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Addgene inc pmx-klf4 retroviral vector

(A) Nuclear extracts from primary human keratinocytes (HKC) and HeLa cells were immunoprecipitated with antibodies against methylated DNA, followed by PCR analysis of the precipitated DNA with primers specific for the indicated regions of the Notch1 promoter and first intronic region. PCR with primers specific for a known methylated gene was used as positive control. (B) Total RNA samples from primary human keratinocytes (HKC) and the indicated cancer cell lines were analyzed by real time RT-PCR with primers specific for Sp1, Sp3, <t>Klf4,</t> KLF5 and KLF10a. (C) Primary keratinocytes, HeLa and SCC13 cells were analyzed by immunoblotting with antibodies against Sp1, Sp3 and Klf4, with β-actin as equal loading control.

Pmx Klf4 Retroviral Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pmx+klf4+retroviral+vector/pcDNA3%2E1+-+HA-KLF4+FL+(Plasmid+%2334593)/pmc02860992-134-17-23
Average 90 stars, based on 1 article reviews

pmx-klf4 retroviral vector - by Bioz Stars, 2026-09

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1) Product Images from "Differential Control of Notch1 Gene Transcription by Klf4 and Sp3 Transcription Factors in Normal versus Cancer-Derived Keratinocytes"

Article Title: Differential Control of Notch1 Gene Transcription by Klf4 and Sp3 Transcription Factors in Normal versus Cancer-Derived Keratinocytes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0010369

Figure Legend Snippet: (A) Nuclear extracts from primary human keratinocytes (HKC) and HeLa cells were immunoprecipitated with antibodies against methylated DNA, followed by PCR analysis of the precipitated DNA with primers specific for the indicated regions of the Notch1 promoter and first intronic region. PCR with primers specific for a known methylated gene was used as positive control. (B) Total RNA samples from primary human keratinocytes (HKC) and the indicated cancer cell lines were analyzed by real time RT-PCR with primers specific for Sp1, Sp3, Klf4, KLF5 and KLF10a. (C) Primary keratinocytes, HeLa and SCC13 cells were analyzed by immunoblotting with antibodies against Sp1, Sp3 and Klf4, with β-actin as equal loading control.

Techniques Used: Immunoprecipitation, Methylation, Positive Control, Quantitative RT-PCR, Western Blot

(A) Primary keratinocytes were co-transfected with a reporter containing the minimal functional Notch1 promoter (from -392pGL4) together with an expression vector for human Klf4 or empty vector control. Renilla minimal reporter was used for internal normalization, and the promoter activity was measured 48 hours after transfection. Shown are the results of two different experiments. (B) Primary keratinocytes were infected with a retroviral vector expressing KlfF4 (pMSKlf4) or an empty vector control and harvested after 48 hours, followed by determination of Klf4 and Notch1 mRNA expression by real-time PCR. Efficiency of infection with the pMSKlf4 virus was also assessed by the widespread morphological changes with flattening of cells (upper panels). (C) Primary keratinocytes were infected with a KlfF4 expressing retrovirus versus an empty vector control as in the previous panel, followed by immunoblot analysis with antibodies against Notch1, Klf4 and γ-tubulin as equal loading control. Right panel: data were quantified by densitometric scanning of the autoradiograph and expressed as arbitrary units after normalization for γ-tubulin expression. Similar results were obtained in a second independent experiment.

Figure Legend Snippet: (A) Primary keratinocytes were co-transfected with a reporter containing the minimal functional Notch1 promoter (from -392pGL4) together with an expression vector for human Klf4 or empty vector control. Renilla minimal reporter was used for internal normalization, and the promoter activity was measured 48 hours after transfection. Shown are the results of two different experiments. (B) Primary keratinocytes were infected with a retroviral vector expressing KlfF4 (pMSKlf4) or an empty vector control and harvested after 48 hours, followed by determination of Klf4 and Notch1 mRNA expression by real-time PCR. Efficiency of infection with the pMSKlf4 virus was also assessed by the widespread morphological changes with flattening of cells (upper panels). (C) Primary keratinocytes were infected with a KlfF4 expressing retrovirus versus an empty vector control as in the previous panel, followed by immunoblot analysis with antibodies against Notch1, Klf4 and γ-tubulin as equal loading control. Right panel: data were quantified by densitometric scanning of the autoradiograph and expressed as arbitrary units after normalization for γ-tubulin expression. Similar results were obtained in a second independent experiment.

Techniques Used: Transfection, Functional Assay, Expressing, Plasmid Preparation, Activity Assay, Infection, Real-time Polymerase Chain Reaction, Western Blot, Autoradiography

(A) Primary keratinocytes were transfected with two sets of siRNAs for Klf4, Sp1 or Sp3 in parallel with scrambled siRNA controls for 48 hours, followed by expression analysis of the targeted genes by real time RT-PCR and immunoblotting (left and middle panels, respectively) The same RNA samples were also analyzed for levels of Notch1 expression (right panel). (B) Primary keratinocytes, were transfected as in the previous panel with two different sets of siRNAs for Klf4 and Sp3 (siRNA-1, siRNA-2) (left columns) or with siRNAs for Klf4, Sp1 and Sp3 (right columns), followed by real time RT-PCR analysis of Notch1 expression. Shown is the calculated average of four different experiments using 36β4 and 18S RNA for internal normalization. (C) HeLa and SCC13 cells were transfected with two different sets of siRNAs for Klf4 and Sp3 (siRNA-1, siRNA-2) followed by determination of Notch1 expression by real-time RT-PCR as in the previous panel. Primary keratinocytes and SCC13 cells were transfected with siRNAs against the indicated genes followed by immunoblot analysis of Notch1 protein expression with γ-tubulin as equal loading control. Right panel: data were quantified by densitometric scanning of the autoradiograph and expressed as arbitrary units after normalization for γ-tubulin expression.

Figure Legend Snippet: (A) Primary keratinocytes were transfected with two sets of siRNAs for Klf4, Sp1 or Sp3 in parallel with scrambled siRNA controls for 48 hours, followed by expression analysis of the targeted genes by real time RT-PCR and immunoblotting (left and middle panels, respectively) The same RNA samples were also analyzed for levels of Notch1 expression (right panel). (B) Primary keratinocytes, were transfected as in the previous panel with two different sets of siRNAs for Klf4 and Sp3 (siRNA-1, siRNA-2) (left columns) or with siRNAs for Klf4, Sp1 and Sp3 (right columns), followed by real time RT-PCR analysis of Notch1 expression. Shown is the calculated average of four different experiments using 36β4 and 18S RNA for internal normalization. (C) HeLa and SCC13 cells were transfected with two different sets of siRNAs for Klf4 and Sp3 (siRNA-1, siRNA-2) followed by determination of Notch1 expression by real-time RT-PCR as in the previous panel. Primary keratinocytes and SCC13 cells were transfected with siRNAs against the indicated genes followed by immunoblot analysis of Notch1 protein expression with γ-tubulin as equal loading control. Right panel: data were quantified by densitometric scanning of the autoradiograph and expressed as arbitrary units after normalization for γ-tubulin expression.

