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    AllPrep DNA RNA Mini Kit
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    For simultaneous purification of DNA and RNA from cells and tissues Kit contents Qiagen AllPrep DNA RNA Mini Kit 50 preps 30mg Sample 100L Elution Volume Silica Technology Spin Column Format Manual Processing Genomic DNA Total RNA Purification 35 min Time Run Ideal for PCR Real time PCR Microarray Blotting For Simultaneous Purification of DNA and RNA from Cells and Tissues Includes AllPrep DNA Spin Columns RNeasy Mini Spin Columns Collection Tubes RNase free Water and Buffers Benefits High quality DNA and RNA from the same sample Maximal yields of DNA and RNA from precious samples Rapid purification with short streamlined protocol Ready to use DNA and RNA for any downstream analysis
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    AllPrep DNA RNA Mini Kit
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    Qiagen hela cells
    AllPrep DNA RNA Mini Kit
    For simultaneous purification of DNA and RNA from cells and tissues Kit contents Qiagen AllPrep DNA RNA Mini Kit 50 preps 30mg Sample 100L Elution Volume Silica Technology Spin Column Format Manual Processing Genomic DNA Total RNA Purification 35 min Time Run Ideal for PCR Real time PCR Microarray Blotting For Simultaneous Purification of DNA and RNA from Cells and Tissues Includes AllPrep DNA Spin Columns RNeasy Mini Spin Columns Collection Tubes RNase free Water and Buffers Benefits High quality DNA and RNA from the same sample Maximal yields of DNA and RNA from precious samples Rapid purification with short streamlined protocol Ready to use DNA and RNA for any downstream analysis
    https://www.bioz.com/result/hela cells/product/Qiagen
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    hela cells - by Bioz Stars, 2020-07
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    1) Product Images from "The Flow of the Gibbon LAVA Element Is Facilitated by the LINE-1 Retrotransposition Machinery"

    Article Title: The Flow of the Gibbon LAVA Element Is Facilitated by the LINE-1 Retrotransposition Machinery

    Journal: Genome Biology and Evolution

    doi: 10.1093/gbe/evw224

    Analysis of the expression from L1 protein donor and retrotransposition reporter plasmids. ( A ) qRT-PCR analyses to quantify the relative amounts of spliced transcripts expressed from the different retrotransposition reporter cassettes. Total RNA was isolated after 12 days of hygromycin selection following co-transfection of pCEPneo, pAD3/SVA E , pLC5/LAVA C , pLC10/LAVA C , pLC21/LAVA C , or pLC23/LAVA C with the L1 protein donor plasmid pJM101/L1 RP Δneo and co-transfection of pJM101/L1 RP with pCEP4. Each co-transfection was performed in three biological replicates. The used primer/probe combination (see “Materials and Methods” section) is specific for the spliced mneo I-cassette (black box with arrow). Real-time PCR of each biological replicate was conducted in technical triplicates. Relative amounts of mRNA expression refer to the signal obtained from total RNA from pCEP4(mock)-transfected HeLa cells which was set as 1 (pCEP4) and served as negative control. Bars depict arithmetic means ±SD of technical triplicates of three biological replicates. ( B ) Immunoblot analysis of L1 ORF1p (left panel) and L1 ORF2p (right panel) expression after co-transfection of the L1 protein donor plasmid pJM101/L1 RP ΔneoΔORF1 (L1 RP ΔORF1) with the LAVA C retrotransposition reporter plasmids. Whole-cell lysates were prepared 13 days after co-transfection upon completion of hygromycin selection and subjected to immunoblot analysis using antibodies against either L1 ORF1p (αORF1p) or L1 ORF2p (αORF2p). In the case of the anti-L1 ORF1p immunoblot analysis (left panel), 20 µg of the cell lysate isolated from each of the differently transfected HeLa-HA cell cultures, were loaded per lane on a 12% PAA gel. Because ORF2p (Predicted MW∼150 kDa) is expressed at a significantly lower level than ORF1p ( Dai et al. 2014 ), 40 µg of total cell lysate from each of the differently transfected HeLa-HA cells were loaded per lane on a 6% PAA gel to perform anti-L1 ORF2p immunoblot analysis (right panel). Detectable amounts of intact L1 ORF1-encoded proteins are absent from pJM101/L1 RP ΔneoΔORF1-transfected HeLa cells. About 2 µg and 40 µg of the NTERA-2 cell extract were loaded on one lane of the gel used for the anti-ORF1p (left panel) Western blot analysis and the anti-ORF2p (right panel) immunoblot analysis, respectively. Lysates from pCEP4-transfected HeLa cells (HeLa + pCEP4) and from NTERA-2 cells served as negative and positive control for L1 protein detection, respectively. β-actin protein levels (∼42 kDa) were analyzed as loading control.
    Figure Legend Snippet: Analysis of the expression from L1 protein donor and retrotransposition reporter plasmids. ( A ) qRT-PCR analyses to quantify the relative amounts of spliced transcripts expressed from the different retrotransposition reporter cassettes. Total RNA was isolated after 12 days of hygromycin selection following co-transfection of pCEPneo, pAD3/SVA E , pLC5/LAVA C , pLC10/LAVA C , pLC21/LAVA C , or pLC23/LAVA C with the L1 protein donor plasmid pJM101/L1 RP Δneo and co-transfection of pJM101/L1 RP with pCEP4. Each co-transfection was performed in three biological replicates. The used primer/probe combination (see “Materials and Methods” section) is specific for the spliced mneo I-cassette (black box with arrow). Real-time PCR of each biological replicate was conducted in technical triplicates. Relative amounts of mRNA expression refer to the signal obtained from total RNA from pCEP4(mock)-transfected HeLa cells which was set as 1 (pCEP4) and served as negative control. Bars depict arithmetic means ±SD of technical triplicates of three biological replicates. ( B ) Immunoblot analysis of L1 ORF1p (left panel) and L1 ORF2p (right panel) expression after co-transfection of the L1 protein donor plasmid pJM101/L1 RP ΔneoΔORF1 (L1 RP ΔORF1) with the LAVA C retrotransposition reporter plasmids. Whole-cell lysates were prepared 13 days after co-transfection upon completion of hygromycin selection and subjected to immunoblot analysis using antibodies against either L1 ORF1p (αORF1p) or L1 ORF2p (αORF2p). In the case of the anti-L1 ORF1p immunoblot analysis (left panel), 20 µg of the cell lysate isolated from each of the differently transfected HeLa-HA cell cultures, were loaded per lane on a 12% PAA gel. Because ORF2p (Predicted MW∼150 kDa) is expressed at a significantly lower level than ORF1p ( Dai et al. 2014 ), 40 µg of total cell lysate from each of the differently transfected HeLa-HA cells were loaded per lane on a 6% PAA gel to perform anti-L1 ORF2p immunoblot analysis (right panel). Detectable amounts of intact L1 ORF1-encoded proteins are absent from pJM101/L1 RP ΔneoΔORF1-transfected HeLa cells. About 2 µg and 40 µg of the NTERA-2 cell extract were loaded on one lane of the gel used for the anti-ORF1p (left panel) Western blot analysis and the anti-ORF2p (right panel) immunoblot analysis, respectively. Lysates from pCEP4-transfected HeLa cells (HeLa + pCEP4) and from NTERA-2 cells served as negative and positive control for L1 protein detection, respectively. β-actin protein levels (∼42 kDa) were analyzed as loading control.

    Techniques Used: Expressing, Quantitative RT-PCR, Isolation, Selection, Cotransfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Transfection, Negative Control, Western Blot, Positive Control

    Trans -mobilization of mneo I-tagged LAVA C reporter elements by the human L1 protein machinery requires the presence of L1 ORF1p. ( A ) Immunoblot analysis of L1 protein expression in HeLa-HA cells after co-transfection of the L1 protein donor plasmid pJM101/L1 RP Δneo (L1 RP ) with the LAVA C , SVA E or pCEPneo reporter plasmids. Cell lysates were isolated 13 days after co-transfection upon completion of hyg R selection and subjected to immunoblot analysis using anti-L1 ORF1p antibodies (αORF1p). About 20 µg of cell lysates from the differently transfected cells were loaded per lane. Only 2 µg of NTERA-2 cell lysates were separated on one lane of the gel. β-actin protein levels (∼42 kDa) served as loading control. Lysates from pCEP4-transfected HeLa cells (HeLa + pCEP4) and from the human embryonal carcinoma cell line NTERA-2 served as negative and positive control for L1 ORF1p detection, respectively. ( B ) LAVA C retrotransposition reporter assay after hyg R selection for the presence of expression plasmids. LAVA C reporter plasmids pLC5/LAVA C , pLC10/LAVA C , pLC21/LAVA C , and pLC23/LAVA C , were co-transfected with the L1 protein donors pJM101/L1 RP Δneo (L1 RP ) or pJM101/L1 RP ΔneoΔORF (ΔORF) or the empty vector pCEP4. Control constructs pAD3/SVA E and pCEPneo were co-transfected with either pJM101/L1 RP Δneo or pCEP4. After hyg R selection, G418 R selection for retrotransposition events followed and retrotransposition rates were determined by counting G418 R HeLa colonies. To quantify retrotransposition frequencies, each co-transfection was performed in biological triplicates with each biological replicate being executed on a separate day. Each biological replicate was conducted in technical triplicates. Absolute retrotransposition frequencies per 10 6 cells are listed and relative retrotransposition rates are presented as bar diagrams. Cis retrotransposition frequency of the L1 reporter element encoded by pJM101/L1 RP after its co-transfection with pCEP4 was set as 100%. Each bar depicts the arithmetic mean ± SD of the relative retrotransposition frequencies that resulted from nine individual co-transfection experiments ( n = 9).
    Figure Legend Snippet: Trans -mobilization of mneo I-tagged LAVA C reporter elements by the human L1 protein machinery requires the presence of L1 ORF1p. ( A ) Immunoblot analysis of L1 protein expression in HeLa-HA cells after co-transfection of the L1 protein donor plasmid pJM101/L1 RP Δneo (L1 RP ) with the LAVA C , SVA E or pCEPneo reporter plasmids. Cell lysates were isolated 13 days after co-transfection upon completion of hyg R selection and subjected to immunoblot analysis using anti-L1 ORF1p antibodies (αORF1p). About 20 µg of cell lysates from the differently transfected cells were loaded per lane. Only 2 µg of NTERA-2 cell lysates were separated on one lane of the gel. β-actin protein levels (∼42 kDa) served as loading control. Lysates from pCEP4-transfected HeLa cells (HeLa + pCEP4) and from the human embryonal carcinoma cell line NTERA-2 served as negative and positive control for L1 ORF1p detection, respectively. ( B ) LAVA C retrotransposition reporter assay after hyg R selection for the presence of expression plasmids. LAVA C reporter plasmids pLC5/LAVA C , pLC10/LAVA C , pLC21/LAVA C , and pLC23/LAVA C , were co-transfected with the L1 protein donors pJM101/L1 RP Δneo (L1 RP ) or pJM101/L1 RP ΔneoΔORF (ΔORF) or the empty vector pCEP4. Control constructs pAD3/SVA E and pCEPneo were co-transfected with either pJM101/L1 RP Δneo or pCEP4. After hyg R selection, G418 R selection for retrotransposition events followed and retrotransposition rates were determined by counting G418 R HeLa colonies. To quantify retrotransposition frequencies, each co-transfection was performed in biological triplicates with each biological replicate being executed on a separate day. Each biological replicate was conducted in technical triplicates. Absolute retrotransposition frequencies per 10 6 cells are listed and relative retrotransposition rates are presented as bar diagrams. Cis retrotransposition frequency of the L1 reporter element encoded by pJM101/L1 RP after its co-transfection with pCEP4 was set as 100%. Each bar depicts the arithmetic mean ± SD of the relative retrotransposition frequencies that resulted from nine individual co-transfection experiments ( n = 9).

    Techniques Used: Expressing, Cotransfection, Plasmid Preparation, Isolation, Selection, Transfection, Positive Control, Reporter Assay, Construct

