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Addgene inc pcdna3 myr ha akt1 plasmid
Pcdna3 Myr Ha Akt1 Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pcdna3 myr ha akt1 plasmid - by Bioz Stars, 2024-04
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Addgene inc 1036 pcdna3 myr ha akt1
1036 Pcdna3 Myr Ha Akt1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/1036 pcdna3 myr ha akt1/product/Addgene inc
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
1036 pcdna3 myr ha akt1 - by Bioz Stars, 2024-04
86/100 stars

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Structured Review

Addgene inc 1036 pcdna3 myr ha akt1
1036 Pcdna3 Myr Ha Akt1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/1036 pcdna3 myr ha akt1/product/Addgene inc
Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
1036 pcdna3 myr ha akt1 - by Bioz Stars, 2024-04
93/100 stars

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Addgene inc active akt
(A) Human CRC HCT-116 cells were cultured in RPMI-1640 medium with CAPE and CAPPE (at concentrations of 0, 5, 10, 20, 50 and 100 µM) in the presence or absence of compound C (10 µM) for 24 h. Transfections of constitutively active <t>Akt</t> <t>(Myr-Akt1)</t> and empty vector (pcDNA3) were conducted before the treatment of CA derivatives. The cell proliferation was measured by MTT assay as described in . Data are the mean ± SD (standard deviation) of three independent experiments. The different symbols (??? for CAPE and ▵ for CAPPE) represent a statistically significant difference compared to the CA derivative -untreated control group in each group, respectively, at P<0.05. The different symbols (# for CAPE_Akt, § for CAPE_compound C, ▴ for CAPPE_Akt, and ▪ for CAPPE_compound C) represent a statistically significant difference compared to each corresponding CA derivative- treated control group in each dosage subgroup, respectively, at P<0.05. (B–C) Cytoplasmic proteins were prepared for Western blotting analysis using monoclonal antibodies against anti-phosphorylation Akt (S473), total-Akt, anti-phosphorylation AMPKα (T172) and total-AMPKα.
Active Akt, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
active akt - by Bioz Stars, 2024-04
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1) Product Images from "Caffeic Acid Derivatives Inhibit the Growth of Colon Cancer: Involvement of the PI3-K/Akt and AMPK Signaling Pathways"

Article Title: Caffeic Acid Derivatives Inhibit the Growth of Colon Cancer: Involvement of the PI3-K/Akt and AMPK Signaling Pathways

Journal: PLoS ONE

doi: 10.1371/journal.pone.0099631

(A) Human CRC HCT-116 cells were cultured in RPMI-1640 medium with CAPE and CAPPE (at concentrations of 0, 5, 10, 20, 50 and 100 µM) in the presence or absence of compound C (10 µM) for 24 h. Transfections of constitutively active Akt (Myr-Akt1) and empty vector (pcDNA3) were conducted before the treatment of CA derivatives. The cell proliferation was measured by MTT assay as described in . Data are the mean ± SD (standard deviation) of three independent experiments. The different symbols (??? for CAPE and ▵ for CAPPE) represent a statistically significant difference compared to the CA derivative -untreated control group in each group, respectively, at P<0.05. The different symbols (# for CAPE_Akt, § for CAPE_compound C, ▴ for CAPPE_Akt, and ▪ for CAPPE_compound C) represent a statistically significant difference compared to each corresponding CA derivative- treated control group in each dosage subgroup, respectively, at P<0.05. (B–C) Cytoplasmic proteins were prepared for Western blotting analysis using monoclonal antibodies against anti-phosphorylation Akt (S473), total-Akt, anti-phosphorylation AMPKα (T172) and total-AMPKα.
Figure Legend Snippet: (A) Human CRC HCT-116 cells were cultured in RPMI-1640 medium with CAPE and CAPPE (at concentrations of 0, 5, 10, 20, 50 and 100 µM) in the presence or absence of compound C (10 µM) for 24 h. Transfections of constitutively active Akt (Myr-Akt1) and empty vector (pcDNA3) were conducted before the treatment of CA derivatives. The cell proliferation was measured by MTT assay as described in . Data are the mean ± SD (standard deviation) of three independent experiments. The different symbols (??? for CAPE and ▵ for CAPPE) represent a statistically significant difference compared to the CA derivative -untreated control group in each group, respectively, at P<0.05. The different symbols (# for CAPE_Akt, § for CAPE_compound C, ▴ for CAPPE_Akt, and ▪ for CAPPE_compound C) represent a statistically significant difference compared to each corresponding CA derivative- treated control group in each dosage subgroup, respectively, at P<0.05. (B–C) Cytoplasmic proteins were prepared for Western blotting analysis using monoclonal antibodies against anti-phosphorylation Akt (S473), total-Akt, anti-phosphorylation AMPKα (T172) and total-AMPKα.

Techniques Used: Cell Culture, Transfection, Plasmid Preparation, MTT Assay, Standard Deviation, Western Blot

(A) Human CRC SW-480 cells were cultured in RPMI-1640 medium with CAPE and CAPPE (at concentrations of 0, 5, 10, 20, 50 and 100 µM) in the presence or absence of compound C (10 µM) for 24 h. Transfections of constitutively active Akt (Myr-Akt1) and empty vector (pcDNA3) were conducted before the treatment of CA derivatives. The cell proliferation was measured by MTT assay as described in . Data are the mean ± SD (standard deviation) of three independent experiments. The different symbols (??? for CAPE and ▵ for CAPPE) represent a statistically significant difference compared to the CA derivative -untreated control group in each group, respectively, at P<0.05. The different symbols (# for CAPE_Akt, § for CAPE_compound C, ▴ for CAPPE_Akt, and ▪ for CAPPE_compound C) represent a statistically significant difference compared to each corresponding CA derivative- treated control group in each dosage subgroup, respectively, at P<0.05. (B–C) Cytoplasmic proteins were prepared for Western blotting analysis using monoclonal antibodies against anti-phosphorylation Akt (S473), total-Akt, anti-phosphorylation AMPKα (T172) and total-AMPKα.
Figure Legend Snippet: (A) Human CRC SW-480 cells were cultured in RPMI-1640 medium with CAPE and CAPPE (at concentrations of 0, 5, 10, 20, 50 and 100 µM) in the presence or absence of compound C (10 µM) for 24 h. Transfections of constitutively active Akt (Myr-Akt1) and empty vector (pcDNA3) were conducted before the treatment of CA derivatives. The cell proliferation was measured by MTT assay as described in . Data are the mean ± SD (standard deviation) of three independent experiments. The different symbols (??? for CAPE and ▵ for CAPPE) represent a statistically significant difference compared to the CA derivative -untreated control group in each group, respectively, at P<0.05. The different symbols (# for CAPE_Akt, § for CAPE_compound C, ▴ for CAPPE_Akt, and ▪ for CAPPE_compound C) represent a statistically significant difference compared to each corresponding CA derivative- treated control group in each dosage subgroup, respectively, at P<0.05. (B–C) Cytoplasmic proteins were prepared for Western blotting analysis using monoclonal antibodies against anti-phosphorylation Akt (S473), total-Akt, anti-phosphorylation AMPKα (T172) and total-AMPKα.