Techniques Used: Transfection, Expressing, Quantitative RT-PCR, Western Blot, Autoradiography

(A) Predicted Klf4- and Sp1/Sp3 binding sites in the −340/−300 bp Notch1 promoter region. Shown is the nucleotide sequence of this region with, on top, the predicted binding sites for Klf4- and Sp1/Sp3. (B and C) Primary keratinocytes (HKC) and HeLa cells were processed for chromatin immunoprecipitation (ChIP) analysis with antibodies against Sp1, Sp3 (B), Klf4 (C) or Maz, as indicated, followed by amplification of two Notch1 promoter regions located between bp −740/−262 (Chip 1) and around −6600 bp (Chip 2), which contain and lack, respectively, putative Sp1/Sp3 and Klf4 binding sites. Amplification of the proximal promoter region of the p21 WAF1/Cip1 gene (Chip p21) was used as positive control. Un-precipitated chromatin preparations were used for parallel amplification reactions as ‘input’ DNA controls. (D) Chip assays with anti-Sp3 and Klf4 antibodies and non immune IgGs as in the previous panels were followed by real time PCR amplification of region1 of the Notch1 promoter. The amount of precipitated DNA was calculated relative to the total input chromatin and expressed as a percentage of the total according to the following formula : percentage total = 2ΔCt×5, where ΔCt = Ct (input) − Ct (immunoprecipitation), and Ct is the cycle threshold.

Figure Legend Snippet: (A) Predicted Klf4- and Sp1/Sp3 binding sites in the −340/−300 bp Notch1 promoter region. Shown is the nucleotide sequence of this region with, on top, the predicted binding sites for Klf4- and Sp1/Sp3. (B and C) Primary keratinocytes (HKC) and HeLa cells were processed for chromatin immunoprecipitation (ChIP) analysis with antibodies against Sp1, Sp3 (B), Klf4 (C) or Maz, as indicated, followed by amplification of two Notch1 promoter regions located between bp −740/−262 (Chip 1) and around −6600 bp (Chip 2), which contain and lack, respectively, putative Sp1/Sp3 and Klf4 binding sites. Amplification of the proximal promoter region of the p21 WAF1/Cip1 gene (Chip p21) was used as positive control. Un-precipitated chromatin preparations were used for parallel amplification reactions as ‘input’ DNA controls. (D) Chip assays with anti-Sp3 and Klf4 antibodies and non immune IgGs as in the previous panels were followed by real time PCR amplification of region1 of the Notch1 promoter. The amount of precipitated DNA was calculated relative to the total input chromatin and expressed as a percentage of the total according to the following formula : percentage total = 2ΔCt×5, where ΔCt = Ct (input) − Ct (immunoprecipitation), and Ct is the cycle threshold.

Techniques Used: Binding Assay, Sequencing, Chromatin Immunoprecipitation, Amplification, Positive Control, Real-time Polymerase Chain Reaction, Immunoprecipitation

(A) HeLa cells were infected with a recombinant adenovirus expressing wild-type p53 (Adp53) or GFP control (AdGFP) and processed for ChIP assays with antibodies against RNA polymerase II (α-PolII) and non-immune IgGs as control. PCR amplification of the indicated regions of the Notch1 gene was performed, in parallel with similar amplification of the input chromatin DNA. Right panels: for quantification of the results, the chromatin immunoprecipitated material was also analyzed by real time PCR amplification of the indicated regions of the Notch1 promoter, in parallel with input chromatin DNA, followed by a calculation of binding according to the same formula utilized in . (B) Primary keratinocytes (HKC) were infected with a retroviral vector over-expressing Klf4 (pMSKlf4) or empty vector control (Ctrl) for 48 hours followed by ChIP assays for PolII binding as in the previous panel, including quantification of the results by real time PCR (right panels).

Figure Legend Snippet: (A) HeLa cells were infected with a recombinant adenovirus expressing wild-type p53 (Adp53) or GFP control (AdGFP) and processed for ChIP assays with antibodies against RNA polymerase II (α-PolII) and non-immune IgGs as control. PCR amplification of the indicated regions of the Notch1 gene was performed, in parallel with similar amplification of the input chromatin DNA. Right panels: for quantification of the results, the chromatin immunoprecipitated material was also analyzed by real time PCR amplification of the indicated regions of the Notch1 promoter, in parallel with input chromatin DNA, followed by a calculation of binding according to the same formula utilized in . (B) Primary keratinocytes (HKC) were infected with a retroviral vector over-expressing Klf4 (pMSKlf4) or empty vector control (Ctrl) for 48 hours followed by ChIP assays for PolII binding as in the previous panel, including quantification of the results by real time PCR (right panels).

Techniques Used: Infection, Recombinant, Expressing, Amplification, Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Plasmid Preparation

(A) HeLa cells were transfected with siRNAs for Sp3 and Klf4 in parallel with scrambled siRNA controls followed, 48 hours after transfection, by infection with a p53 expressing adenovirus (Adp53) or GFP control (AdGFP), for 24 hours. Levels of Notch1 mRNA expression were determined by real-time RT-PCR. The expected changes of p53, Sp3 and Klf4 expression were also confirmed by real time RT-PCR, with results similar to those shown in previous figures. (B) HeLa cells were transfected with siRNAs for UBE3A, Klf4 and Sp3 alone or in combinations as indicated, in parallel with scrambled siRNA controls. UBE3A and Notch1 expression was assessed by real-time RT-PCR. (C and D) Primary keratinocytes (HKC) (C) and HeLa and SCC13 cells (D were infected with a Klf4 expressing retrovirus or empty vector control for 48 hours, followed by infection with the Adp53 or AdGFP viruses for 24 hours. Notch1 mRNA levels were assessed by real-time RT-PCR.

Figure Legend Snippet: (A) HeLa cells were transfected with siRNAs for Sp3 and Klf4 in parallel with scrambled siRNA controls followed, 48 hours after transfection, by infection with a p53 expressing adenovirus (Adp53) or GFP control (AdGFP), for 24 hours. Levels of Notch1 mRNA expression were determined by real-time RT-PCR. The expected changes of p53, Sp3 and Klf4 expression were also confirmed by real time RT-PCR, with results similar to those shown in previous figures. (B) HeLa cells were transfected with siRNAs for UBE3A, Klf4 and Sp3 alone or in combinations as indicated, in parallel with scrambled siRNA controls. UBE3A and Notch1 expression was assessed by real-time RT-PCR. (C and D) Primary keratinocytes (HKC) (C) and HeLa and SCC13 cells (D were infected with a Klf4 expressing retrovirus or empty vector control for 48 hours, followed by infection with the Adp53 or AdGFP viruses for 24 hours. Notch1 mRNA levels were assessed by real-time RT-PCR.

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

Virus:

Article Title: Differential control of Notch1 gene transcription by Klf4 and Sp3 transcription factors in normal versus cancer-derived keratinocytes.
Article Snippet: .. The Ad-p53 virus was a gift of Dr. S. Lee (Massachusetts General Hospital, Charleston, MA), and the pMX-Klf4 retroviral vector [52] was obtained from Addgene. ..

Retroviral:

Plasmid Preparation:

Image Search Results

Journal: PLoS ONE

Article Title: Differential Control of Notch1 Gene Transcription by Klf4 and Sp3 Transcription Factors in Normal versus Cancer-Derived Keratinocytes

doi: 10.1371/journal.pone.0010369

Figure Lengend Snippet: (A) Nuclear extracts from primary human keratinocytes (HKC) and HeLa cells were immunoprecipitated with antibodies against methylated DNA, followed by PCR analysis of the precipitated DNA with primers specific for the indicated regions of the Notch1 promoter and first intronic region. PCR with primers specific for a known methylated gene was used as positive control. (B) Total RNA samples from primary human keratinocytes (HKC) and the indicated cancer cell lines were analyzed by real time RT-PCR with primers specific for Sp1, Sp3, Klf4, KLF5 and KLF10a. (C) Primary keratinocytes, HeLa and SCC13 cells were analyzed by immunoblotting with antibodies against Sp1, Sp3 and Klf4, with β-actin as equal loading control.

