Structured Review

OriGene pcmv6 vector
HuR protein may stabilize PLAGL2 mRNA. (A) Correlation analysis between PLAGL2 expression and HuR expression in CRC was analysed using starBase v2.0. (B) HuR expression in CRC samples and normal samples was analysed using starBase v2.0. (C) HuR protein expression in 6 randomly selected, paired CRC tissues and normal tissues, was detected by western blotting. (D) Co-precipitated RNAs put down by HuR protein and IgG (negative control) were detected by RT-qPCR. (E) Following transfection with empty vector (negative control) and <t>pCMV6-HuR</t> (HuR-expressing plasmid), the mRNA expression of HuR and PLAGL2 was detected by RT-qPCR and the protein expression of PLAGL2 were detected by western blotting. Data are presented as the mean ± standard deviation. ** p
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1) Product Images from "Studying the mechanism of PLAGL2 overexpression and its carcinogenic characteristics based on 3′-untranslated region in colorectal cancer"

Article Title: Studying the mechanism of PLAGL2 overexpression and its carcinogenic characteristics based on 3′-untranslated region in colorectal cancer

Journal: International Journal of Oncology

doi: 10.3892/ijo.2018.4305

HuR protein may stabilize PLAGL2 mRNA. (A) Correlation analysis between PLAGL2 expression and HuR expression in CRC was analysed using starBase v2.0. (B) HuR expression in CRC samples and normal samples was analysed using starBase v2.0. (C) HuR protein expression in 6 randomly selected, paired CRC tissues and normal tissues, was detected by western blotting. (D) Co-precipitated RNAs put down by HuR protein and IgG (negative control) were detected by RT-qPCR. (E) Following transfection with empty vector (negative control) and pCMV6-HuR (HuR-expressing plasmid), the mRNA expression of HuR and PLAGL2 was detected by RT-qPCR and the protein expression of PLAGL2 were detected by western blotting. Data are presented as the mean ± standard deviation. ** p
Figure Legend Snippet: HuR protein may stabilize PLAGL2 mRNA. (A) Correlation analysis between PLAGL2 expression and HuR expression in CRC was analysed using starBase v2.0. (B) HuR expression in CRC samples and normal samples was analysed using starBase v2.0. (C) HuR protein expression in 6 randomly selected, paired CRC tissues and normal tissues, was detected by western blotting. (D) Co-precipitated RNAs put down by HuR protein and IgG (negative control) were detected by RT-qPCR. (E) Following transfection with empty vector (negative control) and pCMV6-HuR (HuR-expressing plasmid), the mRNA expression of HuR and PLAGL2 was detected by RT-qPCR and the protein expression of PLAGL2 were detected by western blotting. Data are presented as the mean ± standard deviation. ** p

Techniques Used: Expressing, Western Blot, Negative Control, Quantitative RT-PCR, Transfection, Plasmid Preparation, Standard Deviation

2) Product Images from "SPHK1 (sphingosine kinase 1) induces epithelial-mesenchymal transition by promoting the autophagy-linked lysosomal degradation of CDH1/E-cadherin in hepatoma cells"

Article Title: SPHK1 (sphingosine kinase 1) induces epithelial-mesenchymal transition by promoting the autophagy-linked lysosomal degradation of CDH1/E-cadherin in hepatoma cells

Journal: Autophagy

doi: 10.1080/15548627.2017.1291479

SPHK1 promotes the lysosomal degradation of CDH1. (A) SPHK1 decreased the expression of CDH1. Cells were transfected with the indicated concentrations of MYC-SPHK1 or vector and protein lysates were analyzed by immunoblotting with the indicated antibodies. (B) SPHK1 did not affect the level of CDH1 mRNA in HepG2 cells. Cells were transfected with the indicated concentrations of MYC-SPHK1 or vector, and total RNA was isolated. CDH1 mRNA was analyzed by fluorescent quantitative RT-PCR, as indicated in Materials and Methods. (C) SPHK1 inhibits the degradation of CDH1 in HepG2 cells. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with pCMV6-CDH1 for 24 h and then treated with CHX (20 μmol/L) for the indicated times. The cell lysates were detected by western blotting using an anti-CDH1 antibody. (D) SPHK1 did not affect the proteasomal degradation of CDH1. HepG2 cells were transfected with pCMV6-CDH1 for 24 h. Cells were treated with MG132 (10 μmol/L) for 2 h, and then also treated with CHX for the indicated times. Immunoblotting was performed with the indicated antibody. (E) SPHK1 accelerated the lysosomal degradation of CDH1. Cells were transfected with pCMV6-CDH1 for 24 h. HepG2 cells were treated with CQ (100 μmol/L) for 12 h, and then CHX was added for the indicated times. Immunoblotting was performed with the indicated antibody. Data are presented as the mean ± SE (n = 4). NS, nonsignificant; CHX, cycloheximide; CQ, chloroquine.
Figure Legend Snippet: SPHK1 promotes the lysosomal degradation of CDH1. (A) SPHK1 decreased the expression of CDH1. Cells were transfected with the indicated concentrations of MYC-SPHK1 or vector and protein lysates were analyzed by immunoblotting with the indicated antibodies. (B) SPHK1 did not affect the level of CDH1 mRNA in HepG2 cells. Cells were transfected with the indicated concentrations of MYC-SPHK1 or vector, and total RNA was isolated. CDH1 mRNA was analyzed by fluorescent quantitative RT-PCR, as indicated in Materials and Methods. (C) SPHK1 inhibits the degradation of CDH1 in HepG2 cells. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with pCMV6-CDH1 for 24 h and then treated with CHX (20 μmol/L) for the indicated times. The cell lysates were detected by western blotting using an anti-CDH1 antibody. (D) SPHK1 did not affect the proteasomal degradation of CDH1. HepG2 cells were transfected with pCMV6-CDH1 for 24 h. Cells were treated with MG132 (10 μmol/L) for 2 h, and then also treated with CHX for the indicated times. Immunoblotting was performed with the indicated antibody. (E) SPHK1 accelerated the lysosomal degradation of CDH1. Cells were transfected with pCMV6-CDH1 for 24 h. HepG2 cells were treated with CQ (100 μmol/L) for 12 h, and then CHX was added for the indicated times. Immunoblotting was performed with the indicated antibody. Data are presented as the mean ± SE (n = 4). NS, nonsignificant; CHX, cycloheximide; CQ, chloroquine.

Techniques Used: Expressing, Transfection, Plasmid Preparation, Isolation, Quantitative RT-PCR, Stable Transfection, Western Blot

SPHK1 induces the EMT by stimulating BECN1 in HepG2 cells. (A, B) Silencing BECN1 blocked cell migration and invasion in SPHK1-overexpressing cells. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with control-siRNA or si- BECN1 and plated in the upper chamber of the filters for 24 h. Then cells migrating to the underside of the transwell insert were counted. (C) SPHK1 overexpression regulated the expression of EMT-related markers through stimulating BECN1. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with control-siRNA or si- BECN1 for 24 h and then harvested for cell lysate extraction. The level of epithelial or mesenchymal markers was detected by western blot analysis. (D) Silencing BECN1 blocked the degradation of CDH1 in SPHK1-overexpressing cells. HepG2 cells stably expressing MYC-SPHK1 were cotransfected with pCMV6-CDH1 and si- BECN1 (or control-siRNA) for 24 h and then treated with CHX (20 μmol/L) for the indicated times. The cell lysates were analyzed by western blotting using an anti-CDH1 antibody. (E) Schematic diagram of the mechanism of SPHK1-mediated autophagy and lysosomal CDH1 degradation that stimulates the EMT in HepG2 cells. Con-siRNA, control-siRNA.
Figure Legend Snippet: SPHK1 induces the EMT by stimulating BECN1 in HepG2 cells. (A, B) Silencing BECN1 blocked cell migration and invasion in SPHK1-overexpressing cells. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with control-siRNA or si- BECN1 and plated in the upper chamber of the filters for 24 h. Then cells migrating to the underside of the transwell insert were counted. (C) SPHK1 overexpression regulated the expression of EMT-related markers through stimulating BECN1. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with control-siRNA or si- BECN1 for 24 h and then harvested for cell lysate extraction. The level of epithelial or mesenchymal markers was detected by western blot analysis. (D) Silencing BECN1 blocked the degradation of CDH1 in SPHK1-overexpressing cells. HepG2 cells stably expressing MYC-SPHK1 were cotransfected with pCMV6-CDH1 and si- BECN1 (or control-siRNA) for 24 h and then treated with CHX (20 μmol/L) for the indicated times. The cell lysates were analyzed by western blotting using an anti-CDH1 antibody. (E) Schematic diagram of the mechanism of SPHK1-mediated autophagy and lysosomal CDH1 degradation that stimulates the EMT in HepG2 cells. Con-siRNA, control-siRNA.

Techniques Used: Migration, Stable Transfection, Expressing, Plasmid Preparation, Transfection, Over Expression, Western Blot

3) Product Images from "B-cell Translocation Gene 2 (BTG2) Stimulates Cellular Antioxidant Defenses through the Antioxidant Transcription Factor NFE2L2 in Human Mammary Epithelial Cells *"

Article Title: B-cell Translocation Gene 2 (BTG2) Stimulates Cellular Antioxidant Defenses through the Antioxidant Transcription Factor NFE2L2 in Human Mammary Epithelial Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.367433

BTG2 physically associates with NFE2L2. A and B , MCF-7 cells were transfected with FLAG-BTG2 or empty pCMV6-FLAG vector; postincubated for 24 h; and subjected to anti-FLAG ( A ) or anti-NFE2L2 ( B ) IP. Precipitated proteins were Western blotted using anti-FLAG
Figure Legend Snippet: BTG2 physically associates with NFE2L2. A and B , MCF-7 cells were transfected with FLAG-BTG2 or empty pCMV6-FLAG vector; postincubated for 24 h; and subjected to anti-FLAG ( A ) or anti-NFE2L2 ( B ) IP. Precipitated proteins were Western blotted using anti-FLAG

Techniques Used: Transfection, Plasmid Preparation, Western Blot

4) Product Images from "Studying the mechanism of PLAGL2 overexpression and its carcinogenic characteristics based on 3′-untranslated region in colorectal cancer"

Article Title: Studying the mechanism of PLAGL2 overexpression and its carcinogenic characteristics based on 3′-untranslated region in colorectal cancer

Journal: International Journal of Oncology

doi: 10.3892/ijo.2018.4305

HuR protein may stabilize PLAGL2 mRNA. (A) Correlation analysis between PLAGL2 expression and HuR expression in CRC was analysed using starBase v2.0. (B) HuR expression in CRC samples and normal samples was analysed using starBase v2.0. (C) HuR protein expression in 6 randomly selected, paired CRC tissues and normal tissues, was detected by western blotting. (D) Co-precipitated RNAs put down by HuR protein and IgG (negative control) were detected by RT-qPCR. (E) Following transfection with empty vector (negative control) and pCMV6-HuR (HuR-expressing plasmid), the mRNA expression of HuR and PLAGL2 was detected by RT-qPCR and the protein expression of PLAGL2 were detected by western blotting. Data are presented as the mean ± standard deviation. ** p
Figure Legend Snippet: HuR protein may stabilize PLAGL2 mRNA. (A) Correlation analysis between PLAGL2 expression and HuR expression in CRC was analysed using starBase v2.0. (B) HuR expression in CRC samples and normal samples was analysed using starBase v2.0. (C) HuR protein expression in 6 randomly selected, paired CRC tissues and normal tissues, was detected by western blotting. (D) Co-precipitated RNAs put down by HuR protein and IgG (negative control) were detected by RT-qPCR. (E) Following transfection with empty vector (negative control) and pCMV6-HuR (HuR-expressing plasmid), the mRNA expression of HuR and PLAGL2 was detected by RT-qPCR and the protein expression of PLAGL2 were detected by western blotting. Data are presented as the mean ± standard deviation. ** p