Techniques Used: Cell Culture, Transfection, Plasmid Preparation, MTT Assay, Standard Deviation, Western Blot


Structured Review

Addgene inc pcdna3 myr ha akt1
A) DLD-1 cells were sorted by flow cytometry and different populations with CD44 positive /CD133 negative (Q1), CD44 positive /CD133 positive (Q2), CD44 negative CD133 negative (Q3), were collected. B) The sorted cells were further analyzed with western blot for total AKT, <t>AKT1</t> or AKT2 and betaactin expression.
Pcdna3 Myr Ha Akt1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
pcdna3 myr ha akt1 - by Bioz Stars, 2024-04
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1) Product Images from "Evaluation of Cancer Stem Cell Markers CD133, CD44, CD24: Association with AKT Isoforms and Radiation Resistance in Colon Cancer Cells"

Article Title: Evaluation of Cancer Stem Cell Markers CD133, CD44, CD24: Association with AKT Isoforms and Radiation Resistance in Colon Cancer Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0094621

A) DLD-1 cells were sorted by flow cytometry and different populations with CD44 positive /CD133 negative (Q1), CD44 positive /CD133 positive (Q2), CD44 negative CD133 negative (Q3), were collected. B) The sorted cells were further analyzed with western blot for total AKT, AKT1 or AKT2 and betaactin expression.
Figure Legend Snippet: A) DLD-1 cells were sorted by flow cytometry and different populations with CD44 positive /CD133 negative (Q1), CD44 positive /CD133 positive (Q2), CD44 negative CD133 negative (Q3), were collected. B) The sorted cells were further analyzed with western blot for total AKT, AKT1 or AKT2 and betaactin expression.

Techniques Used: Flow Cytometry, Western Blot, Expressing


Structured Review

Addgene inc plasmid pcdna3 myr ha akt1
(A) RBP2 is overexpressed in human NSCLC cell lines SK-MES-1, A549, SPCA-1 and H1975 compared to the human bronchial epithelial cell line BEAS2B cells. (B) Effects of RBP2 siRNA1, RBP2 siRNA2 and <t>pcDNA3-HA-RBP2</t> on the expression of the RBP2 protein.
Plasmid Pcdna3 Myr Ha Akt1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/plasmid pcdna3 myr ha akt1/product/Addgene inc
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plasmid pcdna3 myr ha akt1 - by Bioz Stars, 2024-04
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1) Product Images from "Retinoblastoma Binding Protein 2 (RBP2) Promotes HIF-1α–VEGF-Induced Angiogenesis of Non-Small Cell Lung Cancer via the Akt Pathway"

Article Title: Retinoblastoma Binding Protein 2 (RBP2) Promotes HIF-1α–VEGF-Induced Angiogenesis of Non-Small Cell Lung Cancer via the Akt Pathway

Journal: PLoS ONE

doi: 10.1371/journal.pone.0106032

(A) RBP2 is overexpressed in human NSCLC cell lines SK-MES-1, A549, SPCA-1 and H1975 compared to the human bronchial epithelial cell line BEAS2B cells. (B) Effects of RBP2 siRNA1, RBP2 siRNA2 and pcDNA3-HA-RBP2 on the expression of the RBP2 protein.
Figure Legend Snippet: (A) RBP2 is overexpressed in human NSCLC cell lines SK-MES-1, A549, SPCA-1 and H1975 compared to the human bronchial epithelial cell line BEAS2B cells. (B) Effects of RBP2 siRNA1, RBP2 siRNA2 and pcDNA3-HA-RBP2 on the expression of the RBP2 protein.

Techniques Used: Expressing

(A) Silencing RBP2 expression in H1975 cells significantly decreased the phosphorylation of Akt, and the forced expression of RBP2 in SK-MES-1 cells increased the activity of Akt. (B) When Akt was constitutively activated in RBP2-siRNA2 H1975, the expression of HIF-1α and VEGF were increased compared to the control. The PI3K/Akt inhibitor LY294002 significantly inhibited the expression of HIF-1α and VEGF in pcDNA3-HA-RBP2 SK-MES-1 cells. (C) Westernblots showing the time course of Akt phosphorylation in RBP2-siRNA2 H1975 cells due to VEGF-165 (25 ng/mL). (D) In the presence of recombinant human VEGF-165 stimulation, the activation of Akt was increased in RBP2-siRNA2 H1975 cells and RBP2-overexpressing SK-MES-1 cells (25 ng/mL, 30 minutes).
Figure Legend Snippet: (A) Silencing RBP2 expression in H1975 cells significantly decreased the phosphorylation of Akt, and the forced expression of RBP2 in SK-MES-1 cells increased the activity of Akt. (B) When Akt was constitutively activated in RBP2-siRNA2 H1975, the expression of HIF-1α and VEGF were increased compared to the control. The PI3K/Akt inhibitor LY294002 significantly inhibited the expression of HIF-1α and VEGF in pcDNA3-HA-RBP2 SK-MES-1 cells. (C) Westernblots showing the time course of Akt phosphorylation in RBP2-siRNA2 H1975 cells due to VEGF-165 (25 ng/mL). (D) In the presence of recombinant human VEGF-165 stimulation, the activation of Akt was increased in RBP2-siRNA2 H1975 cells and RBP2-overexpressing SK-MES-1 cells (25 ng/mL, 30 minutes).

Techniques Used: Expressing, Activity Assay, Recombinant, Activation Assay


Structured Review

Addgene inc pcdna3 myr ha akt1
A, <t>AKT1,</t> 2, 3 and actin as control mRNA expression in A549 and REN cells was evaluated by RT-PCR as reported in in “ ” section. B , AKT1 and 3 protein expression was evaluated by Western blot analysis in REN cells with isoform specific antibodies, tubulin blot confirmed equal loading. Representative of three independent experiments. C , REN cells were transfected with 1 µg/plate of plasmid coding for the constitutive activated form of AKT, Myr-AKT1 or 3. 24 hours post transfection REN cells were treated with 23 µM perifosine for 1 hour, then were lysed and equal amounts of proteins were subjected to SDS-PAGE followed by immuno-blotting with specific antibodies to phosphorylated AKT, HA to confirm transfection and tubulin for equal loading. D , The effect of perifosine on cells transfected with Myr- AKT1 or 3 was also assessed on cell proliferation. REN cells were transfected with 1 µg/plate of plasmid coding Myr-AKT1 or 3 and 24 hours post transfection were treated with 23 µM perifosine for 24 hours. At the end of treatment viable cell count was determined. Points , means ± SD of three individual measurements. Perifosine markedly alters AKT membrane translocation, so we next asked whether forced expression of an exogenous Myr-AKT1 or Myr-AKT3 could abrogate the effect of perifosine on AKT activating phosphorylations and cell growth. Contradictory data are available in literature on the capability of a constitutively active AKT to overcome perifosine action. While in prostate cancer cells inhibition of AKT phosphorylation was substantially relieved by introduction of a Myr-AKT1, which bypassed the requirement for PH domain-mediated membrane recruitment, in myeloma cells it has been described that perifosine overcome constitutive highly active AKT1 , . We transiently transfected REN cells either with an HA-tagged Myr-AKT1 or Myr-AKT3 and treated them for 24 hours with the IC50 dose of perifosine. Western-blot analysis reported in shows that transfection of both Myr-AKT1 and Myr-AKT3 overcomes perifosine inhibition of AKT phosphorylation in REN cells; only a slight reduction of phosphorylation, probably due to the block of the endogenous proteins, was observed. Cell growth analysis reported in demonstrates that both transfected Myr-AKTs rescued perifosine induced block of cell proliferation.
Pcdna3 Myr Ha Akt1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pcdna3 myr ha akt1/product/Addgene inc
Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
pcdna3 myr ha akt1 - by Bioz Stars, 2024-04
93/100 stars