Article Snippet: The Ad-p53 virus was a gift of Dr. S. Lee (Massachusetts General Hospital, Charleston, MA), and the pMX-Klf4 retroviral vector was obtained from Addgene.

Techniques: Immunoprecipitation, Methylation, Positive Control, Quantitative RT-PCR, Western Blot

Journal: PLoS ONE

Article Title: Differential Control of Notch1 Gene Transcription by Klf4 and Sp3 Transcription Factors in Normal versus Cancer-Derived Keratinocytes

doi: 10.1371/journal.pone.0010369

Figure Lengend Snippet: (A) Primary keratinocytes were co-transfected with a reporter containing the minimal functional Notch1 promoter (from -392pGL4) together with an expression vector for human Klf4 or empty vector control. Renilla minimal reporter was used for internal normalization, and the promoter activity was measured 48 hours after transfection. Shown are the results of two different experiments. (B) Primary keratinocytes were infected with a retroviral vector expressing KlfF4 (pMSKlf4) or an empty vector control and harvested after 48 hours, followed by determination of Klf4 and Notch1 mRNA expression by real-time PCR. Efficiency of infection with the pMSKlf4 virus was also assessed by the widespread morphological changes with flattening of cells (upper panels). (C) Primary keratinocytes were infected with a KlfF4 expressing retrovirus versus an empty vector control as in the previous panel, followed by immunoblot analysis with antibodies against Notch1, Klf4 and γ-tubulin as equal loading control. Right panel: data were quantified by densitometric scanning of the autoradiograph and expressed as arbitrary units after normalization for γ-tubulin expression. Similar results were obtained in a second independent experiment.

Article Snippet: The Ad-p53 virus was a gift of Dr. S. Lee (Massachusetts General Hospital, Charleston, MA), and the pMX-Klf4 retroviral vector was obtained from Addgene.

Techniques: Transfection, Functional Assay, Expressing, Plasmid Preparation, Activity Assay, Infection, Real-time Polymerase Chain Reaction, Western Blot, Autoradiography

Journal: PLoS ONE

Article Title: Differential Control of Notch1 Gene Transcription by Klf4 and Sp3 Transcription Factors in Normal versus Cancer-Derived Keratinocytes

doi: 10.1371/journal.pone.0010369

Figure Lengend Snippet: (A) Primary keratinocytes were transfected with two sets of siRNAs for Klf4, Sp1 or Sp3 in parallel with scrambled siRNA controls for 48 hours, followed by expression analysis of the targeted genes by real time RT-PCR and immunoblotting (left and middle panels, respectively) The same RNA samples were also analyzed for levels of Notch1 expression (right panel). (B) Primary keratinocytes, were transfected as in the previous panel with two different sets of siRNAs for Klf4 and Sp3 (siRNA-1, siRNA-2) (left columns) or with siRNAs for Klf4, Sp1 and Sp3 (right columns), followed by real time RT-PCR analysis of Notch1 expression. Shown is the calculated average of four different experiments using 36β4 and 18S RNA for internal normalization. (C) HeLa and SCC13 cells were transfected with two different sets of siRNAs for Klf4 and Sp3 (siRNA-1, siRNA-2) followed by determination of Notch1 expression by real-time RT-PCR as in the previous panel. Primary keratinocytes and SCC13 cells were transfected with siRNAs against the indicated genes followed by immunoblot analysis of Notch1 protein expression with γ-tubulin as equal loading control. Right panel: data were quantified by densitometric scanning of the autoradiograph and expressed as arbitrary units after normalization for γ-tubulin expression.

Article Snippet: The Ad-p53 virus was a gift of Dr. S. Lee (Massachusetts General Hospital, Charleston, MA), and the pMX-Klf4 retroviral vector was obtained from Addgene.

Techniques: Transfection, Expressing, Quantitative RT-PCR, Western Blot, Autoradiography

Journal: PLoS ONE

Article Title: Differential Control of Notch1 Gene Transcription by Klf4 and Sp3 Transcription Factors in Normal versus Cancer-Derived Keratinocytes

doi: 10.1371/journal.pone.0010369

Figure Lengend Snippet: (A) Predicted Klf4- and Sp1/Sp3 binding sites in the −340/−300 bp Notch1 promoter region. Shown is the nucleotide sequence of this region with, on top, the predicted binding sites for Klf4- and Sp1/Sp3. (B and C) Primary keratinocytes (HKC) and HeLa cells were processed for chromatin immunoprecipitation (ChIP) analysis with antibodies against Sp1, Sp3 (B), Klf4 (C) or Maz, as indicated, followed by amplification of two Notch1 promoter regions located between bp −740/−262 (Chip 1) and around −6600 bp (Chip 2), which contain and lack, respectively, putative Sp1/Sp3 and Klf4 binding sites. Amplification of the proximal promoter region of the p21 WAF1/Cip1 gene (Chip p21) was used as positive control. Un-precipitated chromatin preparations were used for parallel amplification reactions as ‘input’ DNA controls. (D) Chip assays with anti-Sp3 and Klf4 antibodies and non immune IgGs as in the previous panels were followed by real time PCR amplification of region1 of the Notch1 promoter. The amount of precipitated DNA was calculated relative to the total input chromatin and expressed as a percentage of the total according to the following formula : percentage total = 2ΔCt×5, where ΔCt = Ct (input) − Ct (immunoprecipitation), and Ct is the cycle threshold.

Article Snippet: The Ad-p53 virus was a gift of Dr. S. Lee (Massachusetts General Hospital, Charleston, MA), and the pMX-Klf4 retroviral vector was obtained from Addgene.

Techniques: Binding Assay, Sequencing, Chromatin Immunoprecipitation, Amplification, Positive Control, Real-time Polymerase Chain Reaction, Immunoprecipitation

Journal: PLoS ONE

Article Title: Differential Control of Notch1 Gene Transcription by Klf4 and Sp3 Transcription Factors in Normal versus Cancer-Derived Keratinocytes

doi: 10.1371/journal.pone.0010369

Figure Lengend Snippet: (A) HeLa cells were infected with a recombinant adenovirus expressing wild-type p53 (Adp53) or GFP control (AdGFP) and processed for ChIP assays with antibodies against RNA polymerase II (α-PolII) and non-immune IgGs as control. PCR amplification of the indicated regions of the Notch1 gene was performed, in parallel with similar amplification of the input chromatin DNA. Right panels: for quantification of the results, the chromatin immunoprecipitated material was also analyzed by real time PCR amplification of the indicated regions of the Notch1 promoter, in parallel with input chromatin DNA, followed by a calculation of binding according to the same formula utilized in . (B) Primary keratinocytes (HKC) were infected with a retroviral vector over-expressing Klf4 (pMSKlf4) or empty vector control (Ctrl) for 48 hours followed by ChIP assays for PolII binding as in the previous panel, including quantification of the results by real time PCR (right panels).

Article Snippet: The Ad-p53 virus was a gift of Dr. S. Lee (Massachusetts General Hospital, Charleston, MA), and the pMX-Klf4 retroviral vector was obtained from Addgene.