Techniques Used: Expressing, Western Blot, Negative Control, Quantitative RT-PCR, Transfection, Plasmid Preparation, Standard Deviation

5) Product Images from "B-cell Translocation Gene 2 (BTG2) Stimulates Cellular Antioxidant Defenses through the Antioxidant Transcription Factor NFE2L2 in Human Mammary Epithelial Cells *"

Article Title: B-cell Translocation Gene 2 (BTG2) Stimulates Cellular Antioxidant Defenses through the Antioxidant Transcription Factor NFE2L2 in Human Mammary Epithelial Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.367433

BTG2 physically associates with NFE2L2. A and B , MCF-7 cells were transfected with FLAG-BTG2 or empty pCMV6-FLAG vector; postincubated for 24 h; and subjected to anti-FLAG ( A ) or anti-NFE2L2 ( B ) IP. Precipitated proteins were Western blotted using anti-FLAG
Figure Legend Snippet: BTG2 physically associates with NFE2L2. A and B , MCF-7 cells were transfected with FLAG-BTG2 or empty pCMV6-FLAG vector; postincubated for 24 h; and subjected to anti-FLAG ( A ) or anti-NFE2L2 ( B ) IP. Precipitated proteins were Western blotted using anti-FLAG

Techniques Used: Transfection, Plasmid Preparation, Western Blot

6) Product Images from "The pregnane X receptor (PXR) and the nuclear receptor corepressor 2 (NCoR2) modulate cell growth in head and neck squamous cell carcinoma"

Article Title: The pregnane X receptor (PXR) and the nuclear receptor corepressor 2 (NCoR2) modulate cell growth in head and neck squamous cell carcinoma

Journal: PLoS ONE

doi: 10.1371/journal.pone.0193242

Effect of NCoR2 over-expression on PXR activity and cell growth in HNO97, HNO124 and HNO210 cells. Cells were transfected with the pCMV6-NCoR2 plasmid (or the empty vector pCMV6). NCoR2 over-expression was quantified at the mRNA level through qRT-PCR at 12 h post-transfection in HNO97 (a) , HNO124 (b) and HNO210 (c) cells. RPII was used as housekeeping gene. mRNA data were normalized to NCoR2 expression in cells transfected with the pCMV6 empty vector, set to 1. NCoR2 over-expression was verified at the protein level at the same time point through western blot analysis using histone H1 as a loading control. Representative images are shown for each cell line (d) . PXR activity was measured at 48 h post-transfection in HNO97 (e) , HNO124 (f) and HNO210 (g) cells. Cell growth was measured at 72 h post-transfection in HNO97 (h) , HNO124 (i) and HNO210 (j) cells using the crystal violet method. Results were normalized to PXR activity or cell growth in cells transfected with the pCMV6 empty vector, set to 1. All data are expressed as mean ± S.D. *significantly different from pCMV6-transfected cells, p
Figure Legend Snippet: Effect of NCoR2 over-expression on PXR activity and cell growth in HNO97, HNO124 and HNO210 cells. Cells were transfected with the pCMV6-NCoR2 plasmid (or the empty vector pCMV6). NCoR2 over-expression was quantified at the mRNA level through qRT-PCR at 12 h post-transfection in HNO97 (a) , HNO124 (b) and HNO210 (c) cells. RPII was used as housekeeping gene. mRNA data were normalized to NCoR2 expression in cells transfected with the pCMV6 empty vector, set to 1. NCoR2 over-expression was verified at the protein level at the same time point through western blot analysis using histone H1 as a loading control. Representative images are shown for each cell line (d) . PXR activity was measured at 48 h post-transfection in HNO97 (e) , HNO124 (f) and HNO210 (g) cells. Cell growth was measured at 72 h post-transfection in HNO97 (h) , HNO124 (i) and HNO210 (j) cells using the crystal violet method. Results were normalized to PXR activity or cell growth in cells transfected with the pCMV6 empty vector, set to 1. All data are expressed as mean ± S.D. *significantly different from pCMV6-transfected cells, p

Techniques Used: Over Expression, Activity Assay, Transfection, Plasmid Preparation, Quantitative RT-PCR, Expressing, Western Blot

Cleaved caspase 3 and cleaved PARP expression in NCoR2 over-expressing HNO97, HNO124 and HNO210 cells. Cells were transfected with pCMV6-NCoR2 plasmid (or the empty vector pCMV6). Cleaved caspase 3 and cleaved PARP protein levels were evaluated at 24 h post-transfection in HNO97 (a) , HNO124 (b) and HNO210 (c) cells through western blot. β-actin was used as a loading control. Representative western blot detections are shown for each cell line. *significantly different from pCMV6-transfected cells, p
Figure Legend Snippet: Cleaved caspase 3 and cleaved PARP expression in NCoR2 over-expressing HNO97, HNO124 and HNO210 cells. Cells were transfected with pCMV6-NCoR2 plasmid (or the empty vector pCMV6). Cleaved caspase 3 and cleaved PARP protein levels were evaluated at 24 h post-transfection in HNO97 (a) , HNO124 (b) and HNO210 (c) cells through western blot. β-actin was used as a loading control. Representative western blot detections are shown for each cell line. *significantly different from pCMV6-transfected cells, p

Techniques Used: Expressing, Transfection, Plasmid Preparation, Western Blot

Caspase activity in NCoR2 over-expressing HNO97, HNO124 and HNO210 cells. Cells were transfected with pCMV6-NCoR2 plasmid (or the empty vector pCMV6). Caspase activity was assessed at 24 h post-transfection in HNO97 (a) , HNO124 (b) and HNO210 (c) cells. Results were normalized to the corresponding activity in cells transfected with the pCMV6 empty vector, set to 1. *significantly different from pCMV6-transfected cells, p
Figure Legend Snippet: Caspase activity in NCoR2 over-expressing HNO97, HNO124 and HNO210 cells. Cells were transfected with pCMV6-NCoR2 plasmid (or the empty vector pCMV6). Caspase activity was assessed at 24 h post-transfection in HNO97 (a) , HNO124 (b) and HNO210 (c) cells. Results were normalized to the corresponding activity in cells transfected with the pCMV6 empty vector, set to 1. *significantly different from pCMV6-transfected cells, p

Techniques Used: Activity Assay, Expressing, Transfection, Plasmid Preparation

7) Product Images from "Glycerol-3-Phosphate Acyltranferase-2 Behaves as a Cancer Testis Gene and Promotes Growth and Tumorigenicity of the Breast Cancer MDA-MB-231 Cell Line"

Article Title: Glycerol-3-Phosphate Acyltranferase-2 Behaves as a Cancer Testis Gene and Promotes Growth and Tumorigenicity of the Breast Cancer MDA-MB-231 Cell Line

Journal: PLoS ONE

doi: 10.1371/journal.pone.0100896

Phenotipic consequences of human and murine GPAT2 overexpression. A) Total RNA from pCMV6 and pCMV6-GPAT2 cells was extracted, subjected to cDNA synthesis and amplified by quantitative RT-PCR using primers for human GPAT2 gene, normalizing its expression level to that of TBP and β-actin housekeeping genes *** p
Figure Legend Snippet: Phenotipic consequences of human and murine GPAT2 overexpression. A) Total RNA from pCMV6 and pCMV6-GPAT2 cells was extracted, subjected to cDNA synthesis and amplified by quantitative RT-PCR using primers for human GPAT2 gene, normalizing its expression level to that of TBP and β-actin housekeeping genes *** p

Techniques Used: Over Expression, Amplification, Quantitative RT-PCR, Expressing

8) Product Images from "The catalytic, stem, and transmembrane portions of matriptase-2 are required for suppressing the expression of the iron-regulatory hormone hepcidin"

Article Title: The catalytic, stem, and transmembrane portions of matriptase-2 are required for suppressing the expression of the iron-regulatory hormone hepcidin

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA118.006468

HAI-2 inhibits the substrate cleavage by MT2 and truncated MT2. A–D, HAI-2 inhibition of cleavage for ALK2 ( A ), ALK3 ( B ), ActRIIA ( C ), and Bmpr2 ( D ). pCMV6-ALK2, ALK3, ActRIIA, or Bmpr2 were co-transfected into HEK293 cells with pCMV6-MT2 or truncates and pCMV6-HAI-2 or pEGFP-N1 at 1:1:1 ratios of plasmid DNA. After 48 h of transfection, ∼150 μg of cell lysate proteins were subjected to SDS-PAGE and immunodetection using anti-FLAG (for MT2, HAI2, ALK2, ALK3, ActRIIA, and Bmpr2), β-actin, and GFP antibodies. E, HAI-2 inhibition of cleavage for Hfe. HEK293 cells were co-transfected with pCMV6-Hfe, pJB-1-B2M, pCMV6-MT2, or truncates, and HAI-2 or pEGFP-N1 at 1:1:1:1 ratios of plasmid DNA. Analysis was performed at 48 h post-transfection as described above in A–D. F, HAI-2 inhibition of cleavage for Tfr2. Co-transfection of Tfr2 with MT2 truncates and HAI-2 or pEGFP-N1 were performed essentially the same as described above for A–D . At about 30 h post-transfection, medium was changed to Opti-MEM, 1% FCS. Analysis was performed after another 18 h of incubation. About 150 μg of cell-lysate proteins and the TCA-precipitated proteins from ∼600 μl of CM were subjected to SDS and immunodetection. Tfr2 was detected by using a rabbit anti-Tfr2 antibody. G, HAI-2 inhibition of cleavage for Hjv. Co-transfection of HEK293 cells with Hjv, truncated MT2, and HAI-2 was performed as described above in A–D . At about 30 h post-transfection, medium was changed to Opti-MEM, 1% FCS. After another 18 h of incubation, about 150 μg of cell-lysate proteins and the TCA-precipitated proteins from ∼600 μl of CM were subjected to SDS-PAGE and immunodetection using anti-FLAG (for MT2, Hjv, and HAI-2), β-actin, GPF, and HJV antibodies. n.s ., nonspecific bands. All experiments were repeated at least three times with consistent results.
Figure Legend Snippet: HAI-2 inhibits the substrate cleavage by MT2 and truncated MT2. A–D, HAI-2 inhibition of cleavage for ALK2 ( A ), ALK3 ( B ), ActRIIA ( C ), and Bmpr2 ( D ). pCMV6-ALK2, ALK3, ActRIIA, or Bmpr2 were co-transfected into HEK293 cells with pCMV6-MT2 or truncates and pCMV6-HAI-2 or pEGFP-N1 at 1:1:1 ratios of plasmid DNA. After 48 h of transfection, ∼150 μg of cell lysate proteins were subjected to SDS-PAGE and immunodetection using anti-FLAG (for MT2, HAI2, ALK2, ALK3, ActRIIA, and Bmpr2), β-actin, and GFP antibodies. E, HAI-2 inhibition of cleavage for Hfe. HEK293 cells were co-transfected with pCMV6-Hfe, pJB-1-B2M, pCMV6-MT2, or truncates, and HAI-2 or pEGFP-N1 at 1:1:1:1 ratios of plasmid DNA. Analysis was performed at 48 h post-transfection as described above in A–D. F, HAI-2 inhibition of cleavage for Tfr2. Co-transfection of Tfr2 with MT2 truncates and HAI-2 or pEGFP-N1 were performed essentially the same as described above for A–D . At about 30 h post-transfection, medium was changed to Opti-MEM, 1% FCS. Analysis was performed after another 18 h of incubation. About 150 μg of cell-lysate proteins and the TCA-precipitated proteins from ∼600 μl of CM were subjected to SDS and immunodetection. Tfr2 was detected by using a rabbit anti-Tfr2 antibody. G, HAI-2 inhibition of cleavage for Hjv. Co-transfection of HEK293 cells with Hjv, truncated MT2, and HAI-2 was performed as described above in A–D . At about 30 h post-transfection, medium was changed to Opti-MEM, 1% FCS. After another 18 h of incubation, about 150 μg of cell-lysate proteins and the TCA-precipitated proteins from ∼600 μl of CM were subjected to SDS-PAGE and immunodetection using anti-FLAG (for MT2, Hjv, and HAI-2), β-actin, GPF, and HJV antibodies. n.s ., nonspecific bands. All experiments were repeated at least three times with consistent results.

