned above were evaluated using real-time PCR. The relative mRNA levels were normalized to that of GAPDH and shown as a histogram (left of panels a - d ). Meanwhile, whole cell lysates were prepared from cells and analyzed by western blotting for the indicated proteins. Representative immunoblots are shown in the middle of panels a - d . β-actin was used as loading control. Relative protein signal intensities were quantitatively analyzed using Image Lab software and shown as histograms (right of panels a - d ). Values are presented as the mean ± SD; * p

Techniques Used: Over Expression, Transfection, Multiple Displacement Amplification, Real-time Polymerase Chain Reaction, Western Blot, Software

Representative immunohistochemical staining images in both HER2 − breast cancer tissues. a , i Cancer tissues staining without primary antibody as negative control. b , j β2M immunoreactivity. c , k HIF-1α immunoreactivity. d , l p-CREB immunoreactivity. e , m VEGF immunoreactivity. f , n p-SGK1 immunoreactivity. g , o p-ERK1/2 immunoreactivity. h , p Bcl-2 immunoreactivity. Magnification, 100×. Black boxes, a magnification of the tumor areas. Arrows, positive staining. Scale bars, 200 μm
Figure Legend Snippet: Representative immunohistochemical staining images in both HER2 − breast cancer tissues. a , i Cancer tissues staining without primary antibody as negative control. b , j β2M immunoreactivity. c , k HIF-1α immunoreactivity. d , l p-CREB immunoreactivity. e , m VEGF immunoreactivity. f , n p-SGK1 immunoreactivity. g , o p-ERK1/2 immunoreactivity. h , p Bcl-2 immunoreactivity. Magnification, 100×. Black boxes, a magnification of the tumor areas. Arrows, positive staining. Scale bars, 200 μm

Techniques Used: Immunohistochemistry, Staining, Negative Control

Effects of β2M silencing in two types of HER2 − breast cancer cell lines. Cells (MCF-7 (a) , T47D (b) , MDA-MB-231 (c) , and Hs578T (d) ) were transfected with the siβ2M or control siRNA for 48 h. Total RNAs were extracted and β2M, HER2, HIF-1α, VEGF, and Bcl-2 mRNA levels were evaluated using real-time PCR. The relative mRNA levels were normalized to that of GAPDH and shown as a histogram (left of panels a - d ). Whole cell lysates were prepared from cells and analyzed by western blotting for the indicated proteins. Representative immunoblots are shown in the middle of panels a-d . β-actin was used as a loading control. The relative protein signal intensity was quantitatively analyzed using Image Lab software and shown as histograms (right of panels a-d ). Values were presented as the mean ± SD; * p
Figure Legend Snippet: Effects of β2M silencing in two types of HER2 − breast cancer cell lines. Cells (MCF-7 (a) , T47D (b) , MDA-MB-231 (c) , and Hs578T (d) ) were transfected with the siβ2M or control siRNA for 48 h. Total RNAs were extracted and β2M, HER2, HIF-1α, VEGF, and Bcl-2 mRNA levels were evaluated using real-time PCR. The relative mRNA levels were normalized to that of GAPDH and shown as a histogram (left of panels a - d ). Whole cell lysates were prepared from cells and analyzed by western blotting for the indicated proteins. Representative immunoblots are shown in the middle of panels a-d . β-actin was used as a loading control. The relative protein signal intensity was quantitatively analyzed using Image Lab software and shown as histograms (right of panels a-d ). Values were presented as the mean ± SD; * p

Techniques Used: Multiple Displacement Amplification, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Software

14) Product Images from "β2-microglobulin has a different regulatory molecular mechanism between ER+ and ER− breast cancer with HER2−"

Article Title: β2-microglobulin has a different regulatory molecular mechanism between ER+ and ER− breast cancer with HER2−

Journal: BMC Cancer

doi: 10.1186/s12885-019-5410-1

Effects of β2M overexpression in two types of HER2 − breast cancer cell lines. pEGFP-C1 (control) or pEGFP-C1-β2M (β2M overexpression group) plasmids were transfected into cells (MCF-7 ( a ), T47D ( b ), MDA-MB-231 ( c ), and Hs578T ( d )) for 36 h. Total RNAs were extracted, and the mRNA levels mentioned above were evaluated using real-time PCR. The relative mRNA levels were normalized to that of GAPDH and shown as a histogram (left of panels a - d ). Meanwhile, whole cell lysates were prepared from cells and analyzed by western blotting for the indicated proteins. Representative immunoblots are shown in the middle of panels a - d . β-actin was used as loading control. Relative protein signal intensities were quantitatively analyzed using Image Lab software and shown as histograms (right of panels a - d ). Values are presented as the mean ± SD; * p
Figure Legend Snippet: Effects of β2M overexpression in two types of HER2 − breast cancer cell lines. pEGFP-C1 (control) or pEGFP-C1-β2M (β2M overexpression group) plasmids were transfected into cells (MCF-7 ( a ), T47D ( b ), MDA-MB-231 ( c ), and Hs578T ( d )) for 36 h. Total RNAs were extracted, and the mRNA levels mentioned above were evaluated using real-time PCR. The relative mRNA levels were normalized to that of GAPDH and shown as a histogram (left of panels a - d ). Meanwhile, whole cell lysates were prepared from cells and analyzed by western blotting for the indicated proteins. Representative immunoblots are shown in the middle of panels a - d . β-actin was used as loading control. Relative protein signal intensities were quantitatively analyzed using Image Lab software and shown as histograms (right of panels a - d ). Values are presented as the mean ± SD; * p

Techniques Used: Over Expression, Transfection, Multiple Displacement Amplification, Real-time Polymerase Chain Reaction, Western Blot, Software

Representative immunohistochemical staining images in both HER2 − breast cancer tissues. a , i Cancer tissues staining without primary antibody as negative control. b , j β2M immunoreactivity. c , k HIF-1α immunoreactivity. d , l p-CREB immunoreactivity. e , m VEGF immunoreactivity. f , n p-SGK1 immunoreactivity. g , o p-ERK1/2 immunoreactivity. h , p Bcl-2 immunoreactivity. Magnification, 100×. Black boxes, a magnification of the tumor areas. Arrows, positive staining. Scale bars, 200 μm
Figure Legend Snippet: Representative immunohistochemical staining images in both HER2 − breast cancer tissues. a , i Cancer tissues staining without primary antibody as negative control. b , j β2M immunoreactivity. c , k HIF-1α immunoreactivity. d , l p-CREB immunoreactivity. e , m VEGF immunoreactivity. f , n p-SGK1 immunoreactivity. g , o p-ERK1/2 immunoreactivity. h , p Bcl-2 immunoreactivity. Magnification, 100×. Black boxes, a magnification of the tumor areas. Arrows, positive staining. Scale bars, 200 μm

Techniques Used: Immunohistochemistry, Staining, Negative Control

Effects of β2M silencing in two types of HER2 − breast cancer cell lines. Cells (MCF-7 (a) , T47D (b) , MDA-MB-231 (c) , and Hs578T (d) ) were transfected with the siβ2M or control siRNA for 48 h. Total RNAs were extracted and β2M, HER2, HIF-1α, VEGF, and Bcl-2 mRNA levels were evaluated using real-time PCR. The relative mRNA levels were normalized to that of GAPDH and shown as a histogram (left of panels a - d ). Whole cell lysates were prepared from cells and analyzed by western blotting for the indicated proteins. Representative immunoblots are shown in the middle of panels a-d . β-actin was used as a loading control. The relative protein signal intensity was quantitatively analyzed using Image Lab software and shown as histograms (right of panels a-d ). Values were presented as the mean ± SD; * p
Figure Legend Snippet: Effects of β2M silencing in two types of HER2 − breast cancer cell lines. Cells (MCF-7 (a) , T47D (b) , MDA-MB-231 (c) , and Hs578T (d) ) were transfected with the siβ2M or control siRNA for 48 h. Total RNAs were extracted and β2M, HER2, HIF-1α, VEGF, and Bcl-2 mRNA levels were evaluated using real-time PCR. The relative mRNA levels were normalized to that of GAPDH and shown as a histogram (left of panels a - d ). Whole cell lysates were prepared from cells and analyzed by western blotting for the indicated proteins. Representative immunoblots are shown in the middle of panels a-d . β-actin was used as a loading control. The relative protein signal intensity was quantitatively analyzed using Image Lab software and shown as histograms (right of panels a-d ). Values were presented as the mean ± SD; * p

Techniques Used: Multiple Displacement Amplification, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Software

15) Product Images from "β2-microglobulin has a different regulatory molecular mechanism between ER+ and ER− breast cancer with HER2−"

Article Title: β2-microglobulin has a different regulatory molecular mechanism between ER+ and ER− breast cancer with HER2−

Journal: BMC Cancer

doi: 10.1186/s12885-019-5410-1

Effects of β2M overexpression in two types of HER2 − breast cancer cell lines. pEGFP-C1 (control) or pEGFP-C1-β2M (β2M overexpression group) plasmids were transfected into cells (MCF-7 ( a ), T47D ( b ), MDA-MB-231 ( c ), and Hs578T ( d )) for 36 h. Total RNAs were extracted, and the mRNA levels mentioned above were evaluated using real-time PCR. The relative mRNA levels were normalized to that of GAPDH and shown as a histogram (left of panels a - d ). Meanwhile, whole cell lysates were prepared from cells and analyzed by western blotting for the indicated proteins. Representative immunoblots are shown in the middle of panels a - d . β-actin was used as loading control. Relative protein signal intensities were quantitatively analyzed using Image Lab software and shown as histograms (right of panels a - d ). Values are presented as the mean ± SD; * p
Figure Legend Snippet: Effects of β2M overexpression in two types of HER2 − breast cancer cell lines. pEGFP-C1 (control) or pEGFP-C1-β2M (β2M overexpression group) plasmids were transfected into cells (MCF-7 ( a ), T47D ( b ), MDA-MB-231 ( c ), and Hs578T ( d )) for 36 h. Total RNAs were extracted, and the mRNA levels mentioned above were evaluated using real-time PCR. The relative mRNA levels were normalized to that of GAPDH and shown as a histogram (left of panels a - d ). Meanwhile, whole cell lysates were prepared from cells and analyzed by western blotting for the indicated proteins. Representative immunoblots are shown in the middle of panels a - d . β-actin was used as loading control. Relative protein signal intensities were quantitatively analyzed using Image Lab software and shown as histograms (right of panels a - d ). Values are presented as the mean ± SD; * p

Techniques Used: Over Expression, Transfection, Multiple Displacement Amplification, Real-time Polymerase Chain Reaction, Western Blot, Software

Representative immunohistochemical staining images in both HER2 − breast cancer tissues. a , i Cancer tissues staining without primary antibody as negative control. b , j β2M immunoreactivity. c , k HIF-1α immunoreactivity. d , l p-CREB immunoreactivity. e , m VEGF immunoreactivity. f , n p-SGK1 immunoreactivity. g , o p-ERK1/2 immunoreactivity. h , p Bcl-2 immunoreactivity. Magnification, 100×. Black boxes, a magnification of the tumor areas. Arrows, positive staining. Scale bars, 200 μm
Figure Legend Snippet: Representative immunohistochemical staining images in both HER2 − breast cancer tissues. a , i Cancer tissues staining without primary antibody as negative control. b , j β2M immunoreactivity. c , k HIF-1α immunoreactivity. d , l p-CREB immunoreactivity. e , m VEGF immunoreactivity. f , n p-SGK1 immunoreactivity. g , o p-ERK1/2 immunoreactivity. h , p Bcl-2 immunoreactivity. Magnification, 100×. Black boxes, a magnification of the tumor areas. Arrows, positive staining. Scale bars, 200 μm

Techniques Used: Immunohistochemistry, Staining, Negative Control

Effects of β2M silencing in two types of HER2 − breast cancer cell lines. Cells (MCF-7 (a) , T47D (b) , MDA-MB-231 (c) , and Hs578T (d) ) were transfected with the siβ2M or control siRNA for 48 h. Total RNAs were extracted and β2M, HER2, HIF-1α, VEGF, and Bcl-2 mRNA levels were evaluated using real-time PCR. The relative mRNA levels were normalized to that of GAPDH and shown as a histogram (left of panels a - d ). Whole cell lysates were prepared from cells and analyzed by western blotting for the indicated proteins. Representative immunoblots are shown in the middle of panels a-d . β-actin was used as a loading control. The relative protein signal intensity was quantitatively analyzed using Image Lab software and shown as histograms (right of panels a-d ). Values were presented as the mean ± SD; * p
Figure Legend Snippet: Effects of β2M silencing in two types of HER2 − breast cancer cell lines. Cells (MCF-7 (a) , T47D (b) , MDA-MB-231 (c) , and Hs578T (d) ) were transfected with the siβ2M or control siRNA for 48 h. Total RNAs were extracted and β2M, HER2, HIF-1α, VEGF, and Bcl-2 mRNA levels were evaluated using real-time PCR. The relative mRNA levels were normalized to that of GAPDH and shown as a histogram (left of panels a - d ). Whole cell lysates were prepared from cells and analyzed by western blotting for the indicated proteins. Representative immunoblots are shown in the middle of panels a-d . β-actin was used as a loading control. The relative protein signal intensity was quantitatively analyzed using Image Lab software and shown as histograms (right of panels a-d ). Values were presented as the mean ± SD; * p

Techniques Used: Multiple Displacement Amplification, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Software

16) Product Images from "β2-microglobulin has a different regulatory molecular mechanism between ER+ and ER− breast cancer with HER2−"

Article Title: β2-microglobulin has a different regulatory molecular mechanism between ER+ and ER− breast cancer with HER2−

Journal: BMC Cancer

doi: 10.1186/s12885-019-5410-1

Effects of β2M overexpression in two types of HER2 − breast cancer cell lines. pEGFP-C1 (control) or pEGFP-C1-β2M (β2M overexpression group) plasmids were transfected into cells (MCF-7 ( a ), T47D ( b ), MDA-MB-231 ( c ), and Hs578T ( d )) for 36 h. Total RNAs were extracted, and the mRNA levels mentioned above were evaluated using real-time PCR. The relative mRNA levels were normalized to that of GAPDH and shown as a histogram (left of panels a - d ). Meanwhile, whole cell lysates were prepared from cells and analyzed by western blotting for the indicated proteins. Representative immunoblots are shown in the middle of panels a - d . β-actin was used as loading control. Relative protein signal intensities were quantitatively analyzed using Image Lab software and shown as histograms (right of panels a - d ). Values are presented as the mean ± SD; * p
Figure Legend Snippet: Effects of β2M overexpression in two types of HER2 − breast cancer cell lines. pEGFP-C1 (control) or pEGFP-C1-β2M (β2M overexpression group) plasmids were transfected into cells (MCF-7 ( a ), T47D ( b ), MDA-MB-231 ( c ), and Hs578T ( d )) for 36 h. Total RNAs were extracted, and the mRNA levels mentioned above were evaluated using real-time PCR. The relative mRNA levels were normalized to that of GAPDH and shown as a histogram (left of panels a - d ). Meanwhile, whole cell lysates were prepared from cells and analyzed by western blotting for the indicated proteins. Representative immunoblots are shown in the middle of panels a - d . β-actin was used as loading control. Relative protein signal intensities were quantitatively analyzed using Image Lab software and shown as histograms (right of panels a - d ). Values are presented as the mean ± SD; * p

Techniques Used: Over Expression, Transfection, Multiple Displacement Amplification, Real-time Polymerase Chain Reaction, Western Blot, Software