Techniques: Infection, Recombinant, Expressing, Amplification, Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Plasmid Preparation

Journal: PLoS ONE

Article Title: Differential Control of Notch1 Gene Transcription by Klf4 and Sp3 Transcription Factors in Normal versus Cancer-Derived Keratinocytes

doi: 10.1371/journal.pone.0010369

Figure Lengend Snippet: (A) HeLa cells were transfected with siRNAs for Sp3 and Klf4 in parallel with scrambled siRNA controls followed, 48 hours after transfection, by infection with a p53 expressing adenovirus (Adp53) or GFP control (AdGFP), for 24 hours. Levels of Notch1 mRNA expression were determined by real-time RT-PCR. The expected changes of p53, Sp3 and Klf4 expression were also confirmed by real time RT-PCR, with results similar to those shown in previous figures. (B) HeLa cells were transfected with siRNAs for UBE3A, Klf4 and Sp3 alone or in combinations as indicated, in parallel with scrambled siRNA controls. UBE3A and Notch1 expression was assessed by real-time RT-PCR. (C and D) Primary keratinocytes (HKC) (C) and HeLa and SCC13 cells (D were infected with a Klf4 expressing retrovirus or empty vector control for 48 hours, followed by infection with the Adp53 or AdGFP viruses for 24 hours. Notch1 mRNA levels were assessed by real-time RT-PCR.

Article Snippet: The Ad-p53 virus was a gift of Dr. S. Lee (Massachusetts General Hospital, Charleston, MA), and the pMX-Klf4 retroviral vector was obtained from Addgene.

Techniques: Transfection, Infection, Expressing, Quantitative RT-PCR, Plasmid Preparation

( A ) Lipid profiles of MEFs, mESCs, and MEFs undergoing Sox2, Klf4, and Oct4 (SKO) reprogramming on days 2, 4, 6, and 8. n = 6. Lipid species identified are listed in table S1. ( B ) Schematic of key phospholipid synthesis pathways. ( C ) qRT-PCR analysis of expression of genes encoding rate-limiting enzymes in phospholipid synthesis pathways on days 0, 2, and 4 in MEFs transduced with SKO in iCD1 medium. Data are represented as mean ± SD ( n = 3). ** P < 0.01 and *** P < 0.001. ( D ) Cellular PE levels were measured in MEFs transduced with SKO in iCD1 medium on days 0, 2, and 4. Data are represented as mean ± SD ( n = 7). ** P < 0.01 and *** P < 0.001. ( E ) Knockdown of Etnk1 and Etnk2 (shEtnk1/2) or Pcyt2 (shPcyt2) impaired reprogramming efficiency. The numbers of Oct4 -GFP + colonies were counted on day 6. shRNA against luciferase (shLuc) was used as control. Data are represented as mean ± SD ( n = 4). *** P < 0.001. ( F ) The numbers of Oct4 -GFP + colonies were counted on day 6 in MEFs transduced with SKO in iCD1 medium or iCD1 medium without Etn (ΔiCD1). Data are represented as mean ± SD ( n = 4). *** P < 0.001. ( G ) The numbers of Oct4 -GFP + colonies were counted on day 6 in MEFs transduced with SKO in ΔiCD1 medium supplemented with vehicle, Etn, CDP-Etn, or L-α-PE. Data are represented as mean ± SD ( n = 3). * P < 0.05 and *** P < 0.001. ( H ) Etn had no effect on reprogramming when Etnk1/2 or Pcyt2 was silenced by shRNAs. The numbers of Oct4 -GFP + colonies were counted on day 6. shLuc was used as control. Data are represented as mean ± SD ( n = 3). *** P < 0.001. ( I ) Suppression of the CDP-Etn pathway by shRNA against Etnk1/2 or Pcyt2 or Etn deprivation had no effect on the proliferation of MEFs undergoing reprogramming. Data are represented as mean ± SD ( n = 3). ns, not significant. ( J ) Etn deprivation inhibited ESC growth. Representative phase-contrast and Oct4-GFP images (left) and growth curves (right) of ESCs in complete medium (control) or medium deprived of Etn. Scale bar, 250 μm. Data are represented as mean ± SD ( n = 3). * P < 0.05. ( K ) Knockout (KO) of Pcyt2 significantly impaired ESC growth. Representative phase-contrast and Oct4-GFP images of wild-type (WT) and Pcyt2 knockout ESCs. Scale bar, 100 μm.

Journal: Science Advances

Article Title: Phospholipid remodeling is critical for stem cell pluripotency by facilitating mesenchymal-to-epithelial transition

doi: 10.1126/sciadv.aax7525

Figure Lengend Snippet: ( A ) Lipid profiles of MEFs, mESCs, and MEFs undergoing Sox2, Klf4, and Oct4 (SKO) reprogramming on days 2, 4, 6, and 8. n = 6. Lipid species identified are listed in table S1. ( B ) Schematic of key phospholipid synthesis pathways. ( C ) qRT-PCR analysis of expression of genes encoding rate-limiting enzymes in phospholipid synthesis pathways on days 0, 2, and 4 in MEFs transduced with SKO in iCD1 medium. Data are represented as mean ± SD ( n = 3). ** P < 0.01 and *** P < 0.001. ( D ) Cellular PE levels were measured in MEFs transduced with SKO in iCD1 medium on days 0, 2, and 4. Data are represented as mean ± SD ( n = 7). ** P < 0.01 and *** P < 0.001. ( E ) Knockdown of Etnk1 and Etnk2 (shEtnk1/2) or Pcyt2 (shPcyt2) impaired reprogramming efficiency. The numbers of Oct4 -GFP + colonies were counted on day 6. shRNA against luciferase (shLuc) was used as control. Data are represented as mean ± SD ( n = 4). *** P < 0.001. ( F ) The numbers of Oct4 -GFP + colonies were counted on day 6 in MEFs transduced with SKO in iCD1 medium or iCD1 medium without Etn (ΔiCD1). Data are represented as mean ± SD ( n = 4). *** P < 0.001. ( G ) The numbers of Oct4 -GFP + colonies were counted on day 6 in MEFs transduced with SKO in ΔiCD1 medium supplemented with vehicle, Etn, CDP-Etn, or L-α-PE. Data are represented as mean ± SD ( n = 3). * P < 0.05 and *** P < 0.001. ( H ) Etn had no effect on reprogramming when Etnk1/2 or Pcyt2 was silenced by shRNAs. The numbers of Oct4 -GFP + colonies were counted on day 6. shLuc was used as control. Data are represented as mean ± SD ( n = 3). *** P < 0.001. ( I ) Suppression of the CDP-Etn pathway by shRNA against Etnk1/2 or Pcyt2 or Etn deprivation had no effect on the proliferation of MEFs undergoing reprogramming. Data are represented as mean ± SD ( n = 3). ns, not significant. ( J ) Etn deprivation inhibited ESC growth. Representative phase-contrast and Oct4-GFP images (left) and growth curves (right) of ESCs in complete medium (control) or medium deprived of Etn. Scale bar, 250 μm. Data are represented as mean ± SD ( n = 3). * P < 0.05. ( K ) Knockout (KO) of Pcyt2 significantly impaired ESC growth. Representative phase-contrast and Oct4-GFP images of wild-type (WT) and Pcyt2 knockout ESCs. Scale bar, 100 μm.

Article Snippet: The pMX retroviral vector expressing Oct4, Sox2, or Klf4 and the pX330 vector were purchased from Addgene. shRNA inserts were cloned into pRetroSuper retroviral vectors. shRNA target sequences were listed in table S2.