Techniques Used: Inhibition, Transfection, Plasmid Preparation, SDS Page, Immunodetection, Cotransfection, Incubation

The stem region of MT2 is required for an efficient cleavage of ALK3. A, an equal amount of pCMV6-ALK3 (2 μg) was co-transfected with an increasing amount of pCMV6-MT2 or MT2 truncates (0.25, 1.0, and 2.0 μg), and decreasing amount of pEGFP-N1 (1.75, 1.0, and 0 μg) into HEK293 cells in 12-well plates. Co-transfection with 2 μg of pEGFP-N1 ( GFP ) was included as negative controls. After 48 h of transfection, ∼150 μg of cell lysate proteins was subjected to SDS-PAGE and immunodetection by using anti-FLAG (for MT2 and ALK3), β-actin, and GFP antibodies. Each panel w as cropped from the same image. B, effects of truncated MT2 on cell-surface ALK3. pCMV6-ALK3 was co-transfected with an equal amount of pEGFP-N1 ( GFP ), pCMV9-MT2, ΔCyto, mask, Ecto, or S/P plasmid DNA into HEK293 cells. At about 48 h after transfection, cell-surface proteins were biotinylated at 4 °C, followed by pulldown of the biotinylated proteins using streptavidin-agarose beads. The entire eluted cell-surface proteins and ∼25% of input lysate were subjected to SDS-PAGE and immunodetection using anti-FLAG (for ALK3), NaK-ATPase, and TfR1 antibodies. n.s ., nonspecific bands. All experiments were repeated at least three times with consistent results.
Figure Legend Snippet: The stem region of MT2 is required for an efficient cleavage of ALK3. A, an equal amount of pCMV6-ALK3 (2 μg) was co-transfected with an increasing amount of pCMV6-MT2 or MT2 truncates (0.25, 1.0, and 2.0 μg), and decreasing amount of pEGFP-N1 (1.75, 1.0, and 0 μg) into HEK293 cells in 12-well plates. Co-transfection with 2 μg of pEGFP-N1 ( GFP ) was included as negative controls. After 48 h of transfection, ∼150 μg of cell lysate proteins was subjected to SDS-PAGE and immunodetection by using anti-FLAG (for MT2 and ALK3), β-actin, and GFP antibodies. Each panel w as cropped from the same image. B, effects of truncated MT2 on cell-surface ALK3. pCMV6-ALK3 was co-transfected with an equal amount of pEGFP-N1 ( GFP ), pCMV9-MT2, ΔCyto, mask, Ecto, or S/P plasmid DNA into HEK293 cells. At about 48 h after transfection, cell-surface proteins were biotinylated at 4 °C, followed by pulldown of the biotinylated proteins using streptavidin-agarose beads. The entire eluted cell-surface proteins and ∼25% of input lysate were subjected to SDS-PAGE and immunodetection using anti-FLAG (for ALK3), NaK-ATPase, and TfR1 antibodies. n.s ., nonspecific bands. All experiments were repeated at least three times with consistent results.

Techniques Used: Transfection, Cotransfection, SDS Page, Immunodetection, Plasmid Preparation

9) Product Images from "Regulation of c-Maf and αA-Crystallin in Ocular Lens by Fibroblast Growth Factor Signaling *"

Article Title: Regulation of c-Maf and αA-Crystallin in Ocular Lens by Fibroblast Growth Factor Signaling *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.705103

Transcriptional regulation of c-Maf by c-Jun and Etv5/ERM. A , distribution of c-Jun and Etv5/ERM factors and H3 K4me3 promoter marker along the mouse c-Maf locus in lens chromatin. The locations of the qChIP amplicons covering 11 kb (−5 kb/+6 kb) c-Maf locus is shown at top . c-Jun, Etv5/ERM, and H3K4me3 are presented in the middle and lower panel , respectively. Statistically significant enrichment of binding signals is indicated above the horizontal dotted line . The relative enrichments are shown as 1% of the input. B , diagram of firefly luciferase reporter constructs without promoter (pGL3-Basic), with the intact −494/+210 c-Maf promoter (WT), with the c-Maf promoter lacking the −272/-70 FRE region (ΔFRE), and with three copies of the FRE followed by a minimal E4TATA promoter (3XFRE). C , results of transient co-transfection/reporter assays. The firefly luciferase activities were normalized using Renilla luciferase as an internal control. The results shown are from two independent experiments with duplicates. The relative luciferase activities were calculated using the “WT reporter with pCMV6 cDNA vector” value set as 1.
Figure Legend Snippet: Transcriptional regulation of c-Maf by c-Jun and Etv5/ERM. A , distribution of c-Jun and Etv5/ERM factors and H3 K4me3 promoter marker along the mouse c-Maf locus in lens chromatin. The locations of the qChIP amplicons covering 11 kb (−5 kb/+6 kb) c-Maf locus is shown at top . c-Jun, Etv5/ERM, and H3K4me3 are presented in the middle and lower panel , respectively. Statistically significant enrichment of binding signals is indicated above the horizontal dotted line . The relative enrichments are shown as 1% of the input. B , diagram of firefly luciferase reporter constructs without promoter (pGL3-Basic), with the intact −494/+210 c-Maf promoter (WT), with the c-Maf promoter lacking the −272/-70 FRE region (ΔFRE), and with three copies of the FRE followed by a minimal E4TATA promoter (3XFRE). C , results of transient co-transfection/reporter assays. The firefly luciferase activities were normalized using Renilla luciferase as an internal control. The results shown are from two independent experiments with duplicates. The relative luciferase activities were calculated using the “WT reporter with pCMV6 cDNA vector” value set as 1.

Techniques Used: Marker, Binding Assay, Luciferase, Construct, Cotransfection, Plasmid Preparation

10) Product Images from "FAM83G Is a Novel Inducer of Apoptosis"

Article Title: FAM83G Is a Novel Inducer of Apoptosis

Journal: Molecules

doi: 10.3390/molecules25122810

Effect of FAM83G overexpression on HCT116 cells. ( a ) Estimation of FAM83G overexpression in HCT116 cells. The amount of overexpressed wild type (WT)-FAM83G in HCT116 cells was ~10-fold greater than that of endogenous FAM83G (upper panel). α-tubulin blotting proved that equal protein amounts were loaded in each sample (lower panel). The experiments were independently performed in triplicate. ( b ) Overexpressed WT-FAM83G reduces the number of live HCT116 cells; relative live HCT116 cell numbers were quantified using the LIVE/DEAD cell assay following the expression of an empty pCMV6 vector (control) or pCMV6-WT-FAM83G. The results of the statistical analyses are shown: WT-FAM83G overexpression significantly reduced the number of live cells. The experiments were independently performed in triplicate.
Figure Legend Snippet: Effect of FAM83G overexpression on HCT116 cells. ( a ) Estimation of FAM83G overexpression in HCT116 cells. The amount of overexpressed wild type (WT)-FAM83G in HCT116 cells was ~10-fold greater than that of endogenous FAM83G (upper panel). α-tubulin blotting proved that equal protein amounts were loaded in each sample (lower panel). The experiments were independently performed in triplicate. ( b ) Overexpressed WT-FAM83G reduces the number of live HCT116 cells; relative live HCT116 cell numbers were quantified using the LIVE/DEAD cell assay following the expression of an empty pCMV6 vector (control) or pCMV6-WT-FAM83G. The results of the statistical analyses are shown: WT-FAM83G overexpression significantly reduced the number of live cells. The experiments were independently performed in triplicate.

Techniques Used: Over Expression, Expressing, Plasmid Preparation

Effect of overexpressed FAM83G with or without S356 phosphorylation on cell numbers. ( a ) Interaction between FAM83G and PKD1/PKCμ; pCMV6 and pCMV6-wild type (WT)-FAM83G were expressed in CHO cells, FAM83G was immunoprecipitated, and then the immunoprecipitated FAM83G was immunoblotted with an antibody specific for FAM83G and PKD1/PKCμ. Endogenous FAM83G was co-immunoprecipitated with endogenous PKD1/PKCμ (pCMV6 lane, control sample). Overexpressed FAM83G was co-immunoprecipitated with increased PKD1/PKCμ levels (pCMV6-WT-FAM83G lane). The experiments were independently performed in triplicate. ( b ) Phosphorylation of the HSP27 S82 residue is inversely correlated with the levels of FAM83G expression. Upper panel: exogenously expressed FAM83G and endogenous FAM83G protein levels in CHO cells following immunoblotting (IB) with a FAM83G-specific antibody. Middle panel: the relative levels of S82-phosphorylated HSP27 are shown by IB with a specific anti-phospho-(S82) HSP27 antibody. Lower panel: relative HSP27 protein levels following IB with a specific anti-phospho-(S82) HSP27 antibody (strip and re-probe technique). The experiments were independently performed in triplicate. ( c ) Cell survival following the expression of pCMV6, WT-FAM83G, Y586A-FAM83G, and S356A-FAM83G in CHO cells; cell survival (relative number of live cells), as measured by the XTT assay. An empty pCMV6 plasmid transfected into the CHO cells acted as the control. The results of the statistical analyses, which are shown to the right of the bar graph, indicated that phosphorylation at S356 was required for WT-FAM83G overexpression to significantly reduce live cell numbers. The experiments were independently performed in triplicate. ( d ) Expression levels of WT-FAM83G and the FAM83G mutants; exogenously expressed FAM83G protein levels were approximately 7-fold greater than the endogenous FAM83G level. The relative levels of endogenous-tubulin are shown as loading controls. The experiments were independently performed in triplicate.
Figure Legend Snippet: Effect of overexpressed FAM83G with or without S356 phosphorylation on cell numbers. ( a ) Interaction between FAM83G and PKD1/PKCμ; pCMV6 and pCMV6-wild type (WT)-FAM83G were expressed in CHO cells, FAM83G was immunoprecipitated, and then the immunoprecipitated FAM83G was immunoblotted with an antibody specific for FAM83G and PKD1/PKCμ. Endogenous FAM83G was co-immunoprecipitated with endogenous PKD1/PKCμ (pCMV6 lane, control sample). Overexpressed FAM83G was co-immunoprecipitated with increased PKD1/PKCμ levels (pCMV6-WT-FAM83G lane). The experiments were independently performed in triplicate. ( b ) Phosphorylation of the HSP27 S82 residue is inversely correlated with the levels of FAM83G expression. Upper panel: exogenously expressed FAM83G and endogenous FAM83G protein levels in CHO cells following immunoblotting (IB) with a FAM83G-specific antibody. Middle panel: the relative levels of S82-phosphorylated HSP27 are shown by IB with a specific anti-phospho-(S82) HSP27 antibody. Lower panel: relative HSP27 protein levels following IB with a specific anti-phospho-(S82) HSP27 antibody (strip and re-probe technique). The experiments were independently performed in triplicate. ( c ) Cell survival following the expression of pCMV6, WT-FAM83G, Y586A-FAM83G, and S356A-FAM83G in CHO cells; cell survival (relative number of live cells), as measured by the XTT assay. An empty pCMV6 plasmid transfected into the CHO cells acted as the control. The results of the statistical analyses, which are shown to the right of the bar graph, indicated that phosphorylation at S356 was required for WT-FAM83G overexpression to significantly reduce live cell numbers. The experiments were independently performed in triplicate. ( d ) Expression levels of WT-FAM83G and the FAM83G mutants; exogenously expressed FAM83G protein levels were approximately 7-fold greater than the endogenous FAM83G level. The relative levels of endogenous-tubulin are shown as loading controls. The experiments were independently performed in triplicate.