    Structures of LAVA C retrotransposition reporter plasmids and rationale of the LAVA trans -mobilization assay. ( A ) Organization of the non-autonomous non-LTR retrotransposon LAVA in the gibbon genome. The SVA-derived module consists of CT-rich (green), Alu -like (light blue), and VNTR (banded) region. It is separated from the Alu Sz-derived module (black) by Unique Region 1 (U1). The L1ME5-derived module (pink) is separated from the Alu Sz module by Unique Region 2 (U2). Black arrows in boxed Alu -like region and L1ME5 module indicate antisense orientation. Poly(A), poly(A) stretch; TSD, target site duplication. ( B ) Schematics of the retrotransposition reporter plasmid pLC10/LAVA C carrying the full-length LAVA C element, the expression cassettes of the reporter plasmids pLC5/LAVA C , pLC21/LAVA C , pLC23/LAVA C expressing truncated LAVA C versions, and pAD3/SVA E ( Raiz et al. 2012 ). pCEPneo is used to measure processed pseudogene formation frequency. Each of the LAVA and SVA reporter elements and the processed pseudogene formation cassette were tagged with the indicator gene mneo I, and set under transcriptional control of the human CMV immediate early enhancer/promoter (CMV P ). Splice donor (SD) and splice acceptor (SA) sites of the oppositely oriented γ-globin intron are indicated. mneo I is flanked by an SV40 promoter (P’) and polyadenylation signal (A’).Transcripts starting from CMV P driving LAVA mneo I, SVA mneo I or pCEP mneo I transcription, can splice the intron, but contain an antisense copy of the neo R gene. G418 resistant (G418 R ) colonies accrue only if this transcript is reverse transcribed, integrated into chromosomal DNA, and expressed from its own promoter P’. LAVA or SVA sequences were inserted between CMV P and the mneo I cassette. pLC5/LAVA C differs from pLC10/LAVA C exclusively in the absence of the L1ME5 and U2 modules covering 322 bp. pLC21/LAVA C and pLC23/LAVA C encode the 5′-terminal 819 bp of LAVA C covering TSD, CT-rich and Alu -like region, and the 3′-terminal 473 bp covering U1, Alu Sz and L1ME5 modules, respectively. pAD3/SVA E ( Raiz et al. 2012 ) serves as positive control construct for trans -mobilization. Transcriptional termination signals at the 3′ ends of the L1ME5 (pLC10/LAVA C , pLC23/LAVA C ) and SINE-R (pAD3/SVA E ) modules were deleted from the LAVA and SVA reporter cassettes (ΔAATAAA) to ensure transcriptional read-through into the mneo I cassette and polyadenylation at the pCEP4-encoded SV40 polyadenylation signal (pA). pCEPneo is distinguished from the remaining presented reporter constructs by the absence of any LAVA or SVA sequence. CMV P sequences are highlighted in grey. CMV P major and minor transcription start sites ( Isomura et al. 2008 ) are indicated by arrows. TSD, target site duplication; CT-rich, Alu -like, VNTR ( V ariable n umber of t andem r epeats), U1, Alu Sz, U2 and L1ME5 represent repeat modules, LAVA is composed of; pA, poly(A) tail. Hyg R , hygromycin resistance gene serving as selectable marker for eukaryotic cells. ( C ) Design of the experimental approach to test for trans -mobilization of the mneo I-tagged LAVA C element by the human L1 protein machinery. LAVA or SVA retrotransposition reporter plasmids, or pCEPneo were each co-transfected with L1 protein donor plasmid pJM101/L1 RP Δneo or pJM101/L1 RP ΔneoΔORF1 (blue) into HeLa-HA cells that were subsequently selected for hygromycin resistance for 12 days. Hyg R cells were assayed for retrotransposition events by selecting for 9–12 days for G418 R HeLa colonies.
    Figure Legend Snippet: Structures of LAVA C retrotransposition reporter plasmids and rationale of the LAVA trans -mobilization assay. ( A ) Organization of the non-autonomous non-LTR retrotransposon LAVA in the gibbon genome. The SVA-derived module consists of CT-rich (green), Alu -like (light blue), and VNTR (banded) region. It is separated from the Alu Sz-derived module (black) by Unique Region 1 (U1). The L1ME5-derived module (pink) is separated from the Alu Sz module by Unique Region 2 (U2). Black arrows in boxed Alu -like region and L1ME5 module indicate antisense orientation. Poly(A), poly(A) stretch; TSD, target site duplication. ( B ) Schematics of the retrotransposition reporter plasmid pLC10/LAVA C carrying the full-length LAVA C element, the expression cassettes of the reporter plasmids pLC5/LAVA C , pLC21/LAVA C , pLC23/LAVA C expressing truncated LAVA C versions, and pAD3/SVA E ( Raiz et al. 2012 ). pCEPneo is used to measure processed pseudogene formation frequency. Each of the LAVA and SVA reporter elements and the processed pseudogene formation cassette were tagged with the indicator gene mneo I, and set under transcriptional control of the human CMV immediate early enhancer/promoter (CMV P ). Splice donor (SD) and splice acceptor (SA) sites of the oppositely oriented γ-globin intron are indicated. mneo I is flanked by an SV40 promoter (P’) and polyadenylation signal (A’).Transcripts starting from CMV P driving LAVA mneo I, SVA mneo I or pCEP mneo I transcription, can splice the intron, but contain an antisense copy of the neo R gene. G418 resistant (G418 R ) colonies accrue only if this transcript is reverse transcribed, integrated into chromosomal DNA, and expressed from its own promoter P’. LAVA or SVA sequences were inserted between CMV P and the mneo I cassette. pLC5/LAVA C differs from pLC10/LAVA C exclusively in the absence of the L1ME5 and U2 modules covering 322 bp. pLC21/LAVA C and pLC23/LAVA C encode the 5′-terminal 819 bp of LAVA C covering TSD, CT-rich and Alu -like region, and the 3′-terminal 473 bp covering U1, Alu Sz and L1ME5 modules, respectively. pAD3/SVA E ( Raiz et al. 2012 ) serves as positive control construct for trans -mobilization. Transcriptional termination signals at the 3′ ends of the L1ME5 (pLC10/LAVA C , pLC23/LAVA C ) and SINE-R (pAD3/SVA E ) modules were deleted from the LAVA and SVA reporter cassettes (ΔAATAAA) to ensure transcriptional read-through into the mneo I cassette and polyadenylation at the pCEP4-encoded SV40 polyadenylation signal (pA). pCEPneo is distinguished from the remaining presented reporter constructs by the absence of any LAVA or SVA sequence. CMV P sequences are highlighted in grey. CMV P major and minor transcription start sites ( Isomura et al. 2008 ) are indicated by arrows. TSD, target site duplication; CT-rich, Alu -like, VNTR ( V ariable n umber of t andem r epeats), U1, Alu Sz, U2 and L1ME5 represent repeat modules, LAVA is composed of; pA, poly(A) tail. Hyg R , hygromycin resistance gene serving as selectable marker for eukaryotic cells. ( C ) Design of the experimental approach to test for trans -mobilization of the mneo I-tagged LAVA C element by the human L1 protein machinery. LAVA or SVA retrotransposition reporter plasmids, or pCEPneo were each co-transfected with L1 protein donor plasmid pJM101/L1 RP Δneo or pJM101/L1 RP ΔneoΔORF1 (blue) into HeLa-HA cells that were subsequently selected for hygromycin resistance for 12 days. Hyg R cells were assayed for retrotransposition events by selecting for 9–12 days for G418 R HeLa colonies.

    Techniques Used: Derivative Assay, Plasmid Preparation, Expressing, Positive Control, Construct, Sequencing, Marker, Transfection

    2) Product Images from "RPE and neuronal differentiation of allotransplantated porcine ciliary epithelium-derived cells"

    Article Title: RPE and neuronal differentiation of allotransplantated porcine ciliary epithelium-derived cells

    Journal: Molecular Vision

    doi:

    Quantitative real time PCR data for ciliary epithelium (CE)-derived cell cultures following in vitro differentiation. RNA was isolated from CE-derived cells after in vitro differentiation on poly-D-Lysine, laminin coated plates in the presence of 1% serum and 10 ng/ml basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) for 20 days. The data was analyzed using REST software for the relative quantification. The expression ratio represents the ratio of expression in differentiated compared to undifferentiated cultures. After differentiation, protein kinase α (PKCα; p
    Figure Legend Snippet: Quantitative real time PCR data for ciliary epithelium (CE)-derived cell cultures following in vitro differentiation. RNA was isolated from CE-derived cells after in vitro differentiation on poly-D-Lysine, laminin coated plates in the presence of 1% serum and 10 ng/ml basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) for 20 days. The data was analyzed using REST software for the relative quantification. The expression ratio represents the ratio of expression in differentiated compared to undifferentiated cultures. After differentiation, protein kinase α (PKCα; p

    Techniques Used: Real-time Polymerase Chain Reaction, Derivative Assay, In Vitro, Isolation, Software, Expressing

    Gene expression of ciliary epithelium (CE)-derived cells determined by RT–PCR. RNA was isolated from passage 1 and was subjected to conventional RT–PCR. PCR products were resolved on 1.5% agarose gel. A : Amplification of mRNA for pluripotency markers. B : Amplification of mRNA for retinal progenitor genes. Sizes in base pairs for the corresponding marker bands (M) are shown on the left, adjacent to the gel images. PCR reactions performed with cDNA template are shown in lanes marked as '+' and negative control reactions performed with templates from the RT where reverse transcriptase was omitted are shown in lanes marked as '–'. Amplicons for a housekeeping gene (HPRT) under the same conditions are shown in the bottom panels.
    Figure Legend Snippet: Gene expression of ciliary epithelium (CE)-derived cells determined by RT–PCR. RNA was isolated from passage 1 and was subjected to conventional RT–PCR. PCR products were resolved on 1.5% agarose gel. A : Amplification of mRNA for pluripotency markers. B : Amplification of mRNA for retinal progenitor genes. Sizes in base pairs for the corresponding marker bands (M) are shown on the left, adjacent to the gel images. PCR reactions performed with cDNA template are shown in lanes marked as '+' and negative control reactions performed with templates from the RT where reverse transcriptase was omitted are shown in lanes marked as '–'. Amplicons for a housekeeping gene (HPRT) under the same conditions are shown in the bottom panels.

    Techniques Used: Expressing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Isolation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, Marker, Negative Control

    3) Product Images from "ZBTB48 is both a vertebrate telomere‐binding protein and a transcriptional activator"

    Article Title: ZBTB48 is both a vertebrate telomere‐binding protein and a transcriptional activator

    Journal: EMBO Reports

    doi: 10.15252/embr.201744095

    ZBTB 48 binds telomeres via its ZnF11 domain in vitro and in vivo Domain structure of wild‐type (WT) ZBTB48 containing a N‐terminal BTB domain and 11 zinc fingers (ZnF) at the C‐terminus of the 688‐amino‐acid protein. Note that ZnF2 is degenerate. Below is a schematic of the deletion construct lacking ZnF1‐10 used in (B). Sequence‐specific DNA pull‐downs with either telomeric (TTAGGG) or a control sequence (GTGAGT) for FLAG‐ZBTB48 WT, point mutants for the ten functional zinc fingers and a domain deletion construct for ZnF1‐10. Sequence‐specific DNA pull‐downs for FLAG‐ZBTB48 WT and ZnF11 point mutant for telomeric and subtelomeric variant repeat sequences (green) and their respective scrambled controls (blue). Co‐localization analysis of endogenous ZBTB48 and TRF2 in U2OS cells by immunofluorescence (IF) staining. A representative image illustrating the co‐localization between ZBTB48 (green) and TRF2 (red) as a marker for telomeres is shown with DAPI (blue) used as a nuclear counterstain. The quantification of frequency of co‐localization events (right) was done after 3D reconstruction of the acquired z ‐stacks ( n = 104 cells). The average value is indicated by a red bar. IF stainings for exogenous FLAG‐ZBTB48 WT and point mutants for ZnF10 and ZnF11 in U2OS cells. The same analysis as in (D) was performed and average co‐localization frequencies are shown ( n = 24–33 cells). Error bars indicate standard deviations, and P ‐values are based on Student's t ‐test. Co‐localization analysis between ZBTB48 (green) and PML (red) as a marker for PML bodies analogous to (D) in U2OS cells ( n = 102 cells). Data information: (D–F) Co‐localization events are indicated by white arrows. Scale bars represent 5 μm. Source data are available online for this figure.
    Figure Legend Snippet: ZBTB 48 binds telomeres via its ZnF11 domain in vitro and in vivo Domain structure of wild‐type (WT) ZBTB48 containing a N‐terminal BTB domain and 11 zinc fingers (ZnF) at the C‐terminus of the 688‐amino‐acid protein. Note that ZnF2 is degenerate. Below is a schematic of the deletion construct lacking ZnF1‐10 used in (B). Sequence‐specific DNA pull‐downs with either telomeric (TTAGGG) or a control sequence (GTGAGT) for FLAG‐ZBTB48 WT, point mutants for the ten functional zinc fingers and a domain deletion construct for ZnF1‐10. Sequence‐specific DNA pull‐downs for FLAG‐ZBTB48 WT and ZnF11 point mutant for telomeric and subtelomeric variant repeat sequences (green) and their respective scrambled controls (blue). Co‐localization analysis of endogenous ZBTB48 and TRF2 in U2OS cells by immunofluorescence (IF) staining. A representative image illustrating the co‐localization between ZBTB48 (green) and TRF2 (red) as a marker for telomeres is shown with DAPI (blue) used as a nuclear counterstain. The quantification of frequency of co‐localization events (right) was done after 3D reconstruction of the acquired z ‐stacks ( n = 104 cells). The average value is indicated by a red bar. IF stainings for exogenous FLAG‐ZBTB48 WT and point mutants for ZnF10 and ZnF11 in U2OS cells. The same analysis as in (D) was performed and average co‐localization frequencies are shown ( n = 24–33 cells). Error bars indicate standard deviations, and P ‐values are based on Student's t ‐test. Co‐localization analysis between ZBTB48 (green) and PML (red) as a marker for PML bodies analogous to (D) in U2OS cells ( n = 102 cells). Data information: (D–F) Co‐localization events are indicated by white arrows. Scale bars represent 5 μm. Source data are available online for this figure.

    Techniques Used: In Vitro, In Vivo, Zinc-Fingers, Construct, Sequencing, Functional Assay, Mutagenesis, Variant Assay, Immunofluorescence, Staining, Marker

    ZBTB48 and HOT1 KO clones show depleted expression Depiction of the ZBTB48 TALEN binding sites located in exon 2. Genotypes of HeLa and U2OS WT and ZBTB48 KO clones. The grey boxes represent the TALEN binding sites; insertions (blue) and deletions (red) are marked. The numbers in brackets represent the numbers of clones found with the specific genotype. Gel image of T7E1 assay showing ZBTB48 TALEN activity in HeLa cells. Western blot confirmation of depleted ZBTB48 expression in each five HeLa and U2OS ZBTB48 KO clones compared to parental cells. IF confirmation of depleted ZBTB48 expression (green) with DAPI (blue) as nuclear counterstain in U2OS ZBTB48 KO cells. ZBTB48 signals are reduced to background levels. Scale bars represent 5 μm. Depiction of the HOT1 TALEN binding sites located in exon 3. Genotypes of HeLa and U2OS WT and HOT1 KO clones. The grey boxes represent the TALEN binding sites; insertions (blue) and deletions (red) are marked. The numbers in brackets represent the numbers of clones found with the specific genotype. Gel image of T7E1 assay showing HOT1 TALEN activity in HeLa cells. IF confirmation of depleted HOT1 expression (green) with DAPI (blue) as nuclear counterstain in a representative HeLa HOT1 KO clone. Scale bars represent 5 μm. Source data are available online for this figure.
    Figure Legend Snippet: ZBTB48 and HOT1 KO clones show depleted expression Depiction of the ZBTB48 TALEN binding sites located in exon 2. Genotypes of HeLa and U2OS WT and ZBTB48 KO clones. The grey boxes represent the TALEN binding sites; insertions (blue) and deletions (red) are marked. The numbers in brackets represent the numbers of clones found with the specific genotype. Gel image of T7E1 assay showing ZBTB48 TALEN activity in HeLa cells. Western blot confirmation of depleted ZBTB48 expression in each five HeLa and U2OS ZBTB48 KO clones compared to parental cells. IF confirmation of depleted ZBTB48 expression (green) with DAPI (blue) as nuclear counterstain in U2OS ZBTB48 KO cells. ZBTB48 signals are reduced to background levels. Scale bars represent 5 μm. Depiction of the HOT1 TALEN binding sites located in exon 3. Genotypes of HeLa and U2OS WT and HOT1 KO clones. The grey boxes represent the TALEN binding sites; insertions (blue) and deletions (red) are marked. The numbers in brackets represent the numbers of clones found with the specific genotype. Gel image of T7E1 assay showing HOT1 TALEN activity in HeLa cells. IF confirmation of depleted HOT1 expression (green) with DAPI (blue) as nuclear counterstain in a representative HeLa HOT1 KO clone. Scale bars represent 5 μm. Source data are available online for this figure.