Representative immunohistochemical staining images in both HER2 − breast cancer tissues. a , i Cancer tissues staining without primary antibody as negative control. b , j β2M immunoreactivity. c , k HIF-1α immunoreactivity. d , l p-CREB immunoreactivity. e , m VEGF immunoreactivity. f , n p-SGK1 immunoreactivity. g , o p-ERK1/2 immunoreactivity. h , p Bcl-2 immunoreactivity. Magnification, 100×. Black boxes, a magnification of the tumor areas. Arrows, positive staining. Scale bars, 200 μm
Figure Legend Snippet: Representative immunohistochemical staining images in both HER2 − breast cancer tissues. a , i Cancer tissues staining without primary antibody as negative control. b , j β2M immunoreactivity. c , k HIF-1α immunoreactivity. d , l p-CREB immunoreactivity. e , m VEGF immunoreactivity. f , n p-SGK1 immunoreactivity. g , o p-ERK1/2 immunoreactivity. h , p Bcl-2 immunoreactivity. Magnification, 100×. Black boxes, a magnification of the tumor areas. Arrows, positive staining. Scale bars, 200 μm

Techniques Used: Immunohistochemistry, Staining, Negative Control

Effects of β2M silencing in two types of HER2 − breast cancer cell lines. Cells (MCF-7 (a) , T47D (b) , MDA-MB-231 (c) , and Hs578T (d) ) were transfected with the siβ2M or control siRNA for 48 h. Total RNAs were extracted and β2M, HER2, HIF-1α, VEGF, and Bcl-2 mRNA levels were evaluated using real-time PCR. The relative mRNA levels were normalized to that of GAPDH and shown as a histogram (left of panels a - d ). Whole cell lysates were prepared from cells and analyzed by western blotting for the indicated proteins. Representative immunoblots are shown in the middle of panels a-d . β-actin was used as a loading control. The relative protein signal intensity was quantitatively analyzed using Image Lab software and shown as histograms (right of panels a-d ). Values were presented as the mean ± SD; * p
Figure Legend Snippet: Effects of β2M silencing in two types of HER2 − breast cancer cell lines. Cells (MCF-7 (a) , T47D (b) , MDA-MB-231 (c) , and Hs578T (d) ) were transfected with the siβ2M or control siRNA for 48 h. Total RNAs were extracted and β2M, HER2, HIF-1α, VEGF, and Bcl-2 mRNA levels were evaluated using real-time PCR. The relative mRNA levels were normalized to that of GAPDH and shown as a histogram (left of panels a - d ). Whole cell lysates were prepared from cells and analyzed by western blotting for the indicated proteins. Representative immunoblots are shown in the middle of panels a-d . β-actin was used as a loading control. The relative protein signal intensity was quantitatively analyzed using Image Lab software and shown as histograms (right of panels a-d ). Values were presented as the mean ± SD; * p

Techniques Used: Multiple Displacement Amplification, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Software

17) Product Images from "β2-microglobulin has a different regulatory molecular mechanism between ER+ and ER− breast cancer with HER2−"

Article Title: β2-microglobulin has a different regulatory molecular mechanism between ER+ and ER− breast cancer with HER2−

Journal: BMC Cancer

doi: 10.1186/s12885-019-5410-1

Effects of β2M overexpression in two types of HER2 − breast cancer cell lines. pEGFP-C1 (control) or pEGFP-C1-β2M (β2M overexpression group) plasmids were transfected into cells (MCF-7 ( a ), T47D ( b ), MDA-MB-231 ( c ), and Hs578T ( d )) for 36 h. Total RNAs were extracted, and the mRNA levels mentioned above were evaluated using real-time PCR. The relative mRNA levels were normalized to that of GAPDH and shown as a histogram (left of panels a - d ). Meanwhile, whole cell lysates were prepared from cells and analyzed by western blotting for the indicated proteins. Representative immunoblots are shown in the middle of panels a - d . β-actin was used as loading control. Relative protein signal intensities were quantitatively analyzed using Image Lab software and shown as histograms (right of panels a - d ). Values are presented as the mean ± SD; * p
Figure Legend Snippet: Effects of β2M overexpression in two types of HER2 − breast cancer cell lines. pEGFP-C1 (control) or pEGFP-C1-β2M (β2M overexpression group) plasmids were transfected into cells (MCF-7 ( a ), T47D ( b ), MDA-MB-231 ( c ), and Hs578T ( d )) for 36 h. Total RNAs were extracted, and the mRNA levels mentioned above were evaluated using real-time PCR. The relative mRNA levels were normalized to that of GAPDH and shown as a histogram (left of panels a - d ). Meanwhile, whole cell lysates were prepared from cells and analyzed by western blotting for the indicated proteins. Representative immunoblots are shown in the middle of panels a - d . β-actin was used as loading control. Relative protein signal intensities were quantitatively analyzed using Image Lab software and shown as histograms (right of panels a - d ). Values are presented as the mean ± SD; * p

Techniques Used: Over Expression, Transfection, Multiple Displacement Amplification, Real-time Polymerase Chain Reaction, Western Blot, Software

Representative immunohistochemical staining images in both HER2 − breast cancer tissues. a , i Cancer tissues staining without primary antibody as negative control. b , j β2M immunoreactivity. c , k HIF-1α immunoreactivity. d , l p-CREB immunoreactivity. e , m VEGF immunoreactivity. f , n p-SGK1 immunoreactivity. g , o p-ERK1/2 immunoreactivity. h , p Bcl-2 immunoreactivity. Magnification, 100×. Black boxes, a magnification of the tumor areas. Arrows, positive staining. Scale bars, 200 μm
Figure Legend Snippet: Representative immunohistochemical staining images in both HER2 − breast cancer tissues. a , i Cancer tissues staining without primary antibody as negative control. b , j β2M immunoreactivity. c , k HIF-1α immunoreactivity. d , l p-CREB immunoreactivity. e , m VEGF immunoreactivity. f , n p-SGK1 immunoreactivity. g , o p-ERK1/2 immunoreactivity. h , p Bcl-2 immunoreactivity. Magnification, 100×. Black boxes, a magnification of the tumor areas. Arrows, positive staining. Scale bars, 200 μm

Techniques Used: Immunohistochemistry, Staining, Negative Control

Effects of β2M silencing in two types of HER2 − breast cancer cell lines. Cells (MCF-7 (a) , T47D (b) , MDA-MB-231 (c) , and Hs578T (d) ) were transfected with the siβ2M or control siRNA for 48 h. Total RNAs were extracted and β2M, HER2, HIF-1α, VEGF, and Bcl-2 mRNA levels were evaluated using real-time PCR. The relative mRNA levels were normalized to that of GAPDH and shown as a histogram (left of panels a - d ). Whole cell lysates were prepared from cells and analyzed by western blotting for the indicated proteins. Representative immunoblots are shown in the middle of panels a-d . β-actin was used as a loading control. The relative protein signal intensity was quantitatively analyzed using Image Lab software and shown as histograms (right of panels a-d ). Values were presented as the mean ± SD; * p
Figure Legend Snippet: Effects of β2M silencing in two types of HER2 − breast cancer cell lines. Cells (MCF-7 (a) , T47D (b) , MDA-MB-231 (c) , and Hs578T (d) ) were transfected with the siβ2M or control siRNA for 48 h. Total RNAs were extracted and β2M, HER2, HIF-1α, VEGF, and Bcl-2 mRNA levels were evaluated using real-time PCR. The relative mRNA levels were normalized to that of GAPDH and shown as a histogram (left of panels a - d ). Whole cell lysates were prepared from cells and analyzed by western blotting for the indicated proteins. Representative immunoblots are shown in the middle of panels a-d . β-actin was used as a loading control. The relative protein signal intensity was quantitatively analyzed using Image Lab software and shown as histograms (right of panels a-d ). Values were presented as the mean ± SD; * p

Techniques Used: Multiple Displacement Amplification, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Software

18) Product Images from "β2-microglobulin has a different regulatory molecular mechanism between ER+ and ER− breast cancer with HER2−"

Article Title: β2-microglobulin has a different regulatory molecular mechanism between ER+ and ER− breast cancer with HER2−

Journal: BMC Cancer

doi: 10.1186/s12885-019-5410-1

Effects of β2M overexpression in two types of HER2 − breast cancer cell lines. pEGFP-C1 (control) or pEGFP-C1-β2M (β2M overexpression group) plasmids were transfected into cells (MCF-7 ( a ), T47D ( b ), MDA-MB-231 ( c ), and Hs578T ( d )) for 36 h. Total RNAs were extracted, and the mRNA levels mentioned above were evaluated using real-time PCR. The relative mRNA levels were normalized to that of GAPDH and shown as a histogram (left of panels a - d ). Meanwhile, whole cell lysates were prepared from cells and analyzed by western blotting for the indicated proteins. Representative immunoblots are shown in the middle of panels a - d . β-actin was used as loading control. Relative protein signal intensities were quantitatively analyzed using Image Lab software and shown as histograms (right of panels a - d ). Values are presented as the mean ± SD; * p
Figure Legend Snippet: Effects of β2M overexpression in two types of HER2 − breast cancer cell lines. pEGFP-C1 (control) or pEGFP-C1-β2M (β2M overexpression group) plasmids were transfected into cells (MCF-7 ( a ), T47D ( b ), MDA-MB-231 ( c ), and Hs578T ( d )) for 36 h. Total RNAs were extracted, and the mRNA levels mentioned above were evaluated using real-time PCR. The relative mRNA levels were normalized to that of GAPDH and shown as a histogram (left of panels a - d ). Meanwhile, whole cell lysates were prepared from cells and analyzed by western blotting for the indicated proteins. Representative immunoblots are shown in the middle of panels a - d . β-actin was used as loading control. Relative protein signal intensities were quantitatively analyzed using Image Lab software and shown as histograms (right of panels a - d ). Values are presented as the mean ± SD; * p

Techniques Used: Over Expression, Transfection, Multiple Displacement Amplification, Real-time Polymerase Chain Reaction, Western Blot, Software

Representative immunohistochemical staining images in both HER2 − breast cancer tissues. a , i Cancer tissues staining without primary antibody as negative control. b , j β2M immunoreactivity. c , k HIF-1α immunoreactivity. d , l p-CREB immunoreactivity. e , m VEGF immunoreactivity. f , n p-SGK1 immunoreactivity. g , o p-ERK1/2 immunoreactivity. h , p Bcl-2 immunoreactivity. Magnification, 100×. Black boxes, a magnification of the tumor areas. Arrows, positive staining. Scale bars, 200 μm
Figure Legend Snippet: Representative immunohistochemical staining images in both HER2 − breast cancer tissues. a , i Cancer tissues staining without primary antibody as negative control. b , j β2M immunoreactivity. c , k HIF-1α immunoreactivity. d , l p-CREB immunoreactivity. e , m VEGF immunoreactivity. f , n p-SGK1 immunoreactivity. g , o p-ERK1/2 immunoreactivity. h , p Bcl-2 immunoreactivity. Magnification, 100×. Black boxes, a magnification of the tumor areas. Arrows, positive staining. Scale bars, 200 μm

Techniques Used: Immunohistochemistry, Staining, Negative Control

Effects of β2M silencing in two types of HER2 − breast cancer cell lines. Cells (MCF-7 (a) , T47D (b) , MDA-MB-231 (c) , and Hs578T (d) ) were transfected with the siβ2M or control siRNA for 48 h. Total RNAs were extracted and β2M, HER2, HIF-1α, VEGF, and Bcl-2 mRNA levels were evaluated using real-time PCR. The relative mRNA levels were normalized to that of GAPDH and shown as a histogram (left of panels a - d ). Whole cell lysates were prepared from cells and analyzed by western blotting for the indicated proteins. Representative immunoblots are shown in the middle of panels a-d . β-actin was used as a loading control. The relative protein signal intensity was quantitatively analyzed using Image Lab software and shown as histograms (right of panels a-d ). Values were presented as the mean ± SD; * p
Figure Legend Snippet: Effects of β2M silencing in two types of HER2 − breast cancer cell lines. Cells (MCF-7 (a) , T47D (b) , MDA-MB-231 (c) , and Hs578T (d) ) were transfected with the siβ2M or control siRNA for 48 h. Total RNAs were extracted and β2M, HER2, HIF-1α, VEGF, and Bcl-2 mRNA levels were evaluated using real-time PCR. The relative mRNA levels were normalized to that of GAPDH and shown as a histogram (left of panels a - d ). Whole cell lysates were prepared from cells and analyzed by western blotting for the indicated proteins. Representative immunoblots are shown in the middle of panels a-d . β-actin was used as a loading control. The relative protein signal intensity was quantitatively analyzed using Image Lab software and shown as histograms (right of panels a-d ). Values were presented as the mean ± SD; * p

Techniques Used: Multiple Displacement Amplification, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Software

19) Product Images from "β2-microglobulin has a different regulatory molecular mechanism between ER+ and ER− breast cancer with HER2−"

Article Title: β2-microglobulin has a different regulatory molecular mechanism between ER+ and ER− breast cancer with HER2−

Journal: BMC Cancer

doi: 10.1186/s12885-019-5410-1

Effects of β2M overexpression in two types of HER2 − breast cancer cell lines. pEGFP-C1 (control) or pEGFP-C1-β2M (β2M overexpression group) plasmids were transfected into cells (MCF-7 ( a ), T47D ( b ), MDA-MB-231 ( c ), and Hs578T ( d )) for 36 h. Total RNAs were extracted, and the mRNA levels mentioned above were evaluated using real-time PCR. The relative mRNA levels were normalized to that of GAPDH and shown as a histogram (left of panels a - d ). Meanwhile, whole cell lysates were prepared from cells and analyzed by western blotting for the indicated proteins. Representative immunoblots are shown in the middle of panels a - d . β-actin was used as loading control. Relative protein signal intensities were quantitatively analyzed using Image Lab software and shown as histograms (right of panels a - d ). Values are presented as the mean ± SD; * p
Figure Legend Snippet: Effects of β2M overexpression in two types of HER2 − breast cancer cell lines. pEGFP-C1 (control) or pEGFP-C1-β2M (β2M overexpression group) plasmids were transfected into cells (MCF-7 ( a ), T47D ( b ), MDA-MB-231 ( c ), and Hs578T ( d )) for 36 h. Total RNAs were extracted, and the mRNA levels mentioned above were evaluated using real-time PCR. The relative mRNA levels were normalized to that of GAPDH and shown as a histogram (left of panels a - d ). Meanwhile, whole cell lysates were prepared from cells and analyzed by western blotting for the indicated proteins. Representative immunoblots are shown in the middle of panels a - d . β-actin was used as loading control. Relative protein signal intensities were quantitatively analyzed using Image Lab software and shown as histograms (right of panels a - d ). Values are presented as the mean ± SD; * p

Techniques Used: Over Expression, Transfection, Multiple Displacement Amplification, Real-time Polymerase Chain Reaction, Western Blot, Software

Representative immunohistochemical staining images in both HER2 − breast cancer tissues. a , i Cancer tissues staining without primary antibody as negative control. b , j β2M immunoreactivity. c , k HIF-1α immunoreactivity. d , l p-CREB immunoreactivity. e , m VEGF immunoreactivity. f , n p-SGK1 immunoreactivity. g , o p-ERK1/2 immunoreactivity. h , p Bcl-2 immunoreactivity. Magnification, 100×. Black boxes, a magnification of the tumor areas. Arrows, positive staining. Scale bars, 200 μm
Figure Legend Snippet: Representative immunohistochemical staining images in both HER2 − breast cancer tissues. a , i Cancer tissues staining without primary antibody as negative control. b , j β2M immunoreactivity. c , k HIF-1α immunoreactivity. d , l p-CREB immunoreactivity. e , m VEGF immunoreactivity. f , n p-SGK1 immunoreactivity. g , o p-ERK1/2 immunoreactivity. h , p Bcl-2 immunoreactivity. Magnification, 100×. Black boxes, a magnification of the tumor areas. Arrows, positive staining. Scale bars, 200 μm