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1) Product Images from "Perifosine as a Potential Novel Anti-Cancer Agent Inhibits EGFR/MET-AKT Axis in Malignant Pleural Mesothelioma"

Article Title: Perifosine as a Potential Novel Anti-Cancer Agent Inhibits EGFR/MET-AKT Axis in Malignant Pleural Mesothelioma

Journal: PLoS ONE

doi: 10.1371/journal.pone.0036856

A, AKT1, 2, 3 and actin as control mRNA expression in A549 and REN cells was evaluated by RT-PCR as reported in in “ ” section. B , AKT1 and 3 protein expression was evaluated by Western blot analysis in REN cells with isoform specific antibodies, tubulin blot confirmed equal loading. Representative of three independent experiments. C , REN cells were transfected with 1 µg/plate of plasmid coding for the constitutive activated form of AKT, Myr-AKT1 or 3. 24 hours post transfection REN cells were treated with 23 µM perifosine for 1 hour, then were lysed and equal amounts of proteins were subjected to SDS-PAGE followed by immuno-blotting with specific antibodies to phosphorylated AKT, HA to confirm transfection and tubulin for equal loading. D , The effect of perifosine on cells transfected with Myr- AKT1 or 3 was also assessed on cell proliferation. REN cells were transfected with 1 µg/plate of plasmid coding Myr-AKT1 or 3 and 24 hours post transfection were treated with 23 µM perifosine for 24 hours. At the end of treatment viable cell count was determined. Points , means ± SD of three individual measurements. Perifosine markedly alters AKT membrane translocation, so we next asked whether forced expression of an exogenous Myr-AKT1 or Myr-AKT3 could abrogate the effect of perifosine on AKT activating phosphorylations and cell growth. Contradictory data are available in literature on the capability of a constitutively active AKT to overcome perifosine action. While in prostate cancer cells inhibition of AKT phosphorylation was substantially relieved by introduction of a Myr-AKT1, which bypassed the requirement for PH domain-mediated membrane recruitment, in myeloma cells it has been described that perifosine overcome constitutive highly active AKT1 , . We transiently transfected REN cells either with an HA-tagged Myr-AKT1 or Myr-AKT3 and treated them for 24 hours with the IC50 dose of perifosine. Western-blot analysis reported in shows that transfection of both Myr-AKT1 and Myr-AKT3 overcomes perifosine inhibition of AKT phosphorylation in REN cells; only a slight reduction of phosphorylation, probably due to the block of the endogenous proteins, was observed. Cell growth analysis reported in demonstrates that both transfected Myr-AKTs rescued perifosine induced block of cell proliferation.
Figure Legend Snippet: A, AKT1, 2, 3 and actin as control mRNA expression in A549 and REN cells was evaluated by RT-PCR as reported in in “ ” section. B , AKT1 and 3 protein expression was evaluated by Western blot analysis in REN cells with isoform specific antibodies, tubulin blot confirmed equal loading. Representative of three independent experiments. C , REN cells were transfected with 1 µg/plate of plasmid coding for the constitutive activated form of AKT, Myr-AKT1 or 3. 24 hours post transfection REN cells were treated with 23 µM perifosine for 1 hour, then were lysed and equal amounts of proteins were subjected to SDS-PAGE followed by immuno-blotting with specific antibodies to phosphorylated AKT, HA to confirm transfection and tubulin for equal loading. D , The effect of perifosine on cells transfected with Myr- AKT1 or 3 was also assessed on cell proliferation. REN cells were transfected with 1 µg/plate of plasmid coding Myr-AKT1 or 3 and 24 hours post transfection were treated with 23 µM perifosine for 24 hours. At the end of treatment viable cell count was determined. Points , means ± SD of three individual measurements. Perifosine markedly alters AKT membrane translocation, so we next asked whether forced expression of an exogenous Myr-AKT1 or Myr-AKT3 could abrogate the effect of perifosine on AKT activating phosphorylations and cell growth. Contradictory data are available in literature on the capability of a constitutively active AKT to overcome perifosine action. While in prostate cancer cells inhibition of AKT phosphorylation was substantially relieved by introduction of a Myr-AKT1, which bypassed the requirement for PH domain-mediated membrane recruitment, in myeloma cells it has been described that perifosine overcome constitutive highly active AKT1 , . We transiently transfected REN cells either with an HA-tagged Myr-AKT1 or Myr-AKT3 and treated them for 24 hours with the IC50 dose of perifosine. Western-blot analysis reported in shows that transfection of both Myr-AKT1 and Myr-AKT3 overcomes perifosine inhibition of AKT phosphorylation in REN cells; only a slight reduction of phosphorylation, probably due to the block of the endogenous proteins, was observed. Cell growth analysis reported in demonstrates that both transfected Myr-AKTs rescued perifosine induced block of cell proliferation.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Plasmid Preparation, SDS Page, Cell Counting, Translocation Assay, Inhibition, Blocking Assay


Structured Review

Addgene inc pcdna3 myr ha akt1
In A. SiHa and CaSki cell lines 48 hours after transient transfection of OGDHL and empty vector; and B. HeLa and ME180 cell lines after OGDHL siRNA and scramble siRNA transfection; OGDHL inhibition by OGDHL siRNA has dramatic effect on total AKT and phospho-AKT level C. Immunoblotting analysis of phospo-NF-κB and total-NF-κB of SiHa cell lines 48 hours after OGDHL and empty vector transfection; β-actin and lamin was used as a loading control for cytosolic and nuclear fraction respectively; NF-κB translocation from cytoplasm towards the nucleus was decreased after OGDHL overexpression in SiHa cells; D. NF-κB gel shift assay in SiHa and HeLa cell lines; OGDHL overexpression in SiHa cells decreased the DNA binding activity of NF-κB compared to that of empty vector control; OGDHL siRNA increase the binding activity of NF-κB in HeLa cell; E. NF-κB luciferase assay in SiHa (upper panel) and CaSki (lower panel) cell lines after transient over-expression of OGDHL with or without <t>AKT1</t> over-expression. It is evident that OGDHL suppresses AKT activity and lead to inhibition of NF-κB-dependent gene transcription (* p<0.001). F. Immunoblotting analysis of phospho- NF-κB, total- NF-κB, β-actin and lamin in nuclear and cytosolic fractions of SiHa cell lines after OGDHL over-expression with or without AKT1 overexpression. The data showed that co-transfection of OGDHL and AKT1 partially increased the translocation of NF-κB from the cytoplasm to the nucleus.
Pcdna3 Myr Ha Akt1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pcdna3 myr ha akt1 - by Bioz Stars, 2024-04
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1) Product Images from "OGDHL Is a Modifier of AKT-Dependent Signaling and NF-κB Function"