Techniques Used: Immunoprecipitation, Expressing, Stripping Membranes, XTT Assay, Plasmid Preparation, Transfection, Over Expression

Related Articles

Clone Assay:

Article Title: Glycerol-3-Phosphate Acyltranferase-2 Behaves as a Cancer Testis Gene and Promotes Growth and Tumorigenicity of the Breast Cancer MDA-MB-231 Cell Line
Article Snippet: .. To obtain stable cell lines, MDA-MB-231 cells were grown in 60-mm dishes to 90% confluence and then transfected with 5 µg of the cDNA encoding the complete open reading frame of human GPAT2 cloned in the pCMV6 vector (TrueORF, Origene) or with the empty vector as control. .. Both plasmids also contain a sequence coding for green fluorescent protein driven by an IRE translational element.

Transfection:

Article Title: The pregnane X receptor (PXR) and the nuclear receptor corepressor 2 (NCoR2) modulate cell growth in head and neck squamous cell carcinoma
Article Snippet: .. Control cells were transfected with 1 μg of the empty pCMV6 vector (Clone PS100001, Origene, Rockville, USA). ..

Article Title: Glycerol-3-Phosphate Acyltranferase-2 Behaves as a Cancer Testis Gene and Promotes Growth and Tumorigenicity of the Breast Cancer MDA-MB-231 Cell Line
Article Snippet: .. To obtain stable cell lines, MDA-MB-231 cells were grown in 60-mm dishes to 90% confluence and then transfected with 5 µg of the cDNA encoding the complete open reading frame of human GPAT2 cloned in the pCMV6 vector (TrueORF, Origene) or with the empty vector as control. .. Both plasmids also contain a sequence coding for green fluorescent protein driven by an IRE translational element.

Luciferase:

Article Title: Studying the mechanism of PLAGL2 overexpression and its carcinogenic characteristics based on 3′-untranslated region in colorectal cancer
Article Snippet: .. The wt 3′-UTR (422 bp) and mut 3′-UTR (422 bp) of PLAGL2 in the putative miR-486-5p binding sites were inserted downstream of the Firefly luciferase gene in the pmirGLO vector (Promega Corporation), and the complement sequence of HuR DNA were inserted into the pCMV6 vector (OriGene Technologies, Inc.). .. Lentiviral expression vectors, RNAi-Mate, and lentiviral packaging vectors, including pGag/Pol, pRev and pVSV-G, were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China), and a blank lentiviral expression vector was used as a control.

Article Title: Studying the mechanism of PLAGL2 overexpression and its carcinogenic characteristics based on 3′-untranslated region in colorectal cancer
Article Snippet: .. Plasmid and lentivirus constructs The wt 3′-UTR (422 bp) and mut 3′-UTR (422 bp) of PLAGL2 in the putative miR-486-5p binding sites were inserted downstream of the Firefly luciferase gene in the pmirGLO vector (Promega Corporation), and the complement sequence of HuR DNA were inserted into the pCMV6 vector (OriGene Technologies, Inc.). .. Lentiviral expression vectors, RNAi-Mate, and lentiviral packaging vectors, including pGag/Pol, pRev and pVSV-G, were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China), and a blank lentiviral expression vector was used as a control.

Stable Transfection:

Article Title: Glycerol-3-Phosphate Acyltranferase-2 Behaves as a Cancer Testis Gene and Promotes Growth and Tumorigenicity of the Breast Cancer MDA-MB-231 Cell Line
Article Snippet: .. To obtain stable cell lines, MDA-MB-231 cells were grown in 60-mm dishes to 90% confluence and then transfected with 5 µg of the cDNA encoding the complete open reading frame of human GPAT2 cloned in the pCMV6 vector (TrueORF, Origene) or with the empty vector as control. .. Both plasmids also contain a sequence coding for green fluorescent protein driven by an IRE translational element.

Immunoprecipitation:

Article Title: B-cell Translocation Gene 2 (BTG2) Stimulates Cellular Antioxidant Defenses through the Antioxidant Transcription Factor NFE2L2 in Human Mammary Epithelial Cells *
Article Snippet: .. For immunoprecipitation and chromatin immunoprecipitation experiments, we utilized BTG2 cDNA in the pCMV6 vector, which contains an NH2 -terminal FLAG epitope tag (FLAG-BTG2) and the empty pCMV6 vector (OriGene Technologies, Rockville, MD). .. For domain mapping studies, we used expression vectors encoding HA-tagged human wild-type BTG2 (wt-HA-BTG2) and three HA-tagged deletion mutants: HA-BTG2-ΔA, HA-BTG2-ΔB, and HA-BTG2-ΔC, which were a gift from Dr. Shyamala Maheswaran (Massachusetts General Hospital, Boston, MA).

Multiple Displacement Amplification:

Article Title: Glycerol-3-Phosphate Acyltranferase-2 Behaves as a Cancer Testis Gene and Promotes Growth and Tumorigenicity of the Breast Cancer MDA-MB-231 Cell Line
Article Snippet: .. To obtain stable cell lines, MDA-MB-231 cells were grown in 60-mm dishes to 90% confluence and then transfected with 5 µg of the cDNA encoding the complete open reading frame of human GPAT2 cloned in the pCMV6 vector (TrueORF, Origene) or with the empty vector as control. .. Both plasmids also contain a sequence coding for green fluorescent protein driven by an IRE translational element.

Subcloning:

Article Title: SPHK1 (sphingosine kinase 1) induces epithelial-mesenchymal transition by promoting the autophagy-linked lysosomal degradation of CDH1/E-cadherin in hepatoma cells
Article Snippet: .. CDH1 was constructed in the pCMV6 vector (PS100001, Origene) by standard subcloning. .. HA-tagged BECN1 was constructed in the pCMV6-AC-HA vector (PS100004, Origene) by standard subcloning.

Construct:

Article Title: SPHK1 (sphingosine kinase 1) induces epithelial-mesenchymal transition by promoting the autophagy-linked lysosomal degradation of CDH1/E-cadherin in hepatoma cells
Article Snippet: .. CDH1 was constructed in the pCMV6 vector (PS100001, Origene) by standard subcloning. .. HA-tagged BECN1 was constructed in the pCMV6-AC-HA vector (PS100004, Origene) by standard subcloning.

Article Title: Studying the mechanism of PLAGL2 overexpression and its carcinogenic characteristics based on 3′-untranslated region in colorectal cancer
Article Snippet: .. Plasmid and lentivirus constructs The wt 3′-UTR (422 bp) and mut 3′-UTR (422 bp) of PLAGL2 in the putative miR-486-5p binding sites were inserted downstream of the Firefly luciferase gene in the pmirGLO vector (Promega Corporation), and the complement sequence of HuR DNA were inserted into the pCMV6 vector (OriGene Technologies, Inc.). .. Lentiviral expression vectors, RNAi-Mate, and lentiviral packaging vectors, including pGag/Pol, pRev and pVSV-G, were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China), and a blank lentiviral expression vector was used as a control.

FLAG-tag:

Article Title: B-cell Translocation Gene 2 (BTG2) Stimulates Cellular Antioxidant Defenses through the Antioxidant Transcription Factor NFE2L2 in Human Mammary Epithelial Cells *
Article Snippet: .. For immunoprecipitation and chromatin immunoprecipitation experiments, we utilized BTG2 cDNA in the pCMV6 vector, which contains an NH2 -terminal FLAG epitope tag (FLAG-BTG2) and the empty pCMV6 vector (OriGene Technologies, Rockville, MD). .. For domain mapping studies, we used expression vectors encoding HA-tagged human wild-type BTG2 (wt-HA-BTG2) and three HA-tagged deletion mutants: HA-BTG2-ΔA, HA-BTG2-ΔB, and HA-BTG2-ΔC, which were a gift from Dr. Shyamala Maheswaran (Massachusetts General Hospital, Boston, MA).

Sequencing:

Article Title: Studying the mechanism of PLAGL2 overexpression and its carcinogenic characteristics based on 3′-untranslated region in colorectal cancer
Article Snippet: .. The wt 3′-UTR (422 bp) and mut 3′-UTR (422 bp) of PLAGL2 in the putative miR-486-5p binding sites were inserted downstream of the Firefly luciferase gene in the pmirGLO vector (Promega Corporation), and the complement sequence of HuR DNA were inserted into the pCMV6 vector (OriGene Technologies, Inc.). .. Lentiviral expression vectors, RNAi-Mate, and lentiviral packaging vectors, including pGag/Pol, pRev and pVSV-G, were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China), and a blank lentiviral expression vector was used as a control.

Article Title: Studying the mechanism of PLAGL2 overexpression and its carcinogenic characteristics based on 3′-untranslated region in colorectal cancer
Article Snippet: .. Plasmid and lentivirus constructs The wt 3′-UTR (422 bp) and mut 3′-UTR (422 bp) of PLAGL2 in the putative miR-486-5p binding sites were inserted downstream of the Firefly luciferase gene in the pmirGLO vector (Promega Corporation), and the complement sequence of HuR DNA were inserted into the pCMV6 vector (OriGene Technologies, Inc.). .. Lentiviral expression vectors, RNAi-Mate, and lentiviral packaging vectors, including pGag/Pol, pRev and pVSV-G, were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China), and a blank lentiviral expression vector was used as a control.

Binding Assay:

Article Title: Studying the mechanism of PLAGL2 overexpression and its carcinogenic characteristics based on 3′-untranslated region in colorectal cancer
Article Snippet: .. The wt 3′-UTR (422 bp) and mut 3′-UTR (422 bp) of PLAGL2 in the putative miR-486-5p binding sites were inserted downstream of the Firefly luciferase gene in the pmirGLO vector (Promega Corporation), and the complement sequence of HuR DNA were inserted into the pCMV6 vector (OriGene Technologies, Inc.). .. Lentiviral expression vectors, RNAi-Mate, and lentiviral packaging vectors, including pGag/Pol, pRev and pVSV-G, were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China), and a blank lentiviral expression vector was used as a control.