    Techniques Used: Clone Assay, Expressing, Binding Assay, Activity Assay, Western Blot

    ZBTB48 is a direct telomere‐binding protein but TRAP activity is not affected in ZBTB48 KO clones TTAGGG content of telomeric reads in ChIPseq samples from U2OS WT, U2OS ZBTB48 KO and U2OS HOT1 KO clones using IgG, TRF2, ZBTB48 Atlas, ZBTB48 GeneTex and HOT1 antibodies as well as FLAG antibody for FLAG‐ZBTB48 WT and point mutants for ZnF10 and ZnF11. The percentage contribution to all reads containing 1–8× TTAGGG repeats is shown. All reactions were performed in technical replicates on two independent WT and KO clones each. These reactions are biological replicates of Fig 2 A, performed with independent U2OS ZBTB48 WT and KO clones for ZBTB48 and with a second HOT1 antibody for HOT1. Telomerase activity was determined based on a quantitative TRAP assay. Heat‐inactivated HeLa extracts were used as a threshold to determine non‐specific background signal. Differences in C t values (∆∆ C t ) from the quantitative PCR measurements are displayed relative to the heat‐inactivation control. Error bars represent standard deviations ( n = 3). The P ‐value is based on Student's t ‐test.
    Figure Legend Snippet: ZBTB48 is a direct telomere‐binding protein but TRAP activity is not affected in ZBTB48 KO clones TTAGGG content of telomeric reads in ChIPseq samples from U2OS WT, U2OS ZBTB48 KO and U2OS HOT1 KO clones using IgG, TRF2, ZBTB48 Atlas, ZBTB48 GeneTex and HOT1 antibodies as well as FLAG antibody for FLAG‐ZBTB48 WT and point mutants for ZnF10 and ZnF11. The percentage contribution to all reads containing 1–8× TTAGGG repeats is shown. All reactions were performed in technical replicates on two independent WT and KO clones each. These reactions are biological replicates of Fig 2 A, performed with independent U2OS ZBTB48 WT and KO clones for ZBTB48 and with a second HOT1 antibody for HOT1. Telomerase activity was determined based on a quantitative TRAP assay. Heat‐inactivated HeLa extracts were used as a threshold to determine non‐specific background signal. Differences in C t values (∆∆ C t ) from the quantitative PCR measurements are displayed relative to the heat‐inactivation control. Error bars represent standard deviations ( n = 3). The P ‐value is based on Student's t ‐test.

    Techniques Used: Binding Assay, Activity Assay, Clone Assay, TRAP Assay, Real-time Polymerase Chain Reaction

    ZBTB 48 preferentially binds to promoter regions Peak calling of ZBTB48 ChIPseq reactions from two U2OS ZBTB48 WT clones (top) and two U2OS ZBTB48 KO clones (bottom). ChIP reactions were performed with two independent ZBT48 antibodies (ab1 and ab2) each in two independent clones (WT1 and WT2 or KO1 and KO2). Only peaks that are enriched by at least eightfold in the WT over the KO clones and have a FDR
    Figure Legend Snippet: ZBTB 48 preferentially binds to promoter regions Peak calling of ZBTB48 ChIPseq reactions from two U2OS ZBTB48 WT clones (top) and two U2OS ZBTB48 KO clones (bottom). ChIP reactions were performed with two independent ZBT48 antibodies (ab1 and ab2) each in two independent clones (WT1 and WT2 or KO1 and KO2). Only peaks that are enriched by at least eightfold in the WT over the KO clones and have a FDR

    Techniques Used: Chromatin Immunoprecipitation, Clone Assay

    ZBTB 48 associates with short and long telomeres in vivo TTAGGG content of telomeric reads in ChIPseq samples from U2OS WT, U2OS ZBTB48 KO and U2OS HOT1 KO clones using IgG, TRF2, ZBTB48 Atlas, ZBTB48 GeneTex and HOT1 antibodies as well as FLAG antibody for FLAG‐ZBTB48 WT and point mutants for ZnF10 and ZnF11. The percentage contribution to all reads containing 1–8× TTAGGG repeats is shown. All reactions were performed in technical replicates on two independent WT and KO clones each. MEME sequence logo and bit score for the top 500 extratelomeric ChIPseq peaks for ZBTB48 Atlas, ZBTB48 GeneTex, TRF2 and HOT1 antibodies compared to IgG samples in U2OS. For each antibody, the most frequent motif is shown. TTAGGG enrichment in ChIPseq samples with reads with 7× or 8× TTAGGG repeats. Absolute read counts from ChIPseq reactions from each two HeLa and U2OS WT and KO clones with two independent antibodies for ZBTB48 and HOT1 are shown (left). The corresponding fold enrichments are calculated (right) and compared to TRF2 ChIPseq reactions for which fold enrichments are calculated relative to IgG samples. Error bars represent standard deviations ( n = 4), and P ‐values are based on Student's t ‐test with ** indicating P
    Figure Legend Snippet: ZBTB 48 associates with short and long telomeres in vivo TTAGGG content of telomeric reads in ChIPseq samples from U2OS WT, U2OS ZBTB48 KO and U2OS HOT1 KO clones using IgG, TRF2, ZBTB48 Atlas, ZBTB48 GeneTex and HOT1 antibodies as well as FLAG antibody for FLAG‐ZBTB48 WT and point mutants for ZnF10 and ZnF11. The percentage contribution to all reads containing 1–8× TTAGGG repeats is shown. All reactions were performed in technical replicates on two independent WT and KO clones each. MEME sequence logo and bit score for the top 500 extratelomeric ChIPseq peaks for ZBTB48 Atlas, ZBTB48 GeneTex, TRF2 and HOT1 antibodies compared to IgG samples in U2OS. For each antibody, the most frequent motif is shown. TTAGGG enrichment in ChIPseq samples with reads with 7× or 8× TTAGGG repeats. Absolute read counts from ChIPseq reactions from each two HeLa and U2OS WT and KO clones with two independent antibodies for ZBTB48 and HOT1 are shown (left). The corresponding fold enrichments are calculated (right) and compared to TRF2 ChIPseq reactions for which fold enrichments are calculated relative to IgG samples. Error bars represent standard deviations ( n = 4), and P ‐values are based on Student's t ‐test with ** indicating P

    Techniques Used: In Vivo, Clone Assay, Sequencing

    ZBTB48 ZnF11 is necessary to bind to telomeres Sequence‐specific DNA pull‐downs with either telomeric (TTAGGG) or a control sequence (GTGAGT) for FLAG‐ZBTB48 WT, domain deletion constructs for different zinc finger and combinations of deletion constructs with ZnF10 or 11 point mutants. Domain structures are indicated on the right. Sequence‐specific DNA pull‐downs for FLAG‐ZBTB48 WT and ZnF11 point mutant for telomeric repeat sequences of different phyla (green) and their respective scrambled controls (blue). Protein expression analysis of ZBTB48 by Western blot for the cell lines used in this study. GAPDH serves as a loading control. IF stainings for exogenous FLAG‐ZBTB48 WT and point mutants for ZnF10 and ZnF11 in U2OS cells. The same analysis as in Fig 1 E was performed and average co‐localization frequencies are shown ( n = 24–37 cells). Co‐localization analysis of endogenous ZBTB48 or exogenous FLAG‐ZBTB48 WT with TRF2 in HeLa cells by immunofluorescence (IF) staining. A representative image illustrating the co‐localization between ZBTB48 or FLAG‐ZBTB48 WT (green) and TRF2 (red) as a marker for telomeres is shown with DAPI (blue) used as a nuclear counterstain. Co‐localization events are indicated by white arrows. The quantification of frequency of co‐localization events (right) was done after 3D reconstruction of the acquired z ‐stacks ( n = 30 cells). Co‐localization analysis of endogenous ZBTB48 or exogenous FLAG‐ZBTB48 WT with TRF2 in HeLa 1.3 cells by immunofluorescence (IF) staining analogous to (E) ( n = 30 cells). Co‐localization analysis of endogenous ZBTB48 or exogenous FLAG‐ZBTB48 WT with TRF2 in HT1080 super‐telomerase cells by immunofluorescence (IF) staining analogous to (E) ( n = 30 cells). Data information: (D–G) Scale bars represent 5 μm. Error bars indicate standard deviations, and P ‐values are based on Student's t ‐test. Source data are available online for this figure.
    Figure Legend Snippet: ZBTB48 ZnF11 is necessary to bind to telomeres Sequence‐specific DNA pull‐downs with either telomeric (TTAGGG) or a control sequence (GTGAGT) for FLAG‐ZBTB48 WT, domain deletion constructs for different zinc finger and combinations of deletion constructs with ZnF10 or 11 point mutants. Domain structures are indicated on the right. Sequence‐specific DNA pull‐downs for FLAG‐ZBTB48 WT and ZnF11 point mutant for telomeric repeat sequences of different phyla (green) and their respective scrambled controls (blue). Protein expression analysis of ZBTB48 by Western blot for the cell lines used in this study. GAPDH serves as a loading control. IF stainings for exogenous FLAG‐ZBTB48 WT and point mutants for ZnF10 and ZnF11 in U2OS cells. The same analysis as in Fig 1 E was performed and average co‐localization frequencies are shown ( n = 24–37 cells). Co‐localization analysis of endogenous ZBTB48 or exogenous FLAG‐ZBTB48 WT with TRF2 in HeLa cells by immunofluorescence (IF) staining. A representative image illustrating the co‐localization between ZBTB48 or FLAG‐ZBTB48 WT (green) and TRF2 (red) as a marker for telomeres is shown with DAPI (blue) used as a nuclear counterstain. Co‐localization events are indicated by white arrows. The quantification of frequency of co‐localization events (right) was done after 3D reconstruction of the acquired z ‐stacks ( n = 30 cells). Co‐localization analysis of endogenous ZBTB48 or exogenous FLAG‐ZBTB48 WT with TRF2 in HeLa 1.3 cells by immunofluorescence (IF) staining analogous to (E) ( n = 30 cells). Co‐localization analysis of endogenous ZBTB48 or exogenous FLAG‐ZBTB48 WT with TRF2 in HT1080 super‐telomerase cells by immunofluorescence (IF) staining analogous to (E) ( n = 30 cells). Data information: (D–G) Scale bars represent 5 μm. Error bars indicate standard deviations, and P ‐values are based on Student's t ‐test. Source data are available online for this figure.

    Techniques Used: Sequencing, Construct, Mutagenesis, Expressing, Western Blot, Immunofluorescence, Staining, Marker

    ZBTB48 is a transcriptional activator Differential expression analysis of the RNA sequencing (RNAseq) gene quantitation, comparing each five WT and ZBTB48 KO clones for U2OS (left) and HeLa (right). Cut‐offs for significant differential expression were set to log 2 (fold change) > |1| and −log 10 (adjusted P ‐value) > 2 (FDR
    Figure Legend Snippet: ZBTB48 is a transcriptional activator Differential expression analysis of the RNA sequencing (RNAseq) gene quantitation, comparing each five WT and ZBTB48 KO clones for U2OS (left) and HeLa (right). Cut‐offs for significant differential expression were set to log 2 (fold change) > |1| and −log 10 (adjusted P ‐value) > 2 (FDR

    Techniques Used: Expressing, RNA Sequencing Assay, Quantitation Assay, Clone Assay

    ZBTB 48 is a negative regulator of telomere length Terminal restriction fragment (TRF) analysis for each five independent HeLa WT and HeLa ZBTB48 KO clones at passage 20. The average telomere length was determined from the telomeric distribution (left) and used for average quantification of WT vs. KO clones (right). Average telomere length is indicated for all WT samples (dotted line) and individually for all samples (red dots). Error bars indicate standard deviations, and the P ‐value is based on Student's t ‐test. TRF analysis using pulsed‐field gel electrophoresis (PFGE) of parental U2OS WT cells compared to five independent U2OS ZBTB48 KO clones at passage 37. Average telomere length is indicated for all WT samples (dotted line) and individually for all samples (red dots). Quantification of C‐circles in U2OS WT cells compared to five independent U2OS ZBTB48 KO clones. C‐circle reactions were carried out and spotted in triplicate (top), and average quantifications are displayed (bottom). No Φ indicates negative control reactions without the ΦDNA polymerase. 15 ng DNA was used as input material per reaction. The dashed line indicates cropping of the membrane between KO clones 4 and 5. Error bars indicate standard deviations, and P ‐values are based on Student's t ‐test with * indicating P
    Figure Legend Snippet: ZBTB 48 is a negative regulator of telomere length Terminal restriction fragment (TRF) analysis for each five independent HeLa WT and HeLa ZBTB48 KO clones at passage 20. The average telomere length was determined from the telomeric distribution (left) and used for average quantification of WT vs. KO clones (right). Average telomere length is indicated for all WT samples (dotted line) and individually for all samples (red dots). Error bars indicate standard deviations, and the P ‐value is based on Student's t ‐test. TRF analysis using pulsed‐field gel electrophoresis (PFGE) of parental U2OS WT cells compared to five independent U2OS ZBTB48 KO clones at passage 37. Average telomere length is indicated for all WT samples (dotted line) and individually for all samples (red dots). Quantification of C‐circles in U2OS WT cells compared to five independent U2OS ZBTB48 KO clones. C‐circle reactions were carried out and spotted in triplicate (top), and average quantifications are displayed (bottom). No Φ indicates negative control reactions without the ΦDNA polymerase. 15 ng DNA was used as input material per reaction. The dashed line indicates cropping of the membrane between KO clones 4 and 5. Error bars indicate standard deviations, and P ‐values are based on Student's t ‐test with * indicating P

    Techniques Used: Clone Assay, Pulsed-Field Gel, Electrophoresis, Negative Control

    ZBTB48 acts as a transcription factor in contrast to HOT1 Differential expression analysis of the RNA sequencing (RNAseq) gene quantitation, comparing each five WT and ZBTB48 KO clones for U2OS (left) and HeLa (right). Cut‐offs for significant differential expression were set to log 2 (fold change) > |1| and −log 10 (adjusted P ‐value) > 2 (FDR
    Figure Legend Snippet: ZBTB48 acts as a transcription factor in contrast to HOT1 Differential expression analysis of the RNA sequencing (RNAseq) gene quantitation, comparing each five WT and ZBTB48 KO clones for U2OS (left) and HeLa (right). Cut‐offs for significant differential expression were set to log 2 (fold change) > |1| and −log 10 (adjusted P ‐value) > 2 (FDR

    Techniques Used: Expressing, RNA Sequencing Assay, Quantitation Assay, Clone Assay

    ZBTB48‐dependent loss of MTFP1 phenocopies MTFP1 depletion Fluorescence microscopy analysis of the structure and localization of the mitochondrial network in HeLa WT and ZBTB48 KO clones. Mitochondria are marked with the MitoTracker dye (red), and nuclei are counterstained with DAPI (blue). Scale bars represent 20 μm. The same analysis as in (A) for U2OS WT and ZBTB48 KO clones.
    Figure Legend Snippet: ZBTB48‐dependent loss of MTFP1 phenocopies MTFP1 depletion Fluorescence microscopy analysis of the structure and localization of the mitochondrial network in HeLa WT and ZBTB48 KO clones. Mitochondria are marked with the MitoTracker dye (red), and nuclei are counterstained with DAPI (blue). Scale bars represent 20 μm. The same analysis as in (A) for U2OS WT and ZBTB48 KO clones.

    Techniques Used: Fluorescence, Microscopy, Clone Assay

    ZBTB48 is required for MTFP1 expression Western blot confirmation of reduced MTFP1 expression in each five HeLa and U2OS ZBTB48 KO clones compared to five WT clones each. Similar to ZBTB48, no detectable MTFP1 protein is found in the KO cells. Super‐resolution fluorescence microscopy analysis of the structure and localization of the mitochondrial network in HeLa WT and ZBTB48 KO clones. Mitochondria are marked with the MitoTracker dye (red), and nuclei are counterstained with DAPI (blue). Scale bars represent 5 μm. mtDNA level quantification comparing five HeLa ZBTB48 WT and KO clones. mtDNA levels were quantified based on three mtDNA loci and normalized to two genomic regions. Error bars represent standard deviations ( n = 3). The P ‐value is based on Student's t ‐test.
    Figure Legend Snippet: ZBTB48 is required for MTFP1 expression Western blot confirmation of reduced MTFP1 expression in each five HeLa and U2OS ZBTB48 KO clones compared to five WT clones each. Similar to ZBTB48, no detectable MTFP1 protein is found in the KO cells. Super‐resolution fluorescence microscopy analysis of the structure and localization of the mitochondrial network in HeLa WT and ZBTB48 KO clones. Mitochondria are marked with the MitoTracker dye (red), and nuclei are counterstained with DAPI (blue). Scale bars represent 5 μm. mtDNA level quantification comparing five HeLa ZBTB48 WT and KO clones. mtDNA levels were quantified based on three mtDNA loci and normalized to two genomic regions. Error bars represent standard deviations ( n = 3). The P ‐value is based on Student's t ‐test.