Article Title: OGDHL Is a Modifier of AKT-Dependent Signaling and NF-κB Function

Journal: PLoS ONE

doi: 10.1371/journal.pone.0048770

In A. SiHa and CaSki cell lines 48 hours after transient transfection of OGDHL and empty vector; and B. HeLa and ME180 cell lines after OGDHL siRNA and scramble siRNA transfection; OGDHL inhibition by OGDHL siRNA has dramatic effect on total AKT and phospho-AKT level C. Immunoblotting analysis of phospo-NF-κB and total-NF-κB of SiHa cell lines 48 hours after OGDHL and empty vector transfection; β-actin and lamin was used as a loading control for cytosolic and nuclear fraction respectively; NF-κB translocation from cytoplasm towards the nucleus was decreased after OGDHL overexpression in SiHa cells; D. NF-κB gel shift assay in SiHa and HeLa cell lines; OGDHL overexpression in SiHa cells decreased the DNA binding activity of NF-κB compared to that of empty vector control; OGDHL siRNA increase the binding activity of NF-κB in HeLa cell; E. NF-κB luciferase assay in SiHa (upper panel) and CaSki (lower panel) cell lines after transient over-expression of OGDHL with or without AKT1 over-expression. It is evident that OGDHL suppresses AKT activity and lead to inhibition of NF-κB-dependent gene transcription (* p<0.001). F. Immunoblotting analysis of phospho- NF-κB, total- NF-κB, β-actin and lamin in nuclear and cytosolic fractions of SiHa cell lines after OGDHL over-expression with or without AKT1 overexpression. The data showed that co-transfection of OGDHL and AKT1 partially increased the translocation of NF-κB from the cytoplasm to the nucleus.
Figure Legend Snippet: In A. SiHa and CaSki cell lines 48 hours after transient transfection of OGDHL and empty vector; and B. HeLa and ME180 cell lines after OGDHL siRNA and scramble siRNA transfection; OGDHL inhibition by OGDHL siRNA has dramatic effect on total AKT and phospho-AKT level C. Immunoblotting analysis of phospo-NF-κB and total-NF-κB of SiHa cell lines 48 hours after OGDHL and empty vector transfection; β-actin and lamin was used as a loading control for cytosolic and nuclear fraction respectively; NF-κB translocation from cytoplasm towards the nucleus was decreased after OGDHL overexpression in SiHa cells; D. NF-κB gel shift assay in SiHa and HeLa cell lines; OGDHL overexpression in SiHa cells decreased the DNA binding activity of NF-κB compared to that of empty vector control; OGDHL siRNA increase the binding activity of NF-κB in HeLa cell; E. NF-κB luciferase assay in SiHa (upper panel) and CaSki (lower panel) cell lines after transient over-expression of OGDHL with or without AKT1 over-expression. It is evident that OGDHL suppresses AKT activity and lead to inhibition of NF-κB-dependent gene transcription (* p<0.001). F. Immunoblotting analysis of phospho- NF-κB, total- NF-κB, β-actin and lamin in nuclear and cytosolic fractions of SiHa cell lines after OGDHL over-expression with or without AKT1 overexpression. The data showed that co-transfection of OGDHL and AKT1 partially increased the translocation of NF-κB from the cytoplasm to the nucleus.

Techniques Used: Transfection, Plasmid Preparation, Inhibition, Western Blot, Translocation Assay, Over Expression, Electrophoretic Mobility Shift Assay, Binding Assay, Activity Assay, Luciferase, Cotransfection

A. Immunoblotting analysis of total-AKT (T-AKT), total NF-κB (T -NF-κB), pro and cleaved caspase 3, cleaved PARP1/2 in SiHa cell line 48 hours after OGDHL over-expression in the presence or absence of ROS (N-acetyl l-cysteine or NAC, 2.5 mM), Lipid-peroxidation (butylated hydroxytoluene or BHT, 0.2 mM) and caspase 3 (DEVD-FMK, 25 µM) inhibitor. β-actin was used as a loading control. All the inhibitors protect AKT from getting cleaved. ROS and lipid peroxidation inhibitors have protective effect on caspase 3 activation; B. MTT assay of SiHa cell line 48 hours after OGDHL or AKT1 over-expression in the presence or absence of NAC (ROS inhibitor), BHT (Lipid peroxidation inhibitor) or DEVD-FMK (caspase 3 inhibitor); NAC, BHT and DEVD-FMK significantly prevented cell death induced by OGDHL; C. MTT assay of HeLa cell line after OGDHL knockdown with or without AKT1 knockdown. AKT1 down-regulation by AKT1 siRNA decreased HeLa cell survival that was induced by knockdown of OGDHL (* P <0.05).
Figure Legend Snippet: A. Immunoblotting analysis of total-AKT (T-AKT), total NF-κB (T -NF-κB), pro and cleaved caspase 3, cleaved PARP1/2 in SiHa cell line 48 hours after OGDHL over-expression in the presence or absence of ROS (N-acetyl l-cysteine or NAC, 2.5 mM), Lipid-peroxidation (butylated hydroxytoluene or BHT, 0.2 mM) and caspase 3 (DEVD-FMK, 25 µM) inhibitor. β-actin was used as a loading control. All the inhibitors protect AKT from getting cleaved. ROS and lipid peroxidation inhibitors have protective effect on caspase 3 activation; B. MTT assay of SiHa cell line 48 hours after OGDHL or AKT1 over-expression in the presence or absence of NAC (ROS inhibitor), BHT (Lipid peroxidation inhibitor) or DEVD-FMK (caspase 3 inhibitor); NAC, BHT and DEVD-FMK significantly prevented cell death induced by OGDHL; C. MTT assay of HeLa cell line after OGDHL knockdown with or without AKT1 knockdown. AKT1 down-regulation by AKT1 siRNA decreased HeLa cell survival that was induced by knockdown of OGDHL (* P <0.05).

Techniques Used: Western Blot, Over Expression, Activation Assay, MTT Assay

A. RT-PCR analysis of SiHa cells stably transfected with the mammalian expression vector pcDNA3 with full-length OGDHL cDNA and empty pcDNA3 vector (mock). All the three OGDHL clones (clone number at the bottom) containing full-length OGDHL cDNA expressed OGDHL at various levels. There is a very low level endogenous OGDHL expression in mock-transfected clones. B. Immunoblot analysis of Flag, OGDHL and β-actin. All the three OGDHL over-expressing clones showing Flag tagged with OGDHL and OGDHL expression while no Flag-OGDHL and OGDHL expression in empty vector stable clones (clone number at the bottom). C. Soft agar assay: number of soft agar colonies was significantly lower in the OGDHL over-expressed cells compared with empty vector-transfected cells ( *P <0.001) (Bottom). Magnification, ×100 in representative photograph (Top). D. Invasion assay: Compared with empty vector, stable OGDHL over-expressing SiHa cell clone showed significantly less number of invading cells (* P <0.001) (right). Magnification, ×100 in representative photograph (left. Each experiment was repeated twice. In general all the findings are consistent with the transient transfection data; E. Immunoblotting analysis of phospho-AKT, total-AKT, phospho-PI3K, total-NF-κB and β-actin in empty vector (left lane) and OGDHL over-expressing stable SiHa cell clone (right lane) and the findings are similar to transient transfection. F. NF-κB gel shift assay in SiHa cell lines stably transfected with empty vector or vector containing OGDHL-Flag indicate that OGDHL inhibited the binding of NF-κB to the nuclear DNA. G. Inhibition of OGDHL mediated ROS generation by over-expression of Mn-SOD increases the viable cells determined by MTT assay (* P <0.05) (left panel) and this increase number of cells is partially due to decrease ROS production as shown in right panel (* P <0.05).
Figure Legend Snippet: A. RT-PCR analysis of SiHa cells stably transfected with the mammalian expression vector pcDNA3 with full-length OGDHL cDNA and empty pcDNA3 vector (mock). All the three OGDHL clones (clone number at the bottom) containing full-length OGDHL cDNA expressed OGDHL at various levels. There is a very low level endogenous OGDHL expression in mock-transfected clones. B. Immunoblot analysis of Flag, OGDHL and β-actin. All the three OGDHL over-expressing clones showing Flag tagged with OGDHL and OGDHL expression while no Flag-OGDHL and OGDHL expression in empty vector stable clones (clone number at the bottom). C. Soft agar assay: number of soft agar colonies was significantly lower in the OGDHL over-expressed cells compared with empty vector-transfected cells ( *P <0.001) (Bottom). Magnification, ×100 in representative photograph (Top). D. Invasion assay: Compared with empty vector, stable OGDHL over-expressing SiHa cell clone showed significantly less number of invading cells (* P <0.001) (right). Magnification, ×100 in representative photograph (left. Each experiment was repeated twice. In general all the findings are consistent with the transient transfection data; E. Immunoblotting analysis of phospho-AKT, total-AKT, phospho-PI3K, total-NF-κB and β-actin in empty vector (left lane) and OGDHL over-expressing stable SiHa cell clone (right lane) and the findings are similar to transient transfection. F. NF-κB gel shift assay in SiHa cell lines stably transfected with empty vector or vector containing OGDHL-Flag indicate that OGDHL inhibited the binding of NF-κB to the nuclear DNA. G. Inhibition of OGDHL mediated ROS generation by over-expression of Mn-SOD increases the viable cells determined by MTT assay (* P <0.05) (left panel) and this increase number of cells is partially due to decrease ROS production as shown in right panel (* P <0.05).