Article Title: Studying the mechanism of PLAGL2 overexpression and its carcinogenic characteristics based on 3′-untranslated region in colorectal cancer
Article Snippet: .. Plasmid and lentivirus constructs The wt 3′-UTR (422 bp) and mut 3′-UTR (422 bp) of PLAGL2 in the putative miR-486-5p binding sites were inserted downstream of the Firefly luciferase gene in the pmirGLO vector (Promega Corporation), and the complement sequence of HuR DNA were inserted into the pCMV6 vector (OriGene Technologies, Inc.). .. Lentiviral expression vectors, RNAi-Mate, and lentiviral packaging vectors, including pGag/Pol, pRev and pVSV-G, were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China), and a blank lentiviral expression vector was used as a control.

Chromatin Immunoprecipitation:

Article Title: B-cell Translocation Gene 2 (BTG2) Stimulates Cellular Antioxidant Defenses through the Antioxidant Transcription Factor NFE2L2 in Human Mammary Epithelial Cells *
Article Snippet: .. For immunoprecipitation and chromatin immunoprecipitation experiments, we utilized BTG2 cDNA in the pCMV6 vector, which contains an NH2 -terminal FLAG epitope tag (FLAG-BTG2) and the empty pCMV6 vector (OriGene Technologies, Rockville, MD). .. For domain mapping studies, we used expression vectors encoding HA-tagged human wild-type BTG2 (wt-HA-BTG2) and three HA-tagged deletion mutants: HA-BTG2-ΔA, HA-BTG2-ΔB, and HA-BTG2-ΔC, which were a gift from Dr. Shyamala Maheswaran (Massachusetts General Hospital, Boston, MA).

Plasmid Preparation:

Article Title: The pregnane X receptor (PXR) and the nuclear receptor corepressor 2 (NCoR2) modulate cell growth in head and neck squamous cell carcinoma
Article Snippet: .. Control cells were transfected with 1 μg of the empty pCMV6 vector (Clone PS100001, Origene, Rockville, USA). ..

Article Title: The catalytic, stem, and transmembrane portions of matriptase-2 are required for suppressing the expression of the iron-regulatory hormone hepcidin
Article Snippet: .. Murine HAI-2 ( ) ORF with a C-terminal FLAG/MYC epitope in the pCMV6 vector was purchased from Origene ( ). ..

Article Title: Studying the mechanism of PLAGL2 overexpression and its carcinogenic characteristics based on 3′-untranslated region in colorectal cancer
Article Snippet: .. The wt 3′-UTR (422 bp) and mut 3′-UTR (422 bp) of PLAGL2 in the putative miR-486-5p binding sites were inserted downstream of the Firefly luciferase gene in the pmirGLO vector (Promega Corporation), and the complement sequence of HuR DNA were inserted into the pCMV6 vector (OriGene Technologies, Inc.). .. Lentiviral expression vectors, RNAi-Mate, and lentiviral packaging vectors, including pGag/Pol, pRev and pVSV-G, were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China), and a blank lentiviral expression vector was used as a control.

Article Title: B-cell Translocation Gene 2 (BTG2) Stimulates Cellular Antioxidant Defenses through the Antioxidant Transcription Factor NFE2L2 in Human Mammary Epithelial Cells *
Article Snippet: .. For immunoprecipitation and chromatin immunoprecipitation experiments, we utilized BTG2 cDNA in the pCMV6 vector, which contains an NH2 -terminal FLAG epitope tag (FLAG-BTG2) and the empty pCMV6 vector (OriGene Technologies, Rockville, MD). .. For domain mapping studies, we used expression vectors encoding HA-tagged human wild-type BTG2 (wt-HA-BTG2) and three HA-tagged deletion mutants: HA-BTG2-ΔA, HA-BTG2-ΔB, and HA-BTG2-ΔC, which were a gift from Dr. Shyamala Maheswaran (Massachusetts General Hospital, Boston, MA).

Article Title: SPHK1 (sphingosine kinase 1) induces epithelial-mesenchymal transition by promoting the autophagy-linked lysosomal degradation of CDH1/E-cadherin in hepatoma cells
Article Snippet: .. CDH1 was constructed in the pCMV6 vector (PS100001, Origene) by standard subcloning. .. HA-tagged BECN1 was constructed in the pCMV6-AC-HA vector (PS100004, Origene) by standard subcloning.

Article Title: Glycerol-3-Phosphate Acyltranferase-2 Behaves as a Cancer Testis Gene and Promotes Growth and Tumorigenicity of the Breast Cancer MDA-MB-231 Cell Line
Article Snippet: .. To obtain stable cell lines, MDA-MB-231 cells were grown in 60-mm dishes to 90% confluence and then transfected with 5 µg of the cDNA encoding the complete open reading frame of human GPAT2 cloned in the pCMV6 vector (TrueORF, Origene) or with the empty vector as control. .. Both plasmids also contain a sequence coding for green fluorescent protein driven by an IRE translational element.

Article Title: Studying the mechanism of PLAGL2 overexpression and its carcinogenic characteristics based on 3′-untranslated region in colorectal cancer
Article Snippet: .. Plasmid and lentivirus constructs The wt 3′-UTR (422 bp) and mut 3′-UTR (422 bp) of PLAGL2 in the putative miR-486-5p binding sites were inserted downstream of the Firefly luciferase gene in the pmirGLO vector (Promega Corporation), and the complement sequence of HuR DNA were inserted into the pCMV6 vector (OriGene Technologies, Inc.). .. Lentiviral expression vectors, RNAi-Mate, and lentiviral packaging vectors, including pGag/Pol, pRev and pVSV-G, were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China), and a blank lentiviral expression vector was used as a control.

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    OriGene ezh2
    DZNep treatment in diffuse cutaneous SSc fibroblasts and animal model led to an antifibrotic phenotype. ( A ) Inhibiting <t>EZH2</t> by DZNep in SSc dermal fibroblasts resulted in a dose-dependent reduction in COL1A1 , TGFB , and FRA2 . ( B ) Selected hypomethylated and hypermethylated genes affected by EZH2 inhibition that were implicated in fibrosis or fibrosis-related processes. ( C ) Inhibition of EZH2 by GSK126 dose-dependently relaxed gel contraction in SSc fibroblasts. ( D ) Scratch-wound assay showing reduced wound closure (cell migration) at 48 h in SSc fibroblasts treated with DZNep (5 μM) compared with PBS control. ( E ) EZH2 overexpression in fibroblasts from healthy individuals increased cell migration at 48 h. ( F ) Increased cell migration by EZH2 overexpression in normal fibroblasts was reversed by knocking down LRRC16A simultaneously, implicating LRRC16A in EZH2-mediated cell migration. ( G and H ) Skin fibrosis was induced in mice by daily bleomycin injection. Mice treated with DZNep showed a significant reduction in dermal thickness compared with vehicle-treated mice. ( I ) Hydroxyproline content was significantly reduced in DZNep-treated mice. Results are expressed as mean ± SD. Kruskal–Wallis test or Mann–Whitney U test was performed, and * P
    Ezh2, supplied by OriGene, used in various techniques. Bioz Stars score: 93/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    OriGene empty pcmv6 entry vector
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    OriGene igfbp1 expression vectors
    Silencing of FOXO3a overcame UA-induced cell growth inhibition and exogenous expressed FOXO3a enhanced UA-induced phosphorylation of p38 MAPK through <t>IGFBP1.</t> a , Bel-7402 and HepG2 cells were transfected with control and FOXO3a siRNAs for 24 h before exposing the cells to UA (25 μM) for an additional 24 h. Afterwards, FOXO3a and IGFBP1 protein expressions were determined by Western blot. b , Bel-7402 and HepG2 cells were transfected with control or FOXO3a siRNAs (up to 50 nM each) for 24 h prior to exposure of the cells to UA (25 μM) for an additional 24 h. Afterwards, FOXO3a protein expression and cell viability were determined by Western blot and MTT assays. Insert represents the protein expression of FOXO3a. c - d, Bel-7402 and HepG2 cells were transfected with control and FOXO3a overexpression vectors for 24 h before exposing the cells to UA (25 μM) for an additional 2 and 24 h, respectively. Afterwards, the protein levels of FOXO3a and p-p38 MAPK, and IGFBP1 protein expression were examined by Western blot. e , Bel-7402 and HepG2 cells silenced of IGFBP1 by siRNA previously were transfected with control and FOXO3a overexpression vector for 24 h before exposing the cells to UA (25 μM) for an additional 2 and 24 h, respectively. Afterwards, IGFBP1, FOXO3a protein and phosphorylation of p38 MAPK were determined by Western blot. Values in bar graphs were given as the mean ± SD from three independent experiments performed in triplicate. *Indicates significant difference as compared to the untreated control group ( P
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    OriGene pd l1 orf
    Manipulation of PD-L1 modulates EMT status of breast cancer cells. EMT status determined by the expression of CD44, CD24, and vimentin or CD44/CD24 combination as measured by flow cytometry following PD-L1 knockdown using specific Sh-RNA mesenchymal-like (MDA-MB-231) breast cancer cells ( a b ). or PD-L1 overexpression by transfection with <t>PD-L1</t> ORF in the luminal-like (T47D) breast cancer cells ( c ). Bars in a represent the means of 3 three different clones and 3 different experiments ± standard deviation ( n = 9) while histograms in b c are representative of one of the experiments. Lines in each histograms represent threshold of positivity as determined by isotype control except of CD44 in MDA-MB-231 cells where it is an arbitrary line to show CD44 high status
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    DZNep treatment in diffuse cutaneous SSc fibroblasts and animal model led to an antifibrotic phenotype. ( A ) Inhibiting EZH2 by DZNep in SSc dermal fibroblasts resulted in a dose-dependent reduction in COL1A1 , TGFB , and FRA2 . ( B ) Selected hypomethylated and hypermethylated genes affected by EZH2 inhibition that were implicated in fibrosis or fibrosis-related processes. ( C ) Inhibition of EZH2 by GSK126 dose-dependently relaxed gel contraction in SSc fibroblasts. ( D ) Scratch-wound assay showing reduced wound closure (cell migration) at 48 h in SSc fibroblasts treated with DZNep (5 μM) compared with PBS control. ( E ) EZH2 overexpression in fibroblasts from healthy individuals increased cell migration at 48 h. ( F ) Increased cell migration by EZH2 overexpression in normal fibroblasts was reversed by knocking down LRRC16A simultaneously, implicating LRRC16A in EZH2-mediated cell migration. ( G and H ) Skin fibrosis was induced in mice by daily bleomycin injection. Mice treated with DZNep showed a significant reduction in dermal thickness compared with vehicle-treated mice. ( I ) Hydroxyproline content was significantly reduced in DZNep-treated mice. Results are expressed as mean ± SD. Kruskal–Wallis test or Mann–Whitney U test was performed, and * P

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

    Article Title: Inhibition of EZH2 prevents fibrosis and restores normal angiogenesis in scleroderma

    doi: 10.1073/pnas.1813006116

    Figure Lengend Snippet: DZNep treatment in diffuse cutaneous SSc fibroblasts and animal model led to an antifibrotic phenotype. ( A ) Inhibiting EZH2 by DZNep in SSc dermal fibroblasts resulted in a dose-dependent reduction in COL1A1 , TGFB , and FRA2 . ( B ) Selected hypomethylated and hypermethylated genes affected by EZH2 inhibition that were implicated in fibrosis or fibrosis-related processes. ( C ) Inhibition of EZH2 by GSK126 dose-dependently relaxed gel contraction in SSc fibroblasts. ( D ) Scratch-wound assay showing reduced wound closure (cell migration) at 48 h in SSc fibroblasts treated with DZNep (5 μM) compared with PBS control. ( E ) EZH2 overexpression in fibroblasts from healthy individuals increased cell migration at 48 h. ( F ) Increased cell migration by EZH2 overexpression in normal fibroblasts was reversed by knocking down LRRC16A simultaneously, implicating LRRC16A in EZH2-mediated cell migration. ( G and H ) Skin fibrosis was induced in mice by daily bleomycin injection. Mice treated with DZNep showed a significant reduction in dermal thickness compared with vehicle-treated mice. ( I ) Hydroxyproline content was significantly reduced in DZNep-treated mice. Results are expressed as mean ± SD. Kruskal–Wallis test or Mann–Whitney U test was performed, and * P

    Article Snippet: To evaluate the effect of EZH2 on angiogenesis in ECs, we used 75 nM EZH2 siRNA (Santa Cruz Biotechnology) to transfect ECs for 48 h. Overexpression of EZH2 in ECs was achieved by transfecting 0.33 μg of EZH2 (control vector pCMV6-XL5; Origene) using Lipofectamine 2000 (Invitrogen) in T12.5 flasks.