Techniques Used: Immunohistochemistry, Staining, Negative Control

Effects of β2M silencing in two types of HER2 − breast cancer cell lines. Cells (MCF-7 (a) , T47D (b) , MDA-MB-231 (c) , and Hs578T (d) ) were transfected with the siβ2M or control siRNA for 48 h. Total RNAs were extracted and β2M, HER2, HIF-1α, VEGF, and Bcl-2 mRNA levels were evaluated using real-time PCR. The relative mRNA levels were normalized to that of GAPDH and shown as a histogram (left of panels a - d ). Whole cell lysates were prepared from cells and analyzed by western blotting for the indicated proteins. Representative immunoblots are shown in the middle of panels a-d . β-actin was used as a loading control. The relative protein signal intensity was quantitatively analyzed using Image Lab software and shown as histograms (right of panels a-d ). Values were presented as the mean ± SD; * p
Figure Legend Snippet: Effects of β2M silencing in two types of HER2 − breast cancer cell lines. Cells (MCF-7 (a) , T47D (b) , MDA-MB-231 (c) , and Hs578T (d) ) were transfected with the siβ2M or control siRNA for 48 h. Total RNAs were extracted and β2M, HER2, HIF-1α, VEGF, and Bcl-2 mRNA levels were evaluated using real-time PCR. The relative mRNA levels were normalized to that of GAPDH and shown as a histogram (left of panels a - d ). Whole cell lysates were prepared from cells and analyzed by western blotting for the indicated proteins. Representative immunoblots are shown in the middle of panels a-d . β-actin was used as a loading control. The relative protein signal intensity was quantitatively analyzed using Image Lab software and shown as histograms (right of panels a-d ). Values were presented as the mean ± SD; * p

Techniques Used: Multiple Displacement Amplification, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Software

20) Product Images from "Hsc70 Regulates Accumulation of Cyclin D1 and Cyclin D1-Dependent Protein Kinase"

Article Title: Hsc70 Regulates Accumulation of Cyclin D1 and Cyclin D1-Dependent Protein Kinase

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.5.1764-1774.2003

Hsc70 maintains cyclin D1-dependent cell cycle progression in the presence of inhibitory levels of p21 Cip1 . (A) NIH 3T3 cells were transfected with vectors encoding Flag-D1 and CDK4, Flag-D1, CDK4, and p21 Cip1 , or Flag-D1, CDK4, p21 Cip1 , and Hsc70; 24 h posttransfection, cells were labeled with bromodeoxyuridine (BrdU) for 20 h. Cells were fixed and stained with monoclonal M2 antibody (red), bromodeoxyuridine (green), and Hoechst dye (blue). (B) Quantitation of panel A, representing three independent experiments; error bars represent standard errors between experiments. (C) Whole-cell lysates prepared from cells transfected as for panel A were resolved by SDS-PAGE, and levels of p21 Cip1 were monitored by immunoblot analysis with a p21-specific antibody.
Figure Legend Snippet: Hsc70 maintains cyclin D1-dependent cell cycle progression in the presence of inhibitory levels of p21 Cip1 . (A) NIH 3T3 cells were transfected with vectors encoding Flag-D1 and CDK4, Flag-D1, CDK4, and p21 Cip1 , or Flag-D1, CDK4, p21 Cip1 , and Hsc70; 24 h posttransfection, cells were labeled with bromodeoxyuridine (BrdU) for 20 h. Cells were fixed and stained with monoclonal M2 antibody (red), bromodeoxyuridine (green), and Hoechst dye (blue). (B) Quantitation of panel A, representing three independent experiments; error bars represent standard errors between experiments. (C) Whole-cell lysates prepared from cells transfected as for panel A were resolved by SDS-PAGE, and levels of p21 Cip1 were monitored by immunoblot analysis with a p21-specific antibody.

Techniques Used: Transfection, Labeling, Staining, Quantitation Assay, SDS Page

Reconstitution of a 158-kDa cyclin-CDK complex. (A) Sf9 insect cells were infected with baculoviruses encoding cyclin D1, CDK4, and p21 Cip1 with (lanes 1 to 6) or without (lanes 7 to 12) Hsc70. Lysates prepared from these cells were resolved by gel filtration chromatography and subjected to immunoblot analysis for Hsc70 (top panel) or cyclin D1 (bottom panel). The elution positions of molecular weight standards are indicated at the top of each panel. (B) Fractions corresponding to lanes 3 and 4 of part A were precipitated with the cyclin D1 monoclonal antibody. Coprecipitation of Hsc70 was monitored by immunoblotting with the Hsc70 monoclonal antibody. (C) Lysates prepared from Sf9 insect cells infected with baculoviruses encoding either cyclin D1 and CDK4 (lane 1), cyclin D1, CDK4, and Hsc70 (lane 2), cyclin D1, CDK4, and p21 Cip1 (lane 3), or all four (lane 4) and metabolically labeled with [ 35 S]methionine were precipitated with the cyclin D1 monoclonal antibody. Positions of Hsc70, cyclin D1, CDK4, and p21 Cip1 are indicated to the left of the panel and were verified independently by immunoprecipitation with antigen-specific antibodies (data not shown). Labeled proteins were visualized by autoradiography.
Figure Legend Snippet: Reconstitution of a 158-kDa cyclin-CDK complex. (A) Sf9 insect cells were infected with baculoviruses encoding cyclin D1, CDK4, and p21 Cip1 with (lanes 1 to 6) or without (lanes 7 to 12) Hsc70. Lysates prepared from these cells were resolved by gel filtration chromatography and subjected to immunoblot analysis for Hsc70 (top panel) or cyclin D1 (bottom panel). The elution positions of molecular weight standards are indicated at the top of each panel. (B) Fractions corresponding to lanes 3 and 4 of part A were precipitated with the cyclin D1 monoclonal antibody. Coprecipitation of Hsc70 was monitored by immunoblotting with the Hsc70 monoclonal antibody. (C) Lysates prepared from Sf9 insect cells infected with baculoviruses encoding either cyclin D1 and CDK4 (lane 1), cyclin D1, CDK4, and Hsc70 (lane 2), cyclin D1, CDK4, and p21 Cip1 (lane 3), or all four (lane 4) and metabolically labeled with [ 35 S]methionine were precipitated with the cyclin D1 monoclonal antibody. Positions of Hsc70, cyclin D1, CDK4, and p21 Cip1 are indicated to the left of the panel and were verified independently by immunoprecipitation with antigen-specific antibodies (data not shown). Labeled proteins were visualized by autoradiography.

Techniques Used: Infection, Filtration, Chromatography, Molecular Weight, Metabolic Labelling, Labeling, Immunoprecipitation, Autoradiography

Mitogen-dependent regulation of cyclin D1-Hsc70 binding. (A) Wild-type NIH 3T3 cells were used as an asynchronous culture (asyn) or synchronized by serum deprivation for 36 h and stimulated to reenter the cell cycle by addition of 10% fetal calf serum. Cell lysates were prepared at the indicated intervals (bottom) and immunoprecipitated (IP) with normal rabbit serum (NRS) or a cyclin D1-specific monoclonal antibody (D1). Precipitated proteins were then subjected to immunoblot analysis with antibodies specific for Hsc70 (top) or cyclin D1 (bottom). (B) D1-3T3 cells were synchronized as in panel A. Cell lysates were prepared at the indicated intervals (bottom) and precipitated with normal rabbit serum (NRS) or the M2 monoclonal antibody and subjected to immunoblot analysis with antibodies specific for Hsc70 (top panel) or cyclin D1 (bottom panel). (C) Cell lysates prepared as for panel A were subjected to direct Western analysis with an Hsc70-specific antibody. (D) Whole-cell lysates prepared from asynchronous D1-3T3 cells or D1-3T3 cells cultured in medium containing 0.1% fetal calf serum for the indicated intervals were precipitated with normal rabbit antiserum (NRS) or with the M2 monoclonal antibody. Precipitates were immunoblotted with either Hsc70 (top panel) or cyclin D1 (lower panel) antibodies. The arrow indicates the point at which cells were placed in medium containing 0.1% serum.
Figure Legend Snippet: Mitogen-dependent regulation of cyclin D1-Hsc70 binding. (A) Wild-type NIH 3T3 cells were used as an asynchronous culture (asyn) or synchronized by serum deprivation for 36 h and stimulated to reenter the cell cycle by addition of 10% fetal calf serum. Cell lysates were prepared at the indicated intervals (bottom) and immunoprecipitated (IP) with normal rabbit serum (NRS) or a cyclin D1-specific monoclonal antibody (D1). Precipitated proteins were then subjected to immunoblot analysis with antibodies specific for Hsc70 (top) or cyclin D1 (bottom). (B) D1-3T3 cells were synchronized as in panel A. Cell lysates were prepared at the indicated intervals (bottom) and precipitated with normal rabbit serum (NRS) or the M2 monoclonal antibody and subjected to immunoblot analysis with antibodies specific for Hsc70 (top panel) or cyclin D1 (bottom panel). (C) Cell lysates prepared as for panel A were subjected to direct Western analysis with an Hsc70-specific antibody. (D) Whole-cell lysates prepared from asynchronous D1-3T3 cells or D1-3T3 cells cultured in medium containing 0.1% fetal calf serum for the indicated intervals were precipitated with normal rabbit antiserum (NRS) or with the M2 monoclonal antibody. Precipitates were immunoblotted with either Hsc70 (top panel) or cyclin D1 (lower panel) antibodies. The arrow indicates the point at which cells were placed in medium containing 0.1% serum.

Techniques Used: Binding Assay, Immunoprecipitation, Western Blot, Cell Culture

Hsc70 copurifies with cyclin D1. (A) Lysates were fractionated by gel filtration chromatography, and 5% of each fraction was resolved by SDS-PAGE; elution of cyclin D1 (top panel) and CDK4 (bottom panel) was visualized by immunoblot analysis. The positions of eluting molecular weight standards are indicated at the top. (B) Fractions corresponding to lanes 4 to 8 in panel A were immunoprecipitated (IP) with a monoclonal antibody specific for cyclin D1 and either blotted for associated CDK4 (top) or assayed for their ability to phosphorylate recombinant GST-Rb (bottom). (C) Detection of cyclin D1 and cyclin D1-associated proteins by silver stain. Lane 1 contains molecular weight markers (sizes shown in kilodaltons), lane 2 contains proteins that nonspecifically bind to M2 beads from control NIH 3T3 lysates, and lane 3 contains cyclin D1 complexes isolated from FlagD1-T286A-3T3 lysates (an essentially identical pattern was recovered from Flag-D1-3T3 cells; data not shown). Proteins were eluted with excess Flag peptide. Positions of Flag-D1, CDK4, and p21 Cip1 are indicated to the right, as is the position of Hsc70, along with peptides identified by mass spectrometry.
Figure Legend Snippet: Hsc70 copurifies with cyclin D1. (A) Lysates were fractionated by gel filtration chromatography, and 5% of each fraction was resolved by SDS-PAGE; elution of cyclin D1 (top panel) and CDK4 (bottom panel) was visualized by immunoblot analysis. The positions of eluting molecular weight standards are indicated at the top. (B) Fractions corresponding to lanes 4 to 8 in panel A were immunoprecipitated (IP) with a monoclonal antibody specific for cyclin D1 and either blotted for associated CDK4 (top) or assayed for their ability to phosphorylate recombinant GST-Rb (bottom). (C) Detection of cyclin D1 and cyclin D1-associated proteins by silver stain. Lane 1 contains molecular weight markers (sizes shown in kilodaltons), lane 2 contains proteins that nonspecifically bind to M2 beads from control NIH 3T3 lysates, and lane 3 contains cyclin D1 complexes isolated from FlagD1-T286A-3T3 lysates (an essentially identical pattern was recovered from Flag-D1-3T3 cells; data not shown). Proteins were eluted with excess Flag peptide. Positions of Flag-D1, CDK4, and p21 Cip1 are indicated to the right, as is the position of Hsc70, along with peptides identified by mass spectrometry.

Techniques Used: Filtration, Chromatography, SDS Page, Molecular Weight, Immunoprecipitation, Recombinant, Silver Staining, Isolation, Mass Spectrometry

Cyclin D1 and Hsc70 are components of multiple complexes. (A) Affinity-purified cyclin D1 complexes were resolved by gel filtration. Ten percent of each fraction was subjected to immunoblot analysis with antibodies specific for Hsc70 (top panel), cyclin D1 (second panel), CDK4 (bottom panel), or p21 Cip1 (bottom panel). (B and C) The fraction corresponding to lane 5 in panel A was precipitated with a p21-specific antiserum (B) or an Hsc70 monoclonal antibody (C). Associated proteins were monitored by immunoblot analysis with antibodies specific for the proteins indicated to the left of each panel. (D) The fraction corresponding to lane 5 of part A (158 kDa) was subjected to two rounds of immunodepletion with either normal rabbit antiserum (mock) or the Hsc70-specific monoclonal antibody. Codepletion of cyclin D1 (top panel), CDK4 (middle panel), and p21 Cip1 (lower panel) was monitored by immunoblot analysis with the respective antibodies. (E) Asynchronous NIH 3T3 cells were pulse-labeled with [ 35 S]methionine for 15 min (lanes 1 to 6) or pulse-labeled and subsequently chased for 10 min with excess unlabeled methionine for 10 min (lanes 7 to 12). Lysates prepared from these cells were resolved by gel filtration chromatography, and cyclin D1 was precipitated from fractions ranging from 670 kDa to 100 kDa (indicated at the top). Proteins were then analyzed by SDS-PAGE, and cyclin D1 was visualized by autoradiography.
Figure Legend Snippet: Cyclin D1 and Hsc70 are components of multiple complexes. (A) Affinity-purified cyclin D1 complexes were resolved by gel filtration. Ten percent of each fraction was subjected to immunoblot analysis with antibodies specific for Hsc70 (top panel), cyclin D1 (second panel), CDK4 (bottom panel), or p21 Cip1 (bottom panel). (B and C) The fraction corresponding to lane 5 in panel A was precipitated with a p21-specific antiserum (B) or an Hsc70 monoclonal antibody (C). Associated proteins were monitored by immunoblot analysis with antibodies specific for the proteins indicated to the left of each panel. (D) The fraction corresponding to lane 5 of part A (158 kDa) was subjected to two rounds of immunodepletion with either normal rabbit antiserum (mock) or the Hsc70-specific monoclonal antibody. Codepletion of cyclin D1 (top panel), CDK4 (middle panel), and p21 Cip1 (lower panel) was monitored by immunoblot analysis with the respective antibodies. (E) Asynchronous NIH 3T3 cells were pulse-labeled with [ 35 S]methionine for 15 min (lanes 1 to 6) or pulse-labeled and subsequently chased for 10 min with excess unlabeled methionine for 10 min (lanes 7 to 12). Lysates prepared from these cells were resolved by gel filtration chromatography, and cyclin D1 was precipitated from fractions ranging from 670 kDa to 100 kDa (indicated at the top). Proteins were then analyzed by SDS-PAGE, and cyclin D1 was visualized by autoradiography.