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Clone Assay, Western Blot, Soft Agar Assay, Invasion Assay, Electrophoretic Mobility Shift Assay, Binding Assay, Inhibition, Over Expression, MTT Assay


Structured Review

Addgene inc myr akt
( A ) OCI-LY3 cells were treated with indicated concentrations of PCI-24781 for 6 or 16 hr followed by cell lysis and western blotting for indicated proteins. ( B ) SUDHL4 and OCI-LY3 cells were pre-incubated with PD98059 (PD) or LY294002 (LY) for 2hr followed by 48 hr. incubation with PCI-24781. Cells were stained with AnnexinV/PI and analyzed by flow cytometry. ( C ) Malignant B- cells obtained from a CLL patient blood sample using Histopaque were incubated with PCI for 44 hr followed by Annexin V/PI staining and analyzed by flowcytometry. ( D ) SUDHL6 and OCI-LY3 cells were transfected with <t>Myr-Akt</t> or empty vector using Amaxa kit V and L respectively followed by selection in neomycin for one week. OCI-LY3 Cells were analyzed by western blotting for constitutive activation of AKT by western blotting and incubated with PCI-24781 for 48 hr followed by annexinV/PI staining and analyzed by flow cytometry. ( E ) SUDHL6 expressing constitutively active AKT (Myr-Akt) and wt -AKT were incubated with indicated concentrations of PCI-24781, SAHA and TSA for 48 hr followed by Annexin V/PI staining and analyzed by flow cytometry. (ns= not significant, * = P<0.05; **= p<0.01; *** = P<0.001).
Myr Akt, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Paradoxical Regulation of Hypoxia Inducible Factor-1α (HIF-1α) by Histone Deacetylase Inhibitor in Diffuse Large B-Cell Lymphoma"

Article Title: Paradoxical Regulation of Hypoxia Inducible Factor-1α (HIF-1α) by Histone Deacetylase Inhibitor in Diffuse Large B-Cell Lymphoma

Journal: PLoS ONE

doi: 10.1371/journal.pone.0081333

( A ) OCI-LY3 cells were treated with indicated concentrations of PCI-24781 for 6 or 16 hr followed by cell lysis and western blotting for indicated proteins. ( B ) SUDHL4 and OCI-LY3 cells were pre-incubated with PD98059 (PD) or LY294002 (LY) for 2hr followed by 48 hr. incubation with PCI-24781. Cells were stained with AnnexinV/PI and analyzed by flow cytometry. ( C ) Malignant B- cells obtained from a CLL patient blood sample using Histopaque were incubated with PCI for 44 hr followed by Annexin V/PI staining and analyzed by flowcytometry. ( D ) SUDHL6 and OCI-LY3 cells were transfected with Myr-Akt or empty vector using Amaxa kit V and L respectively followed by selection in neomycin for one week. OCI-LY3 Cells were analyzed by western blotting for constitutive activation of AKT by western blotting and incubated with PCI-24781 for 48 hr followed by annexinV/PI staining and analyzed by flow cytometry. ( E ) SUDHL6 expressing constitutively active AKT (Myr-Akt) and wt -AKT were incubated with indicated concentrations of PCI-24781, SAHA and TSA for 48 hr followed by Annexin V/PI staining and analyzed by flow cytometry. (ns= not significant, * = P<0.05; **= p<0.01; *** = P<0.001).
Figure Legend Snippet: ( A ) OCI-LY3 cells were treated with indicated concentrations of PCI-24781 for 6 or 16 hr followed by cell lysis and western blotting for indicated proteins. ( B ) SUDHL4 and OCI-LY3 cells were pre-incubated with PD98059 (PD) or LY294002 (LY) for 2hr followed by 48 hr. incubation with PCI-24781. Cells were stained with AnnexinV/PI and analyzed by flow cytometry. ( C ) Malignant B- cells obtained from a CLL patient blood sample using Histopaque were incubated with PCI for 44 hr followed by Annexin V/PI staining and analyzed by flowcytometry. ( D ) SUDHL6 and OCI-LY3 cells were transfected with Myr-Akt or empty vector using Amaxa kit V and L respectively followed by selection in neomycin for one week. OCI-LY3 Cells were analyzed by western blotting for constitutive activation of AKT by western blotting and incubated with PCI-24781 for 48 hr followed by annexinV/PI staining and analyzed by flow cytometry. ( E ) SUDHL6 expressing constitutively active AKT (Myr-Akt) and wt -AKT were incubated with indicated concentrations of PCI-24781, SAHA and TSA for 48 hr followed by Annexin V/PI staining and analyzed by flow cytometry. (ns= not significant, * = P<0.05; **= p<0.01; *** = P<0.001).

Techniques Used: Lysis, Western Blot, Incubation, Staining, Flow Cytometry, Transfection, Plasmid Preparation, Selection, Activation Assay, Expressing

( A ) To check the expression of HIF-1α in SUDHL6 and OCI-LY3 cells expressing Myr-Akt and wt-Akt, cells were subjected to lysis followed by western blotting using HIF-1α antibody. ( B ) SUDHL4-and OCI-LY3 cells were incubated with indicated concentration of LY294002 (LY) for 24 hr followed by cell lysis and Western blotting using HIF-1α antibody. ( C ) Malignant cells obtained from blood sample of CLL patient were incubated with indicated concentration of PCI or LY alone or in combination for 16 hr followed by cell lysis and western blotting using specific antibodies as indicated. ( D ) SUDHL4 and OCI-LY3 cells were treated with PCI and LY either alone or in combination for 24 hr followed by cell lysis and western blotting using specific antibodies for pAkt and Akt. Actin is used as an internal control.
Figure Legend Snippet: ( A ) To check the expression of HIF-1α in SUDHL6 and OCI-LY3 cells expressing Myr-Akt and wt-Akt, cells were subjected to lysis followed by western blotting using HIF-1α antibody. ( B ) SUDHL4-and OCI-LY3 cells were incubated with indicated concentration of LY294002 (LY) for 24 hr followed by cell lysis and Western blotting using HIF-1α antibody. ( C ) Malignant cells obtained from blood sample of CLL patient were incubated with indicated concentration of PCI or LY alone or in combination for 16 hr followed by cell lysis and western blotting using specific antibodies as indicated. ( D ) SUDHL4 and OCI-LY3 cells were treated with PCI and LY either alone or in combination for 24 hr followed by cell lysis and western blotting using specific antibodies for pAkt and Akt. Actin is used as an internal control.