    Techniques: Animal Model, Inhibition, Scratch Wound Assay Assay, Migration, Over Expression, Mouse Assay, Injection, MANN-WHITNEY

    The expression of EZH2 and H3K27me3 in SSc fibroblasts and ECs. ( A ) EZH2 mRNA levels were significantly elevated in fibroblasts from diffuse cutaneous SSc patients compared with healthy fibroblasts, but not in limited cutaneous SSc fibroblasts. EZH2 protein levels ( B ) and H3K27me3 levels ( C ) were significantly increased in diffuse cutaneous SSc fibroblasts compared with healthy controls. ( D ) EZH2 mRNA levels were significantly lower in diffuse cutaneous SSc ECs compared with healthy ECs. ( E ) EZH2 protein levels were significantly higher in diffuse cutaneous SSc ECs compared with healthy ECs. ( F ) The expression of H3K27me3 was not significantly different between diffuse cutaneous SSc ECs and healthy controls. Results are expressed as mean ± SD. The Mann–Whitney U test was performed, and * P

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

    Article Title: Inhibition of EZH2 prevents fibrosis and restores normal angiogenesis in scleroderma

    doi: 10.1073/pnas.1813006116

    Figure Lengend Snippet: The expression of EZH2 and H3K27me3 in SSc fibroblasts and ECs. ( A ) EZH2 mRNA levels were significantly elevated in fibroblasts from diffuse cutaneous SSc patients compared with healthy fibroblasts, but not in limited cutaneous SSc fibroblasts. EZH2 protein levels ( B ) and H3K27me3 levels ( C ) were significantly increased in diffuse cutaneous SSc fibroblasts compared with healthy controls. ( D ) EZH2 mRNA levels were significantly lower in diffuse cutaneous SSc ECs compared with healthy ECs. ( E ) EZH2 protein levels were significantly higher in diffuse cutaneous SSc ECs compared with healthy ECs. ( F ) The expression of H3K27me3 was not significantly different between diffuse cutaneous SSc ECs and healthy controls. Results are expressed as mean ± SD. The Mann–Whitney U test was performed, and * P

    Article Snippet: To evaluate the effect of EZH2 on angiogenesis in ECs, we used 75 nM EZH2 siRNA (Santa Cruz Biotechnology) to transfect ECs for 48 h. Overexpression of EZH2 in ECs was achieved by transfecting 0.33 μg of EZH2 (control vector pCMV6-XL5; Origene) using Lipofectamine 2000 (Invitrogen) in T12.5 flasks.

    Techniques: Expressing, MANN-WHITNEY

    Manipulation of EZH2 expression affected EC angiogenesis in vitro. ( A ) Overexpression of EZH2 in normal ECs led to a decreased number of nodes and tubes in Matrigel tube formation assay. Knockdown of EZH2 in normal ECs ( B ) and diffuse cutaneous SSc ECs ( C ) resulted in increased angiogenesis. ( D ) Inhibition of EZH2 by DZNep similarly increased angiogenesis in diffuse cutaneous SSc ECs compared with cells treated with PBS control. Results are expressed as mean ± SD. Mann–Whitney U test was performed, and * P

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

    Article Title: Inhibition of EZH2 prevents fibrosis and restores normal angiogenesis in scleroderma

    doi: 10.1073/pnas.1813006116

    Figure Lengend Snippet: Manipulation of EZH2 expression affected EC angiogenesis in vitro. ( A ) Overexpression of EZH2 in normal ECs led to a decreased number of nodes and tubes in Matrigel tube formation assay. Knockdown of EZH2 in normal ECs ( B ) and diffuse cutaneous SSc ECs ( C ) resulted in increased angiogenesis. ( D ) Inhibition of EZH2 by DZNep similarly increased angiogenesis in diffuse cutaneous SSc ECs compared with cells treated with PBS control. Results are expressed as mean ± SD. Mann–Whitney U test was performed, and * P

    Article Snippet: To evaluate the effect of EZH2 on angiogenesis in ECs, we used 75 nM EZH2 siRNA (Santa Cruz Biotechnology) to transfect ECs for 48 h. Overexpression of EZH2 in ECs was achieved by transfecting 0.33 μg of EZH2 (control vector pCMV6-XL5; Origene) using Lipofectamine 2000 (Invitrogen) in T12.5 flasks.

    Techniques: Expressing, In Vitro, Over Expression, Tube Formation Assay, Inhibition, MANN-WHITNEY

    EZH2 affected diffuse cutaneous SSc EC angiogenesis through the Notch signaling pathway. ( A ) Heat map of angiogenesis-associated genes in SSc ECs after PBS or DZNep treatment. Only HEY2 , a Notch target gene, was significantly up-regulated after DZNep treatment. ( B ) Relative mRNA expression of genes involved in Notch signaling after EZH2 inhibition by DZNep (5 μM, 48 h) in SSc ECs. Data are expressed as fold change versus PBS-treated cells. ( C ) Increased angiogenesis mediated by DZNep in SSc ECs was mediated by increased expression of Notch ligand DLL4, as DLL4 knockdown led to significant decrease in angiogenesis when cells were treated simultaneously with DZNep. ( D ) Genome browser tracks of the DLL4 locus along with ChIP-seq data for EZH2 and H3K27me3 in human umbilical endothelial cells generated by the ENCODE project. Results are expressed as mean ± SD. Unpaired t test or Kruskal–Wallis test were performed, and * P

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

    Article Title: Inhibition of EZH2 prevents fibrosis and restores normal angiogenesis in scleroderma

    doi: 10.1073/pnas.1813006116

    Figure Lengend Snippet: EZH2 affected diffuse cutaneous SSc EC angiogenesis through the Notch signaling pathway. ( A ) Heat map of angiogenesis-associated genes in SSc ECs after PBS or DZNep treatment. Only HEY2 , a Notch target gene, was significantly up-regulated after DZNep treatment. ( B ) Relative mRNA expression of genes involved in Notch signaling after EZH2 inhibition by DZNep (5 μM, 48 h) in SSc ECs. Data are expressed as fold change versus PBS-treated cells. ( C ) Increased angiogenesis mediated by DZNep in SSc ECs was mediated by increased expression of Notch ligand DLL4, as DLL4 knockdown led to significant decrease in angiogenesis when cells were treated simultaneously with DZNep. ( D ) Genome browser tracks of the DLL4 locus along with ChIP-seq data for EZH2 and H3K27me3 in human umbilical endothelial cells generated by the ENCODE project. Results are expressed as mean ± SD. Unpaired t test or Kruskal–Wallis test were performed, and * P

    Article Snippet: To evaluate the effect of EZH2 on angiogenesis in ECs, we used 75 nM EZH2 siRNA (Santa Cruz Biotechnology) to transfect ECs for 48 h. Overexpression of EZH2 in ECs was achieved by transfecting 0.33 μg of EZH2 (control vector pCMV6-XL5; Origene) using Lipofectamine 2000 (Invitrogen) in T12.5 flasks.

    Techniques: Expressing, Inhibition, Chromatin Immunoprecipitation, Generated

    Quantitative RT-PCR analysis of selected pro-apoptotic genes in TCam-2 cells overexpressed the empty pCMV6-entry vector, the wild-type (WT) or mutated (MUT) NANOS1 p.[(Pro34Thr);(Ser83del)]. Repression of 7 mRNAs by the wild type NANOS1 is shown in the grey box, while disruption of repression of 4 mRNAs by the mutated NANOS1 is shown in the white box. The P value

    Journal: bioRxiv

    Article Title: A male infertility mutation reverts NANOS1 activity from anti-apoptotic to pro-apoptotic by disrupting repression of GADD45A, GADD45B, GADD45G and RHOB genes

    doi: 10.1101/858654

    Figure Lengend Snippet: Quantitative RT-PCR analysis of selected pro-apoptotic genes in TCam-2 cells overexpressed the empty pCMV6-entry vector, the wild-type (WT) or mutated (MUT) NANOS1 p.[(Pro34Thr);(Ser83del)]. Repression of 7 mRNAs by the wild type NANOS1 is shown in the grey box, while disruption of repression of 4 mRNAs by the mutated NANOS1 is shown in the white box. The P value

    Article Snippet: Reverse transcription and quantitative PCR 2×105 TCam-2 cells were transfected with 1.5 µg of constructs encoding the wild-type, mutated NANOS1 or empty pCMV6-entry vector in three biological replicates.

    Techniques: Quantitative RT-PCR, Plasmid Preparation

    Analysis of the effect of the wild-type and mutated NANOS1 p.[(Pro34Thr);(Ser83del)] on confluency and cell proliferation. TCam-2 cells were transfected with plasmids encoding the wild-type (WT), mutated (MUT) NANOS1 or empty pCMV6-entry vector. A – Scheme of the NANOS1 protein. NOT1 interacting motif (NIM) domain, zinc-finger (ZNF) domain and amino acid position of the double mutation p.[(Pro34Thr);(Ser83del)] are indicated. B – Confluency was measured 24 h after transfection using JuLI FL, a fluorescence live cell movie analyzer. C – The MTS assay was used to analyse cell proliferation of TCam-2 cells 24, 48 and 72 h after transfection. A similar number of cells counted 12 h after transfection was used for normalization of each experiment. This experiment was performed in three biological replicates. D – The expression of the wild-type and mutated NANOS1 constructs after transfection compared to the negative control (transfection with empty plasmid) were analysed by western blot analysis using an anti-DDK antibody (OriGene). As loading control VCL (vinculin) protein was used, in each sample shown in lower panel. The P value

    Journal: bioRxiv

    Article Title: A male infertility mutation reverts NANOS1 activity from anti-apoptotic to pro-apoptotic by disrupting repression of GADD45A, GADD45B, GADD45G and RHOB genes

    doi: 10.1101/858654

    Figure Lengend Snippet: Analysis of the effect of the wild-type and mutated NANOS1 p.[(Pro34Thr);(Ser83del)] on confluency and cell proliferation. TCam-2 cells were transfected with plasmids encoding the wild-type (WT), mutated (MUT) NANOS1 or empty pCMV6-entry vector. A – Scheme of the NANOS1 protein. NOT1 interacting motif (NIM) domain, zinc-finger (ZNF) domain and amino acid position of the double mutation p.[(Pro34Thr);(Ser83del)] are indicated. B – Confluency was measured 24 h after transfection using JuLI FL, a fluorescence live cell movie analyzer. C – The MTS assay was used to analyse cell proliferation of TCam-2 cells 24, 48 and 72 h after transfection. A similar number of cells counted 12 h after transfection was used for normalization of each experiment. This experiment was performed in three biological replicates. D – The expression of the wild-type and mutated NANOS1 constructs after transfection compared to the negative control (transfection with empty plasmid) were analysed by western blot analysis using an anti-DDK antibody (OriGene). As loading control VCL (vinculin) protein was used, in each sample shown in lower panel. The P value

    Article Snippet: Reverse transcription and quantitative PCR 2×105 TCam-2 cells were transfected with 1.5 µg of constructs encoding the wild-type, mutated NANOS1 or empty pCMV6-entry vector in three biological replicates.