Techniques: Quantitative RT-PCR, Expressing, Transduction, shRNA, Luciferase, Knock-Out

( A ) Experimental design of RNA-seq for profiling of genes regulated by the CDP-Etn pathway during reprogramming. shLuc was used as control. ( B ) Venn diagram showing the substantial overlap of Etn-, shEtnk1/2-, and shPcyt2-regulated genes on day 2 of SKO reprogramming. ( C ) GO analysis of genes regulated by the CDP-Etn pathway on day 2 of SKO reprogramming. ( D ) Heatmap showing the expression of selected epithelial genes ( Cdh1 , Epcam , Krt7 , Krt8 , Ocln , and Tjp2 ) and mesenchymal genes ( Snail , Twist1 , Twist2 , Zeb1 , Vim , Itga7 , Col1a1 , Col1a2 , Col5a1 , and Col5a2 ) on day 2 of SKO reprogramming. ( E ) qRT-PCR analysis of expression of selected epithelial and mesenchymal genes on day 2 in MEFs transduced with SKO and shLuc, shEtnk1/2, or shPcyt2 in iCD1 medium (left) and in MEFs transduced with SKO in ΔiCD1 or iCD1 medium (right). Data are represented as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, and *** P < 0.001. ( F ) Western blot analysis (left) and corresponding quantification (right) of Cdh1 expression on day 2 in MEFs transduced with SKO and shLuc, shEtnk1/2, or shPcyt2 in iCD1 medium or in MEFs transduced with SKO in ΔiCD1 or iCD1 medium. Data are represented as mean ± SD ( n = 3). ** P < 0.01 and *** P < 0.001. ( G and H ) Representative images (G) and quantification (H) of scratch assays on day 2 in MEFs transduced with SKO and shLuc, shEtnk1/2, or shPcyt2 in iCD1 medium (left) or in MEFs transduced with SKO in ΔiCD1 or iCD1 medium (right). Scale bar, 100 μm. Data are represented as mean ± SEM ( n = 4). ** P < 0.01 and *** P < 0.001. ( I ) Flow cytometry analysis of Cdh1 in MEFs and MEFs transduced with SKO in ΔiCD1 or iCD1 medium. Representative images of immunofluorescence staining of Cdh1 (green) are shown. Scale bar, 100 μm. ( J ) Etn had no effect on reprogramming in the presence of TGF-β1, TGF-β2, or TGF-β3. The numbers of Oct4 -GFP + colonies were counted on day 6. Data are represented as mean ± SD ( n = 3). *** P < 0.001.

Journal: Science Advances

Article Title: Phospholipid remodeling is critical for stem cell pluripotency by facilitating mesenchymal-to-epithelial transition

doi: 10.1126/sciadv.aax7525

Figure Lengend Snippet: ( A ) Experimental design of RNA-seq for profiling of genes regulated by the CDP-Etn pathway during reprogramming. shLuc was used as control. ( B ) Venn diagram showing the substantial overlap of Etn-, shEtnk1/2-, and shPcyt2-regulated genes on day 2 of SKO reprogramming. ( C ) GO analysis of genes regulated by the CDP-Etn pathway on day 2 of SKO reprogramming. ( D ) Heatmap showing the expression of selected epithelial genes ( Cdh1 , Epcam , Krt7 , Krt8 , Ocln , and Tjp2 ) and mesenchymal genes ( Snail , Twist1 , Twist2 , Zeb1 , Vim , Itga7 , Col1a1 , Col1a2 , Col5a1 , and Col5a2 ) on day 2 of SKO reprogramming. ( E ) qRT-PCR analysis of expression of selected epithelial and mesenchymal genes on day 2 in MEFs transduced with SKO and shLuc, shEtnk1/2, or shPcyt2 in iCD1 medium (left) and in MEFs transduced with SKO in ΔiCD1 or iCD1 medium (right). Data are represented as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, and *** P < 0.001. ( F ) Western blot analysis (left) and corresponding quantification (right) of Cdh1 expression on day 2 in MEFs transduced with SKO and shLuc, shEtnk1/2, or shPcyt2 in iCD1 medium or in MEFs transduced with SKO in ΔiCD1 or iCD1 medium. Data are represented as mean ± SD ( n = 3). ** P < 0.01 and *** P < 0.001. ( G and H ) Representative images (G) and quantification (H) of scratch assays on day 2 in MEFs transduced with SKO and shLuc, shEtnk1/2, or shPcyt2 in iCD1 medium (left) or in MEFs transduced with SKO in ΔiCD1 or iCD1 medium (right). Scale bar, 100 μm. Data are represented as mean ± SEM ( n = 4). ** P < 0.01 and *** P < 0.001. ( I ) Flow cytometry analysis of Cdh1 in MEFs and MEFs transduced with SKO in ΔiCD1 or iCD1 medium. Representative images of immunofluorescence staining of Cdh1 (green) are shown. Scale bar, 100 μm. ( J ) Etn had no effect on reprogramming in the presence of TGF-β1, TGF-β2, or TGF-β3. The numbers of Oct4 -GFP + colonies were counted on day 6. Data are represented as mean ± SD ( n = 3). *** P < 0.001.

Techniques: RNA Sequencing Assay, Expressing, Quantitative RT-PCR, Transduction, Western Blot, Flow Cytometry, Immunofluorescence, Staining

( A ) qRT-PCR analysis (left) and Western blot analysis (right) of expression of Pebp1 on days 0, 2, 4, and 6 in MEFs transduced with SKO in iCD1 medium. Data are represented as mean ± SD ( n = 3). ** P < 0.01 and *** P < 0.001. ( B ) Knockdown of Pebp1 (shPebp1) impaired reprogramming efficiency. The numbers of Oct4 -GFP + colonies were counted on day 6. Data are represented as mean ± SD ( n = 3). *** P < 0.001. ( C ) Venn diagram showing the overlap of genes regulated by the CDP-Etn pathway and Pebp1 on day 2 of SKO reprogramming. ( D ) GO analysis of genes regulated by both the CDP-Etn pathway and Pebp1 on day 2 of SKO reprogramming. ( E ) qRT-PCR analysis of expression of selected epithelial and mesenchymal genes on day 2 in MEFs transduced with SKO and shLuc or shPebp1 in iCD1 medium. Data are represented as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, and *** P < 0.001. ( F ) Flow cytometry analysis of Cdh1 in MEFs and MEFs transduced with SKO and shLuc or shPebp1 in iCD1 medium. Representative images of immunofluorescence staining of Cdh1 (green) are shown. Scale bar, 100 μm. ( G and H ) No significant differences in cell migration were observed between reprogramming in ΔiCD1 or iCD1 medium when Pebp1 was knocked down. Representative images (G) and quantification (H) of scratch assays were analyzed on day 2. Scale bar, 100 μm. Data are represented as mean ± SEM ( n = 4). ** P < 0.01. ( I ) No significant differences in reprogramming efficiency were observed between reprogramming in ΔiCD1 or iCD1 medium when Pebp1 was knocked down. The numbers of Oct4 -GFP + colonies were counted on day 6. Data are represented as mean ± SD ( n = 3). *** P < 0.001.