Techniques Used: Expressing, Lysis, Western Blot, Incubation, Concentration Assay


Structured Review

Addgene inc pcdna3 myr ha akt1 template
Pcdna3 Myr Ha Akt1 Template, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
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    Addgene inc pcdna3 myr ha akt1 plasmid
    Pcdna3 Myr Ha Akt1 Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc active akt
    (A) Human CRC HCT-116 cells were cultured in RPMI-1640 medium with CAPE and CAPPE (at concentrations of 0, 5, 10, 20, 50 and 100 µM) in the presence or absence of compound C (10 µM) for 24 h. Transfections of constitutively active <t>Akt</t> <t>(Myr-Akt1)</t> and empty vector (pcDNA3) were conducted before the treatment of CA derivatives. The cell proliferation was measured by MTT assay as described in . Data are the mean ± SD (standard deviation) of three independent experiments. The different symbols (??? for CAPE and ▵ for CAPPE) represent a statistically significant difference compared to the CA derivative -untreated control group in each group, respectively, at P<0.05. The different symbols (# for CAPE_Akt, § for CAPE_compound C, ▴ for CAPPE_Akt, and ▪ for CAPPE_compound C) represent a statistically significant difference compared to each corresponding CA derivative- treated control group in each dosage subgroup, respectively, at P<0.05. (B–C) Cytoplasmic proteins were prepared for Western blotting analysis using monoclonal antibodies against anti-phosphorylation Akt (S473), total-Akt, anti-phosphorylation AMPKα (T172) and total-AMPKα.
    Active Akt, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    A) DLD-1 cells were sorted by flow cytometry and different populations with CD44 positive /CD133 negative (Q1), CD44 positive /CD133 positive (Q2), CD44 negative CD133 negative (Q3), were collected. B) The sorted cells were further analyzed with western blot for total AKT, <t>AKT1</t> or AKT2 and betaactin expression.
    Pcdna3 Myr Ha Akt1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (A) RBP2 is overexpressed in human NSCLC cell lines SK-MES-1, A549, SPCA-1 and H1975 compared to the human bronchial epithelial cell line BEAS2B cells. (B) Effects of RBP2 siRNA1, RBP2 siRNA2 and <t>pcDNA3-HA-RBP2</t> on the expression of the RBP2 protein.
    Plasmid Pcdna3 Myr Ha Akt1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc myr akt
    ( A ) OCI-LY3 cells were treated with indicated concentrations of PCI-24781 for 6 or 16 hr followed by cell lysis and western blotting for indicated proteins. ( B ) SUDHL4 and OCI-LY3 cells were pre-incubated with PD98059 (PD) or LY294002 (LY) for 2hr followed by 48 hr. incubation with PCI-24781. Cells were stained with AnnexinV/PI and analyzed by flow cytometry. ( C ) Malignant B- cells obtained from a CLL patient blood sample using Histopaque were incubated with PCI for 44 hr followed by Annexin V/PI staining and analyzed by flowcytometry. ( D ) SUDHL6 and OCI-LY3 cells were transfected with <t>Myr-Akt</t> or empty vector using Amaxa kit V and L respectively followed by selection in neomycin for one week. OCI-LY3 Cells were analyzed by western blotting for constitutive activation of AKT by western blotting and incubated with PCI-24781 for 48 hr followed by annexinV/PI staining and analyzed by flow cytometry. ( E ) SUDHL6 expressing constitutively active AKT (Myr-Akt) and wt -AKT were incubated with indicated concentrations of PCI-24781, SAHA and TSA for 48 hr followed by Annexin V/PI staining and analyzed by flow cytometry. (ns= not significant, * = P<0.05; **= p<0.01; *** = P<0.001).
    Myr Akt, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/myr akt/product/Addgene inc
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    Addgene inc pcdna3 myr ha akt1 template
    ( A ) OCI-LY3 cells were treated with indicated concentrations of PCI-24781 for 6 or 16 hr followed by cell lysis and western blotting for indicated proteins. ( B ) SUDHL4 and OCI-LY3 cells were pre-incubated with PD98059 (PD) or LY294002 (LY) for 2hr followed by 48 hr. incubation with PCI-24781. Cells were stained with AnnexinV/PI and analyzed by flow cytometry. ( C ) Malignant B- cells obtained from a CLL patient blood sample using Histopaque were incubated with PCI for 44 hr followed by Annexin V/PI staining and analyzed by flowcytometry. ( D ) SUDHL6 and OCI-LY3 cells were transfected with <t>Myr-Akt</t> or empty vector using Amaxa kit V and L respectively followed by selection in neomycin for one week. OCI-LY3 Cells were analyzed by western blotting for constitutive activation of AKT by western blotting and incubated with PCI-24781 for 48 hr followed by annexinV/PI staining and analyzed by flow cytometry. ( E ) SUDHL6 expressing constitutively active AKT (Myr-Akt) and wt -AKT were incubated with indicated concentrations of PCI-24781, SAHA and TSA for 48 hr followed by Annexin V/PI staining and analyzed by flow cytometry. (ns= not significant, * = P<0.05; **= p<0.01; *** = P<0.001).
    Pcdna3 Myr Ha Akt1 Template, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (A) Human CRC HCT-116 cells were cultured in RPMI-1640 medium with CAPE and CAPPE (at concentrations of 0, 5, 10, 20, 50 and 100 µM) in the presence or absence of compound C (10 µM) for 24 h. Transfections of constitutively active Akt (Myr-Akt1) and empty vector (pcDNA3) were conducted before the treatment of CA derivatives. The cell proliferation was measured by MTT assay as described in . Data are the mean ± SD (standard deviation) of three independent experiments. The different symbols (??? for CAPE and ▵ for CAPPE) represent a statistically significant difference compared to the CA derivative -untreated control group in each group, respectively, at P<0.05. The different symbols (# for CAPE_Akt, § for CAPE_compound C, ▴ for CAPPE_Akt, and ▪ for CAPPE_compound C) represent a statistically significant difference compared to each corresponding CA derivative- treated control group in each dosage subgroup, respectively, at P<0.05. (B–C) Cytoplasmic proteins were prepared for Western blotting analysis using monoclonal antibodies against anti-phosphorylation Akt (S473), total-Akt, anti-phosphorylation AMPKα (T172) and total-AMPKα.