    Techniques: Transfection, Plasmid Preparation, Mutagenesis, Fluorescence, MTS Assay, Expressing, Construct, Negative Control, Western Blot

    Analysis of the influence of the wild-type and mutated NANOS1 p.[(Pro34Thr);(Ser83del)] on apoptosis of TCam-2 cells. Apoptosis was measured by using Annexin V staining and flow cytometry. A – The representation of dot plots generated by flow cytometric analysis of apoptosis in TCam-2 cells upon transfection of the wild-type (WT), mutated (MUT) NANOS1 or empty pCMV6-entry plasmid. The percentages of apoptotic cells are shown in each plot. B – Quantitation of the data from the dot plots that represents the proportion of apoptotic cells in TCam-2 cells transfected with the wild-type or mutated NANOS1, relative to cells transfected with the empty vector (baseline). C – Western blot analysis after transfection of TCam-2 cells with pCMV6-entry vector, the wild-type or mutated NANOS1. As loading control was used ACTB (actin beta) protein, in each sample shown in lower panel. These experiment was performed in three biological replicates. The P value

    Journal: bioRxiv

    Article Title: A male infertility mutation reverts NANOS1 activity from anti-apoptotic to pro-apoptotic by disrupting repression of GADD45A, GADD45B, GADD45G and RHOB genes

    doi: 10.1101/858654

    Figure Lengend Snippet: Analysis of the influence of the wild-type and mutated NANOS1 p.[(Pro34Thr);(Ser83del)] on apoptosis of TCam-2 cells. Apoptosis was measured by using Annexin V staining and flow cytometry. A – The representation of dot plots generated by flow cytometric analysis of apoptosis in TCam-2 cells upon transfection of the wild-type (WT), mutated (MUT) NANOS1 or empty pCMV6-entry plasmid. The percentages of apoptotic cells are shown in each plot. B – Quantitation of the data from the dot plots that represents the proportion of apoptotic cells in TCam-2 cells transfected with the wild-type or mutated NANOS1, relative to cells transfected with the empty vector (baseline). C – Western blot analysis after transfection of TCam-2 cells with pCMV6-entry vector, the wild-type or mutated NANOS1. As loading control was used ACTB (actin beta) protein, in each sample shown in lower panel. These experiment was performed in three biological replicates. The P value

    Article Snippet: Reverse transcription and quantitative PCR 2×105 TCam-2 cells were transfected with 1.5 µg of constructs encoding the wild-type, mutated NANOS1 or empty pCMV6-entry vector in three biological replicates.

    Techniques: Staining, Flow Cytometry, Generated, Transfection, Plasmid Preparation, Quantitation Assay, Western Blot

    Silencing of FOXO3a overcame UA-induced cell growth inhibition and exogenous expressed FOXO3a enhanced UA-induced phosphorylation of p38 MAPK through IGFBP1. a , Bel-7402 and HepG2 cells were transfected with control and FOXO3a siRNAs for 24 h before exposing the cells to UA (25 μM) for an additional 24 h. Afterwards, FOXO3a and IGFBP1 protein expressions were determined by Western blot. b , Bel-7402 and HepG2 cells were transfected with control or FOXO3a siRNAs (up to 50 nM each) for 24 h prior to exposure of the cells to UA (25 μM) for an additional 24 h. Afterwards, FOXO3a protein expression and cell viability were determined by Western blot and MTT assays. Insert represents the protein expression of FOXO3a. c - d, Bel-7402 and HepG2 cells were transfected with control and FOXO3a overexpression vectors for 24 h before exposing the cells to UA (25 μM) for an additional 2 and 24 h, respectively. Afterwards, the protein levels of FOXO3a and p-p38 MAPK, and IGFBP1 protein expression were examined by Western blot. e , Bel-7402 and HepG2 cells silenced of IGFBP1 by siRNA previously were transfected with control and FOXO3a overexpression vector for 24 h before exposing the cells to UA (25 μM) for an additional 2 and 24 h, respectively. Afterwards, IGFBP1, FOXO3a protein and phosphorylation of p38 MAPK were determined by Western blot. Values in bar graphs were given as the mean ± SD from three independent experiments performed in triplicate. *Indicates significant difference as compared to the untreated control group ( P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Inter-regulation of IGFBP1 and FOXO3a unveils novel mechanism in ursolic acid-inhibited growth of hepatocellular carcinoma cells

    doi: 10.1186/s13046-016-0330-2

    Figure Lengend Snippet: Silencing of FOXO3a overcame UA-induced cell growth inhibition and exogenous expressed FOXO3a enhanced UA-induced phosphorylation of p38 MAPK through IGFBP1. a , Bel-7402 and HepG2 cells were transfected with control and FOXO3a siRNAs for 24 h before exposing the cells to UA (25 μM) for an additional 24 h. Afterwards, FOXO3a and IGFBP1 protein expressions were determined by Western blot. b , Bel-7402 and HepG2 cells were transfected with control or FOXO3a siRNAs (up to 50 nM each) for 24 h prior to exposure of the cells to UA (25 μM) for an additional 24 h. Afterwards, FOXO3a protein expression and cell viability were determined by Western blot and MTT assays. Insert represents the protein expression of FOXO3a. c - d, Bel-7402 and HepG2 cells were transfected with control and FOXO3a overexpression vectors for 24 h before exposing the cells to UA (25 μM) for an additional 2 and 24 h, respectively. Afterwards, the protein levels of FOXO3a and p-p38 MAPK, and IGFBP1 protein expression were examined by Western blot. e , Bel-7402 and HepG2 cells silenced of IGFBP1 by siRNA previously were transfected with control and FOXO3a overexpression vector for 24 h before exposing the cells to UA (25 μM) for an additional 2 and 24 h, respectively. Afterwards, IGFBP1, FOXO3a protein and phosphorylation of p38 MAPK were determined by Western blot. Values in bar graphs were given as the mean ± SD from three independent experiments performed in triplicate. *Indicates significant difference as compared to the untreated control group ( P

    Article Snippet: For each well, 2 μg of the desired N1-GFP or FOXO3a-GFP plasmid DNA, kindly provided by Frank M. J. Jacobs (Rudolf Magnus Institute of Neuroscience, Department of Pharmacology and Anatomy, University Medical Center, Utrecht, Netherlands) and was reported previously [ ] and the control (pCMV-6) or IGFBP1 expression vectors (IGFBP1- pCMV6 ) purchased from OriGene Technologies, Inc. (Rockville, MD, USA) at a final concentration of 2 μg/mL were transfected into the cells using the lipofectamine 3000 reagent according to the manufacturer’s instructions for up to 24 h, followed by treating with UA for an additional 24 h. In separated experiment, cell were transfected with pEZX-PG04 -IGFBP1 promoter construct linked Gaussia luciferase (GLuc) gene and secreted alkaline phosphatase (SEAP) internal control obtained from GeneCopoeia, Inc. (GeneCopoeia, Inc., Rockville, MD, USA).

    Techniques: Inhibition, Transfection, Western Blot, Expressing, MTT Assay, Over Expression, Plasmid Preparation

    Overexpression of IGFBP1 enhanced the effect of UA on FOXO3a expression and phosphorylation of p38 MAPK, and restored UA-inhibited cell growth in cells silencing of endogenous IGFBP1 gene. a , Bel-7402 and HepG2 cells were transfected with control or IGFBP1 siRNAs (50 nM each) for 24 h prior to exposure of the cells to UA (25 μM) for an additional 24 h. Afterwards, IGFBP1 protein expression and cell viability were determined by Western blot and MTT assays. b - c , Bel-7402 and HepG2 cells were transfected with control and IGFBP1 overexpression vectors for 24 h before exposing the cells to UA (25 μM) for an additional 2 and 24 h, respectively. Afterwards, IGFBP1, FOXO3a protein levels and phosphorylation of p38 MAPK were determined by Western blot. d , Bel-7402 and HepG2 cells silenced of IGFBP1 by siRNA previously were transfected with control and IGFBP1 overexpression vectors for 24 h before exposing the cells to UA (25 μM) for an additional 24 h. Afterwards, IGFBP1 protein expressions and cell viability were determined by Western blot and MTT assays. Values in bar graphs were given as the mean ± SD from three independent experiments performed in triplicate. *Indicates significant difference as compared to the untreated control group ( P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Inter-regulation of IGFBP1 and FOXO3a unveils novel mechanism in ursolic acid-inhibited growth of hepatocellular carcinoma cells

    doi: 10.1186/s13046-016-0330-2

    Figure Lengend Snippet: Overexpression of IGFBP1 enhanced the effect of UA on FOXO3a expression and phosphorylation of p38 MAPK, and restored UA-inhibited cell growth in cells silencing of endogenous IGFBP1 gene. a , Bel-7402 and HepG2 cells were transfected with control or IGFBP1 siRNAs (50 nM each) for 24 h prior to exposure of the cells to UA (25 μM) for an additional 24 h. Afterwards, IGFBP1 protein expression and cell viability were determined by Western blot and MTT assays. b - c , Bel-7402 and HepG2 cells were transfected with control and IGFBP1 overexpression vectors for 24 h before exposing the cells to UA (25 μM) for an additional 2 and 24 h, respectively. Afterwards, IGFBP1, FOXO3a protein levels and phosphorylation of p38 MAPK were determined by Western blot. d , Bel-7402 and HepG2 cells silenced of IGFBP1 by siRNA previously were transfected with control and IGFBP1 overexpression vectors for 24 h before exposing the cells to UA (25 μM) for an additional 24 h. Afterwards, IGFBP1 protein expressions and cell viability were determined by Western blot and MTT assays. Values in bar graphs were given as the mean ± SD from three independent experiments performed in triplicate. *Indicates significant difference as compared to the untreated control group ( P

    Article Snippet: For each well, 2 μg of the desired N1-GFP or FOXO3a-GFP plasmid DNA, kindly provided by Frank M. J. Jacobs (Rudolf Magnus Institute of Neuroscience, Department of Pharmacology and Anatomy, University Medical Center, Utrecht, Netherlands) and was reported previously [ ] and the control (pCMV-6) or IGFBP1 expression vectors (IGFBP1- pCMV6 ) purchased from OriGene Technologies, Inc. (Rockville, MD, USA) at a final concentration of 2 μg/mL were transfected into the cells using the lipofectamine 3000 reagent according to the manufacturer’s instructions for up to 24 h, followed by treating with UA for an additional 24 h. In separated experiment, cell were transfected with pEZX-PG04 -IGFBP1 promoter construct linked Gaussia luciferase (GLuc) gene and secreted alkaline phosphatase (SEAP) internal control obtained from GeneCopoeia, Inc. (GeneCopoeia, Inc., Rockville, MD, USA).