Journal: Science Advances

Article Title: Phospholipid remodeling is critical for stem cell pluripotency by facilitating mesenchymal-to-epithelial transition

doi: 10.1126/sciadv.aax7525

Figure Lengend Snippet: ( A ) qRT-PCR analysis (left) and Western blot analysis (right) of expression of Pebp1 on days 0, 2, 4, and 6 in MEFs transduced with SKO in iCD1 medium. Data are represented as mean ± SD ( n = 3). ** P < 0.01 and *** P < 0.001. ( B ) Knockdown of Pebp1 (shPebp1) impaired reprogramming efficiency. The numbers of Oct4 -GFP + colonies were counted on day 6. Data are represented as mean ± SD ( n = 3). *** P < 0.001. ( C ) Venn diagram showing the overlap of genes regulated by the CDP-Etn pathway and Pebp1 on day 2 of SKO reprogramming. ( D ) GO analysis of genes regulated by both the CDP-Etn pathway and Pebp1 on day 2 of SKO reprogramming. ( E ) qRT-PCR analysis of expression of selected epithelial and mesenchymal genes on day 2 in MEFs transduced with SKO and shLuc or shPebp1 in iCD1 medium. Data are represented as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, and *** P < 0.001. ( F ) Flow cytometry analysis of Cdh1 in MEFs and MEFs transduced with SKO and shLuc or shPebp1 in iCD1 medium. Representative images of immunofluorescence staining of Cdh1 (green) are shown. Scale bar, 100 μm. ( G and H ) No significant differences in cell migration were observed between reprogramming in ΔiCD1 or iCD1 medium when Pebp1 was knocked down. Representative images (G) and quantification (H) of scratch assays were analyzed on day 2. Scale bar, 100 μm. Data are represented as mean ± SEM ( n = 4). ** P < 0.01. ( I ) No significant differences in reprogramming efficiency were observed between reprogramming in ΔiCD1 or iCD1 medium when Pebp1 was knocked down. The numbers of Oct4 -GFP + colonies were counted on day 6. Data are represented as mean ± SD ( n = 3). *** P < 0.001.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Transduction, Flow Cytometry, Immunofluorescence, Staining, Migration

(A) Morphology of the prostatic ductal system of the dorsal prostate showing proximal (Prox) and distal regions. U, urethra. Scale bar, 0.5 mm. (B and C) The relative expression levels of Klf4 (B) mRNA and (C) protein in the proximal stem cell niche and the distal ductal region. Experiments were repeated 2 times. Data represent means ± SDs. p = 0.0005. (D) Western blot indicating Klf4 expression in proximal and distal ducts. (E) Duct-forming ability of proximal cells (3 × 10 4 ) in collagen gels infected with shCon or shKlf4 lentivirus. Experiments were repeated at least 3 times. Data represent means ± SDs. p < 0.03. (F) Proximal cells were infected with shCon or shKlf4 lentivirus and inoculated (5 × 10 4 ) sub-RC with UGM (2 × 10 5 ). Experiments were repeated at least 3 times. Data represent means ± SDs. p < 0.03. (G) Prostatic tissue generated by shCon or shKlf4 cells was examined for CK5, CK8, and Ki67 expression. Scale bars, 35 μm. (H) Proximal cells were infected with Akt lentiviruses combined with control (shCon + Akt) or shKlf4 (shKlf4 + Akt) lentiviruses and seeded (3 × 10 4 ) in collagen gels. Experiments were repeated at least 3 times. Data represent means ± SDs. p < 0.02. (I) Proximal cells were infected with shCon + Akt or shKlf4 + Akt lentiviruses and inoculated sub-RC (5 × 10 4 ) with UGM (2 × 10 5 ). Experiments were repeated 3 times. Data represent means ± SDs. p < 0.02. (J) PIN and carcinoma-containing tubules in shCon + Akt or shKlf4 + Akt grafts were determined in all fields in 3 sections from each graft (3 grafts per condition). p < 0.00001. (K) Grafts from shCon + Akt and shKlf4 + Akt cells were examined by H&E and for expression of CK5, CK8, pAkt, Ki67, and Klf4. Scale bars, 30 μm. See also Figures and .

Journal: Cell reports

Article Title: KLF4, A Gene Regulating Prostate Stem Cell Homeostasis, Is a Barrier to Malignant Progression and Predictor of Good Prognosis in Prostate Cancer

doi: 10.1016/j.celrep.2018.11.065

Figure Lengend Snippet: (A) Morphology of the prostatic ductal system of the dorsal prostate showing proximal (Prox) and distal regions. U, urethra. Scale bar, 0.5 mm. (B and C) The relative expression levels of Klf4 (B) mRNA and (C) protein in the proximal stem cell niche and the distal ductal region. Experiments were repeated 2 times. Data represent means ± SDs. p = 0.0005. (D) Western blot indicating Klf4 expression in proximal and distal ducts. (E) Duct-forming ability of proximal cells (3 × 10 4 ) in collagen gels infected with shCon or shKlf4 lentivirus. Experiments were repeated at least 3 times. Data represent means ± SDs. p < 0.03. (F) Proximal cells were infected with shCon or shKlf4 lentivirus and inoculated (5 × 10 4 ) sub-RC with UGM (2 × 10 5 ). Experiments were repeated at least 3 times. Data represent means ± SDs. p < 0.03. (G) Prostatic tissue generated by shCon or shKlf4 cells was examined for CK5, CK8, and Ki67 expression. Scale bars, 35 μm. (H) Proximal cells were infected with Akt lentiviruses combined with control (shCon + Akt) or shKlf4 (shKlf4 + Akt) lentiviruses and seeded (3 × 10 4 ) in collagen gels. Experiments were repeated at least 3 times. Data represent means ± SDs. p < 0.02. (I) Proximal cells were infected with shCon + Akt or shKlf4 + Akt lentiviruses and inoculated sub-RC (5 × 10 4 ) with UGM (2 × 10 5 ). Experiments were repeated 3 times. Data represent means ± SDs. p < 0.02. (J) PIN and carcinoma-containing tubules in shCon + Akt or shKlf4 + Akt grafts were determined in all fields in 3 sections from each graft (3 grafts per condition). p < 0.00001. (K) Grafts from shCon + Akt and shKlf4 + Akt cells were examined by H&E and for expression of CK5, CK8, pAkt, Ki67, and Klf4. Scale bars, 30 μm. See also Figures and .

Article Snippet: pMXs-Klf4-IP retroviral vector , Addgene , Plasmid ID: 15920.

Techniques: Expressing, Western Blot, Infection, Generated

(A) Expression of Sca-1 and CD49f by the Akt cell line (right); cells with control antibodies (left). (B) Sphere-forming ability of the Akt cell line (3,000 cells/well) in Matrigel after infection with control (shCon) or shKlf4 lentivirus. Scale bars, 30 μm. Experiments were repeated 3 times. Data represent means ± SDs. p = 0.001. (C) Spheres from shKlf4 and shCon Akt cell line were dissociated, and equal numbers of cells (2,000) were passaged 6 times in triplicate in Matrigel. The sphere number diminished with passage as cells became more migratory and infiltrated the Matrigel (bottom). Scale bars, 30 μm. (D) Sphere-forming ability of the Akt cell line (3,000 cells/well) in Matrigel after infection with control or Klf4-expressing constructs. Scale bars, 30 μm. Experiments were repeated 3 times. Data represent means ± SDs. p < 0.006.

Journal: Cell reports

Article Title: KLF4, A Gene Regulating Prostate Stem Cell Homeostasis, Is a Barrier to Malignant Progression and Predictor of Good Prognosis in Prostate Cancer

doi: 10.1016/j.celrep.2018.11.065

Figure Lengend Snippet: (A) Expression of Sca-1 and CD49f by the Akt cell line (right); cells with control antibodies (left). (B) Sphere-forming ability of the Akt cell line (3,000 cells/well) in Matrigel after infection with control (shCon) or shKlf4 lentivirus. Scale bars, 30 μm. Experiments were repeated 3 times. Data represent means ± SDs. p = 0.001. (C) Spheres from shKlf4 and shCon Akt cell line were dissociated, and equal numbers of cells (2,000) were passaged 6 times in triplicate in Matrigel. The sphere number diminished with passage as cells became more migratory and infiltrated the Matrigel (bottom). Scale bars, 30 μm. (D) Sphere-forming ability of the Akt cell line (3,000 cells/well) in Matrigel after infection with control or Klf4-expressing constructs. Scale bars, 30 μm. Experiments were repeated 3 times. Data represent means ± SDs. p < 0.006.