    Techniques Used: Expressing, Western Blot, Clone Assay, Fluorescence, Microscopy

    4) Product Images from "ZBTB48 is both a vertebrate telomere‐binding protein and a transcriptional activator"

    Article Title: ZBTB48 is both a vertebrate telomere‐binding protein and a transcriptional activator

    Journal: EMBO Reports

    doi: 10.15252/embr.201744095

    ZBTB 48 binds telomeres via its ZnF11 domain in vitro and in vivo Domain structure of wild‐type (WT) ZBTB48 containing a N‐terminal BTB domain and 11 zinc fingers (ZnF) at the C‐terminus of the 688‐amino‐acid protein. Note that ZnF2 is degenerate. Below is a schematic of the deletion construct lacking ZnF1‐10 used in (B). Sequence‐specific DNA pull‐downs with either telomeric (TTAGGG) or a control sequence (GTGAGT) for FLAG‐ZBTB48 WT, point mutants for the ten functional zinc fingers and a domain deletion construct for ZnF1‐10. Sequence‐specific DNA pull‐downs for FLAG‐ZBTB48 WT and ZnF11 point mutant for telomeric and subtelomeric variant repeat sequences (green) and their respective scrambled controls (blue). Co‐localization analysis of endogenous ZBTB48 and TRF2 in U2OS cells by immunofluorescence (IF) staining. A representative image illustrating the co‐localization between ZBTB48 (green) and TRF2 (red) as a marker for telomeres is shown with DAPI (blue) used as a nuclear counterstain. The quantification of frequency of co‐localization events (right) was done after 3D reconstruction of the acquired z ‐stacks ( n = 104 cells). The average value is indicated by a red bar. IF stainings for exogenous FLAG‐ZBTB48 WT and point mutants for ZnF10 and ZnF11 in U2OS cells. The same analysis as in (D) was performed and average co‐localization frequencies are shown ( n = 24–33 cells). Error bars indicate standard deviations, and P ‐values are based on Student's t ‐test. Co‐localization analysis between ZBTB48 (green) and PML (red) as a marker for PML bodies analogous to (D) in U2OS cells ( n = 102 cells). Data information: (D–F) Co‐localization events are indicated by white arrows. Scale bars represent 5 μm. Source data are available online for this figure.
    Figure Legend Snippet: ZBTB 48 binds telomeres via its ZnF11 domain in vitro and in vivo Domain structure of wild‐type (WT) ZBTB48 containing a N‐terminal BTB domain and 11 zinc fingers (ZnF) at the C‐terminus of the 688‐amino‐acid protein. Note that ZnF2 is degenerate. Below is a schematic of the deletion construct lacking ZnF1‐10 used in (B). Sequence‐specific DNA pull‐downs with either telomeric (TTAGGG) or a control sequence (GTGAGT) for FLAG‐ZBTB48 WT, point mutants for the ten functional zinc fingers and a domain deletion construct for ZnF1‐10. Sequence‐specific DNA pull‐downs for FLAG‐ZBTB48 WT and ZnF11 point mutant for telomeric and subtelomeric variant repeat sequences (green) and their respective scrambled controls (blue). Co‐localization analysis of endogenous ZBTB48 and TRF2 in U2OS cells by immunofluorescence (IF) staining. A representative image illustrating the co‐localization between ZBTB48 (green) and TRF2 (red) as a marker for telomeres is shown with DAPI (blue) used as a nuclear counterstain. The quantification of frequency of co‐localization events (right) was done after 3D reconstruction of the acquired z ‐stacks ( n = 104 cells). The average value is indicated by a red bar. IF stainings for exogenous FLAG‐ZBTB48 WT and point mutants for ZnF10 and ZnF11 in U2OS cells. The same analysis as in (D) was performed and average co‐localization frequencies are shown ( n = 24–33 cells). Error bars indicate standard deviations, and P ‐values are based on Student's t ‐test. Co‐localization analysis between ZBTB48 (green) and PML (red) as a marker for PML bodies analogous to (D) in U2OS cells ( n = 102 cells). Data information: (D–F) Co‐localization events are indicated by white arrows. Scale bars represent 5 μm. Source data are available online for this figure.

    Techniques Used: In Vitro, In Vivo, Zinc-Fingers, Construct, Sequencing, Functional Assay, Mutagenesis, Variant Assay, Immunofluorescence, Staining, Marker

    ZBTB48 and HOT1 KO clones show depleted expression Depiction of the ZBTB48 TALEN binding sites located in exon 2. Genotypes of HeLa and U2OS WT and ZBTB48 KO clones. The grey boxes represent the TALEN binding sites; insertions (blue) and deletions (red) are marked. The numbers in brackets represent the numbers of clones found with the specific genotype. Gel image of T7E1 assay showing ZBTB48 TALEN activity in HeLa cells. Western blot confirmation of depleted ZBTB48 expression in each five HeLa and U2OS ZBTB48 KO clones compared to parental cells. IF confirmation of depleted ZBTB48 expression (green) with DAPI (blue) as nuclear counterstain in U2OS ZBTB48 KO cells. ZBTB48 signals are reduced to background levels. Scale bars represent 5 μm. Depiction of the HOT1 TALEN binding sites located in exon 3. Genotypes of HeLa and U2OS WT and HOT1 KO clones. The grey boxes represent the TALEN binding sites; insertions (blue) and deletions (red) are marked. The numbers in brackets represent the numbers of clones found with the specific genotype. Gel image of T7E1 assay showing HOT1 TALEN activity in HeLa cells. IF confirmation of depleted HOT1 expression (green) with DAPI (blue) as nuclear counterstain in a representative HeLa HOT1 KO clone. Scale bars represent 5 μm. Source data are available online for this figure.
    Figure Legend Snippet: ZBTB48 and HOT1 KO clones show depleted expression Depiction of the ZBTB48 TALEN binding sites located in exon 2. Genotypes of HeLa and U2OS WT and ZBTB48 KO clones. The grey boxes represent the TALEN binding sites; insertions (blue) and deletions (red) are marked. The numbers in brackets represent the numbers of clones found with the specific genotype. Gel image of T7E1 assay showing ZBTB48 TALEN activity in HeLa cells. Western blot confirmation of depleted ZBTB48 expression in each five HeLa and U2OS ZBTB48 KO clones compared to parental cells. IF confirmation of depleted ZBTB48 expression (green) with DAPI (blue) as nuclear counterstain in U2OS ZBTB48 KO cells. ZBTB48 signals are reduced to background levels. Scale bars represent 5 μm. Depiction of the HOT1 TALEN binding sites located in exon 3. Genotypes of HeLa and U2OS WT and HOT1 KO clones. The grey boxes represent the TALEN binding sites; insertions (blue) and deletions (red) are marked. The numbers in brackets represent the numbers of clones found with the specific genotype. Gel image of T7E1 assay showing HOT1 TALEN activity in HeLa cells. IF confirmation of depleted HOT1 expression (green) with DAPI (blue) as nuclear counterstain in a representative HeLa HOT1 KO clone. Scale bars represent 5 μm. Source data are available online for this figure.

    Techniques Used: Clone Assay, Expressing, Binding Assay, Activity Assay, Western Blot

    ZBTB48 is a direct telomere‐binding protein but TRAP activity is not affected in ZBTB48 KO clones TTAGGG content of telomeric reads in ChIPseq samples from U2OS WT, U2OS ZBTB48 KO and U2OS HOT1 KO clones using IgG, TRF2, ZBTB48 Atlas, ZBTB48 GeneTex and HOT1 antibodies as well as FLAG antibody for FLAG‐ZBTB48 WT and point mutants for ZnF10 and ZnF11. The percentage contribution to all reads containing 1–8× TTAGGG repeats is shown. All reactions were performed in technical replicates on two independent WT and KO clones each. These reactions are biological replicates of Fig 2 A, performed with independent U2OS ZBTB48 WT and KO clones for ZBTB48 and with a second HOT1 antibody for HOT1. Telomerase activity was determined based on a quantitative TRAP assay. Heat‐inactivated HeLa extracts were used as a threshold to determine non‐specific background signal. Differences in C t values (∆∆ C t ) from the quantitative PCR measurements are displayed relative to the heat‐inactivation control. Error bars represent standard deviations ( n = 3). The P ‐value is based on Student's t ‐test.
    Figure Legend Snippet: ZBTB48 is a direct telomere‐binding protein but TRAP activity is not affected in ZBTB48 KO clones TTAGGG content of telomeric reads in ChIPseq samples from U2OS WT, U2OS ZBTB48 KO and U2OS HOT1 KO clones using IgG, TRF2, ZBTB48 Atlas, ZBTB48 GeneTex and HOT1 antibodies as well as FLAG antibody for FLAG‐ZBTB48 WT and point mutants for ZnF10 and ZnF11. The percentage contribution to all reads containing 1–8× TTAGGG repeats is shown. All reactions were performed in technical replicates on two independent WT and KO clones each. These reactions are biological replicates of Fig 2 A, performed with independent U2OS ZBTB48 WT and KO clones for ZBTB48 and with a second HOT1 antibody for HOT1. Telomerase activity was determined based on a quantitative TRAP assay. Heat‐inactivated HeLa extracts were used as a threshold to determine non‐specific background signal. Differences in C t values (∆∆ C t ) from the quantitative PCR measurements are displayed relative to the heat‐inactivation control. Error bars represent standard deviations ( n = 3). The P ‐value is based on Student's t ‐test.

    Techniques Used: Binding Assay, Activity Assay, Clone Assay, TRAP Assay, Real-time Polymerase Chain Reaction

    ZBTB 48 preferentially binds to promoter regions Peak calling of ZBTB48 ChIPseq reactions from two U2OS ZBTB48 WT clones (top) and two U2OS ZBTB48 KO clones (bottom). ChIP reactions were performed with two independent ZBT48 antibodies (ab1 and ab2) each in two independent clones (WT1 and WT2 or KO1 and KO2). Only peaks that are enriched by at least eightfold in the WT over the KO clones and have a FDR
    Figure Legend Snippet: ZBTB 48 preferentially binds to promoter regions Peak calling of ZBTB48 ChIPseq reactions from two U2OS ZBTB48 WT clones (top) and two U2OS ZBTB48 KO clones (bottom). ChIP reactions were performed with two independent ZBT48 antibodies (ab1 and ab2) each in two independent clones (WT1 and WT2 or KO1 and KO2). Only peaks that are enriched by at least eightfold in the WT over the KO clones and have a FDR

    Techniques Used: Chromatin Immunoprecipitation, Clone Assay

    ZBTB 48 associates with short and long telomeres in vivo TTAGGG content of telomeric reads in ChIPseq samples from U2OS WT, U2OS ZBTB48 KO and U2OS HOT1 KO clones using IgG, TRF2, ZBTB48 Atlas, ZBTB48 GeneTex and HOT1 antibodies as well as FLAG antibody for FLAG‐ZBTB48 WT and point mutants for ZnF10 and ZnF11. The percentage contribution to all reads containing 1–8× TTAGGG repeats is shown. All reactions were performed in technical replicates on two independent WT and KO clones each. MEME sequence logo and bit score for the top 500 extratelomeric ChIPseq peaks for ZBTB48 Atlas, ZBTB48 GeneTex, TRF2 and HOT1 antibodies compared to IgG samples in U2OS. For each antibody, the most frequent motif is shown. TTAGGG enrichment in ChIPseq samples with reads with 7× or 8× TTAGGG repeats. Absolute read counts from ChIPseq reactions from each two HeLa and U2OS WT and KO clones with two independent antibodies for ZBTB48 and HOT1 are shown (left). The corresponding fold enrichments are calculated (right) and compared to TRF2 ChIPseq reactions for which fold enrichments are calculated relative to IgG samples. Error bars represent standard deviations ( n = 4), and P ‐values are based on Student's t ‐test with ** indicating P
    Figure Legend Snippet: ZBTB 48 associates with short and long telomeres in vivo TTAGGG content of telomeric reads in ChIPseq samples from U2OS WT, U2OS ZBTB48 KO and U2OS HOT1 KO clones using IgG, TRF2, ZBTB48 Atlas, ZBTB48 GeneTex and HOT1 antibodies as well as FLAG antibody for FLAG‐ZBTB48 WT and point mutants for ZnF10 and ZnF11. The percentage contribution to all reads containing 1–8× TTAGGG repeats is shown. All reactions were performed in technical replicates on two independent WT and KO clones each. MEME sequence logo and bit score for the top 500 extratelomeric ChIPseq peaks for ZBTB48 Atlas, ZBTB48 GeneTex, TRF2 and HOT1 antibodies compared to IgG samples in U2OS. For each antibody, the most frequent motif is shown. TTAGGG enrichment in ChIPseq samples with reads with 7× or 8× TTAGGG repeats. Absolute read counts from ChIPseq reactions from each two HeLa and U2OS WT and KO clones with two independent antibodies for ZBTB48 and HOT1 are shown (left). The corresponding fold enrichments are calculated (right) and compared to TRF2 ChIPseq reactions for which fold enrichments are calculated relative to IgG samples. Error bars represent standard deviations ( n = 4), and P ‐values are based on Student's t ‐test with ** indicating P