Techniques Used: Affinity Purification, Filtration, Labeling, Chromatography, SDS Page, Autoradiography

Hsc70 decreases cyclin D1 proteolysis. (A) NIH 3T3 cells were cotransfected with (lane 2) or without (lane 1) a vector encoding Hsc70 along with vectors encoding either cyclin D1 and CDK4 (top and bottom panels), cyclin D1-T286A and CDK4 (second panel), or Myc-tagged cyclin E and CDK2 (bottom panel). Levels of the indicated proteins were determined by direct Western blot analysis of total cell lysates prepared from cells transfected with the indicated vectors. (B) Whole-cell extracts were prepared from NIH 3T3 cells cotransfected with either Flag-D1 and CDK4 (lanes 1 and 2) or Flag-D1, CDK4, and Hsc70 and precipitated with either normal rabbit serum (lane 1) or the M2 monoclonal antibody (lanes 2 and 3). Cyclin D1 and coprecipitating CDK4 were detected by immunoblot analysis with antigen-specific antibodies. (C) NIH 3T3 cells were cotransfected with a plasmid encoding cyclin D1 with (lane 2) or without (lane 1) a vector encoding Hsc70K71E. Levels of cyclin D1 were monitored by immunoblot analysis. (D) p21/p27 −/− MEFs were transfected with a plasmid encoding cyclin D1 without (lane 1) or with (lane 2) a plasmid encoding Hsc70. Levels of cyclin D1 were monitored by immunoblot analysis. (E and F) NIH 3T3 cells transfected with vectors encoding either Flag-D1 and CDK4 or Flag-D1, CDK4, and Hsc70 were treated with 50 μg of cycloheximide per ml, and lysates prepared from these cells at the intervals indicated at the bottom of each panel were subjected to immunoblot analysis for cyclin D1. The percentage of cyclin D1 remaining at each time point is indicated at the bottom of each lane.
Figure Legend Snippet: Hsc70 decreases cyclin D1 proteolysis. (A) NIH 3T3 cells were cotransfected with (lane 2) or without (lane 1) a vector encoding Hsc70 along with vectors encoding either cyclin D1 and CDK4 (top and bottom panels), cyclin D1-T286A and CDK4 (second panel), or Myc-tagged cyclin E and CDK2 (bottom panel). Levels of the indicated proteins were determined by direct Western blot analysis of total cell lysates prepared from cells transfected with the indicated vectors. (B) Whole-cell extracts were prepared from NIH 3T3 cells cotransfected with either Flag-D1 and CDK4 (lanes 1 and 2) or Flag-D1, CDK4, and Hsc70 and precipitated with either normal rabbit serum (lane 1) or the M2 monoclonal antibody (lanes 2 and 3). Cyclin D1 and coprecipitating CDK4 were detected by immunoblot analysis with antigen-specific antibodies. (C) NIH 3T3 cells were cotransfected with a plasmid encoding cyclin D1 with (lane 2) or without (lane 1) a vector encoding Hsc70K71E. Levels of cyclin D1 were monitored by immunoblot analysis. (D) p21/p27 −/− MEFs were transfected with a plasmid encoding cyclin D1 without (lane 1) or with (lane 2) a plasmid encoding Hsc70. Levels of cyclin D1 were monitored by immunoblot analysis. (E and F) NIH 3T3 cells transfected with vectors encoding either Flag-D1 and CDK4 or Flag-D1, CDK4, and Hsc70 were treated with 50 μg of cycloheximide per ml, and lysates prepared from these cells at the intervals indicated at the bottom of each panel were subjected to immunoblot analysis for cyclin D1. The percentage of cyclin D1 remaining at each time point is indicated at the bottom of each lane.

Techniques Used: Plasmid Preparation, Western Blot, Transfection

Model depicting the participation of chaperones in the regulation of cyclin D1/CDK4 complex maturation (details in text).
Figure Legend Snippet: Model depicting the participation of chaperones in the regulation of cyclin D1/CDK4 complex maturation (details in text).

Techniques Used:

21) Product Images from "Chemoprevention of Pancreatic Cancer: Characterization of Par-4 and its Modulation by 3,3? Diindolylmethane (DIM)"

Article Title: Chemoprevention of Pancreatic Cancer: Characterization of Par-4 and its Modulation by 3,3? Diindolylmethane (DIM)

Journal: Pharmaceutical research

doi: 10.1007/s11095-008-9581-8

Western blot analysis of PAR-4 protein expression in pancreatic cancer cell lines. Panc-1 ( Lane 1 ), L3.6pl ( Lane 2 ), Colo-357 ( Lane 3 ), MiaPaCa ( Lane 4 ), BxPC-3 ( Lane 5 ) and Hs766T ( Lane 6 ). β-actin protein was used as protein loading control as shown for each blot. Cell extracts were prepared according to the procedure described under MATERIALS AND METHODS section.
Figure Legend Snippet: Western blot analysis of PAR-4 protein expression in pancreatic cancer cell lines. Panc-1 ( Lane 1 ), L3.6pl ( Lane 2 ), Colo-357 ( Lane 3 ), MiaPaCa ( Lane 4 ), BxPC-3 ( Lane 5 ) and Hs766T ( Lane 6 ). β-actin protein was used as protein loading control as shown for each blot. Cell extracts were prepared according to the procedure described under MATERIALS AND METHODS section.

Techniques Used: Western Blot, Expressing

PAR-4 expression is up-regulated by B-DIM. ( A ) Western blot analysis of lysates of L3.6pl cells treated with: DMSO (untreated; Lane 1 ); 10 μmol/L B-DIM ( Lane 2 ); 20 μmol/L ( Lane 3 ), respectively. ( B ) Western blot analysis of lysates of Colo-357 cells treated with DMSO (untreated; Lane 1 ); 10 μmol/L B-DIM ( Lane 2 ); 20 μmol/L ( Lane 3 ), respectively. β-actin protein was used as loading control as shown for each blot.
Figure Legend Snippet: PAR-4 expression is up-regulated by B-DIM. ( A ) Western blot analysis of lysates of L3.6pl cells treated with: DMSO (untreated; Lane 1 ); 10 μmol/L B-DIM ( Lane 2 ); 20 μmol/L ( Lane 3 ), respectively. ( B ) Western blot analysis of lysates of Colo-357 cells treated with DMSO (untreated; Lane 1 ); 10 μmol/L B-DIM ( Lane 2 ); 20 μmol/L ( Lane 3 ), respectively. β-actin protein was used as loading control as shown for each blot.

Techniques Used: Expressing, Western Blot

PAR-4 expression is up-regulated by B-DIM in combination with gemcitabine. ( A ) Western blot analysis of lysate extracted from Colo-357 cells treated with; DMSO (untreated; Lane 1 ); B-DIM 20 μmol/L ( Lane 2 ); Gemcitabine 100 nmol/L ( Lane 3 ); B-DIM 20 μmol/L+Gemcitabine 100 nmol/L, respectively. ( B ) Western blot analysis of lysate extracted from L-3.6pl cells treated with; DMSO (untreated; Lane 1 ); B-DIM 20 μmol/L ( Lane 2 ); Gemcitabine 100 nmol/L ( Lane 3 ); B-DIM 20 μmol/L+Gemcitabine 100 nmol/L, respectively. Western blot analysis of lysate extracted from BxPC-3 cells treated with; DMSO (untreated; Lane 1 ); B-DIM 20 μmol/L ( Lane 2 ); Gemcitabine 100 nmol/L ( Lane 3 ); B-DIM 20 μmol/L+ Gemcitabine 100 nmol/L, respectively. Cell lysates were prepared according to the procedure described under MATERIALS AND METHODS section.
Figure Legend Snippet: PAR-4 expression is up-regulated by B-DIM in combination with gemcitabine. ( A ) Western blot analysis of lysate extracted from Colo-357 cells treated with; DMSO (untreated; Lane 1 ); B-DIM 20 μmol/L ( Lane 2 ); Gemcitabine 100 nmol/L ( Lane 3 ); B-DIM 20 μmol/L+Gemcitabine 100 nmol/L, respectively. ( B ) Western blot analysis of lysate extracted from L-3.6pl cells treated with; DMSO (untreated; Lane 1 ); B-DIM 20 μmol/L ( Lane 2 ); Gemcitabine 100 nmol/L ( Lane 3 ); B-DIM 20 μmol/L+Gemcitabine 100 nmol/L, respectively. Western blot analysis of lysate extracted from BxPC-3 cells treated with; DMSO (untreated; Lane 1 ); B-DIM 20 μmol/L ( Lane 2 ); Gemcitabine 100 nmol/L ( Lane 3 ); B-DIM 20 μmol/L+ Gemcitabine 100 nmol/L, respectively. Cell lysates were prepared according to the procedure described under MATERIALS AND METHODS section.

Techniques Used: Expressing, Western Blot

22) Product Images from "In Contrast to Chlamydia trachomatis, Waddlia chondrophila Grows in Human Cells without Inhibiting Apoptosis, Fragmenting the Golgi Apparatus, or Diverting Post-Golgi Sphingomyelin Transport"

Article Title: In Contrast to Chlamydia trachomatis, Waddlia chondrophila Grows in Human Cells without Inhibiting Apoptosis, Fragmenting the Golgi Apparatus, or Diverting Post-Golgi Sphingomyelin Transport

Journal: Infection and Immunity

doi: 10.1128/IAI.00322-15

Impact of Waddlia chondrophila infection on integrity of host cell proteins. (A) HeLa cells were infected with W. chondrophila at an MOI of 1 and whole-cell lysates were prepared with either RIPA (left) or urea (right) extraction buffer (see Materials and Methods) at 30 and 48 h p.i. Ten micrograms of protein was loaded onto each lane. Shown are representative Western blots of uninfected or W. chondrophila -infected cells. The depicted results were reproduced independently. RFX5, n = 3 (urea) and 5 (RIPA); cyclin B1, n = 2 (urea) and 4 (RIPA); Bim EL , n = 4 (urea) and 6 (RIPA); USF-1, n = 3 (urea) and 3 (RIPA); p65, n = 3 (urea) and 4 (RIPA); vimentin, n = 3 (urea) and 6 (RIPA); cytokeratin 8 (CK 8), n = 3 (urea) and 5 (RIPA). Actin, tubulin, and GAPDH were used as loading controls. (B) Representative Western blots of uninfected, W. chondrophila -infected, and C. trachomatis -infected cells. Urea lysates were prepared at 24, 30, or 40 h p.i.
Figure Legend Snippet: Impact of Waddlia chondrophila infection on integrity of host cell proteins. (A) HeLa cells were infected with W. chondrophila at an MOI of 1 and whole-cell lysates were prepared with either RIPA (left) or urea (right) extraction buffer (see Materials and Methods) at 30 and 48 h p.i. Ten micrograms of protein was loaded onto each lane. Shown are representative Western blots of uninfected or W. chondrophila -infected cells. The depicted results were reproduced independently. RFX5, n = 3 (urea) and 5 (RIPA); cyclin B1, n = 2 (urea) and 4 (RIPA); Bim EL , n = 4 (urea) and 6 (RIPA); USF-1, n = 3 (urea) and 3 (RIPA); p65, n = 3 (urea) and 4 (RIPA); vimentin, n = 3 (urea) and 6 (RIPA); cytokeratin 8 (CK 8), n = 3 (urea) and 5 (RIPA). Actin, tubulin, and GAPDH were used as loading controls. (B) Representative Western blots of uninfected, W. chondrophila -infected, and C. trachomatis -infected cells. Urea lysates were prepared at 24, 30, or 40 h p.i.

Techniques Used: Infection, Western Blot

23) Product Images from "Early apoptosis in different models of cardiac hypertrophy induced by high renin-angiotensin system activity involves CaMKII"

Article Title: Early apoptosis in different models of cardiac hypertrophy induced by high renin-angiotensin system activity involves CaMKII

Journal: Journal of Applied Physiology

doi: 10.1152/japplphysiol.01383.2011

Cardiac apoptosis in SHR is prevented by Ena. A : representative blots and average data of the proapoptotic and antiapoptotic proteins Bax and Bcl-2, respectively, and the 17-kDa cleavage product of caspase-3 from control rats, SHR, and SHR treated with Ena. GAPDH signals were used as loading controls. The increased ratio Bax/Bcl-2, used as an apoptotic index, and caspase-3 activation in SHR, are prevented by Ena treatment. B : typical photographs of terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) assay and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining of control, SHR, and SHR treated with Ena. The mean values of TUNEL-positive cells normalized by total DAPI-stained nuclei in the bar graph below indicate that the increment in apoptosis in SHR is prevented by blocking RAAS axis. Values are means ± SE; n , no. of animals in each experimental group (in parentheses). * P
Figure Legend Snippet: Cardiac apoptosis in SHR is prevented by Ena. A : representative blots and average data of the proapoptotic and antiapoptotic proteins Bax and Bcl-2, respectively, and the 17-kDa cleavage product of caspase-3 from control rats, SHR, and SHR treated with Ena. GAPDH signals were used as loading controls. The increased ratio Bax/Bcl-2, used as an apoptotic index, and caspase-3 activation in SHR, are prevented by Ena treatment. B : typical photographs of terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) assay and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining of control, SHR, and SHR treated with Ena. The mean values of TUNEL-positive cells normalized by total DAPI-stained nuclei in the bar graph below indicate that the increment in apoptosis in SHR is prevented by blocking RAAS axis. Values are means ± SE; n , no. of animals in each experimental group (in parentheses). * P

Techniques Used: Activation Assay, End Labeling, TUNEL Assay, Staining, Blocking Assay

Mice with cardiomyocyte-delimited transgenic expression of CaMKII inhibitory peptide are protected from Iso-induced apoptosis. A : lipid peroxidation measured by TBARS indicates that Iso treatment increases oxidative stress in both AC3-C and AC3-I mice. B : representative blots from P-CaMKII and P-Thr 17 , and their respective total proteins, as an index of CaMKII activity, and average results, show that, under Iso treatment, only AC3-C mice increase phosphorylation of CaMKII and PLN at the CaMKII site. C : TUNEL and DAPI photographs of the different groups and mean (SE) values of these experiments, indicating a significant increment in TUNEL-positive cells normalized by total DAPI stained nuclei only in the AC3-C mice treated with Iso (***). D : typical blots of pro- and anti-apoptotic protein Bax, and Bcl-2, and mean (±SE) results in the bar graph below. GAPDH signals were used as loading controls. Iso treatment induced apoptosis only in AC3-C mice. * P
Figure Legend Snippet: Mice with cardiomyocyte-delimited transgenic expression of CaMKII inhibitory peptide are protected from Iso-induced apoptosis. A : lipid peroxidation measured by TBARS indicates that Iso treatment increases oxidative stress in both AC3-C and AC3-I mice. B : representative blots from P-CaMKII and P-Thr 17 , and their respective total proteins, as an index of CaMKII activity, and average results, show that, under Iso treatment, only AC3-C mice increase phosphorylation of CaMKII and PLN at the CaMKII site. C : TUNEL and DAPI photographs of the different groups and mean (SE) values of these experiments, indicating a significant increment in TUNEL-positive cells normalized by total DAPI stained nuclei only in the AC3-C mice treated with Iso (***). D : typical blots of pro- and anti-apoptotic protein Bax, and Bcl-2, and mean (±SE) results in the bar graph below. GAPDH signals were used as loading controls. Iso treatment induced apoptosis only in AC3-C mice. * P

Techniques Used: Mouse Assay, Transgenic Assay, Expressing, Activity Assay, TUNEL Assay, Staining

24) Product Images from "Curcumin suppresses proliferation and induces apoptosis in human biliary cancer cells through modulation of multiple cell signaling pathways"

Article Title: Curcumin suppresses proliferation and induces apoptosis in human biliary cancer cells through modulation of multiple cell signaling pathways

Journal: Carcinogenesis

doi: 10.1093/carcin/bgr032

Curcumin inhibits STAT-3 and JAK-1 phosphorylation in KKU-M156 cells. ( A ) Cells (1 × 10 6 ) were treated with indicated concentrations of curcumin for 6 h, whole cell extracts were prepared and analyzed for STAT-3 phosphorylation using p-STAT-3 (Tyr 705 , Ser 727 ) antibody. ( B and C ) KKU-M156 cells were treated with 50 μM curcumin for the indicated times, and whole cell extracts were prepared and analyzed for STAT-3 phosphorylation (at Tyr 705 and Ser 727 ) and JAK-1 phosphorylation. Data shown are representative of three independent experiments.
Figure Legend Snippet: Curcumin inhibits STAT-3 and JAK-1 phosphorylation in KKU-M156 cells. ( A ) Cells (1 × 10 6 ) were treated with indicated concentrations of curcumin for 6 h, whole cell extracts were prepared and analyzed for STAT-3 phosphorylation using p-STAT-3 (Tyr 705 , Ser 727 ) antibody. ( B and C ) KKU-M156 cells were treated with 50 μM curcumin for the indicated times, and whole cell extracts were prepared and analyzed for STAT-3 phosphorylation (at Tyr 705 and Ser 727 ) and JAK-1 phosphorylation. Data shown are representative of three independent experiments.