    Journal: PLoS ONE

    Article Title: Caffeic Acid Derivatives Inhibit the Growth of Colon Cancer: Involvement of the PI3-K/Akt and AMPK Signaling Pathways

    doi: 10.1371/journal.pone.0099631

    Figure Lengend Snippet: (A) Human CRC HCT-116 cells were cultured in RPMI-1640 medium with CAPE and CAPPE (at concentrations of 0, 5, 10, 20, 50 and 100 µM) in the presence or absence of compound C (10 µM) for 24 h. Transfections of constitutively active Akt (Myr-Akt1) and empty vector (pcDNA3) were conducted before the treatment of CA derivatives. The cell proliferation was measured by MTT assay as described in . Data are the mean ± SD (standard deviation) of three independent experiments. The different symbols (??? for CAPE and ▵ for CAPPE) represent a statistically significant difference compared to the CA derivative -untreated control group in each group, respectively, at P<0.05. The different symbols (# for CAPE_Akt, § for CAPE_compound C, ▴ for CAPPE_Akt, and ▪ for CAPPE_compound C) represent a statistically significant difference compared to each corresponding CA derivative- treated control group in each dosage subgroup, respectively, at P<0.05. (B–C) Cytoplasmic proteins were prepared for Western blotting analysis using monoclonal antibodies against anti-phosphorylation Akt (S473), total-Akt, anti-phosphorylation AMPKα (T172) and total-AMPKα.

    Article Snippet: After 24 h, the culture medium was replaced by media containing CA derivatives at one of five concentrations (i.e.,0, 5, 10, 20, 50 and 100 µM) in the presence or absence of compound C. Transfections of constitutively active Akt (Myr-Akt1, Addgene plasmid 9008) and empty vector (pcDNA3, Addgene plasmid 10792) were conducted by using Lipofectamine LTX transfection reagent.

    Techniques: Cell Culture, Transfection, Plasmid Preparation, MTT Assay, Standard Deviation, Western Blot

    (A) Human CRC SW-480 cells were cultured in RPMI-1640 medium with CAPE and CAPPE (at concentrations of 0, 5, 10, 20, 50 and 100 µM) in the presence or absence of compound C (10 µM) for 24 h. Transfections of constitutively active Akt (Myr-Akt1) and empty vector (pcDNA3) were conducted before the treatment of CA derivatives. The cell proliferation was measured by MTT assay as described in . Data are the mean ± SD (standard deviation) of three independent experiments. The different symbols (??? for CAPE and ▵ for CAPPE) represent a statistically significant difference compared to the CA derivative -untreated control group in each group, respectively, at P<0.05. The different symbols (# for CAPE_Akt, § for CAPE_compound C, ▴ for CAPPE_Akt, and ▪ for CAPPE_compound C) represent a statistically significant difference compared to each corresponding CA derivative- treated control group in each dosage subgroup, respectively, at P<0.05. (B–C) Cytoplasmic proteins were prepared for Western blotting analysis using monoclonal antibodies against anti-phosphorylation Akt (S473), total-Akt, anti-phosphorylation AMPKα (T172) and total-AMPKα.

    Journal: PLoS ONE

    Article Title: Caffeic Acid Derivatives Inhibit the Growth of Colon Cancer: Involvement of the PI3-K/Akt and AMPK Signaling Pathways

    doi: 10.1371/journal.pone.0099631

    Figure Lengend Snippet: (A) Human CRC SW-480 cells were cultured in RPMI-1640 medium with CAPE and CAPPE (at concentrations of 0, 5, 10, 20, 50 and 100 µM) in the presence or absence of compound C (10 µM) for 24 h. Transfections of constitutively active Akt (Myr-Akt1) and empty vector (pcDNA3) were conducted before the treatment of CA derivatives. The cell proliferation was measured by MTT assay as described in . Data are the mean ± SD (standard deviation) of three independent experiments. The different symbols (??? for CAPE and ▵ for CAPPE) represent a statistically significant difference compared to the CA derivative -untreated control group in each group, respectively, at P<0.05. The different symbols (# for CAPE_Akt, § for CAPE_compound C, ▴ for CAPPE_Akt, and ▪ for CAPPE_compound C) represent a statistically significant difference compared to each corresponding CA derivative- treated control group in each dosage subgroup, respectively, at P<0.05. (B–C) Cytoplasmic proteins were prepared for Western blotting analysis using monoclonal antibodies against anti-phosphorylation Akt (S473), total-Akt, anti-phosphorylation AMPKα (T172) and total-AMPKα.

    Article Snippet: After 24 h, the culture medium was replaced by media containing CA derivatives at one of five concentrations (i.e.,0, 5, 10, 20, 50 and 100 µM) in the presence or absence of compound C. Transfections of constitutively active Akt (Myr-Akt1, Addgene plasmid 9008) and empty vector (pcDNA3, Addgene plasmid 10792) were conducted by using Lipofectamine LTX transfection reagent.

    Techniques: Cell Culture, Transfection, Plasmid Preparation, MTT Assay, Standard Deviation, Western Blot

    A) DLD-1 cells were sorted by flow cytometry and different populations with CD44 positive /CD133 negative (Q1), CD44 positive /CD133 positive (Q2), CD44 negative CD133 negative (Q3), were collected. B) The sorted cells were further analyzed with western blot for total AKT, AKT1 or AKT2 and betaactin expression.

    Journal: PLoS ONE

    Article Title: Evaluation of Cancer Stem Cell Markers CD133, CD44, CD24: Association with AKT Isoforms and Radiation Resistance in Colon Cancer Cells

    doi: 10.1371/journal.pone.0094621

    Figure Lengend Snippet: A) DLD-1 cells were sorted by flow cytometry and different populations with CD44 positive /CD133 negative (Q1), CD44 positive /CD133 positive (Q2), CD44 negative CD133 negative (Q3), were collected. B) The sorted cells were further analyzed with western blot for total AKT, AKT1 or AKT2 and betaactin expression.

    Article Snippet: The cells were transfected with pcDNA3 myr HA AKT1 and pcDNA3 myr HA AKT2 plasmids kindly provided by William Sellers (Dana Farber Cancer Institute, Boston, MA, USA) through Addgene (Cambridge, MA).

    Techniques: Flow Cytometry, Western Blot, Expressing

    (A) RBP2 is overexpressed in human NSCLC cell lines SK-MES-1, A549, SPCA-1 and H1975 compared to the human bronchial epithelial cell line BEAS2B cells. (B) Effects of RBP2 siRNA1, RBP2 siRNA2 and pcDNA3-HA-RBP2 on the expression of the RBP2 protein.

    Journal: PLoS ONE

    Article Title: Retinoblastoma Binding Protein 2 (RBP2) Promotes HIF-1α–VEGF-Induced Angiogenesis of Non-Small Cell Lung Cancer via the Akt Pathway

    doi: 10.1371/journal.pone.0106032

    Figure Lengend Snippet: (A) RBP2 is overexpressed in human NSCLC cell lines SK-MES-1, A549, SPCA-1 and H1975 compared to the human bronchial epithelial cell line BEAS2B cells. (B) Effects of RBP2 siRNA1, RBP2 siRNA2 and pcDNA3-HA-RBP2 on the expression of the RBP2 protein.

    Article Snippet: The plasmid pcDNA3 Myr HA Akt1 was purchased from Addgene (Plasmid 9008) .