    Techniques Used: In Vivo, Clone Assay, Sequencing

    ZBTB48 ZnF11 is necessary to bind to telomeres Sequence‐specific DNA pull‐downs with either telomeric (TTAGGG) or a control sequence (GTGAGT) for FLAG‐ZBTB48 WT, domain deletion constructs for different zinc finger and combinations of deletion constructs with ZnF10 or 11 point mutants. Domain structures are indicated on the right. Sequence‐specific DNA pull‐downs for FLAG‐ZBTB48 WT and ZnF11 point mutant for telomeric repeat sequences of different phyla (green) and their respective scrambled controls (blue). Protein expression analysis of ZBTB48 by Western blot for the cell lines used in this study. GAPDH serves as a loading control. IF stainings for exogenous FLAG‐ZBTB48 WT and point mutants for ZnF10 and ZnF11 in U2OS cells. The same analysis as in Fig 1 E was performed and average co‐localization frequencies are shown ( n = 24–37 cells). Co‐localization analysis of endogenous ZBTB48 or exogenous FLAG‐ZBTB48 WT with TRF2 in HeLa cells by immunofluorescence (IF) staining. A representative image illustrating the co‐localization between ZBTB48 or FLAG‐ZBTB48 WT (green) and TRF2 (red) as a marker for telomeres is shown with DAPI (blue) used as a nuclear counterstain. Co‐localization events are indicated by white arrows. The quantification of frequency of co‐localization events (right) was done after 3D reconstruction of the acquired z ‐stacks ( n = 30 cells). Co‐localization analysis of endogenous ZBTB48 or exogenous FLAG‐ZBTB48 WT with TRF2 in HeLa 1.3 cells by immunofluorescence (IF) staining analogous to (E) ( n = 30 cells). Co‐localization analysis of endogenous ZBTB48 or exogenous FLAG‐ZBTB48 WT with TRF2 in HT1080 super‐telomerase cells by immunofluorescence (IF) staining analogous to (E) ( n = 30 cells). Data information: (D–G) Scale bars represent 5 μm. Error bars indicate standard deviations, and P ‐values are based on Student's t ‐test. Source data are available online for this figure.
    Figure Legend Snippet: ZBTB48 ZnF11 is necessary to bind to telomeres Sequence‐specific DNA pull‐downs with either telomeric (TTAGGG) or a control sequence (GTGAGT) for FLAG‐ZBTB48 WT, domain deletion constructs for different zinc finger and combinations of deletion constructs with ZnF10 or 11 point mutants. Domain structures are indicated on the right. Sequence‐specific DNA pull‐downs for FLAG‐ZBTB48 WT and ZnF11 point mutant for telomeric repeat sequences of different phyla (green) and their respective scrambled controls (blue). Protein expression analysis of ZBTB48 by Western blot for the cell lines used in this study. GAPDH serves as a loading control. IF stainings for exogenous FLAG‐ZBTB48 WT and point mutants for ZnF10 and ZnF11 in U2OS cells. The same analysis as in Fig 1 E was performed and average co‐localization frequencies are shown ( n = 24–37 cells). Co‐localization analysis of endogenous ZBTB48 or exogenous FLAG‐ZBTB48 WT with TRF2 in HeLa cells by immunofluorescence (IF) staining. A representative image illustrating the co‐localization between ZBTB48 or FLAG‐ZBTB48 WT (green) and TRF2 (red) as a marker for telomeres is shown with DAPI (blue) used as a nuclear counterstain. Co‐localization events are indicated by white arrows. The quantification of frequency of co‐localization events (right) was done after 3D reconstruction of the acquired z ‐stacks ( n = 30 cells). Co‐localization analysis of endogenous ZBTB48 or exogenous FLAG‐ZBTB48 WT with TRF2 in HeLa 1.3 cells by immunofluorescence (IF) staining analogous to (E) ( n = 30 cells). Co‐localization analysis of endogenous ZBTB48 or exogenous FLAG‐ZBTB48 WT with TRF2 in HT1080 super‐telomerase cells by immunofluorescence (IF) staining analogous to (E) ( n = 30 cells). Data information: (D–G) Scale bars represent 5 μm. Error bars indicate standard deviations, and P ‐values are based on Student's t ‐test. Source data are available online for this figure.

    Techniques Used: Sequencing, Construct, Mutagenesis, Expressing, Western Blot, Immunofluorescence, Staining, Marker

    ZBTB48 is a transcriptional activator Differential expression analysis of the RNA sequencing (RNAseq) gene quantitation, comparing each five WT and ZBTB48 KO clones for U2OS (left) and HeLa (right). Cut‐offs for significant differential expression were set to log 2 (fold change) > |1| and −log 10 (adjusted P ‐value) > 2 (FDR
    Figure Legend Snippet: ZBTB48 is a transcriptional activator Differential expression analysis of the RNA sequencing (RNAseq) gene quantitation, comparing each five WT and ZBTB48 KO clones for U2OS (left) and HeLa (right). Cut‐offs for significant differential expression were set to log 2 (fold change) > |1| and −log 10 (adjusted P ‐value) > 2 (FDR

    Techniques Used: Expressing, RNA Sequencing Assay, Quantitation Assay, Clone Assay

    ZBTB 48 is a negative regulator of telomere length Terminal restriction fragment (TRF) analysis for each five independent HeLa WT and HeLa ZBTB48 KO clones at passage 20. The average telomere length was determined from the telomeric distribution (left) and used for average quantification of WT vs. KO clones (right). Average telomere length is indicated for all WT samples (dotted line) and individually for all samples (red dots). Error bars indicate standard deviations, and the P ‐value is based on Student's t ‐test. TRF analysis using pulsed‐field gel electrophoresis (PFGE) of parental U2OS WT cells compared to five independent U2OS ZBTB48 KO clones at passage 37. Average telomere length is indicated for all WT samples (dotted line) and individually for all samples (red dots). Quantification of C‐circles in U2OS WT cells compared to five independent U2OS ZBTB48 KO clones. C‐circle reactions were carried out and spotted in triplicate (top), and average quantifications are displayed (bottom). No Φ indicates negative control reactions without the ΦDNA polymerase. 15 ng DNA was used as input material per reaction. The dashed line indicates cropping of the membrane between KO clones 4 and 5. Error bars indicate standard deviations, and P ‐values are based on Student's t ‐test with * indicating P
    Figure Legend Snippet: ZBTB 48 is a negative regulator of telomere length Terminal restriction fragment (TRF) analysis for each five independent HeLa WT and HeLa ZBTB48 KO clones at passage 20. The average telomere length was determined from the telomeric distribution (left) and used for average quantification of WT vs. KO clones (right). Average telomere length is indicated for all WT samples (dotted line) and individually for all samples (red dots). Error bars indicate standard deviations, and the P ‐value is based on Student's t ‐test. TRF analysis using pulsed‐field gel electrophoresis (PFGE) of parental U2OS WT cells compared to five independent U2OS ZBTB48 KO clones at passage 37. Average telomere length is indicated for all WT samples (dotted line) and individually for all samples (red dots). Quantification of C‐circles in U2OS WT cells compared to five independent U2OS ZBTB48 KO clones. C‐circle reactions were carried out and spotted in triplicate (top), and average quantifications are displayed (bottom). No Φ indicates negative control reactions without the ΦDNA polymerase. 15 ng DNA was used as input material per reaction. The dashed line indicates cropping of the membrane between KO clones 4 and 5. Error bars indicate standard deviations, and P ‐values are based on Student's t ‐test with * indicating P

    Techniques Used: Clone Assay, Pulsed-Field Gel, Electrophoresis, Negative Control

    ZBTB48 acts as a transcription factor in contrast to HOT1 Differential expression analysis of the RNA sequencing (RNAseq) gene quantitation, comparing each five WT and ZBTB48 KO clones for U2OS (left) and HeLa (right). Cut‐offs for significant differential expression were set to log 2 (fold change) > |1| and −log 10 (adjusted P ‐value) > 2 (FDR
    Figure Legend Snippet: ZBTB48 acts as a transcription factor in contrast to HOT1 Differential expression analysis of the RNA sequencing (RNAseq) gene quantitation, comparing each five WT and ZBTB48 KO clones for U2OS (left) and HeLa (right). Cut‐offs for significant differential expression were set to log 2 (fold change) > |1| and −log 10 (adjusted P ‐value) > 2 (FDR

    Techniques Used: Expressing, RNA Sequencing Assay, Quantitation Assay, Clone Assay

    ZBTB48‐dependent loss of MTFP1 phenocopies MTFP1 depletion Fluorescence microscopy analysis of the structure and localization of the mitochondrial network in HeLa WT and ZBTB48 KO clones. Mitochondria are marked with the MitoTracker dye (red), and nuclei are counterstained with DAPI (blue). Scale bars represent 20 μm. The same analysis as in (A) for U2OS WT and ZBTB48 KO clones.
    Figure Legend Snippet: ZBTB48‐dependent loss of MTFP1 phenocopies MTFP1 depletion Fluorescence microscopy analysis of the structure and localization of the mitochondrial network in HeLa WT and ZBTB48 KO clones. Mitochondria are marked with the MitoTracker dye (red), and nuclei are counterstained with DAPI (blue). Scale bars represent 20 μm. The same analysis as in (A) for U2OS WT and ZBTB48 KO clones.

    Techniques Used: Fluorescence, Microscopy, Clone Assay

    ZBTB48 is required for MTFP1 expression Western blot confirmation of reduced MTFP1 expression in each five HeLa and U2OS ZBTB48 KO clones compared to five WT clones each. Similar to ZBTB48, no detectable MTFP1 protein is found in the KO cells. Super‐resolution fluorescence microscopy analysis of the structure and localization of the mitochondrial network in HeLa WT and ZBTB48 KO clones. Mitochondria are marked with the MitoTracker dye (red), and nuclei are counterstained with DAPI (blue). Scale bars represent 5 μm. mtDNA level quantification comparing five HeLa ZBTB48 WT and KO clones. mtDNA levels were quantified based on three mtDNA loci and normalized to two genomic regions. Error bars represent standard deviations ( n = 3). The P ‐value is based on Student's t ‐test.
    Figure Legend Snippet: ZBTB48 is required for MTFP1 expression Western blot confirmation of reduced MTFP1 expression in each five HeLa and U2OS ZBTB48 KO clones compared to five WT clones each. Similar to ZBTB48, no detectable MTFP1 protein is found in the KO cells. Super‐resolution fluorescence microscopy analysis of the structure and localization of the mitochondrial network in HeLa WT and ZBTB48 KO clones. Mitochondria are marked with the MitoTracker dye (red), and nuclei are counterstained with DAPI (blue). Scale bars represent 5 μm. mtDNA level quantification comparing five HeLa ZBTB48 WT and KO clones. mtDNA levels were quantified based on three mtDNA loci and normalized to two genomic regions. Error bars represent standard deviations ( n = 3). The P ‐value is based on Student's t ‐test.

    Techniques Used: Expressing, Western Blot, Clone Assay, Fluorescence, Microscopy

    5) Product Images from "Targeting Multiple Effector Pathways in Pancreatic Ductal Adenocarcinoma with a G-Quadruplex-Binding Small Molecule"

    Article Title: Targeting Multiple Effector Pathways in Pancreatic Ductal Adenocarcinoma with a G-Quadruplex-Binding Small Molecule

    Journal: Journal of Medicinal Chemistry

    doi: 10.1021/acs.jmedchem.7b01781

    Differentially down-regulated genes common to both PANC-1 and MIA PaCa-2 are enriched in PQs after treatment with 400 nM CM03. (a,b) MIA PaCa-2 and PANC-1 cells were treated with 400 nM CM03 for 6 and 24 h and mRNA extracted for analysis by RNA-Seq. Genes were split into four subgroups according to their fold change upon CM03 treatment versus untreated: Down (Log 2 FC
    Figure Legend Snippet: Differentially down-regulated genes common to both PANC-1 and MIA PaCa-2 are enriched in PQs after treatment with 400 nM CM03. (a,b) MIA PaCa-2 and PANC-1 cells were treated with 400 nM CM03 for 6 and 24 h and mRNA extracted for analysis by RNA-Seq. Genes were split into four subgroups according to their fold change upon CM03 treatment versus untreated: Down (Log 2 FC

    Techniques Used: RNA Sequencing Assay

    Validation of mRNA down regulation by qRT-PCR for a subset of down-regulated genes, selected from RNA-Seq experiments. (a–d) MIA PaCa-2 and PANC-1 cells were treated (a and b) with 400 nM CM03 and (c and d) with 400 nM gemcitabine, all for 6 and 24 h. Total mRNA was extracted, reverse transcribed into cDNA, and then qRT-PCR was performed. The C t values were normalized to the genomic mean of three housekeeping genes ( ACTB , GAPDH , and TUBB ), and the relative gene expression was determined using the Livak method, 2 –ΔΔ C t . The log-fold expression changes (Log 2 FC) for each gene are shown relative to vehicle-treated controls (PBS for CM03 and DMSO for gemcitabine). Student’s t test along with 2 –Δ C t values were used to determine the statistical significance of the observed changes, which are the mean of in each case at least three determinations. Those genes with changes in expression with p
    Figure Legend Snippet: Validation of mRNA down regulation by qRT-PCR for a subset of down-regulated genes, selected from RNA-Seq experiments. (a–d) MIA PaCa-2 and PANC-1 cells were treated (a and b) with 400 nM CM03 and (c and d) with 400 nM gemcitabine, all for 6 and 24 h. Total mRNA was extracted, reverse transcribed into cDNA, and then qRT-PCR was performed. The C t values were normalized to the genomic mean of three housekeeping genes ( ACTB , GAPDH , and TUBB ), and the relative gene expression was determined using the Livak method, 2 –ΔΔ C t . The log-fold expression changes (Log 2 FC) for each gene are shown relative to vehicle-treated controls (PBS for CM03 and DMSO for gemcitabine). Student’s t test along with 2 –Δ C t values were used to determine the statistical significance of the observed changes, which are the mean of in each case at least three determinations. Those genes with changes in expression with p

    Techniques Used: Quantitative RT-PCR, RNA Sequencing Assay, Expressing

    6) Product Images from "Nuclear sensing of mitochondrial DNA breaks enhances immune surveillance"

    Article Title: Nuclear sensing of mitochondrial DNA breaks enhances immune surveillance

    Journal: bioRxiv

    doi: 10.1101/2020.01.31.929075

    Mitochondrial targeted TALENs (mTLNs) trigger an innate immune response. (A) , Heat map representing RNA-seq data for 19 nuclear-encoded mitochondrial genes in ARPE-19 cells analyzed 20 hours after transfection with the indicated mTLNs. A catalytically inactive TALEN (dmTLN) was used as a negative control. Shown are nuclear encoded genes targeted to the mitochondria with statistically significant difference in expression ( > 1.5-fold increase; FDR=0.05) (B) , Real-time PCR (RT-qPCR) to validate top genes identified in (a) (n=3 technical replicates ± SD, two tailed unpaired t-test). Additional experiments and extended statistics in Fig. S2a-c. (C) , Heat map displaying 295 upregulated nuclear genes (FDR=0.05; > 1.5-fold increase) in ARPE-19 cells treated with mTLN DLoop , mTLN ATP8 and a control dmTLN ATP8 . (D) , Graphical representation of gene ontology terms enrichment for genes identified in c. (E) , RT-qPCR to validate candidate genes identified in (c). For each gene, expression levels in mTLN ATP8 treated cells (24 and 48 hours) were normalized to control cells treated with dmTLN ATP8 and harvested 24 hours post-transfection (n=3 technical replicates ± S.D., two tailed unpaired t-test). Additional experiments and extended statistics in Fig. S2d-g.
    Figure Legend Snippet: Mitochondrial targeted TALENs (mTLNs) trigger an innate immune response. (A) , Heat map representing RNA-seq data for 19 nuclear-encoded mitochondrial genes in ARPE-19 cells analyzed 20 hours after transfection with the indicated mTLNs. A catalytically inactive TALEN (dmTLN) was used as a negative control. Shown are nuclear encoded genes targeted to the mitochondria with statistically significant difference in expression ( > 1.5-fold increase; FDR=0.05) (B) , Real-time PCR (RT-qPCR) to validate top genes identified in (a) (n=3 technical replicates ± SD, two tailed unpaired t-test). Additional experiments and extended statistics in Fig. S2a-c. (C) , Heat map displaying 295 upregulated nuclear genes (FDR=0.05; > 1.5-fold increase) in ARPE-19 cells treated with mTLN DLoop , mTLN ATP8 and a control dmTLN ATP8 . (D) , Graphical representation of gene ontology terms enrichment for genes identified in c. (E) , RT-qPCR to validate candidate genes identified in (c). For each gene, expression levels in mTLN ATP8 treated cells (24 and 48 hours) were normalized to control cells treated with dmTLN ATP8 and harvested 24 hours post-transfection (n=3 technical replicates ± S.D., two tailed unpaired t-test). Additional experiments and extended statistics in Fig. S2d-g.