Article Snippet: pMXs-Klf4-IP retroviral vector , Addgene , Plasmid ID: 15920.

Techniques: Expressing, Infection, Construct

(A) Cells from the Akt cell line were infected with non-silencing control (shCon) orshKlf4 (shKlf4) lentiviruses and implanted (2 × 10 6 ) sub-RC. Experiments were repeated at least 3 times. Data represent means ± SDs. p = 0.0006. (B) Gross tumors after implantation of shCon or shKlf4-infected Akt cell line after 4 weeks. T, tumor; G, glomerulus. Scale bars, 35 μm. (C) Sub-RC grafts were examined for expression of E-cadherin (E-cad), vimentin (vim), Slug, and Ki67. Scale bars, 25 μm. (D) Expression of E-cadherin (E-cad), ZO-1, CK5, CK8, and vimentin (vim) in the Akt-transformed cell line after infection with control or shKlf4-expressing lentiviruses. Scale bars, 50 μm. (E) Western blot of Klf4, E-cadherin (E-cad), Slug, Zeb1, and Snail after infection of the Akt cell line with control or shKlf4-expressing lentiviruses. See also .

Journal: Cell reports

Article Title: KLF4, A Gene Regulating Prostate Stem Cell Homeostasis, Is a Barrier to Malignant Progression and Predictor of Good Prognosis in Prostate Cancer

doi: 10.1016/j.celrep.2018.11.065

Figure Lengend Snippet: (A) Cells from the Akt cell line were infected with non-silencing control (shCon) orshKlf4 (shKlf4) lentiviruses and implanted (2 × 10 6 ) sub-RC. Experiments were repeated at least 3 times. Data represent means ± SDs. p = 0.0006. (B) Gross tumors after implantation of shCon or shKlf4-infected Akt cell line after 4 weeks. T, tumor; G, glomerulus. Scale bars, 35 μm. (C) Sub-RC grafts were examined for expression of E-cadherin (E-cad), vimentin (vim), Slug, and Ki67. Scale bars, 25 μm. (D) Expression of E-cadherin (E-cad), ZO-1, CK5, CK8, and vimentin (vim) in the Akt-transformed cell line after infection with control or shKlf4-expressing lentiviruses. Scale bars, 50 μm. (E) Western blot of Klf4, E-cadherin (E-cad), Slug, Zeb1, and Snail after infection of the Akt cell line with control or shKlf4-expressing lentiviruses. See also .

Article Snippet: pMXs-Klf4-IP retroviral vector , Addgene , Plasmid ID: 15920.

Techniques: Infection, Expressing, Transformation Assay, Western Blot

(A) Western blot of KLF4, Slug, vimentin (Vim), Zeb1, and Snail in control cells (shKlf4 + EV) and in cells rescued by expressing KLF4 (shKlf4 + KLF4). (B) Sub-RC grafts of control (shKlf4 + EV) or KLF4-expressing cells (shKlf4 + KLF4) (1 × 10 5 cells). Experiments were repeated 3 times. Data represent means ± SDs. p = 0.002. (C) Sub-RC grafts were examined by H&E (arrowhead indicates duct-like morphology) and for expression of CK8, KLF4, and Ki67. Scale bars, 45 μm. See also .

Journal: Cell reports

Article Title: KLF4, A Gene Regulating Prostate Stem Cell Homeostasis, Is a Barrier to Malignant Progression and Predictor of Good Prognosis in Prostate Cancer

doi: 10.1016/j.celrep.2018.11.065

Figure Lengend Snippet: (A) Western blot of KLF4, Slug, vimentin (Vim), Zeb1, and Snail in control cells (shKlf4 + EV) and in cells rescued by expressing KLF4 (shKlf4 + KLF4). (B) Sub-RC grafts of control (shKlf4 + EV) or KLF4-expressing cells (shKlf4 + KLF4) (1 × 10 5 cells). Experiments were repeated 3 times. Data represent means ± SDs. p = 0.002. (C) Sub-RC grafts were examined by H&E (arrowhead indicates duct-like morphology) and for expression of CK8, KLF4, and Ki67. Scale bars, 45 μm. See also .

Article Snippet: pMXs-Klf4-IP retroviral vector , Addgene , Plasmid ID: 15920.

Techniques: Western Blot, Expressing

(A) Venn diagram of differentially expressed genes (FDR <0.01, FC >2) in the Akt-transformed cell line after Klf4 knockdown (shKlf4) versus KLF4 re-expression (shKlf4 + KLF4). (B) Heatmap of EMT genes regulated by decreased (shKlf4 + empty vector [EV]) or increased KLF expression (shKlf4 + KLF4). (C) Heatmap of stem cell genes regulated by Klf4 knockdown (shKlf4) and KLF4 re-expression. (D) Ingenuity pathway analysis (canonical pathway) reveals pathways regulated by decreased (shKlf4) or increased (shKlf4 + KLF4) KLF4. Left: −log(p value), right: Z score. Pathways highlighted in red represent those that are inversely regulated by knockdown and re-expression of Klf4 (inverse Z score). (E) GSEA reveals positive enrichment of genes in a tumor metastasis gene signature after decreased (shKlf4) Klf4 expression. (F) GSEA reveals positive enrichment of genes in a tumor suppressor gene signature after increased (shKlf4 + KLF4) KLF4 expression. (G) Overlap of genes after Klf4 knockdown in the Akt-transformed cell line with those expressed in a cohort of human metastatic prostate cancer. (H) Overlap of genes after Klf4 knockdown in the Akt-transformed cell line with those expressed in a cohort of aggressive human prostate cancer. See also and Tables , , , and .

Journal: Cell reports

Article Title: KLF4, A Gene Regulating Prostate Stem Cell Homeostasis, Is a Barrier to Malignant Progression and Predictor of Good Prognosis in Prostate Cancer

doi: 10.1016/j.celrep.2018.11.065

Figure Lengend Snippet: (A) Venn diagram of differentially expressed genes (FDR <0.01, FC >2) in the Akt-transformed cell line after Klf4 knockdown (shKlf4) versus KLF4 re-expression (shKlf4 + KLF4). (B) Heatmap of EMT genes regulated by decreased (shKlf4 + empty vector [EV]) or increased KLF expression (shKlf4 + KLF4). (C) Heatmap of stem cell genes regulated by Klf4 knockdown (shKlf4) and KLF4 re-expression. (D) Ingenuity pathway analysis (canonical pathway) reveals pathways regulated by decreased (shKlf4) or increased (shKlf4 + KLF4) KLF4. Left: −log(p value), right: Z score. Pathways highlighted in red represent those that are inversely regulated by knockdown and re-expression of Klf4 (inverse Z score). (E) GSEA reveals positive enrichment of genes in a tumor metastasis gene signature after decreased (shKlf4) Klf4 expression. (F) GSEA reveals positive enrichment of genes in a tumor suppressor gene signature after increased (shKlf4 + KLF4) KLF4 expression. (G) Overlap of genes after Klf4 knockdown in the Akt-transformed cell line with those expressed in a cohort of human metastatic prostate cancer. (H) Overlap of genes after Klf4 knockdown in the Akt-transformed cell line with those expressed in a cohort of aggressive human prostate cancer. See also and Tables , , , and .