Techniques Used:

25) Product Images from "Histone deacetylases mediate the silencing of miR-15a, miR-16, and miR-29b in chronic lymphocytic leukemia"

Article Title: Histone deacetylases mediate the silencing of miR-15a, miR-16, and miR-29b in chronic lymphocytic leukemia

Journal: Blood

doi: 10.1182/blood-2011-05-351510

Action of 10nM LBH589 on Mcl-1, Bcl-2, mitochondrial membrane depolarization and apotosis in CLL cells. Change in Mcl-1 (A), Bcl-2 (B), and mitochondrial membrane depolarization as measured by loss of the mitochondrial dye TMRM (C) and increase in annexin V/propidium iodide positivity after exposure to 10nM LBH589 for 48 hours (D). (E) Influence of pre-exposure to z-vad-fmk on the action of 10nM LBH589 for 48 hours on Mcl-1, appearance of cleaved caspase-3, and apoptosis in primary CLL cells (n = 4).
Figure Legend Snippet: Action of 10nM LBH589 on Mcl-1, Bcl-2, mitochondrial membrane depolarization and apotosis in CLL cells. Change in Mcl-1 (A), Bcl-2 (B), and mitochondrial membrane depolarization as measured by loss of the mitochondrial dye TMRM (C) and increase in annexin V/propidium iodide positivity after exposure to 10nM LBH589 for 48 hours (D). (E) Influence of pre-exposure to z-vad-fmk on the action of 10nM LBH589 for 48 hours on Mcl-1, appearance of cleaved caspase-3, and apoptosis in primary CLL cells (n = 4).

Techniques Used:

miR-15a and miR-16 target Mcl1 but not Bcl-2 in primary CLL cells. (A) Expression of miR-15a and miR-16 in a CLL sample after nucleofection for 48 hours. (B) Levels of Mcl-1 and Bcl-2 in a CLL sample after nucleofection with miR-15a, miR-16, or a scrambled nontargeting control (Scr) miR for 48 hours. (C) Quantitation of the levels of Mcl-1 and Bcl-2 in 10 primary CLL samples after nucleofection with miR-15a, miR-16, or a Scr miR for 48 hours. Statistical significance of the decrease of Mcl-1 and Bcl-2 levels was determined by 1-way ANOVA.
Figure Legend Snippet: miR-15a and miR-16 target Mcl1 but not Bcl-2 in primary CLL cells. (A) Expression of miR-15a and miR-16 in a CLL sample after nucleofection for 48 hours. (B) Levels of Mcl-1 and Bcl-2 in a CLL sample after nucleofection with miR-15a, miR-16, or a scrambled nontargeting control (Scr) miR for 48 hours. (C) Quantitation of the levels of Mcl-1 and Bcl-2 in 10 primary CLL samples after nucleofection with miR-15a, miR-16, or a Scr miR for 48 hours. Statistical significance of the decrease of Mcl-1 and Bcl-2 levels was determined by 1-way ANOVA.

Techniques Used: Expressing, Quantitation Assay

Relation between induction of miR-15a, miR-16, miR-29b, and decrease of Mcl-1 and Bcl-2 in response to 10nM LBH589 in CLL cells. (A) Levels of Mcl-1, Bcl-2, GAPDH, and cleaved caspase-3 at 3, 6, 18, or 24 hours in 3 CLL samples that activated miR-15a, miR-16, or miR-29b in response to 10nM LBH589. (B) Quantitation of Mcl-1 levels at various times (up to 48 hours) in 9 CLL samples that activated miR-15a, miR-16, or miR-29b in response to 10nM LBH589. (C) Quantitation of Bcl-2 levels at various times (up to 48 hours) in 9 CLL samples that activated miR-15a, miR-16, or miR-29b in response to 10nM LBH589. (D) Levels of Mcl-1, Bcl-2, GAPDH, and cleaved caspase-3 at 3, 6, 18, or 24 hours in 3 CLL samples that did not activate miR-15a, miR-16, or miR-29b in response to 10nM LBH589. (E) Quantitation of Mcl-1 levels at various times (up to 48 hours) in 11 CLL samples that did not activate miR-15a, miR-16, or miR-29b in response to 10nM LBH589. (F) Quantitation of Bcl-2 levels at various times (up to 48 hours) in 11 CLL samples that did not activate miR-15a, miR-16, or miR-29b in response to 10nM LBH589.
Figure Legend Snippet: Relation between induction of miR-15a, miR-16, miR-29b, and decrease of Mcl-1 and Bcl-2 in response to 10nM LBH589 in CLL cells. (A) Levels of Mcl-1, Bcl-2, GAPDH, and cleaved caspase-3 at 3, 6, 18, or 24 hours in 3 CLL samples that activated miR-15a, miR-16, or miR-29b in response to 10nM LBH589. (B) Quantitation of Mcl-1 levels at various times (up to 48 hours) in 9 CLL samples that activated miR-15a, miR-16, or miR-29b in response to 10nM LBH589. (C) Quantitation of Bcl-2 levels at various times (up to 48 hours) in 9 CLL samples that activated miR-15a, miR-16, or miR-29b in response to 10nM LBH589. (D) Levels of Mcl-1, Bcl-2, GAPDH, and cleaved caspase-3 at 3, 6, 18, or 24 hours in 3 CLL samples that did not activate miR-15a, miR-16, or miR-29b in response to 10nM LBH589. (E) Quantitation of Mcl-1 levels at various times (up to 48 hours) in 11 CLL samples that did not activate miR-15a, miR-16, or miR-29b in response to 10nM LBH589. (F) Quantitation of Bcl-2 levels at various times (up to 48 hours) in 11 CLL samples that did not activate miR-15a, miR-16, or miR-29b in response to 10nM LBH589.

Techniques Used: Quantitation Assay

26) Product Images from "Histone deacetylases mediate the silencing of miR-15a, miR-16, and miR-29b in chronic lymphocytic leukemia"

Article Title: Histone deacetylases mediate the silencing of miR-15a, miR-16, and miR-29b in chronic lymphocytic leukemia

Journal: Blood

doi: 10.1182/blood-2011-05-351510

Action of 10nM LBH589 on Mcl-1, Bcl-2, mitochondrial membrane depolarization and apotosis in CLL cells. Change in Mcl-1 (A), Bcl-2 (B), and mitochondrial membrane depolarization as measured by loss of the mitochondrial dye TMRM (C) and increase in annexin V/propidium iodide positivity after exposure to 10nM LBH589 for 48 hours (D). (E) Influence of pre-exposure to z-vad-fmk on the action of 10nM LBH589 for 48 hours on Mcl-1, appearance of cleaved caspase-3, and apoptosis in primary CLL cells (n = 4).
Figure Legend Snippet: Action of 10nM LBH589 on Mcl-1, Bcl-2, mitochondrial membrane depolarization and apotosis in CLL cells. Change in Mcl-1 (A), Bcl-2 (B), and mitochondrial membrane depolarization as measured by loss of the mitochondrial dye TMRM (C) and increase in annexin V/propidium iodide positivity after exposure to 10nM LBH589 for 48 hours (D). (E) Influence of pre-exposure to z-vad-fmk on the action of 10nM LBH589 for 48 hours on Mcl-1, appearance of cleaved caspase-3, and apoptosis in primary CLL cells (n = 4).

Techniques Used:

miR-15a and miR-16 target Mcl1 but not Bcl-2 in primary CLL cells. (A) Expression of miR-15a and miR-16 in a CLL sample after nucleofection for 48 hours. (B) Levels of Mcl-1 and Bcl-2 in a CLL sample after nucleofection with miR-15a, miR-16, or a scrambled nontargeting control (Scr) miR for 48 hours. (C) Quantitation of the levels of Mcl-1 and Bcl-2 in 10 primary CLL samples after nucleofection with miR-15a, miR-16, or a Scr miR for 48 hours. Statistical significance of the decrease of Mcl-1 and Bcl-2 levels was determined by 1-way ANOVA.
Figure Legend Snippet: miR-15a and miR-16 target Mcl1 but not Bcl-2 in primary CLL cells. (A) Expression of miR-15a and miR-16 in a CLL sample after nucleofection for 48 hours. (B) Levels of Mcl-1 and Bcl-2 in a CLL sample after nucleofection with miR-15a, miR-16, or a scrambled nontargeting control (Scr) miR for 48 hours. (C) Quantitation of the levels of Mcl-1 and Bcl-2 in 10 primary CLL samples after nucleofection with miR-15a, miR-16, or a Scr miR for 48 hours. Statistical significance of the decrease of Mcl-1 and Bcl-2 levels was determined by 1-way ANOVA.

Techniques Used: Expressing, Quantitation Assay

Relation between induction of miR-15a, miR-16, miR-29b, and decrease of Mcl-1 and Bcl-2 in response to 10nM LBH589 in CLL cells. (A) Levels of Mcl-1, Bcl-2, GAPDH, and cleaved caspase-3 at 3, 6, 18, or 24 hours in 3 CLL samples that activated miR-15a, miR-16, or miR-29b in response to 10nM LBH589. (B) Quantitation of Mcl-1 levels at various times (up to 48 hours) in 9 CLL samples that activated miR-15a, miR-16, or miR-29b in response to 10nM LBH589. (C) Quantitation of Bcl-2 levels at various times (up to 48 hours) in 9 CLL samples that activated miR-15a, miR-16, or miR-29b in response to 10nM LBH589. (D) Levels of Mcl-1, Bcl-2, GAPDH, and cleaved caspase-3 at 3, 6, 18, or 24 hours in 3 CLL samples that did not activate miR-15a, miR-16, or miR-29b in response to 10nM LBH589. (E) Quantitation of Mcl-1 levels at various times (up to 48 hours) in 11 CLL samples that did not activate miR-15a, miR-16, or miR-29b in response to 10nM LBH589. (F) Quantitation of Bcl-2 levels at various times (up to 48 hours) in 11 CLL samples that did not activate miR-15a, miR-16, or miR-29b in response to 10nM LBH589.
Figure Legend Snippet: Relation between induction of miR-15a, miR-16, miR-29b, and decrease of Mcl-1 and Bcl-2 in response to 10nM LBH589 in CLL cells. (A) Levels of Mcl-1, Bcl-2, GAPDH, and cleaved caspase-3 at 3, 6, 18, or 24 hours in 3 CLL samples that activated miR-15a, miR-16, or miR-29b in response to 10nM LBH589. (B) Quantitation of Mcl-1 levels at various times (up to 48 hours) in 9 CLL samples that activated miR-15a, miR-16, or miR-29b in response to 10nM LBH589. (C) Quantitation of Bcl-2 levels at various times (up to 48 hours) in 9 CLL samples that activated miR-15a, miR-16, or miR-29b in response to 10nM LBH589. (D) Levels of Mcl-1, Bcl-2, GAPDH, and cleaved caspase-3 at 3, 6, 18, or 24 hours in 3 CLL samples that did not activate miR-15a, miR-16, or miR-29b in response to 10nM LBH589. (E) Quantitation of Mcl-1 levels at various times (up to 48 hours) in 11 CLL samples that did not activate miR-15a, miR-16, or miR-29b in response to 10nM LBH589. (F) Quantitation of Bcl-2 levels at various times (up to 48 hours) in 11 CLL samples that did not activate miR-15a, miR-16, or miR-29b in response to 10nM LBH589.

Techniques Used: Quantitation Assay

27) Product Images from "VX680 suppresses the growth of HepG2 cells and enhances the chemosensitivity to cisplatin"

Article Title: VX680 suppresses the growth of HepG2 cells and enhances the chemosensitivity to cisplatin

Journal: Oncology Letters

doi: 10.3892/ol.2013.1648

(A) Hepatocellular carcinoma (HepG2) cells were cultured with varying concentrations of VX680 (25 and 50 μmol/l) for 24 h. All cells were collected and analyzed by western blot analysis with an anti-Aurora A antibody. (B) HepG2 cells were treated with 3.125 μmol/l VX680, 0.5 μg/ml cisplatin or a combination of the two for 72 h. Cell lysates were collected and analyzed by western blot analysis with anti-p53, anti-Bcl-2 and anti-β-actin antibodies. The protein level for each group was compared with that of the control group.
Figure Legend Snippet: (A) Hepatocellular carcinoma (HepG2) cells were cultured with varying concentrations of VX680 (25 and 50 μmol/l) for 24 h. All cells were collected and analyzed by western blot analysis with an anti-Aurora A antibody. (B) HepG2 cells were treated with 3.125 μmol/l VX680, 0.5 μg/ml cisplatin or a combination of the two for 72 h. Cell lysates were collected and analyzed by western blot analysis with anti-p53, anti-Bcl-2 and anti-β-actin antibodies. The protein level for each group was compared with that of the control group.