    Techniques Used: TALENs, RNA Sequencing Assay, Transfection, Negative Control, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Two Tailed Test

    Bak-Bax mediated herniation of mitochondria and cytoplasmic sensing of RNA are central to immune activation in response to mtDSBs. (A) Mitochondrial network analysis in ARPE-19 cells treated with mTLN, dmTLN, and CCCP. Representative immunofluorescence highlighting mitochondria in red with mito-RFP, mTLN in green with anti-HA, and nuclei in blue using DAPI. (B) Quantification of mitochondrial mean length and branching 48 hours after transfection with mTLN ATP8 or dmTLN ATP8 performed with Fiji ImageJ and MiNa macro and expressed in μm (52 and 48 independent cells as in (a) were analyzed respectively, two tailed t test). (C) Flow cytometry evaluation of mitochondrial membrane potential measured by import of TMRM in cells expressing either active or dead mTLN DLoop . Cells were treated with 50μM CCCP for 5 minutes as depolarization control. (D) , Flow cytometry evaluation of cellular ROS as oxidation of fluorogenic probe CellROX green in cells were treated as in (c). 100μM menadione (1 hour) treatment was used as a positive control. (E) Deletion of Bak-Bax impeded the activation of ISGs following mtDNA damage. RT-qPCR analysis for RSAD2 and ISG15, 48hrs after mTLN expression in pooled Bax −/− Bak −/− ARPE-19 cells. Displayed is the fold expression ratio for each gene in mTLN Dloop vs . dmTLN DLoop treated cells. Shown is the mean ± S.D (n > 6 independent experiments; one tailed unpaired t-test). (F) RT-qPCR analysis for RSAD2 and ISG15, 48hrs after mTLN treatment in clonally derived MAVS −/− , MDA-5 −/− and STING −/− ARPE-19 cells as well as wildtype cells. Shown is the mean of three biological replicates ± S.D (n=3 independent experiments; one tailed unpaired t-test). (G) Loss of RIG-I hinders ISGs activation in response to mtDSB. RT-qPCR analysis for RSAD2 and ISG15, 48hrs after mTLN treatment in independent clonally derived RIG-I −/− cells. Displayed is the fold expression ratio of mTLN Dloop vs . dmTLN DLoop . Bars represent mean ± S.D. (n=3 independent experiments; one tailed unpaired t-test).
    Figure Legend Snippet: Bak-Bax mediated herniation of mitochondria and cytoplasmic sensing of RNA are central to immune activation in response to mtDSBs. (A) Mitochondrial network analysis in ARPE-19 cells treated with mTLN, dmTLN, and CCCP. Representative immunofluorescence highlighting mitochondria in red with mito-RFP, mTLN in green with anti-HA, and nuclei in blue using DAPI. (B) Quantification of mitochondrial mean length and branching 48 hours after transfection with mTLN ATP8 or dmTLN ATP8 performed with Fiji ImageJ and MiNa macro and expressed in μm (52 and 48 independent cells as in (a) were analyzed respectively, two tailed t test). (C) Flow cytometry evaluation of mitochondrial membrane potential measured by import of TMRM in cells expressing either active or dead mTLN DLoop . Cells were treated with 50μM CCCP for 5 minutes as depolarization control. (D) , Flow cytometry evaluation of cellular ROS as oxidation of fluorogenic probe CellROX green in cells were treated as in (c). 100μM menadione (1 hour) treatment was used as a positive control. (E) Deletion of Bak-Bax impeded the activation of ISGs following mtDNA damage. RT-qPCR analysis for RSAD2 and ISG15, 48hrs after mTLN expression in pooled Bax −/− Bak −/− ARPE-19 cells. Displayed is the fold expression ratio for each gene in mTLN Dloop vs . dmTLN DLoop treated cells. Shown is the mean ± S.D (n > 6 independent experiments; one tailed unpaired t-test). (F) RT-qPCR analysis for RSAD2 and ISG15, 48hrs after mTLN treatment in clonally derived MAVS −/− , MDA-5 −/− and STING −/− ARPE-19 cells as well as wildtype cells. Shown is the mean of three biological replicates ± S.D (n=3 independent experiments; one tailed unpaired t-test). (G) Loss of RIG-I hinders ISGs activation in response to mtDSB. RT-qPCR analysis for RSAD2 and ISG15, 48hrs after mTLN treatment in independent clonally derived RIG-I −/− cells. Displayed is the fold expression ratio of mTLN Dloop vs . dmTLN DLoop . Bars represent mean ± S.D. (n=3 independent experiments; one tailed unpaired t-test).

    Techniques Used: Activation Assay, Immunofluorescence, Transfection, Two Tailed Test, Flow Cytometry, Expressing, Positive Control, Quantitative RT-PCR, One-tailed Test, Derivative Assay, Multiple Displacement Amplification

    mtDNA damage primes an innate immune response following ionizing radiation. (A) RT-qPCR for ISG15 and IFN1-β in cells with the indicated treatment. Shown is the fold change with non-treated cells set as 1 (n=3 technical replicates ± S.D, two tailed unpaired t-test). (B) Western blot for p-STAT1 Y701 in cells with the indicated treatment. Cells were incubated with 5μM ddC for 10 days prior to treatment with 20 Gy IR. Following irradiation, cells were cultured in the presence (closed circle – lane 4) or absence (open circle – lane 5) of ddC and harvested at day 6. (C) Left – Representative immunofluorescence indicating cGAS positive and DAPI-stained micronuclei. Right – quantification of micronuclei positive cells. (Mean ± SD, n=3). (D) Left – Analysis of DNA damage response in pseudo rho 0 cells. Representative immunofluorescence of γ-H2AX indicating DSBs in irradiated cells +/−ddC. Right – Quantification of cells with > 5 γ-H2AX foci per nucleus. (Mean ± SD, n=3). (E) Volcano plot representation of expression profiles for deregulated genes after irradiation of rho 0 MCF10-A cells vs. mtDNA proficient cells. Plotted is the Log2 ratio (Fold change) against the negative Log10 of the p-value from the Student’s t-test. Red dots highlight factors in the RNA sensing pathway. (F) Western blot for p-STAT1 Y701 in clonally derived MCF10-A cells deleted in RIG-I, MAVS, STING and cGAS 6 days after 20 Gy irradiation (quantified in Fig. S8C). (G) Schematic of APEX2 based labeling of cytoplasmic RNA in the vicinity of RIG-I. (H) Graph representing the enrichment of RIG-I proximal mitochondrial RNA 72 hours after IR treatment. Shown is the mean ± SD of the enrichment fold for each mitochondrial primer set over a non-irradiated control (n=3, one tailed ratio paired t-test).
    Figure Legend Snippet: mtDNA damage primes an innate immune response following ionizing radiation. (A) RT-qPCR for ISG15 and IFN1-β in cells with the indicated treatment. Shown is the fold change with non-treated cells set as 1 (n=3 technical replicates ± S.D, two tailed unpaired t-test). (B) Western blot for p-STAT1 Y701 in cells with the indicated treatment. Cells were incubated with 5μM ddC for 10 days prior to treatment with 20 Gy IR. Following irradiation, cells were cultured in the presence (closed circle – lane 4) or absence (open circle – lane 5) of ddC and harvested at day 6. (C) Left – Representative immunofluorescence indicating cGAS positive and DAPI-stained micronuclei. Right – quantification of micronuclei positive cells. (Mean ± SD, n=3). (D) Left – Analysis of DNA damage response in pseudo rho 0 cells. Representative immunofluorescence of γ-H2AX indicating DSBs in irradiated cells +/−ddC. Right – Quantification of cells with > 5 γ-H2AX foci per nucleus. (Mean ± SD, n=3). (E) Volcano plot representation of expression profiles for deregulated genes after irradiation of rho 0 MCF10-A cells vs. mtDNA proficient cells. Plotted is the Log2 ratio (Fold change) against the negative Log10 of the p-value from the Student’s t-test. Red dots highlight factors in the RNA sensing pathway. (F) Western blot for p-STAT1 Y701 in clonally derived MCF10-A cells deleted in RIG-I, MAVS, STING and cGAS 6 days after 20 Gy irradiation (quantified in Fig. S8C). (G) Schematic of APEX2 based labeling of cytoplasmic RNA in the vicinity of RIG-I. (H) Graph representing the enrichment of RIG-I proximal mitochondrial RNA 72 hours after IR treatment. Shown is the mean ± SD of the enrichment fold for each mitochondrial primer set over a non-irradiated control (n=3, one tailed ratio paired t-test).

    Techniques Used: Quantitative RT-PCR, Two Tailed Test, Western Blot, Incubation, Irradiation, Cell Culture, Immunofluorescence, Staining, Expressing, Derivative Assay, Labeling, One-tailed Test

    7) Product Images from "RNA-seq of Human T-Cells After Hematopoietic Stem Cell Transplantation Identifies Linc00402 as a Novel Regulator of T-Cell Alloimmunity"

    Article Title: RNA-seq of Human T-Cells After Hematopoietic Stem Cell Transplantation Identifies Linc00402 as a Novel Regulator of T-Cell Alloimmunity

    Journal: bioRxiv

    doi: 10.1101/2020.04.16.045567

    RNA-seq analysis post HSCT identifies differentially expressed lncRNAs in human allogeneic T cells. Total RNA sequencing was performed on healthy control CD3 + cells or CD3 + cells 30 days after either autologous, MUD, or MMUD HSCT. The experimental and analysis schema are shown in A . Hierarchical clustering analysis is shown in B . Differentially expressed ( p adj
    Figure Legend Snippet: RNA-seq analysis post HSCT identifies differentially expressed lncRNAs in human allogeneic T cells. Total RNA sequencing was performed on healthy control CD3 + cells or CD3 + cells 30 days after either autologous, MUD, or MMUD HSCT. The experimental and analysis schema are shown in A . Hierarchical clustering analysis is shown in B . Differentially expressed ( p adj

    Techniques Used: RNA Sequencing Assay

    Confirmatory expression of protein-coding genes in RNA-seq patient cohort. qRT-PCR was performed on total RNA derived from CD3 + peripheral blood T-cells from patients detailed in Table 1 . The relative expression of ERAP2 ( A ), TXNRD3 ( B ), CDH2 ( C ), IL17RE ( D ), MMP28 ( E ), ZFYVE9 ( F ), KRT73 ( G ), DKK ( I ), NOG ( J ), PRSS50 ( K ), PARK2 ( L ), IL23-R ( M ), FLT4 ( N ), CNN3 ( O ), and SFRP5 ( P ) are shown relative to β-actin. Error bars represent the mean +/− the SEM. * p
    Figure Legend Snippet: Confirmatory expression of protein-coding genes in RNA-seq patient cohort. qRT-PCR was performed on total RNA derived from CD3 + peripheral blood T-cells from patients detailed in Table 1 . The relative expression of ERAP2 ( A ), TXNRD3 ( B ), CDH2 ( C ), IL17RE ( D ), MMP28 ( E ), ZFYVE9 ( F ), KRT73 ( G ), DKK ( I ), NOG ( J ), PRSS50 ( K ), PARK2 ( L ), IL23-R ( M ), FLT4 ( N ), CNN3 ( O ), and SFRP5 ( P ) are shown relative to β-actin. Error bars represent the mean +/− the SEM. * p

    Techniques Used: Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Derivative Assay

    Confirmatory expression of lncRNAs in the RNA-seq patient cohort and their validation in an independent patient cohort. A. qRT-PCR was performed on total RNA derived from CD3 + peripheral blood T cells from patients detailed in Table 1 and the relative expression of the indicated transcripts was measured relative to β-actin. B. qRT-PCR was performed on cryogenically preserved CD3 + peripheral blood T cells from patients detailed in Table 2 . The relative expression of the indicated transcripts is shown relative to β-actin. Error bars represent the mean +/− the SEM. * p
    Figure Legend Snippet: Confirmatory expression of lncRNAs in the RNA-seq patient cohort and their validation in an independent patient cohort. A. qRT-PCR was performed on total RNA derived from CD3 + peripheral blood T cells from patients detailed in Table 1 and the relative expression of the indicated transcripts was measured relative to β-actin. B. qRT-PCR was performed on cryogenically preserved CD3 + peripheral blood T cells from patients detailed in Table 2 . The relative expression of the indicated transcripts is shown relative to β-actin. Error bars represent the mean +/− the SEM. * p

    Techniques Used: Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Derivative Assay

    8) Product Images from "MGA, L3MBTL2 and E2F6 determine genomic binding of the non-canonical Polycomb repressive complex PRC1.6"

    Article Title: MGA, L3MBTL2 and E2F6 determine genomic binding of the non-canonical Polycomb repressive complex PRC1.6

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007193

    The role of PRC1.6 in HEK293 cell function. (A) Reduced proliferation of MGA ko , L3MBTL2 ko and E2F6 ko cells. Shown are growth curves of wildtype, MGA ko , L3MBTL2 ko , E2F6 ko and PCGF6 ko HEK293 cells. Cells were seed at 3x10 5 , and counted and replated at the indicated time points. Cumulative cell numbers were calculated by multiplying the initial cell number with the fold-increase in cell numbers in each interval. (B) Venn diagrams illustrating the overlap of MGA-bound genes and genes down- or up-regulated in MGA ko cells. Left circle, genes with ≥2-fold reduced transcript levels in MGA ko cells; right circle, genes with ≥2-fold increased transcript levels in MGA ko cells. (C) Representative genome browser screenshots of ChIP-seq and RNA-seq tracks illustrating binding of MGA, L3MBTL2, E2F6 and PCGF6 (top tracks) to the CNTD1 and SMC1B promoters, and RNA expression (bottom tracks) of the corresponding genes in three wild type samples (MGA_wt1, MGA_wt2 and MGA_wt3), and in three different MGA ko cell clones (MGA ko _cl26, MGA ko _cl27 and MGA ko _cl30). (D) RT-qPCR-based analysis of expression changes of selected genes in MGA ko , E2F6 ko , L3MBTL2 ko and PCGF6 ko cells. Transcript levels were normalized to B2M transcript levels, and are depicted relative to transcript levels in wild type cells.
    Figure Legend Snippet: The role of PRC1.6 in HEK293 cell function. (A) Reduced proliferation of MGA ko , L3MBTL2 ko and E2F6 ko cells. Shown are growth curves of wildtype, MGA ko , L3MBTL2 ko , E2F6 ko and PCGF6 ko HEK293 cells. Cells were seed at 3x10 5 , and counted and replated at the indicated time points. Cumulative cell numbers were calculated by multiplying the initial cell number with the fold-increase in cell numbers in each interval. (B) Venn diagrams illustrating the overlap of MGA-bound genes and genes down- or up-regulated in MGA ko cells. Left circle, genes with ≥2-fold reduced transcript levels in MGA ko cells; right circle, genes with ≥2-fold increased transcript levels in MGA ko cells. (C) Representative genome browser screenshots of ChIP-seq and RNA-seq tracks illustrating binding of MGA, L3MBTL2, E2F6 and PCGF6 (top tracks) to the CNTD1 and SMC1B promoters, and RNA expression (bottom tracks) of the corresponding genes in three wild type samples (MGA_wt1, MGA_wt2 and MGA_wt3), and in three different MGA ko cell clones (MGA ko _cl26, MGA ko _cl27 and MGA ko _cl30). (D) RT-qPCR-based analysis of expression changes of selected genes in MGA ko , E2F6 ko , L3MBTL2 ko and PCGF6 ko cells. Transcript levels were normalized to B2M transcript levels, and are depicted relative to transcript levels in wild type cells.