    Techniques: Expressing

    (A) Silencing RBP2 expression in H1975 cells significantly decreased the phosphorylation of Akt, and the forced expression of RBP2 in SK-MES-1 cells increased the activity of Akt. (B) When Akt was constitutively activated in RBP2-siRNA2 H1975, the expression of HIF-1α and VEGF were increased compared to the control. The PI3K/Akt inhibitor LY294002 significantly inhibited the expression of HIF-1α and VEGF in pcDNA3-HA-RBP2 SK-MES-1 cells. (C) Westernblots showing the time course of Akt phosphorylation in RBP2-siRNA2 H1975 cells due to VEGF-165 (25 ng/mL). (D) In the presence of recombinant human VEGF-165 stimulation, the activation of Akt was increased in RBP2-siRNA2 H1975 cells and RBP2-overexpressing SK-MES-1 cells (25 ng/mL, 30 minutes).

    Journal: PLoS ONE

    Article Title: Retinoblastoma Binding Protein 2 (RBP2) Promotes HIF-1α–VEGF-Induced Angiogenesis of Non-Small Cell Lung Cancer via the Akt Pathway

    doi: 10.1371/journal.pone.0106032

    Figure Lengend Snippet: (A) Silencing RBP2 expression in H1975 cells significantly decreased the phosphorylation of Akt, and the forced expression of RBP2 in SK-MES-1 cells increased the activity of Akt. (B) When Akt was constitutively activated in RBP2-siRNA2 H1975, the expression of HIF-1α and VEGF were increased compared to the control. The PI3K/Akt inhibitor LY294002 significantly inhibited the expression of HIF-1α and VEGF in pcDNA3-HA-RBP2 SK-MES-1 cells. (C) Westernblots showing the time course of Akt phosphorylation in RBP2-siRNA2 H1975 cells due to VEGF-165 (25 ng/mL). (D) In the presence of recombinant human VEGF-165 stimulation, the activation of Akt was increased in RBP2-siRNA2 H1975 cells and RBP2-overexpressing SK-MES-1 cells (25 ng/mL, 30 minutes).

    Article Snippet: The plasmid pcDNA3 Myr HA Akt1 was purchased from Addgene (Plasmid 9008) .

    Techniques: Expressing, Activity Assay, Recombinant, Activation Assay

    ( A ) OCI-LY3 cells were treated with indicated concentrations of PCI-24781 for 6 or 16 hr followed by cell lysis and western blotting for indicated proteins. ( B ) SUDHL4 and OCI-LY3 cells were pre-incubated with PD98059 (PD) or LY294002 (LY) for 2hr followed by 48 hr. incubation with PCI-24781. Cells were stained with AnnexinV/PI and analyzed by flow cytometry. ( C ) Malignant B- cells obtained from a CLL patient blood sample using Histopaque were incubated with PCI for 44 hr followed by Annexin V/PI staining and analyzed by flowcytometry. ( D ) SUDHL6 and OCI-LY3 cells were transfected with Myr-Akt or empty vector using Amaxa kit V and L respectively followed by selection in neomycin for one week. OCI-LY3 Cells were analyzed by western blotting for constitutive activation of AKT by western blotting and incubated with PCI-24781 for 48 hr followed by annexinV/PI staining and analyzed by flow cytometry. ( E ) SUDHL6 expressing constitutively active AKT (Myr-Akt) and wt -AKT were incubated with indicated concentrations of PCI-24781, SAHA and TSA for 48 hr followed by Annexin V/PI staining and analyzed by flow cytometry. (ns= not significant, * = P<0.05; **= p<0.01; *** = P<0.001).

    Journal: PLoS ONE

    Article Title: Paradoxical Regulation of Hypoxia Inducible Factor-1α (HIF-1α) by Histone Deacetylase Inhibitor in Diffuse Large B-Cell Lymphoma

    doi: 10.1371/journal.pone.0081333

    Figure Lengend Snippet: ( A ) OCI-LY3 cells were treated with indicated concentrations of PCI-24781 for 6 or 16 hr followed by cell lysis and western blotting for indicated proteins. ( B ) SUDHL4 and OCI-LY3 cells were pre-incubated with PD98059 (PD) or LY294002 (LY) for 2hr followed by 48 hr. incubation with PCI-24781. Cells were stained with AnnexinV/PI and analyzed by flow cytometry. ( C ) Malignant B- cells obtained from a CLL patient blood sample using Histopaque were incubated with PCI for 44 hr followed by Annexin V/PI staining and analyzed by flowcytometry. ( D ) SUDHL6 and OCI-LY3 cells were transfected with Myr-Akt or empty vector using Amaxa kit V and L respectively followed by selection in neomycin for one week. OCI-LY3 Cells were analyzed by western blotting for constitutive activation of AKT by western blotting and incubated with PCI-24781 for 48 hr followed by annexinV/PI staining and analyzed by flow cytometry. ( E ) SUDHL6 expressing constitutively active AKT (Myr-Akt) and wt -AKT were incubated with indicated concentrations of PCI-24781, SAHA and TSA for 48 hr followed by Annexin V/PI staining and analyzed by flow cytometry. (ns= not significant, * = P<0.05; **= p<0.01; *** = P<0.001).

    Article Snippet: HIF-1α wt (plasmid no. 18949), HIF-1α mutant (P/A) (plasmid no. 18955), Myr-Akt (plasmid no. 9008) were obtained from Addgene.

    Techniques: Lysis, Western Blot, Incubation, Staining, Flow Cytometry, Transfection, Plasmid Preparation, Selection, Activation Assay, Expressing

    ( A ) To check the expression of HIF-1α in SUDHL6 and OCI-LY3 cells expressing Myr-Akt and wt-Akt, cells were subjected to lysis followed by western blotting using HIF-1α antibody. ( B ) SUDHL4-and OCI-LY3 cells were incubated with indicated concentration of LY294002 (LY) for 24 hr followed by cell lysis and Western blotting using HIF-1α antibody. ( C ) Malignant cells obtained from blood sample of CLL patient were incubated with indicated concentration of PCI or LY alone or in combination for 16 hr followed by cell lysis and western blotting using specific antibodies as indicated. ( D ) SUDHL4 and OCI-LY3 cells were treated with PCI and LY either alone or in combination for 24 hr followed by cell lysis and western blotting using specific antibodies for pAkt and Akt. Actin is used as an internal control.

    Journal: PLoS ONE

    Article Title: Paradoxical Regulation of Hypoxia Inducible Factor-1α (HIF-1α) by Histone Deacetylase Inhibitor in Diffuse Large B-Cell Lymphoma

    doi: 10.1371/journal.pone.0081333

    Figure Lengend Snippet: ( A ) To check the expression of HIF-1α in SUDHL6 and OCI-LY3 cells expressing Myr-Akt and wt-Akt, cells were subjected to lysis followed by western blotting using HIF-1α antibody. ( B ) SUDHL4-and OCI-LY3 cells were incubated with indicated concentration of LY294002 (LY) for 24 hr followed by cell lysis and Western blotting using HIF-1α antibody. ( C ) Malignant cells obtained from blood sample of CLL patient were incubated with indicated concentration of PCI or LY alone or in combination for 16 hr followed by cell lysis and western blotting using specific antibodies as indicated. ( D ) SUDHL4 and OCI-LY3 cells were treated with PCI and LY either alone or in combination for 24 hr followed by cell lysis and western blotting using specific antibodies for pAkt and Akt. Actin is used as an internal control.

    Article Snippet: HIF-1α wt (plasmid no. 18949), HIF-1α mutant (P/A) (plasmid no. 18955), Myr-Akt (plasmid no. 9008) were obtained from Addgene.

    Techniques: Expressing, Lysis, Western Blot, Incubation, Concentration Assay