Techniques Used: Cell Culture, Western Blot

28) Product Images from "Granzyme B triggers a prolonged pressure to die in Bcl-2 overexpressing cells, defining a window of opportunity for effective treatment with ABT-737"

Article Title: Granzyme B triggers a prolonged pressure to die in Bcl-2 overexpressing cells, defining a window of opportunity for effective treatment with ABT-737

Journal: Cell Death & Disease

doi: 10.1038/cddis.2012.73

Bcl-2 sequesters gctBid in the absence of ABT-737 but only binds Bax after treatment with ABT-737. HeLa-Bcl-2 cells were treated with Pfp/GraB for 1 h and a further 30 min±ABT-737 (500 nM) before lysis in 1% digitonin lysis buffer. ( a ) Bcl-2 was immunoprecipitated and immunoblotted for Bcl-2 (upper panel) or Bax (lower panel). ( b ) Similarly Bcl-2 was again immunoprecipitated and immunoblotted for Bcl-2 (top panel) or Bid (both the GraB (gc) and caspase (cc) cleaved forms; third panel). Whole-cell lysates were also immunoblotted for Bcl-2 (second panel) and Bid (bottom panel). 1 After Pfp/GraB treatment cells were incubated for an additional 30 min in the absence of ABT-737. *Is a non-specific band. The experiment was performed twice
Figure Legend Snippet: Bcl-2 sequesters gctBid in the absence of ABT-737 but only binds Bax after treatment with ABT-737. HeLa-Bcl-2 cells were treated with Pfp/GraB for 1 h and a further 30 min±ABT-737 (500 nM) before lysis in 1% digitonin lysis buffer. ( a ) Bcl-2 was immunoprecipitated and immunoblotted for Bcl-2 (upper panel) or Bax (lower panel). ( b ) Similarly Bcl-2 was again immunoprecipitated and immunoblotted for Bcl-2 (top panel) or Bid (both the GraB (gc) and caspase (cc) cleaved forms; third panel). Whole-cell lysates were also immunoblotted for Bcl-2 (second panel) and Bid (bottom panel). 1 After Pfp/GraB treatment cells were incubated for an additional 30 min in the absence of ABT-737. *Is a non-specific band. The experiment was performed twice

Techniques Used: Lysis, Immunoprecipitation, Incubation

Bid cleavage by GraB and translocation of gctBid to the mitochondria is not blocked by Bcl-2 but Bax translocation and Bax/Bak activation are inhibited until after ABT-737 treatment. ( a ) Cells were treated with Pfp/GraB for 1 h. Cytosolic and membrane fractions were harvested and full length (fl) and tBid were detected by western blot analysis. Both GraB-cleaved tBid (gctBid) and caspase-cleaved tBid (cctBid) were detected in treated HeLa cells but only gctBid was detected in the HeLa-Bcl-2 cells. ( b ) HeLa-Bcl-2 cells were treated with Pfp/GraB for 1 h, ABT-737 was added for 30 min as indicated, before fractionation into cytosolic (C) and membrane (M) fractions and immunoblotting for Bax and Bak. Data are representative of three independent experiments. ( c ) HeLa-Bcl-2 cells were treated with Pfp (1 nM)/GraB (25 nM) and zVAD-fmk (100 μ M). ABT-737 (500 nM) was added to the cells indicated after 1 h and incubated for a further 30 min before they were fixed and stained for Bax and cyt c . Images were obtained using an Olympus FV1000 confocal microscope. Cells marked with an arrow indicate cells with active Bax and those with an asterix indicate cells without active Bax. ( d ) HeLa and HeLa-Bcl-2 cells were treated with Pfp (1 nM)/GraB (25 nM) for 1 h. ABT-737 (500 nM) was added for a further 30 min. Flow cytometry was used to detect Bax activation using an antibody specific for the conformational-active form of Bax (clone 6A7). Data are average±S.D. of four independent experiments. ( e ) Conformation change of Bax (upper panels) and Bak (lower panels) was determined by immunoprecipitation with conformation-specific antibodies against Bax (6A7) or Bak (Ab-1) (as described in the Materials and Methods). Lysis in Triton X-100 (TX) was used as a positive control for conformational change of Bax and Bak. 1 Cells were incubated for 30 min in the absence of ABT-737 after Pfp/GraB treatment (for 1 h). The experiment was performed twice
Figure Legend Snippet: Bid cleavage by GraB and translocation of gctBid to the mitochondria is not blocked by Bcl-2 but Bax translocation and Bax/Bak activation are inhibited until after ABT-737 treatment. ( a ) Cells were treated with Pfp/GraB for 1 h. Cytosolic and membrane fractions were harvested and full length (fl) and tBid were detected by western blot analysis. Both GraB-cleaved tBid (gctBid) and caspase-cleaved tBid (cctBid) were detected in treated HeLa cells but only gctBid was detected in the HeLa-Bcl-2 cells. ( b ) HeLa-Bcl-2 cells were treated with Pfp/GraB for 1 h, ABT-737 was added for 30 min as indicated, before fractionation into cytosolic (C) and membrane (M) fractions and immunoblotting for Bax and Bak. Data are representative of three independent experiments. ( c ) HeLa-Bcl-2 cells were treated with Pfp (1 nM)/GraB (25 nM) and zVAD-fmk (100 μ M). ABT-737 (500 nM) was added to the cells indicated after 1 h and incubated for a further 30 min before they were fixed and stained for Bax and cyt c . Images were obtained using an Olympus FV1000 confocal microscope. Cells marked with an arrow indicate cells with active Bax and those with an asterix indicate cells without active Bax. ( d ) HeLa and HeLa-Bcl-2 cells were treated with Pfp (1 nM)/GraB (25 nM) for 1 h. ABT-737 (500 nM) was added for a further 30 min. Flow cytometry was used to detect Bax activation using an antibody specific for the conformational-active form of Bax (clone 6A7). Data are average±S.D. of four independent experiments. ( e ) Conformation change of Bax (upper panels) and Bak (lower panels) was determined by immunoprecipitation with conformation-specific antibodies against Bax (6A7) or Bak (Ab-1) (as described in the Materials and Methods). Lysis in Triton X-100 (TX) was used as a positive control for conformational change of Bax and Bak. 1 Cells were incubated for 30 min in the absence of ABT-737 after Pfp/GraB treatment (for 1 h). The experiment was performed twice

Techniques Used: Translocation Assay, Activation Assay, Western Blot, Fractionation, Incubation, Staining, Microscopy, Flow Cytometry, Cytometry, Immunoprecipitation, Lysis, Positive Control

29) Product Images from "Autophagy and apoptosis-related genes in chronic liver disease and hepatocellular carcinoma"

Article Title: Autophagy and apoptosis-related genes in chronic liver disease and hepatocellular carcinoma

Journal: BMC Gastroenterology

doi: 10.1186/1471-230X-12-118

Schematic representation of the interplay between pro- and anti-apoptotic Bcl-2 family members and Beclin 1, in the endoplasmic reticulum and mitochondria.
Figure Legend Snippet: Schematic representation of the interplay between pro- and anti-apoptotic Bcl-2 family members and Beclin 1, in the endoplasmic reticulum and mitochondria.

Techniques Used:

mRNA analysis by quantitative absolute Real-Time PCR using SYBR Green I. Gene analysis of Beclin 1 ( A ), Bcl-2 ( B ), Bcl-xL ( C ), Bad ( D ) and Bax ( E ). Control: control specimens; CH: chronic hepatitis; CIRR: cirrhosis; PHCC: cirrhotic tissues surrounding hepatocellular carcinoma; HCC: hepatocellular carcinoma. Data are expressed as mean ± SD of the ratio of the gene of interest to that of β-actin.
Figure Legend Snippet: mRNA analysis by quantitative absolute Real-Time PCR using SYBR Green I. Gene analysis of Beclin 1 ( A ), Bcl-2 ( B ), Bcl-xL ( C ), Bad ( D ) and Bax ( E ). Control: control specimens; CH: chronic hepatitis; CIRR: cirrhosis; PHCC: cirrhotic tissues surrounding hepatocellular carcinoma; HCC: hepatocellular carcinoma. Data are expressed as mean ± SD of the ratio of the gene of interest to that of β-actin.

Techniques Used: Real-time Polymerase Chain Reaction, SYBR Green Assay

Protein expression in HCC and the corresponding tissues surrounding HCC. Western blot analysis of: Beclin 1 (A) ; Bcl-2 (B) ; Bcl-xL (C) ; Bad (D) ; Bax (E) . P: cirrhotic liver tissues surrounding hepatocellular carcinoma; HCC: tumoral tissues of 8/13 patients. Data are expressed as the optical density ratio of genes to β-actin. Values are reported as mean values ± SD.
Figure Legend Snippet: Protein expression in HCC and the corresponding tissues surrounding HCC. Western blot analysis of: Beclin 1 (A) ; Bcl-2 (B) ; Bcl-xL (C) ; Bad (D) ; Bax (E) . P: cirrhotic liver tissues surrounding hepatocellular carcinoma; HCC: tumoral tissues of 8/13 patients. Data are expressed as the optical density ratio of genes to β-actin. Values are reported as mean values ± SD.

Techniques Used: Expressing, Western Blot

30) Product Images from "?-Tomatine-Mediated Anti-Cancer Activity In Vitro and In Vivo through Cell Cycle- and Caspase-Independent Pathways"

Article Title: ?-Tomatine-Mediated Anti-Cancer Activity In Vitro and In Vivo through Cell Cycle- and Caspase-Independent Pathways

Journal: PLoS ONE

doi: 10.1371/journal.pone.0044093

α-Tomatine affected mitochondrial apoptotic or anti-apoptotic protein levels. ( A ) α-Tomatine induced Mcl-1s and Bak up-regulations (pro-apoptotic) but did not affect Bcl-2 and Bid protein levels in the HL60 cells. ( B ) In the K562 cells, α-tomatine significantly enhanced the activation of Bak and up-regulated Mcl-1s; however α-tomatine did not influence Bcl-2 and Bid protein expressions. Both cell lines were treated with 5 µM α-tomatine for the indicated intervals. Data are expressed from at least three separate determinations.
Figure Legend Snippet: α-Tomatine affected mitochondrial apoptotic or anti-apoptotic protein levels. ( A ) α-Tomatine induced Mcl-1s and Bak up-regulations (pro-apoptotic) but did not affect Bcl-2 and Bid protein levels in the HL60 cells. ( B ) In the K562 cells, α-tomatine significantly enhanced the activation of Bak and up-regulated Mcl-1s; however α-tomatine did not influence Bcl-2 and Bid protein expressions. Both cell lines were treated with 5 µM α-tomatine for the indicated intervals. Data are expressed from at least three separate determinations.

Techniques Used: Activation Assay

31) Product Images from "Heme Oxygenase-1 Prevents Cardiac Dysfunction in Streptozotocin-Diabetic Mice by Reducing Inflammation, Oxidative Stress, Apoptosis and Enhancing Autophagy"

Article Title: Heme Oxygenase-1 Prevents Cardiac Dysfunction in Streptozotocin-Diabetic Mice by Reducing Inflammation, Oxidative Stress, Apoptosis and Enhancing Autophagy

Journal: PLoS ONE

doi: 10.1371/journal.pone.0075927

Anti-apoptotic effects of HO-1 on cardiomyocytes in diabetic mice. A. Apoptosis assessed by TUNEL assay in the respective groups (left) with quantification on the right. First column: Nuclei of apoptotic cells TUNEL stained in green. Second column: The DAPI-stained nuclei appear in blue. Third column: merged picture of first and second column (scale bar, 20µm). B. Representative immunoblot for p53 and Bcl-2 in the myocardial tissues from the respective groups (left) and densitometric quantification (right). C. Flow cytometric analysis using Annexin V-FITC and propidium iodide stains to evaluate the effects of transfection with HO-1 or empty vector (PC) under control and high-glucose culture conditions (left) with quantification on the right. D. Representative immunoblot for p-Akt, Akt, p-GSK3 and GSK3 in the myocardial tissues from the respective groups (left) and densitometric quantification (right). (n = 5 in each group) Columns and errors bars represent mean ± SD. #p
Figure Legend Snippet: Anti-apoptotic effects of HO-1 on cardiomyocytes in diabetic mice. A. Apoptosis assessed by TUNEL assay in the respective groups (left) with quantification on the right. First column: Nuclei of apoptotic cells TUNEL stained in green. Second column: The DAPI-stained nuclei appear in blue. Third column: merged picture of first and second column (scale bar, 20µm). B. Representative immunoblot for p53 and Bcl-2 in the myocardial tissues from the respective groups (left) and densitometric quantification (right). C. Flow cytometric analysis using Annexin V-FITC and propidium iodide stains to evaluate the effects of transfection with HO-1 or empty vector (PC) under control and high-glucose culture conditions (left) with quantification on the right. D. Representative immunoblot for p-Akt, Akt, p-GSK3 and GSK3 in the myocardial tissues from the respective groups (left) and densitometric quantification (right). (n = 5 in each group) Columns and errors bars represent mean ± SD. #p

Techniques Used: Mouse Assay, TUNEL Assay, Staining, Flow Cytometry, Transfection, Plasmid Preparation

32) Product Images from "Stemness and inducing differentiation of small cell lung cancer NCI-H446 cells"

Article Title: Stemness and inducing differentiation of small cell lung cancer NCI-H446 cells

Journal: Cell Death & Disease

doi: 10.1038/cddis.2013.152

The cancer cells in xenograft tumor expressed multilineage stem cell markers. These stem cell markers included cancer stem cell markers CD133 ( a ) and CD44 ( b ), neural stem cell marker Nestin ( c ), MSC marker Vimentin ( d ), metastasis-associated protein MMP-9 ( e ), stem cell transcription factors Sox-2 ( f ), Sall4 ( g ), and c-Myc ( h ), and proliferation cell nuclear antigen Ki67 ( i ). Immunofluorescence staining with Cy3, counterstaining nuclei with Hoechst 33342. Scale bar, 50 μ m
Figure Legend Snippet: The cancer cells in xenograft tumor expressed multilineage stem cell markers. These stem cell markers included cancer stem cell markers CD133 ( a ) and CD44 ( b ), neural stem cell marker Nestin ( c ), MSC marker Vimentin ( d ), metastasis-associated protein MMP-9 ( e ), stem cell transcription factors Sox-2 ( f ), Sall4 ( g ), and c-Myc ( h ), and proliferation cell nuclear antigen Ki67 ( i ). Immunofluorescence staining with Cy3, counterstaining nuclei with Hoechst 33342. Scale bar, 50 μ m

Techniques Used: Marker, Immunofluorescence, Staining

33) Product Images from "Th Cells Promote CTL Survival and Memory via Acquired pMHC-I and Endogenous IL-2 and CD40L Signaling and by Modulating Apoptosis-Controlling Pathways"

Article Title: Th Cells Promote CTL Survival and Memory via Acquired pMHC-I and Endogenous IL-2 and CD40L Signaling and by Modulating Apoptosis-Controlling Pathways

Journal: PLoS ONE

doi: 10.1371/journal.pone.0064787

Immunoblot analysis of the expression and phosphorylation status of pro-survival and pro-apoptotic proteins. ( a ) Lysates prepared from helped or unhelped CTLs obtained from WT or CD4 + T cell-deficient mice were subjected to SDS-PAGE, and transferred to the nitrocellulose membrane. Western blotting was performed with a panel of Abs specific for β-actin, Bcl-2, Bcl10, Akt1, NF-κB-p65, phosphorylated-Akt1 and -NF-kB-p65, cleaved Caspases-3 and -7, or NFATc1 transcription factor and analyzed by the ODYSSEY densitometer. Densitometric values were normalized on the β-actin control and n-fold changes of normalized targets in the helped CTLs of WT or Ia b-/- mice relative to the unhelped CTLs in Ia b-/- mice are shown below the corresponding lanes. Data are derived from samples pooled from four to six mice in each group of the first experiment. The results of the second experiment are consistent with the first experiment (data not shown). One representative of two independent experiments is shown. ( b ) Representative FACS plots of helped CTLs from WT or CD4-deficient mice, or unhelped CTLs from CD4-deficient mice. Blood samples from helped CTLs in WT and Ia b-/- mice and unhelped CTL in Ia b-/- mice were collected 16 days of adoptive transfer and processed for triple staining. Value in panel a, b and c represents percentage of tetramer + -and CD8 + -specific double-positive cells in the total of CD8 + T cell population. The tetramer + -and CD8 + -specific double-positive cell populations were gated (in the circle) for analysis of TRAIL expression. Histogram overlays (right panels) of CD8 + tetramer + -gated helped CTLs from WT (histogram green filled overlays; right top panel) or CD4-deficient mice (histogram red filled overlays; right bottom panel) and CD8 + tetramer + -gated unhelped CTLs from CD4-deficient mice (histogram grey filled overlays; right top and bottom panel). The mean of fluorescence intensity (MFI) was calculated from triplicate values. The value in each panel represents the mean of MFI ± SD of positive staining cells (grey) versus the controls with MFI of 0.5 for green and MFI of 0.8 for red, respectively. One representative of two independent experiments is shown.
Figure Legend Snippet: Immunoblot analysis of the expression and phosphorylation status of pro-survival and pro-apoptotic proteins. ( a ) Lysates prepared from helped or unhelped CTLs obtained from WT or CD4 + T cell-deficient mice were subjected to SDS-PAGE, and transferred to the nitrocellulose membrane. Western blotting was performed with a panel of Abs specific for β-actin, Bcl-2, Bcl10, Akt1, NF-κB-p65, phosphorylated-Akt1 and -NF-kB-p65, cleaved Caspases-3 and -7, or NFATc1 transcription factor and analyzed by the ODYSSEY densitometer. Densitometric values were normalized on the β-actin control and n-fold changes of normalized targets in the helped CTLs of WT or Ia b-/- mice relative to the unhelped CTLs in Ia b-/- mice are shown below the corresponding lanes. Data are derived from samples pooled from four to six mice in each group of the first experiment. The results of the second experiment are consistent with the first experiment (data not shown). One representative of two independent experiments is shown. ( b ) Representative FACS plots of helped CTLs from WT or CD4-deficient mice, or unhelped CTLs from CD4-deficient mice. Blood samples from helped CTLs in WT and Ia b-/- mice and unhelped CTL in Ia b-/- mice were collected 16 days of adoptive transfer and processed for triple staining. Value in panel a, b and c represents percentage of tetramer + -and CD8 + -specific double-positive cells in the total of CD8 + T cell population. The tetramer + -and CD8 + -specific double-positive cell populations were gated (in the circle) for analysis of TRAIL expression. Histogram overlays (right panels) of CD8 + tetramer + -gated helped CTLs from WT (histogram green filled overlays; right top panel) or CD4-deficient mice (histogram red filled overlays; right bottom panel) and CD8 + tetramer + -gated unhelped CTLs from CD4-deficient mice (histogram grey filled overlays; right top and bottom panel). The mean of fluorescence intensity (MFI) was calculated from triplicate values. The value in each panel represents the mean of MFI ± SD of positive staining cells (grey) versus the controls with MFI of 0.5 for green and MFI of 0.8 for red, respectively. One representative of two independent experiments is shown.