    Techniques: Over Expression, Expressing, Transfection, Western Blot, MTT Assay

    In vivo anti-tumor efficacy of UA in subcutaneous HCC tumor-bearing nude mice. Mice (n = 12/group) were divided to 3 groups [Con (saline), Low (L, 25 mg/kg) and High doses of UA (H, 50 mg/kg)], and UA was given daily around the 10 th day after tumor cells injection by gavages for up to 30 days. a , The xenografts were assessed by in vivobioluminescence imaging at the first and the end of the experiments [on day 1 (D 1) and Day 30 (D 30)]. The tumor growth was monitored by injecting luciferin in the mice followed by measuring bioluminescence using IVIS Imaging System. Imaging and quantification of signals were controlled by the acquisition and analysis software living image as described in the Materials and Methods section. Representative images are shown. b and c , The xenografts were harvested on day 30, and the volume and weight of tumors were measured. d , At the end of the experiments, xenografted tumors in each group were isolated and the tumors lysates were processed for detecting IGFBP1, FOXO3a protein and phosphorylation of p38 MAPK by Western blot. GAPDH was used as loading control. The bar graphs represented the tumor weight and volume of mice results as mean ± SD. *Indicates the significant difference from untreated control ( p

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Inter-regulation of IGFBP1 and FOXO3a unveils novel mechanism in ursolic acid-inhibited growth of hepatocellular carcinoma cells

    doi: 10.1186/s13046-016-0330-2

    Figure Lengend Snippet: In vivo anti-tumor efficacy of UA in subcutaneous HCC tumor-bearing nude mice. Mice (n = 12/group) were divided to 3 groups [Con (saline), Low (L, 25 mg/kg) and High doses of UA (H, 50 mg/kg)], and UA was given daily around the 10 th day after tumor cells injection by gavages for up to 30 days. a , The xenografts were assessed by in vivobioluminescence imaging at the first and the end of the experiments [on day 1 (D 1) and Day 30 (D 30)]. The tumor growth was monitored by injecting luciferin in the mice followed by measuring bioluminescence using IVIS Imaging System. Imaging and quantification of signals were controlled by the acquisition and analysis software living image as described in the Materials and Methods section. Representative images are shown. b and c , The xenografts were harvested on day 30, and the volume and weight of tumors were measured. d , At the end of the experiments, xenografted tumors in each group were isolated and the tumors lysates were processed for detecting IGFBP1, FOXO3a protein and phosphorylation of p38 MAPK by Western blot. GAPDH was used as loading control. The bar graphs represented the tumor weight and volume of mice results as mean ± SD. *Indicates the significant difference from untreated control ( p

    Article Snippet: For each well, 2 μg of the desired N1-GFP or FOXO3a-GFP plasmid DNA, kindly provided by Frank M. J. Jacobs (Rudolf Magnus Institute of Neuroscience, Department of Pharmacology and Anatomy, University Medical Center, Utrecht, Netherlands) and was reported previously [ ] and the control (pCMV-6) or IGFBP1 expression vectors (IGFBP1- pCMV6 ) purchased from OriGene Technologies, Inc. (Rockville, MD, USA) at a final concentration of 2 μg/mL were transfected into the cells using the lipofectamine 3000 reagent according to the manufacturer’s instructions for up to 24 h, followed by treating with UA for an additional 24 h. In separated experiment, cell were transfected with pEZX-PG04 -IGFBP1 promoter construct linked Gaussia luciferase (GLuc) gene and secreted alkaline phosphatase (SEAP) internal control obtained from GeneCopoeia, Inc. (GeneCopoeia, Inc., Rockville, MD, USA).

    Techniques: In Vivo, Mouse Assay, Injection, Imaging, Software, Isolation, Western Blot

    UA increased FOXO3a protein expression through activation of p38 MAPK and expression of IGFBP1. a , Bel-7402 and HepG2 cells were exposed to increased concentrations of UA for 24 h. Afterwards, the expression of FOXO3a protein was detected by Western blot. b , Bel-7402 and HepG2 cells were treated with SB203580 (10 μM) for 2 h before exposure of the cells to UA (25 μM) for an additional 24 h. Afterwards, the expression of FOXO3a protein and phoisphorylation of p38 MAPK were detected by Western blot. c , Bel-7402 and HepG2 cells were transfected with control or IGFBP1 siRNAs (50 nM each) for 24 h prior to exposure of the cells to UA (25 μM) for an additional 24 h. Afterwards, FOXO3a and IGFBP1 protein expressions were determined by Western blot, The bars represent the mean ± SD of at least three independent experiments for each condition. *Indicates significant difference as compared to the untreated control group ( P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Inter-regulation of IGFBP1 and FOXO3a unveils novel mechanism in ursolic acid-inhibited growth of hepatocellular carcinoma cells

    doi: 10.1186/s13046-016-0330-2

    Figure Lengend Snippet: UA increased FOXO3a protein expression through activation of p38 MAPK and expression of IGFBP1. a , Bel-7402 and HepG2 cells were exposed to increased concentrations of UA for 24 h. Afterwards, the expression of FOXO3a protein was detected by Western blot. b , Bel-7402 and HepG2 cells were treated with SB203580 (10 μM) for 2 h before exposure of the cells to UA (25 μM) for an additional 24 h. Afterwards, the expression of FOXO3a protein and phoisphorylation of p38 MAPK were detected by Western blot. c , Bel-7402 and HepG2 cells were transfected with control or IGFBP1 siRNAs (50 nM each) for 24 h prior to exposure of the cells to UA (25 μM) for an additional 24 h. Afterwards, FOXO3a and IGFBP1 protein expressions were determined by Western blot, The bars represent the mean ± SD of at least three independent experiments for each condition. *Indicates significant difference as compared to the untreated control group ( P

    Article Snippet: For each well, 2 μg of the desired N1-GFP or FOXO3a-GFP plasmid DNA, kindly provided by Frank M. J. Jacobs (Rudolf Magnus Institute of Neuroscience, Department of Pharmacology and Anatomy, University Medical Center, Utrecht, Netherlands) and was reported previously [ ] and the control (pCMV-6) or IGFBP1 expression vectors (IGFBP1- pCMV6 ) purchased from OriGene Technologies, Inc. (Rockville, MD, USA) at a final concentration of 2 μg/mL were transfected into the cells using the lipofectamine 3000 reagent according to the manufacturer’s instructions for up to 24 h, followed by treating with UA for an additional 24 h. In separated experiment, cell were transfected with pEZX-PG04 -IGFBP1 promoter construct linked Gaussia luciferase (GLuc) gene and secreted alkaline phosphatase (SEAP) internal control obtained from GeneCopoeia, Inc. (GeneCopoeia, Inc., Rockville, MD, USA).

    Techniques: Expressing, Activation Assay, Western Blot, Transfection

    UA induced the protein, mRNA expression, and promoter activity of IGFBP1, which were blocked by SB203580. a - b , HepG2 and Bel-7402 cells were exposed to increased concentrations of UA or UA (25 μM) for 24 h. Afterwards, the expression of IGFBP1 protein ( a ) and mRNA ( b ) were detected by Western blot and qRT-PCR methods as described in the Materials and Methods section. *Indicates significant difference as compared to the untreated control group ( P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Inter-regulation of IGFBP1 and FOXO3a unveils novel mechanism in ursolic acid-inhibited growth of hepatocellular carcinoma cells

    doi: 10.1186/s13046-016-0330-2

    Figure Lengend Snippet: UA induced the protein, mRNA expression, and promoter activity of IGFBP1, which were blocked by SB203580. a - b , HepG2 and Bel-7402 cells were exposed to increased concentrations of UA or UA (25 μM) for 24 h. Afterwards, the expression of IGFBP1 protein ( a ) and mRNA ( b ) were detected by Western blot and qRT-PCR methods as described in the Materials and Methods section. *Indicates significant difference as compared to the untreated control group ( P

    Article Snippet: For each well, 2 μg of the desired N1-GFP or FOXO3a-GFP plasmid DNA, kindly provided by Frank M. J. Jacobs (Rudolf Magnus Institute of Neuroscience, Department of Pharmacology and Anatomy, University Medical Center, Utrecht, Netherlands) and was reported previously [ ] and the control (pCMV-6) or IGFBP1 expression vectors (IGFBP1- pCMV6 ) purchased from OriGene Technologies, Inc. (Rockville, MD, USA) at a final concentration of 2 μg/mL were transfected into the cells using the lipofectamine 3000 reagent according to the manufacturer’s instructions for up to 24 h, followed by treating with UA for an additional 24 h. In separated experiment, cell were transfected with pEZX-PG04 -IGFBP1 promoter construct linked Gaussia luciferase (GLuc) gene and secreted alkaline phosphatase (SEAP) internal control obtained from GeneCopoeia, Inc. (GeneCopoeia, Inc., Rockville, MD, USA).

    Techniques: Expressing, Activity Assay, Western Blot, Quantitative RT-PCR

    Manipulation of PD-L1 modulates EMT status of breast cancer cells. EMT status determined by the expression of CD44, CD24, and vimentin or CD44/CD24 combination as measured by flow cytometry following PD-L1 knockdown using specific Sh-RNA mesenchymal-like (MDA-MB-231) breast cancer cells ( a b ). or PD-L1 overexpression by transfection with PD-L1 ORF in the luminal-like (T47D) breast cancer cells ( c ). Bars in a represent the means of 3 three different clones and 3 different experiments ± standard deviation ( n = 9) while histograms in b c are representative of one of the experiments. Lines in each histograms represent threshold of positivity as determined by isotype control except of CD44 in MDA-MB-231 cells where it is an arbitrary line to show CD44 high status

    Journal: Molecular Cancer

    Article Title: Bidirectional crosstalk between PD-L1 expression and epithelial to mesenchymal transition: Significance in claudin-low breast cancer cells

    doi: 10.1186/s12943-015-0421-2

    Figure Lengend Snippet: Manipulation of PD-L1 modulates EMT status of breast cancer cells. EMT status determined by the expression of CD44, CD24, and vimentin or CD44/CD24 combination as measured by flow cytometry following PD-L1 knockdown using specific Sh-RNA mesenchymal-like (MDA-MB-231) breast cancer cells ( a b ). or PD-L1 overexpression by transfection with PD-L1 ORF in the luminal-like (T47D) breast cancer cells ( c ). Bars in a represent the means of 3 three different clones and 3 different experiments ± standard deviation ( n = 9) while histograms in b c are representative of one of the experiments. Lines in each histograms represent threshold of positivity as determined by isotype control except of CD44 in MDA-MB-231 cells where it is an arbitrary line to show CD44 high status

    Article Snippet: PD-L1 expression in T-47D was induced by transfecting cells with PD-L1 ORF (pCMV6-AC-GFP vector from Origene) and the selection for PD-L1 ORF transfected cells were made using G418 (500 μg/mL).

    Techniques: Expressing, Flow Cytometry, Cytometry, Multiple Displacement Amplification, Over Expression, Transfection, Clone Assay, Standard Deviation