Article Snippet: pMXs-Klf4-IP retroviral vector , Addgene , Plasmid ID: 15920.

Techniques: Transformation Assay, Expressing, Plasmid Preparation

(A) Primary prostate cancer patients whose tumors express increased levels of KLF4 (21 of 131 cases [red line] have no biochemical recurrence for 10 years. (B) Increased levels of KLF4 predict prolonged survival in primary prostate cancer patients whose tumors have low Gleason scores (3 + 3 and 3 + 4). (C) Expression of KLF4 in 455 patients with primary prostate cancer from TCGA database as a function of Gleason scores. (D) Heatmap indicating the chromatin immunoprecipitation sequencing (ChIP-seq) read density within ±3 kb of the TSS region. (E) Pie chart illustrating the relative distribution of KLF4-bound sequences across the genome. (F) Motif analyses discovered Klf4 consensus motifs at ChIP-seq peak centers (p = 1e–175). (G) Heatmap representing 193 direct Klf4 target genes identified by combinatorial ChIP-seq and RNA-seq analyses of shControl, shKlf4, shKlf4 + EV, and shKlf4 + KLF4. (H) Bar graph illustrating ranked KEGG pathway analysis of 1,528 genes bound by Klf4 and regulated by Klf4 knockdown. See also and Tables and .

Journal: Cell reports

Article Title: KLF4, A Gene Regulating Prostate Stem Cell Homeostasis, Is a Barrier to Malignant Progression and Predictor of Good Prognosis in Prostate Cancer

doi: 10.1016/j.celrep.2018.11.065

Figure Lengend Snippet: (A) Primary prostate cancer patients whose tumors express increased levels of KLF4 (21 of 131 cases [red line] have no biochemical recurrence for 10 years. (B) Increased levels of KLF4 predict prolonged survival in primary prostate cancer patients whose tumors have low Gleason scores (3 + 3 and 3 + 4). (C) Expression of KLF4 in 455 patients with primary prostate cancer from TCGA database as a function of Gleason scores. (D) Heatmap indicating the chromatin immunoprecipitation sequencing (ChIP-seq) read density within ±3 kb of the TSS region. (E) Pie chart illustrating the relative distribution of KLF4-bound sequences across the genome. (F) Motif analyses discovered Klf4 consensus motifs at ChIP-seq peak centers (p = 1e–175). (G) Heatmap representing 193 direct Klf4 target genes identified by combinatorial ChIP-seq and RNA-seq analyses of shControl, shKlf4, shKlf4 + EV, and shKlf4 + KLF4. (H) Bar graph illustrating ranked KEGG pathway analysis of 1,528 genes bound by Klf4 and regulated by Klf4 knockdown. See also and Tables and .

Article Snippet: pMXs-Klf4-IP retroviral vector , Addgene , Plasmid ID: 15920.

Techniques: Expressing, ChIP-sequencing, RNA Sequencing Assay

(A) ChIP-seq (red) and RNA-seq (blue and purple) tracks indicate Klf4 enrichment on gene regulatory elements and transcript changes of good prognosis genes (ATF3, JUND, LIF, TEAD4) after Klf4 knockdown and rescue, respectively. shCon and shKlf4 depicted at scale 0–2.63. shKlf4 + EV and shKlf4 + KLF4 depicted at scale 0–0.46. Asterisks depict Klf4 motif locations. (B) Increased levels of ATF3, JUND, KLF4, LIF, and TEAD4 predict prolonged survival (red line) (p = 0.003) in prostate cancer patients whose tumors have low Gleason scores. (C) Prostate cancer patients whose tumors express increased levels of ATF3, JUND, KLF4, LIF, and TEAD4 (red line) have a good prognosis (p = 0.0002). (D) Increased levels of ATF3, JUND, KLF4, LIF, and TEAD4 predict prolonged survival (red line) (p = 0.008) in prostate cancer patients whose tumors have low Gleason scores. (E) Prostate cancer patients whose tumors express increased levels of ATF3, JUND, KLF4, LIF, and TEAD4 (red line) (p = 0.018) have a good prognosis. (F) Expression of ATF3, JUND, KLF4, LIF, and TEAD4 in 455 patients with prostate cancer from TCGA database as a function of Gleason scores. (G) Cox proportional hazard model on Gleason 6 and 7(3 + 4) comparing the performance of ATF3, JUND, KLF4, LIF, and TEAD4 expression with Gleason score alone or with the D’Amico classification. DF, degree of freedom. See also Figures , , and , and .

Journal: Cell reports

Article Title: KLF4, A Gene Regulating Prostate Stem Cell Homeostasis, Is a Barrier to Malignant Progression and Predictor of Good Prognosis in Prostate Cancer

doi: 10.1016/j.celrep.2018.11.065

Figure Lengend Snippet: (A) ChIP-seq (red) and RNA-seq (blue and purple) tracks indicate Klf4 enrichment on gene regulatory elements and transcript changes of good prognosis genes (ATF3, JUND, LIF, TEAD4) after Klf4 knockdown and rescue, respectively. shCon and shKlf4 depicted at scale 0–2.63. shKlf4 + EV and shKlf4 + KLF4 depicted at scale 0–0.46. Asterisks depict Klf4 motif locations. (B) Increased levels of ATF3, JUND, KLF4, LIF, and TEAD4 predict prolonged survival (red line) (p = 0.003) in prostate cancer patients whose tumors have low Gleason scores. (C) Prostate cancer patients whose tumors express increased levels of ATF3, JUND, KLF4, LIF, and TEAD4 (red line) have a good prognosis (p = 0.0002). (D) Increased levels of ATF3, JUND, KLF4, LIF, and TEAD4 predict prolonged survival (red line) (p = 0.008) in prostate cancer patients whose tumors have low Gleason scores. (E) Prostate cancer patients whose tumors express increased levels of ATF3, JUND, KLF4, LIF, and TEAD4 (red line) (p = 0.018) have a good prognosis. (F) Expression of ATF3, JUND, KLF4, LIF, and TEAD4 in 455 patients with prostate cancer from TCGA database as a function of Gleason scores. (G) Cox proportional hazard model on Gleason 6 and 7(3 + 4) comparing the performance of ATF3, JUND, KLF4, LIF, and TEAD4 expression with Gleason score alone or with the D’Amico classification. DF, degree of freedom. See also Figures , , and , and .

Article Snippet: pMXs-Klf4-IP retroviral vector , Addgene , Plasmid ID: 15920.

Techniques: ChIP-sequencing, RNA Sequencing Assay, Expressing

Journal: Cell reports

Article Title: KLF4, A Gene Regulating Prostate Stem Cell Homeostasis, Is a Barrier to Malignant Progression and Predictor of Good Prognosis in Prostate Cancer

doi: 10.1016/j.celrep.2018.11.065

Figure Lengend Snippet: KEY RESOURCE TABLE

Article Snippet: pMXs-Klf4-IP retroviral vector , Addgene , Plasmid ID: 15920.

Techniques: Plasmid Preparation, Recombinant, SYBR Green Assay, Transformation Assay, shRNA, Software

Review

pmx-klf4 retroviral vector (Addgene inc)

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1) Product Images from "Differential Control of Notch1 Gene Transcription by Klf4 and Sp3 Transcription Factors in Normal versus Cancer-Derived Keratinocytes"

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