Techniques Used: Expressing, Mouse Assay, SDS Page, Western Blot, IA, Derivative Assay, FACS, CTL Assay, Adoptive Transfer Assay, Staining, Fluorescence

34) Product Images from "BH3 Response Profiles From Neuroblastoma Mitochondria Predict Activity of Small Molecule Bcl-2 Family Antagonists"

Article Title: BH3 Response Profiles From Neuroblastoma Mitochondria Predict Activity of Small Molecule Bcl-2 Family Antagonists

Journal: Cell death and differentiation

doi: 10.1038/cdd.2009.171

BH3 profiling predicts NB cell sensitivity to Bcl-2 family small molecule antagonists 30,000 cells were plated in 96 well plates in duplicate, allowed to settle overnight and exposed to increasing concentrations of AT-101, ABT-737 or DMSO control. Cell indexes (viable cell number) for duplicate wells are averaged and plotted as a function of time and normalized separately to the cell index reading of each well at the time just before drug addition (t=0). Cells with Mcl1 dependence were more sensitive to AT-101 (A). Cells with a Bik-dominant mitochondrial profile were exquisitely sensitive to ABT-737 (B) . Cytotoxicity curves represent the average of at least 2 separate experiments. Standard deviations between individual experiments were all less than 5% of the values plotted and are therefore not shown. uM, micromolar; nanoM, nanomolar.
Figure Legend Snippet: BH3 profiling predicts NB cell sensitivity to Bcl-2 family small molecule antagonists 30,000 cells were plated in 96 well plates in duplicate, allowed to settle overnight and exposed to increasing concentrations of AT-101, ABT-737 or DMSO control. Cell indexes (viable cell number) for duplicate wells are averaged and plotted as a function of time and normalized separately to the cell index reading of each well at the time just before drug addition (t=0). Cells with Mcl1 dependence were more sensitive to AT-101 (A). Cells with a Bik-dominant mitochondrial profile were exquisitely sensitive to ABT-737 (B) . Cytotoxicity curves represent the average of at least 2 separate experiments. Standard deviations between individual experiments were all less than 5% of the values plotted and are therefore not shown. uM, micromolar; nanoM, nanomolar.

Techniques Used:

Functional validation of BH3 peptide activity (A) Dose dependent response of LAN5 mitochondria to BH3 peptides BimBH3, BikBH3 and NoxaBH3 showing enhanced sensitivity to activator (Bim) peptides, and demonstrating higher potency for Bik over Noxa in this Bik-dominant NB. (B) Co-immunoprecipitation data confirm that neuroblastomas are primed for death as endogenous activated BH3 protein Bim is bound to Mcl1, Bcl-2, and Bcl-x L at steady state. Reverse pull down, β-tubulin IP and beads lane confirm these to be true BH protein interactions. Bim-EL, “Extra long” Bim isoform; Bim-L, “Long” isoform. ( C) SK-N-AS shows change in BH3 profile to a Noxa-dominant pattern following exposure to sublethal doses of etoposide, identifying Mcl1 as possible contributer to etoposide resistance in this cell line. 2 × 10 8 cells were exposed to 10 uG/mL etoposide, then cells were washed and harvested, mitochondria isolated, and immediately exposed to the panel of BH3 peptides to evaluate cytochrome c release.
Figure Legend Snippet: Functional validation of BH3 peptide activity (A) Dose dependent response of LAN5 mitochondria to BH3 peptides BimBH3, BikBH3 and NoxaBH3 showing enhanced sensitivity to activator (Bim) peptides, and demonstrating higher potency for Bik over Noxa in this Bik-dominant NB. (B) Co-immunoprecipitation data confirm that neuroblastomas are primed for death as endogenous activated BH3 protein Bim is bound to Mcl1, Bcl-2, and Bcl-x L at steady state. Reverse pull down, β-tubulin IP and beads lane confirm these to be true BH protein interactions. Bim-EL, “Extra long” Bim isoform; Bim-L, “Long” isoform. ( C) SK-N-AS shows change in BH3 profile to a Noxa-dominant pattern following exposure to sublethal doses of etoposide, identifying Mcl1 as possible contributer to etoposide resistance in this cell line. 2 × 10 8 cells were exposed to 10 uG/mL etoposide, then cells were washed and harvested, mitochondria isolated, and immediately exposed to the panel of BH3 peptides to evaluate cytochrome c release.

Techniques Used: Functional Assay, Activity Assay, Immunoprecipitation, Isolation

35) Product Images from "Protective effects of total flavonoids from Flos Puerariae on retinal neuronal damage in diabetic mice"

Article Title: Protective effects of total flavonoids from Flos Puerariae on retinal neuronal damage in diabetic mice

Journal: Molecular Vision

doi:

Effect of total flavonoids from Flos Puerariae (TFF) on Bax and Bcl-2 expression in retinas of the diabetic mice. A : The photomicrographs were representatives the retina sections immunohistochemistry stained with Bax and Bcl-2 antibody. n=4 per group. (Magnification: 400×). Bax and Bcl-2 expression were localized in the cytosol of cells mainly in ganglion cell layer (GCL) and inner nuclear layer (INL). B : The photographs were representatives the western blot of Bax and Bcl-2 protein. Five western blots per group were experimented. C : Relative intensities of Bax protein (fold to β-actin). D : Relative intensities of Bcl-2 protein (fold to β-actin). E : Ratio of the relative intensities of Bcl-2 to Bax (Bcl-2 / Bax). Data are expressed as mean ±standard deviation. *p
Figure Legend Snippet: Effect of total flavonoids from Flos Puerariae (TFF) on Bax and Bcl-2 expression in retinas of the diabetic mice. A : The photomicrographs were representatives the retina sections immunohistochemistry stained with Bax and Bcl-2 antibody. n=4 per group. (Magnification: 400×). Bax and Bcl-2 expression were localized in the cytosol of cells mainly in ganglion cell layer (GCL) and inner nuclear layer (INL). B : The photographs were representatives the western blot of Bax and Bcl-2 protein. Five western blots per group were experimented. C : Relative intensities of Bax protein (fold to β-actin). D : Relative intensities of Bcl-2 protein (fold to β-actin). E : Ratio of the relative intensities of Bcl-2 to Bax (Bcl-2 / Bax). Data are expressed as mean ±standard deviation. *p

Techniques Used: Expressing, Mouse Assay, Immunohistochemistry, Staining, Western Blot, Standard Deviation

36) Product Images from "Type II cGMP-dependent protein kinase inhibits epidermal growth factor-induced phosphatidylinositol-3-kinase/Akt signal transduction in gastric cancer cells"

Article Title: Type II cGMP-dependent protein kinase inhibits epidermal growth factor-induced phosphatidylinositol-3-kinase/Akt signal transduction in gastric cancer cells

Journal: Oncology Letters

doi: 10.3892/ol.2013.1630

PKG II inhibits EGF-induced expression of Bcl-2. The AGS cells were treated as in Fig. 5 and harvested and lysed as described in Materials and methods. The cell lysate was subjected to western blotting with an anti Bcl-2 antibody. The results revealed that in the EGF-treated cells, the expression of Bcl-2 increased, and a higher expression and activity of PKG II prevented the EGF-induced increase of Bcl-2 expression. PKG II, cGMP-dependent protein kinase Type II; EGF, epidermal growth factor.
Figure Legend Snippet: PKG II inhibits EGF-induced expression of Bcl-2. The AGS cells were treated as in Fig. 5 and harvested and lysed as described in Materials and methods. The cell lysate was subjected to western blotting with an anti Bcl-2 antibody. The results revealed that in the EGF-treated cells, the expression of Bcl-2 increased, and a higher expression and activity of PKG II prevented the EGF-induced increase of Bcl-2 expression. PKG II, cGMP-dependent protein kinase Type II; EGF, epidermal growth factor.

Techniques Used: Expressing, Western Blot, Activity Assay

37) Product Images from "Overexpression of p65 attenuates celecoxib-induced cell death in MDA-MB-231 human breast cancer cell line"

Article Title: Overexpression of p65 attenuates celecoxib-induced cell death in MDA-MB-231 human breast cancer cell line

Journal: Cancer Cell International

doi: 10.1186/1475-2867-13-14

Effect of p65 overexpression on anti-apoptotic NF-κB target genes including Bcl-2 family and IAP family proteins.
Figure Legend Snippet: Effect of p65 overexpression on anti-apoptotic NF-κB target genes including Bcl-2 family and IAP family proteins.

Techniques Used: Over Expression

38) Product Images from "Neurotoxicity of Perfluorooctane Sulfonate to Hippocampal Cells in Adult Mice"

Article Title: Neurotoxicity of Perfluorooctane Sulfonate to Hippocampal Cells in Adult Mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0054176

Verification of the differentially expressed hippocampal proteins by western blotting. (A) Compared with the control group, Mib1, Tyro3 and Herc5 were found down-regulated due to PFOS-exposure. (B) Compared with the control group, Lig4, Sdha, Plau and Gzma were found up-regulated due to PFOS exposure.
Figure Legend Snippet: Verification of the differentially expressed hippocampal proteins by western blotting. (A) Compared with the control group, Mib1, Tyro3 and Herc5 were found down-regulated due to PFOS-exposure. (B) Compared with the control group, Lig4, Sdha, Plau and Gzma were found up-regulated due to PFOS exposure.

Techniques Used: Western Blot

39) Product Images from "Prognostic Implications of MicoRNA miR-195 Expression in Human Tongue Squamous Cell Carcinoma"

Article Title: Prognostic Implications of MicoRNA miR-195 Expression in Human Tongue Squamous Cell Carcinoma

Journal: PLoS ONE

doi: 10.1371/journal.pone.0056634

Inverse correlation between miR-195 and Cyclin D1 or Bcl-2 protein levels in TSCC. Expression of Cyclin D1 and Bcl-2 was examined by immunohistochemistry (IHC) and miR-195 expression was detected by qRT–PCR and in situ hybridization (ISH). (A), Statistical analysis of the expression of miR-195 in tumor vs nonmalignant tissue. Spearman’s rank correlation analysis was performed, with r and P values as indicated. (B), The concurrence of miR-195 expression and corresponding variation of Cyclin D1 and Bcl-2 was confirmed in human TSCC and nonmalignant specimens by ISH with miR-195 detection probe or Scramble-miR and IHC (200×magnification).
Figure Legend Snippet: Inverse correlation between miR-195 and Cyclin D1 or Bcl-2 protein levels in TSCC. Expression of Cyclin D1 and Bcl-2 was examined by immunohistochemistry (IHC) and miR-195 expression was detected by qRT–PCR and in situ hybridization (ISH). (A), Statistical analysis of the expression of miR-195 in tumor vs nonmalignant tissue. Spearman’s rank correlation analysis was performed, with r and P values as indicated. (B), The concurrence of miR-195 expression and corresponding variation of Cyclin D1 and Bcl-2 was confirmed in human TSCC and nonmalignant specimens by ISH with miR-195 detection probe or Scramble-miR and IHC (200×magnification).

Techniques Used: Expressing, Immunohistochemistry, Quantitative RT-PCR, In Situ Hybridization

Cyclin D1 and Bcl-2 are direct targets of miR-195. (A), Sequence alignments of miR-195 and its target sites in 3′-UTRs of Cyclin D1 or Bcl-2. (B), Targeting of 3′-UTRs of Cyclin D1 or Bcl-2 by miR-195. SCC-15 and CAL27 cells were co-transfected with firefly luciferase reporter plasmids containing wildtype (Wt) and mutant wildtype and mutant (Mut) 3′-UTRs of Cyclin D1 or Bcl-2, and pRL-TK plasmid (a plasmid expressing rellina luciferase) and pcDNA3.0-miR-195 (miR-195) or pcDNA3.0 as indicated. After 48 h, firefly luciferase activities were measured and normalized by renilla luciferase activities. Data were presented as mean ± SD (n = 3) ( ** P
Figure Legend Snippet: Cyclin D1 and Bcl-2 are direct targets of miR-195. (A), Sequence alignments of miR-195 and its target sites in 3′-UTRs of Cyclin D1 or Bcl-2. (B), Targeting of 3′-UTRs of Cyclin D1 or Bcl-2 by miR-195. SCC-15 and CAL27 cells were co-transfected with firefly luciferase reporter plasmids containing wildtype (Wt) and mutant wildtype and mutant (Mut) 3′-UTRs of Cyclin D1 or Bcl-2, and pRL-TK plasmid (a plasmid expressing rellina luciferase) and pcDNA3.0-miR-195 (miR-195) or pcDNA3.0 as indicated. After 48 h, firefly luciferase activities were measured and normalized by renilla luciferase activities. Data were presented as mean ± SD (n = 3) ( ** P

Techniques Used: Sequencing, Transfection, Luciferase, Mutagenesis, Plasmid Preparation, Expressing

Inhibition of Cyclin D1 and Bcl-2 was responsible for the tumor suppressive effects of miR-195. (A), Inhibition of cell cycle progression by knockdown of Cyclin D1. SCC-15 and CAL27 cells were transfected with control RNA (Ctrl RNA) or Cyclin D1 siRNA as indicated. Cells were stained with propidium iodide (PI) at 48 h post-transfection and analyzed with FACS ( * P
Figure Legend Snippet: Inhibition of Cyclin D1 and Bcl-2 was responsible for the tumor suppressive effects of miR-195. (A), Inhibition of cell cycle progression by knockdown of Cyclin D1. SCC-15 and CAL27 cells were transfected with control RNA (Ctrl RNA) or Cyclin D1 siRNA as indicated. Cells were stained with propidium iodide (PI) at 48 h post-transfection and analyzed with FACS ( * P

Techniques Used: Inhibition, Transfection, Staining, FACS

40) Product Images from "Disruption of microRNA Biogenesis Confers Resistance to ER Stress-Induced Cell Death Upstream of the Mitochondrion"

Article Title: Disruption of microRNA Biogenesis Confers Resistance to ER Stress-Induced Cell Death Upstream of the Mitochondrion

Journal: PLoS ONE

doi: 10.1371/journal.pone.0073870

Altered expression of the BCL-2 family of proteins may attenuate cell death in Exn5/Exn5 cells. A) Western blots for BCL-2 family proteins in HCT116 WT and Exn5/Exn5 cells treated with 500 ng/ml of Bfa for 2, 4, 8, 12 and 24 h. Proteins analyzed were MCL-1, BCL-2, BIM, BAD, BAK, BCL-xL, BID and NOXA. ACTIN was used as a loading control. The data is a representative of at least three independent experiments.
Figure Legend Snippet: Altered expression of the BCL-2 family of proteins may attenuate cell death in Exn5/Exn5 cells. A) Western blots for BCL-2 family proteins in HCT116 WT and Exn5/Exn5 cells treated with 500 ng/ml of Bfa for 2, 4, 8, 12 and 24 h. Proteins analyzed were MCL-1, BCL-2, BIM, BAD, BAK, BCL-xL, BID and NOXA. ACTIN was used as a loading control. The data is a representative of at least three independent experiments.