    Techniques Used: Cell Function Assay, Chromatin Immunoprecipitation, RNA Sequencing Assay, Binding Assay, RNA Expression, Clone Assay, Quantitative RT-PCR, Expressing

    9) Product Images from "Alpharetroviral Vector-mediated Gene Therapy for X-CGD: Functional Correction and Lack of Aberrant Splicing"

    Article Title: Alpharetroviral Vector-mediated Gene Therapy for X-CGD: Functional Correction and Lack of Aberrant Splicing

    Journal: Molecular Therapy

    doi: 10.1038/mt.2012.249

    Long-term analysis of gp91 phox expression and functional evaluation in PLB-XCGD cells. X-linked chronic granulomatous disease (X-CGD) PLB-985 cells were transduced with AS.EFS.gp91s at a MOI of 0.1 and transgene expressing cells were enriched by magnetic
    Figure Legend Snippet: Long-term analysis of gp91 phox expression and functional evaluation in PLB-XCGD cells. X-linked chronic granulomatous disease (X-CGD) PLB-985 cells were transduced with AS.EFS.gp91s at a MOI of 0.1 and transgene expressing cells were enriched by magnetic

    Techniques Used: Expressing, Functional Assay, Transduction

    Vector constructs and gp91 phox expression in PLB-XCGD cells. ( a ) Schema of the self-inactivating (SIN) alpharetroviral (SIN-α) and SIN lentiviral (SIN-LV) proviruses used in this study. The transgene cassette contains the elongation factor-1α
    Figure Legend Snippet: Vector constructs and gp91 phox expression in PLB-XCGD cells. ( a ) Schema of the self-inactivating (SIN) alpharetroviral (SIN-α) and SIN lentiviral (SIN-LV) proviruses used in this study. The transgene cassette contains the elongation factor-1α

    Techniques Used: Plasmid Preparation, Construct, Expressing

    Analysis of aberrant transcripts induced by self-inactivating ( SIN) alpharetroviral or SIN lentiviral vectors by RT-PCR and 5′RACE. ( a and b ) Monoclonal PLB-XCGD populations harboring either SIN-α or SIN-lentiviral (LV) integrations in
    Figure Legend Snippet: Analysis of aberrant transcripts induced by self-inactivating ( SIN) alpharetroviral or SIN lentiviral vectors by RT-PCR and 5′RACE. ( a and b ) Monoclonal PLB-XCGD populations harboring either SIN-α or SIN-lentiviral (LV) integrations in

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    10) Product Images from "Host chemokine (C-C motif) ligand-2 (CCL2) is differentially regulated in HIV type 1 (HIV-1)-infected individuals"

    Article Title: Host chemokine (C-C motif) ligand-2 (CCL2) is differentially regulated in HIV type 1 (HIV-1)-infected individuals

    Journal: International Immunology

    doi: 10.1093/intimm/dxl078

    (A) Transcriptional enhancement of CCL2, CXCL10 and IFN-γ in viremic patients. mRNA expression levels of CCL2, CXCL10 and IFN-γ determined by real-time PCR. Patient's RNA samples were analyzed individually. For CCL2 and IFN-γ expression studies, n = 5 each aviremic (
    Figure Legend Snippet: (A) Transcriptional enhancement of CCL2, CXCL10 and IFN-γ in viremic patients. mRNA expression levels of CCL2, CXCL10 and IFN-γ determined by real-time PCR. Patient's RNA samples were analyzed individually. For CCL2 and IFN-γ expression studies, n = 5 each aviremic (

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    11) Product Images from "Sensitivity of Aspergillus nidulans to the Cellulose Synthase Inhibitor Dichlobenil: Insights from Wall-Related Genes' Expression and Ultrastructural Hyphal Morphologies"

    Article Title: Sensitivity of Aspergillus nidulans to the Cellulose Synthase Inhibitor Dichlobenil: Insights from Wall-Related Genes' Expression and Ultrastructural Hyphal Morphologies

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0080038

    DCB does not affect chitin content and triggers the formation of wall “ghosts”. Confocal microscopy pictures of control (A and B) and DCB-treated A. nidulans (C and D). Differential Interference Contrast (DIC) images in (A) and (C) and CFW fluorescence images in (B) and (D). Arrows point to cell wall “ghosts”. Bars refer to 10 µm.
    Figure Legend Snippet: DCB does not affect chitin content and triggers the formation of wall “ghosts”. Confocal microscopy pictures of control (A and B) and DCB-treated A. nidulans (C and D). Differential Interference Contrast (DIC) images in (A) and (C) and CFW fluorescence images in (B) and (D). Arrows point to cell wall “ghosts”. Bars refer to 10 µm.

    Techniques Used: Confocal Microscopy, Fluorescence

    Gene expression analysis in CR and DCB-treated mycelium. Quantitative real-time PCR analysis of cell wall biosynthetic genes in A. nidulans grown in the presence of (A) 100 µM CR and (B) 200 µM DCB for 0, 1, 3, 6 and 24 h. Asterisks indicate significant (*) and very significant (**) changes.
    Figure Legend Snippet: Gene expression analysis in CR and DCB-treated mycelium. Quantitative real-time PCR analysis of cell wall biosynthetic genes in A. nidulans grown in the presence of (A) 100 µM CR and (B) 200 µM DCB for 0, 1, 3, 6 and 24 h. Asterisks indicate significant (*) and very significant (**) changes.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    DCB decreases growth of A. nidulans mycelium. Growth of A. nidulans cultivated on solid MM supplemented with different concentrations of DCB, measured by taking the diameter of the mycelium over 6 days. Inset: images showing the mycelium grown for 2 and 3 days with increasing concentrations of DCB. Bars refer to 1 cm.
    Figure Legend Snippet: DCB decreases growth of A. nidulans mycelium. Growth of A. nidulans cultivated on solid MM supplemented with different concentrations of DCB, measured by taking the diameter of the mycelium over 6 days. Inset: images showing the mycelium grown for 2 and 3 days with increasing concentrations of DCB. Bars refer to 1 cm.

    Techniques Used:

    12) Product Images from "Retinoic Acid Signalling and the Control of Meiotic Entry in the Human Fetal Gonad"

    Article Title: Retinoic Acid Signalling and the Control of Meiotic Entry in the Human Fetal Gonad

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0020249

    Conserved and divergent patterns of expression of STRA8 , CYP26B1 and NANOS1-3 in the human fetal gonad. qRT-PCR analysis of human fetal gonads reveals female-biased and developmentally-regulated expression of STRA8 (A). STRA8 expression increased significantly between 8–9 and 14–16 weeks in the human fetal ovary (a vs b, p
    Figure Legend Snippet: Conserved and divergent patterns of expression of STRA8 , CYP26B1 and NANOS1-3 in the human fetal gonad. qRT-PCR analysis of human fetal gonads reveals female-biased and developmentally-regulated expression of STRA8 (A). STRA8 expression increased significantly between 8–9 and 14–16 weeks in the human fetal ovary (a vs b, p

    Techniques Used: Expressing, Quantitative RT-PCR

    Expression of genes encoding retinaldehyde dehydrogenase enzymes in the human fetal gonad. qRT-PCR analysis reveals developmentally regulated expression of ALDH1A1 (A) in the human fetal testis, with transcript levels increasing significantly between 8–9 weeks gestation and 14–16/17–20 weeks gestation (ANOVA; a,b,c; p
    Figure Legend Snippet: Expression of genes encoding retinaldehyde dehydrogenase enzymes in the human fetal gonad. qRT-PCR analysis reveals developmentally regulated expression of ALDH1A1 (A) in the human fetal testis, with transcript levels increasing significantly between 8–9 weeks gestation and 14–16/17–20 weeks gestation (ANOVA; a,b,c; p

    Techniques Used: Expressing, Quantitative RT-PCR

    Expression of genes encoding retinoic acid and retinoid receptors in the human fetal gonad. qRT-PCR analysis of expression of the genes encoding the retinoic acid (RARα (A), RARβ (B) and RARγ (C) and retinoid (RXRα (D), RXRβ (E) and RXRγ (F)) in the human fetal testis and ovary. No significant differences in levels encoding any of the receptor isoforms were detected between samples of different gestational ages within the same sex, or in gonads of different sexes at the same developmental age, indicating that RAR/RXR receptor expression is not developmentally-regulated in the human fetal gonad. Values denote mean ± s.e.m; 8–9, 14–16 and 17–20 denotes gestational age (in weeks) of specimens.
    Figure Legend Snippet: Expression of genes encoding retinoic acid and retinoid receptors in the human fetal gonad. qRT-PCR analysis of expression of the genes encoding the retinoic acid (RARα (A), RARβ (B) and RARγ (C) and retinoid (RXRα (D), RXRβ (E) and RXRγ (F)) in the human fetal testis and ovary. No significant differences in levels encoding any of the receptor isoforms were detected between samples of different gestational ages within the same sex, or in gonads of different sexes at the same developmental age, indicating that RAR/RXR receptor expression is not developmentally-regulated in the human fetal gonad. Values denote mean ± s.e.m; 8–9, 14–16 and 17–20 denotes gestational age (in weeks) of specimens.

    Techniques Used: Expressing, Quantitative RT-PCR

    13) Product Images from "The Flow of the Gibbon LAVA Element Is Facilitated by the LINE-1 Retrotransposition Machinery"

    Article Title: The Flow of the Gibbon LAVA Element Is Facilitated by the LINE-1 Retrotransposition Machinery

    Journal: Genome Biology and Evolution

    doi: 10.1093/gbe/evw224

    Analysis of the expression from L1 protein donor and retrotransposition reporter plasmids. ( A ) qRT-PCR analyses to quantify the relative amounts of spliced transcripts expressed from the different retrotransposition reporter cassettes. Total RNA was isolated after 12 days of hygromycin selection following co-transfection of pCEPneo, pAD3/SVA E , pLC5/LAVA C , pLC10/LAVA C , pLC21/LAVA C , or pLC23/LAVA C with the L1 protein donor plasmid pJM101/L1 RP Δneo and co-transfection of pJM101/L1 RP with pCEP4. Each co-transfection was performed in three biological replicates. The used primer/probe combination (see “Materials and Methods” section) is specific for the spliced mneo I-cassette (black box with arrow). Real-time PCR of each biological replicate was conducted in technical triplicates. Relative amounts of mRNA expression refer to the signal obtained from total RNA from pCEP4(mock)-transfected HeLa cells which was set as 1 (pCEP4) and served as negative control. Bars depict arithmetic means ±SD of technical triplicates of three biological replicates. ( B ) Immunoblot analysis of L1 ORF1p (left panel) and L1 ORF2p (right panel) expression after co-transfection of the L1 protein donor plasmid pJM101/L1 RP ΔneoΔORF1 (L1 RP ΔORF1) with the LAVA C retrotransposition reporter plasmids. Whole-cell lysates were prepared 13 days after co-transfection upon completion of hygromycin selection and subjected to immunoblot analysis using antibodies against either L1 ORF1p (αORF1p) or L1 ORF2p (αORF2p). In the case of the anti-L1 ORF1p immunoblot analysis (left panel), 20 µg of the cell lysate isolated from each of the differently transfected HeLa-HA cell cultures, were loaded per lane on a 12% PAA gel. Because ORF2p (Predicted MW∼150 kDa) is expressed at a significantly lower level than ORF1p ( Dai et al. 2014 ), 40 µg of total cell lysate from each of the differently transfected HeLa-HA cells were loaded per lane on a 6% PAA gel to perform anti-L1 ORF2p immunoblot analysis (right panel). Detectable amounts of intact L1 ORF1-encoded proteins are absent from pJM101/L1 RP ΔneoΔORF1-transfected HeLa cells. About 2 µg and 40 µg of the NTERA-2 cell extract were loaded on one lane of the gel used for the anti-ORF1p (left panel) Western blot analysis and the anti-ORF2p (right panel) immunoblot analysis, respectively. Lysates from pCEP4-transfected HeLa cells (HeLa + pCEP4) and from NTERA-2 cells served as negative and positive control for L1 protein detection, respectively. β-actin protein levels (∼42 kDa) were analyzed as loading control.
    Figure Legend Snippet: Analysis of the expression from L1 protein donor and retrotransposition reporter plasmids. ( A ) qRT-PCR analyses to quantify the relative amounts of spliced transcripts expressed from the different retrotransposition reporter cassettes. Total RNA was isolated after 12 days of hygromycin selection following co-transfection of pCEPneo, pAD3/SVA E , pLC5/LAVA C , pLC10/LAVA C , pLC21/LAVA C , or pLC23/LAVA C with the L1 protein donor plasmid pJM101/L1 RP Δneo and co-transfection of pJM101/L1 RP with pCEP4. Each co-transfection was performed in three biological replicates. The used primer/probe combination (see “Materials and Methods” section) is specific for the spliced mneo I-cassette (black box with arrow). Real-time PCR of each biological replicate was conducted in technical triplicates. Relative amounts of mRNA expression refer to the signal obtained from total RNA from pCEP4(mock)-transfected HeLa cells which was set as 1 (pCEP4) and served as negative control. Bars depict arithmetic means ±SD of technical triplicates of three biological replicates. ( B ) Immunoblot analysis of L1 ORF1p (left panel) and L1 ORF2p (right panel) expression after co-transfection of the L1 protein donor plasmid pJM101/L1 RP ΔneoΔORF1 (L1 RP ΔORF1) with the LAVA C retrotransposition reporter plasmids. Whole-cell lysates were prepared 13 days after co-transfection upon completion of hygromycin selection and subjected to immunoblot analysis using antibodies against either L1 ORF1p (αORF1p) or L1 ORF2p (αORF2p). In the case of the anti-L1 ORF1p immunoblot analysis (left panel), 20 µg of the cell lysate isolated from each of the differently transfected HeLa-HA cell cultures, were loaded per lane on a 12% PAA gel. Because ORF2p (Predicted MW∼150 kDa) is expressed at a significantly lower level than ORF1p ( Dai et al. 2014 ), 40 µg of total cell lysate from each of the differently transfected HeLa-HA cells were loaded per lane on a 6% PAA gel to perform anti-L1 ORF2p immunoblot analysis (right panel). Detectable amounts of intact L1 ORF1-encoded proteins are absent from pJM101/L1 RP ΔneoΔORF1-transfected HeLa cells. About 2 µg and 40 µg of the NTERA-2 cell extract were loaded on one lane of the gel used for the anti-ORF1p (left panel) Western blot analysis and the anti-ORF2p (right panel) immunoblot analysis, respectively. Lysates from pCEP4-transfected HeLa cells (HeLa + pCEP4) and from NTERA-2 cells served as negative and positive control for L1 protein detection, respectively. β-actin protein levels (∼42 kDa) were analyzed as loading control.