Techniques Used: Expressing, Western Blot

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Pyrolysis Gas Chromatography:

Article Title: Sodium azide induces mitochondria-mediated apoptosis in PC12 cells through Pgc-1α-associated signaling pathway
Article Snippet: .. Following blocking with 5% bovine serum albumin in TBS containing 0.1% Tween-20 (TBST) for 2 h at room temperature, the membranes were incubated with primary antibodies against Pgc-1α (Abcam, Cambridge, MA, USA; 1:500), Nrf-2 (Abcam; 1:500), Cox IV (Abcam; 1:1,000), Tfam (Abcam; 1:1,000), procaspase-3 (Abcam; 1:500), Nrf-1 (Cell Signaling Technology, Inc., Danvers, MA, USA, 1:500), pan-calcineurin A (CaN; Cell Signaling Technology Inc.; 1:1,000), phosphorylated (p)-CaMKII (Cell Signaling Technology; 1:1,000), p-p38 MAPK (Cell Signaling Technology, Inc.; 1:1,000), p-extracellular signal-regulated kinase (Erk)1/2 (Cell Signaling Technology, Inc.; 1:1,000), B-cell lymphoma-2 (Bcl-2)-associated X protein (Bax; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; 1:200), Bcl-2 (Santa Cruz Biotechnology, Inc.; 1:200) and cytochrome c (Santa Cruz Biotechnology, Inc.; 1:200) at 4°C overnight. .. The membranes were then washed with TBST and incubated with horse-radish peroxidase (HRP)-conjugated secondary antibodies (rabbit; cat. no. A0208; 1:1,000; or mouse; cat. no. A0216; 1;1,000; both Beyotime Institute of Biotechnology) for 1 h at room temperature.

Blocking Assay:

Article Title: Sodium azide induces mitochondria-mediated apoptosis in PC12 cells through Pgc-1α-associated signaling pathway
Article Snippet: .. Following blocking with 5% bovine serum albumin in TBS containing 0.1% Tween-20 (TBST) for 2 h at room temperature, the membranes were incubated with primary antibodies against Pgc-1α (Abcam, Cambridge, MA, USA; 1:500), Nrf-2 (Abcam; 1:500), Cox IV (Abcam; 1:1,000), Tfam (Abcam; 1:1,000), procaspase-3 (Abcam; 1:500), Nrf-1 (Cell Signaling Technology, Inc., Danvers, MA, USA, 1:500), pan-calcineurin A (CaN; Cell Signaling Technology Inc.; 1:1,000), phosphorylated (p)-CaMKII (Cell Signaling Technology; 1:1,000), p-p38 MAPK (Cell Signaling Technology, Inc.; 1:1,000), p-extracellular signal-regulated kinase (Erk)1/2 (Cell Signaling Technology, Inc.; 1:1,000), B-cell lymphoma-2 (Bcl-2)-associated X protein (Bax; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; 1:200), Bcl-2 (Santa Cruz Biotechnology, Inc.; 1:200) and cytochrome c (Santa Cruz Biotechnology, Inc.; 1:200) at 4°C overnight. .. The membranes were then washed with TBST and incubated with horse-radish peroxidase (HRP)-conjugated secondary antibodies (rabbit; cat. no. A0208; 1:1,000; or mouse; cat. no. A0216; 1;1,000; both Beyotime Institute of Biotechnology) for 1 h at room temperature.

Mouse Assay:

Article Title: Yes‐associated protein promotes early hepatocyte cell cycle progression in regenerating liver after tissue loss, et al. Yes‐associated protein promotes early hepatocyte cell cycle progression in regenerating liver after tissue loss
Article Snippet: .. Interestingly, LW/BW tended to be reduced (P = 0.086) already at hepatectomy in αYap1 ‐siRNA‐ relative to αAhsa1 ‐siRNA‐treated mice (Figure C), consistent with the role of YAP1 as a physiological regulator of resting liver size. .. Remarkably, liver weight gain was completely suppressed up to 32 hours after sHx; at 48 hours, liver weight still was reduced compared to controls, however, the regain was accelerated toward 48 hours in mice with Yap1 knockdown compared to controls (Figure C).

Incubation:

Article Title: Sodium azide induces mitochondria-mediated apoptosis in PC12 cells through Pgc-1α-associated signaling pathway
Article Snippet: .. Following blocking with 5% bovine serum albumin in TBS containing 0.1% Tween-20 (TBST) for 2 h at room temperature, the membranes were incubated with primary antibodies against Pgc-1α (Abcam, Cambridge, MA, USA; 1:500), Nrf-2 (Abcam; 1:500), Cox IV (Abcam; 1:1,000), Tfam (Abcam; 1:1,000), procaspase-3 (Abcam; 1:500), Nrf-1 (Cell Signaling Technology, Inc., Danvers, MA, USA, 1:500), pan-calcineurin A (CaN; Cell Signaling Technology Inc.; 1:1,000), phosphorylated (p)-CaMKII (Cell Signaling Technology; 1:1,000), p-p38 MAPK (Cell Signaling Technology, Inc.; 1:1,000), p-extracellular signal-regulated kinase (Erk)1/2 (Cell Signaling Technology, Inc.; 1:1,000), B-cell lymphoma-2 (Bcl-2)-associated X protein (Bax; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; 1:200), Bcl-2 (Santa Cruz Biotechnology, Inc.; 1:200) and cytochrome c (Santa Cruz Biotechnology, Inc.; 1:200) at 4°C overnight. .. The membranes were then washed with TBST and incubated with horse-radish peroxidase (HRP)-conjugated secondary antibodies (rabbit; cat. no. A0208; 1:1,000; or mouse; cat. no. A0216; 1;1,000; both Beyotime Institute of Biotechnology) for 1 h at room temperature.

other:

Article Title: Senecavirus A 3C Protease Mediates Host Cell Apoptosis Late in Infection
Article Snippet: Antibodies and ReagentsAntibodies against β-actin (clone C4), NF-κB-p65 (clone C-20; C-terminus), NF-κB-p65 (clone F-6), and IκBα (clone H4) were purchased from Santa Cruz Biotechnology.

Article Title: Yes‐associated protein promotes early hepatocyte cell cycle progression in regenerating liver after tissue loss, et al. Yes‐associated protein promotes early hepatocyte cell cycle progression in regenerating liver after tissue loss
Article Snippet: 4 DISCUSSION Given the role of YAP1 in organ size control, a function for the transcriptional coactivator in liver regeneration has been anticipated.

Article Title: Yes‐associated protein promotes early hepatocyte cell cycle progression in regenerating liver after tissue loss, et al. Yes‐associated protein promotes early hepatocyte cell cycle progression in regenerating liver after tissue loss
Article Snippet: Akin to the mouse liver, YAP1 was induced in human livers with successful regeneration, but not in those with failed regeneration, suggesting YAP1's role in liver regeneration is conserved in mammals.

Western Blot:

Article Title: MCL-1 is a prognostic indicator and drug target in breast cancer
Article Snippet: .. Western blotting Standard western blot procedures were used on whole cell lysates and probed with antibodies specific to MCL-1 (Proteintech, UK), ACTIN (Sigma, UK), PARP (Cell Signaling, UK), BAK (Cell Signaling, UK), BAX (Santa Cruz, CA, USA), HSP70 (Cell Signaling, UK), Active Caspase 3 (Cell Signaling, UK). .. Xenograft experiments For assessment of UMI-77 anti-tumour activity in vivo, 3 million MDA-MB-468 breast cancer cells were injected bilaterally into the inguinal mammary fat pads in 1:1 PBS:matrigel mix into 8-week BALB/c-Nu female mice (Charles River, UK).

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    Santa Cruz Biotechnology bcl 2
    NaN 3 induces mitochondria-mediated apoptosis through the expression of Pgc-1α-associated proteins in PC12 cells. (A) Expression levels of Pgc-1α, Nrf-1, Nrf-2, Tfam and Cox IV detected by western blot analysis. (B) Expression levels of procaspase-3, Bax, <t>Bcl-2</t> and cyt-c detected by western blot analysis. (C) Expression levels of pan-calcineurin A, CaMKII, p-CaMKII, p38 MAPK, p-p38 MAPK, Erk1/2 and p-Erk1/2 detected by western blot analysis. β-actin and GAPDH were used as the internal control. Band intensity ratios for each group are presented as mean ± standard deviation (n=3). *P
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    NaN 3 induces mitochondria-mediated apoptosis through the expression of Pgc-1α-associated proteins in PC12 cells. (A) Expression levels of Pgc-1α, Nrf-1, Nrf-2, Tfam and Cox IV detected by western blot analysis. (B) Expression levels of procaspase-3, Bax, Bcl-2 and cyt-c detected by western blot analysis. (C) Expression levels of pan-calcineurin A, CaMKII, p-CaMKII, p38 MAPK, p-p38 MAPK, Erk1/2 and p-Erk1/2 detected by western blot analysis. β-actin and GAPDH were used as the internal control. Band intensity ratios for each group are presented as mean ± standard deviation (n=3). *P

    Journal: Molecular Medicine Reports

    Article Title: Sodium azide induces mitochondria-mediated apoptosis in PC12 cells through Pgc-1α-associated signaling pathway

    doi: 10.3892/mmr.2019.9853

    Figure Lengend Snippet: NaN 3 induces mitochondria-mediated apoptosis through the expression of Pgc-1α-associated proteins in PC12 cells. (A) Expression levels of Pgc-1α, Nrf-1, Nrf-2, Tfam and Cox IV detected by western blot analysis. (B) Expression levels of procaspase-3, Bax, Bcl-2 and cyt-c detected by western blot analysis. (C) Expression levels of pan-calcineurin A, CaMKII, p-CaMKII, p38 MAPK, p-p38 MAPK, Erk1/2 and p-Erk1/2 detected by western blot analysis. β-actin and GAPDH were used as the internal control. Band intensity ratios for each group are presented as mean ± standard deviation (n=3). *P

    Article Snippet: Following blocking with 5% bovine serum albumin in TBS containing 0.1% Tween-20 (TBST) for 2 h at room temperature, the membranes were incubated with primary antibodies against Pgc-1α (Abcam, Cambridge, MA, USA; 1:500), Nrf-2 (Abcam; 1:500), Cox IV (Abcam; 1:1,000), Tfam (Abcam; 1:1,000), procaspase-3 (Abcam; 1:500), Nrf-1 (Cell Signaling Technology, Inc., Danvers, MA, USA, 1:500), pan-calcineurin A (CaN; Cell Signaling Technology Inc.; 1:1,000), phosphorylated (p)-CaMKII (Cell Signaling Technology; 1:1,000), p-p38 MAPK (Cell Signaling Technology, Inc.; 1:1,000), p-extracellular signal-regulated kinase (Erk)1/2 (Cell Signaling Technology, Inc.; 1:1,000), B-cell lymphoma-2 (Bcl-2)-associated X protein (Bax; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; 1:200), Bcl-2 (Santa Cruz Biotechnology, Inc.; 1:200) and cytochrome c (Santa Cruz Biotechnology, Inc.; 1:200) at 4°C overnight.

    Techniques: Expressing, Pyrolysis Gas Chromatography, Western Blot, Standard Deviation

    Effect of K36 on UVA-induced Bcl-2 expression in human epidermal keratinocytes. The representative image of the western blot ( a ) and the average value of the triplicate experiment ( b ); significant difference versus nonirradiated group: ###, p

    Journal: International Journal of Molecular Sciences

    Article Title: Protective Effects and Mechanisms of N-Phenethyl Caffeamide from UVA-Induced Skin Damage in Human Epidermal Keratinocytes through Nrf2/HO-1 Regulation

    doi: 10.3390/ijms20010164

    Figure Lengend Snippet: Effect of K36 on UVA-induced Bcl-2 expression in human epidermal keratinocytes. The representative image of the western blot ( a ) and the average value of the triplicate experiment ( b ); significant difference versus nonirradiated group: ###, p

    Article Snippet: ERK-1 (sc-93), MMP-1 (sc-12348), MMP-2 (sc-13595), Bcl-2 (sc-7382), c-Fos (sc-7202), COX-2 (sc-19999), JNK (sc-46006), and β-Actin (sc-1616) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing, Western Blot

    Effect of hesperidin on the expression of Bcl-2, caspase-3, TLR-4, and HSP70 protein in the lung tissue of CLP-induced lung injury mice. Mean ±SEM (n=6). ## p

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

    Article Title: Protective Effect of Hesperidin Against Sepsis-Induced Lung Injury by Inducing the Heat-Stable Protein 70 (Hsp70)/Toll-Like Receptor 4 (TLR4)/ Myeloid Differentiation Primary Response 88 (MyD88) Pathway

    doi: 10.12659/MSM.912490

    Figure Lengend Snippet: Effect of hesperidin on the expression of Bcl-2, caspase-3, TLR-4, and HSP70 protein in the lung tissue of CLP-induced lung injury mice. Mean ±SEM (n=6). ## p

    Article Snippet: Proteins were transferred onto nitrocellulose membranes and incubated overnight with caspase-3 (1: 500), Bcl-2 (1: 1000), TLR4 (1: 1000), Hsp70 (1: 1000), and β-Actin antibody (Santa Cruz Biotechnology Inc., Santa Cruz, USA).

    Techniques: Expressing, Mouse Assay

    Effect of bergenin at indicated concentrations on the expression of Bax and Bcl-2 proteins in HeLa cells as shown in the western blot. The experiments were carried out in triplicates. The values were considered significant at *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Anticancer activity of bergenin against cervical cancer cells involves apoptosis, cell cycle arrest, inhibition of cell migration and the STAT3 signalling pathway

    doi: 10.3892/etm.2019.7380

    Figure Lengend Snippet: Effect of bergenin at indicated concentrations on the expression of Bax and Bcl-2 proteins in HeLa cells as shown in the western blot. The experiments were carried out in triplicates. The values were considered significant at *P

    Article Snippet: Afterwards, blocking was done with 5% non-fat milk followed by an incubation at RT for 1 h. The membranes were then subjected to treatment either specific primary antibody (STAT3; cat. no. sc-293151, p-STAT3 (Ser 727); cat. no. sc-8001-R, p-STAT (Tyr705); cat. no. sc-7993-R, Bax; cat. no. sc-20067, Bcl-2; sc-509, Actin; sc-58673 purchased from Santa Cruz Biotechnology Inc.) at 4°C for 20 h. Thereafter, washing in washing buffer was carried out and then the membranes were incubated with secondery antibody (mouse monoclonal secondary antibody conjugated to Horseradish Peroxidase, cat. no. sc-2357) for 1 h. The protein bands were then visualised by an ECL Advanced Western Blot Detection kit.

    Techniques: Expressing, Western Blot