    Techniques Used: Expressing, Quantitative RT-PCR, Isolation, Selection, Cotransfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Transfection, Negative Control, Western Blot, Positive Control

    Structures of LAVA C retrotransposition reporter plasmids and rationale of the LAVA trans -mobilization assay. ( A ) Organization of the non-autonomous non-LTR retrotransposon LAVA in the gibbon genome. The SVA-derived module consists of CT-rich (green), Alu -like (light blue), and VNTR (banded) region. It is separated from the Alu Sz-derived module (black) by Unique Region 1 (U1). The L1ME5-derived module (pink) is separated from the Alu Sz module by Unique Region 2 (U2). Black arrows in boxed Alu -like region and L1ME5 module indicate antisense orientation. Poly(A), poly(A) stretch; TSD, target site duplication. ( B ) Schematics of the retrotransposition reporter plasmid pLC10/LAVA C carrying the full-length LAVA C element, the expression cassettes of the reporter plasmids pLC5/LAVA C , pLC21/LAVA C , pLC23/LAVA C expressing truncated LAVA C versions, and pAD3/SVA E ( Raiz et al. 2012 ). pCEPneo is used to measure processed pseudogene formation frequency. Each of the LAVA and SVA reporter elements and the processed pseudogene formation cassette were tagged with the indicator gene mneo I, and set under transcriptional control of the human CMV immediate early enhancer/promoter (CMV P ). Splice donor (SD) and splice acceptor (SA) sites of the oppositely oriented γ-globin intron are indicated. mneo I is flanked by an SV40 promoter (P’) and polyadenylation signal (A’).Transcripts starting from CMV P driving LAVA mneo I, SVA mneo I or pCEP mneo I transcription, can splice the intron, but contain an antisense copy of the neo R gene. G418 resistant (G418 R ) colonies accrue only if this transcript is reverse transcribed, integrated into chromosomal DNA, and expressed from its own promoter P’. LAVA or SVA sequences were inserted between CMV P and the mneo I cassette. pLC5/LAVA C differs from pLC10/LAVA C exclusively in the absence of the L1ME5 and U2 modules covering 322 bp. pLC21/LAVA C and pLC23/LAVA C encode the 5′-terminal 819 bp of LAVA C covering TSD, CT-rich and Alu -like region, and the 3′-terminal 473 bp covering U1, Alu Sz and L1ME5 modules, respectively. pAD3/SVA E ( Raiz et al. 2012 ) serves as positive control construct for trans -mobilization. Transcriptional termination signals at the 3′ ends of the L1ME5 (pLC10/LAVA C , pLC23/LAVA C ) and SINE-R (pAD3/SVA E ) modules were deleted from the LAVA and SVA reporter cassettes (ΔAATAAA) to ensure transcriptional read-through into the mneo I cassette and polyadenylation at the pCEP4-encoded SV40 polyadenylation signal (pA). pCEPneo is distinguished from the remaining presented reporter constructs by the absence of any LAVA or SVA sequence. CMV P sequences are highlighted in grey. CMV P major and minor transcription start sites ( Isomura et al. 2008 ) are indicated by arrows. TSD, target site duplication; CT-rich, Alu -like, VNTR ( V ariable n umber of t andem r epeats), U1, Alu Sz, U2 and L1ME5 represent repeat modules, LAVA is composed of; pA, poly(A) tail. Hyg R , hygromycin resistance gene serving as selectable marker for eukaryotic cells. ( C ) Design of the experimental approach to test for trans -mobilization of the mneo I-tagged LAVA C element by the human L1 protein machinery. LAVA or SVA retrotransposition reporter plasmids, or pCEPneo were each co-transfected with L1 protein donor plasmid pJM101/L1 RP Δneo or pJM101/L1 RP ΔneoΔORF1 (blue) into HeLa-HA cells that were subsequently selected for hygromycin resistance for 12 days. Hyg R cells were assayed for retrotransposition events by selecting for 9–12 days for G418 R HeLa colonies.
    Figure Legend Snippet: Structures of LAVA C retrotransposition reporter plasmids and rationale of the LAVA trans -mobilization assay. ( A ) Organization of the non-autonomous non-LTR retrotransposon LAVA in the gibbon genome. The SVA-derived module consists of CT-rich (green), Alu -like (light blue), and VNTR (banded) region. It is separated from the Alu Sz-derived module (black) by Unique Region 1 (U1). The L1ME5-derived module (pink) is separated from the Alu Sz module by Unique Region 2 (U2). Black arrows in boxed Alu -like region and L1ME5 module indicate antisense orientation. Poly(A), poly(A) stretch; TSD, target site duplication. ( B ) Schematics of the retrotransposition reporter plasmid pLC10/LAVA C carrying the full-length LAVA C element, the expression cassettes of the reporter plasmids pLC5/LAVA C , pLC21/LAVA C , pLC23/LAVA C expressing truncated LAVA C versions, and pAD3/SVA E ( Raiz et al. 2012 ). pCEPneo is used to measure processed pseudogene formation frequency. Each of the LAVA and SVA reporter elements and the processed pseudogene formation cassette were tagged with the indicator gene mneo I, and set under transcriptional control of the human CMV immediate early enhancer/promoter (CMV P ). Splice donor (SD) and splice acceptor (SA) sites of the oppositely oriented γ-globin intron are indicated. mneo I is flanked by an SV40 promoter (P’) and polyadenylation signal (A’).Transcripts starting from CMV P driving LAVA mneo I, SVA mneo I or pCEP mneo I transcription, can splice the intron, but contain an antisense copy of the neo R gene. G418 resistant (G418 R ) colonies accrue only if this transcript is reverse transcribed, integrated into chromosomal DNA, and expressed from its own promoter P’. LAVA or SVA sequences were inserted between CMV P and the mneo I cassette. pLC5/LAVA C differs from pLC10/LAVA C exclusively in the absence of the L1ME5 and U2 modules covering 322 bp. pLC21/LAVA C and pLC23/LAVA C encode the 5′-terminal 819 bp of LAVA C covering TSD, CT-rich and Alu -like region, and the 3′-terminal 473 bp covering U1, Alu Sz and L1ME5 modules, respectively. pAD3/SVA E ( Raiz et al. 2012 ) serves as positive control construct for trans -mobilization. Transcriptional termination signals at the 3′ ends of the L1ME5 (pLC10/LAVA C , pLC23/LAVA C ) and SINE-R (pAD3/SVA E ) modules were deleted from the LAVA and SVA reporter cassettes (ΔAATAAA) to ensure transcriptional read-through into the mneo I cassette and polyadenylation at the pCEP4-encoded SV40 polyadenylation signal (pA). pCEPneo is distinguished from the remaining presented reporter constructs by the absence of any LAVA or SVA sequence. CMV P sequences are highlighted in grey. CMV P major and minor transcription start sites ( Isomura et al. 2008 ) are indicated by arrows. TSD, target site duplication; CT-rich, Alu -like, VNTR ( V ariable n umber of t andem r epeats), U1, Alu Sz, U2 and L1ME5 represent repeat modules, LAVA is composed of; pA, poly(A) tail. Hyg R , hygromycin resistance gene serving as selectable marker for eukaryotic cells. ( C ) Design of the experimental approach to test for trans -mobilization of the mneo I-tagged LAVA C element by the human L1 protein machinery. LAVA or SVA retrotransposition reporter plasmids, or pCEPneo were each co-transfected with L1 protein donor plasmid pJM101/L1 RP Δneo or pJM101/L1 RP ΔneoΔORF1 (blue) into HeLa-HA cells that were subsequently selected for hygromycin resistance for 12 days. Hyg R cells were assayed for retrotransposition events by selecting for 9–12 days for G418 R HeLa colonies.

    Techniques Used: Derivative Assay, Plasmid Preparation, Expressing, Positive Control, Construct, Sequencing, Marker, Transfection

    14) Product Images from "Targeting Multiple Effector Pathways in Pancreatic Ductal Adenocarcinoma with a G-Quadruplex-Binding Small Molecule"

    Article Title: Targeting Multiple Effector Pathways in Pancreatic Ductal Adenocarcinoma with a G-Quadruplex-Binding Small Molecule

    Journal: Journal of Medicinal Chemistry

    doi: 10.1021/acs.jmedchem.7b01781

    CM03 treatment reduces tumor volume in a MIA PaCa-2 xenograft model of PDAC. (a) Plot showing the tumor volume of MIA PaCa-2 xenografts treated with CM03, MM41, gemcitabine, or saline (control) over 62 days. There are eight CD-1 mice per condition and dosing for all cohorts was stopped on day 28, shown by the red arrow. Standard error of the mean (SEM) is indicated for all growth curves for each tumor volume. * p
    Figure Legend Snippet: CM03 treatment reduces tumor volume in a MIA PaCa-2 xenograft model of PDAC. (a) Plot showing the tumor volume of MIA PaCa-2 xenografts treated with CM03, MM41, gemcitabine, or saline (control) over 62 days. There are eight CD-1 mice per condition and dosing for all cohorts was stopped on day 28, shown by the red arrow. Standard error of the mean (SEM) is indicated for all growth curves for each tumor volume. * p

    Techniques Used: Mouse Assay

    Validation of mRNA down regulation by qRT-PCR for a subset of down-regulated genes, selected from RNA-Seq experiments. (a–d) MIA PaCa-2 and PANC-1 cells were treated (a and b) with 400 nM CM03 and (c and d) with 400 nM gemcitabine, all for 6 and 24 h. Total mRNA was extracted, reverse transcribed into cDNA, and then qRT-PCR was performed. The C t values were normalized to the genomic mean of three housekeeping genes ( ACTB , GAPDH , and TUBB ), and the relative gene expression was determined using the Livak method, 2 –ΔΔ C t . The log-fold expression changes (Log 2 FC) for each gene are shown relative to vehicle-treated controls (PBS for CM03 and DMSO for gemcitabine). Student’s t test along with 2 –Δ C t values were used to determine the statistical significance of the observed changes, which are the mean of in each case at least three determinations. Those genes with changes in expression with p
    Figure Legend Snippet: Validation of mRNA down regulation by qRT-PCR for a subset of down-regulated genes, selected from RNA-Seq experiments. (a–d) MIA PaCa-2 and PANC-1 cells were treated (a and b) with 400 nM CM03 and (c and d) with 400 nM gemcitabine, all for 6 and 24 h. Total mRNA was extracted, reverse transcribed into cDNA, and then qRT-PCR was performed. The C t values were normalized to the genomic mean of three housekeeping genes ( ACTB , GAPDH , and TUBB ), and the relative gene expression was determined using the Livak method, 2 –ΔΔ C t . The log-fold expression changes (Log 2 FC) for each gene are shown relative to vehicle-treated controls (PBS for CM03 and DMSO for gemcitabine). Student’s t test along with 2 –Δ C t values were used to determine the statistical significance of the observed changes, which are the mean of in each case at least three determinations. Those genes with changes in expression with p

    Techniques Used: Quantitative RT-PCR, RNA Sequencing Assay, Expressing

    15) Product Images from "Targeting Multiple Effector Pathways in Pancreatic Ductal Adenocarcinoma with a G-Quadruplex-Binding Small Molecule"

    Article Title: Targeting Multiple Effector Pathways in Pancreatic Ductal Adenocarcinoma with a G-Quadruplex-Binding Small Molecule

    Journal: Journal of Medicinal Chemistry

    doi: 10.1021/acs.jmedchem.7b01781

    Differentially down-regulated genes common to both PANC-1 and MIA PaCa-2 are enriched in PQs after treatment with 400 nM CM03. (a,b) MIA PaCa-2 and PANC-1 cells were treated with 400 nM CM03 for 6 and 24 h and mRNA extracted for analysis by RNA-Seq. Genes were split into four subgroups according to their fold change upon CM03 treatment versus untreated: Down (Log 2 FC
    Figure Legend Snippet: Differentially down-regulated genes common to both PANC-1 and MIA PaCa-2 are enriched in PQs after treatment with 400 nM CM03. (a,b) MIA PaCa-2 and PANC-1 cells were treated with 400 nM CM03 for 6 and 24 h and mRNA extracted for analysis by RNA-Seq. Genes were split into four subgroups according to their fold change upon CM03 treatment versus untreated: Down (Log 2 FC

    Techniques Used: RNA Sequencing Assay

    16) Product Images from "Host chemokine (C-C motif) ligand-2 (CCL2) is differentially regulated in HIV type 1 (HIV-1)-infected individuals"

    Article Title: Host chemokine (C-C motif) ligand-2 (CCL2) is differentially regulated in HIV type 1 (HIV-1)-infected individuals

    Journal: International Immunology

    doi: 10.1093/intimm/dxl078

    Phenotypic analysis of HIV-1 patient-derived PBMC after in vitro culture. (A) Two-color staining was performed on frozen PBMC samples after 24 h of stimulation with PMA. Dot plots represent percentage of CD14+CCL2+ monocytes after gating on monocytes based on forward and side scatter gating. The left panel shows the isotype control in both aviremic and viremic plots and data shown here are representative of three patients from each group. (B) A higher percentage of CD14+ monocytes producing CCL2 is found in viremic (CD4+ T cells 191.8 ± 48.6 and viral load 24 482 ± 16 921, n = 4) as compared with aviremic (CD4+ T cells 377.3 ± 60.0 and viral load
    Figure Legend Snippet: Phenotypic analysis of HIV-1 patient-derived PBMC after in vitro culture. (A) Two-color staining was performed on frozen PBMC samples after 24 h of stimulation with PMA. Dot plots represent percentage of CD14+CCL2+ monocytes after gating on monocytes based on forward and side scatter gating. The left panel shows the isotype control in both aviremic and viremic plots and data shown here are representative of three patients from each group. (B) A higher percentage of CD14+ monocytes producing CCL2 is found in viremic (CD4+ T cells 191.8 ± 48.6 and viral load 24 482 ± 16 921, n = 4) as compared with aviremic (CD4+ T cells 377.3 ± 60.0 and viral load

    Techniques Used: Derivative Assay, In Vitro, Staining

    Quantitative real-time PCR of regulated genes. Pooled PBMC RNAs from HIV-1 viremic and aviremic individuals ( n = 5) were amplified using gene-specific primers as described in Methods. Expression pattern of up-regulated genes CCL2, CXCL10, IFN-γ, GCH1 and CCR1 are shown in (A). Individual patient's inflammatory gene array verification showed similar expression pattern in two HIV-1 viremic and aviremic individuals (B). Av and V stand for aviremic and viremic individuals, respectively. Fold change in mRNA was quantified in relation to internal housekeeper β-actin/GAPDH mRNA as described in Methods.
    Figure Legend Snippet: Quantitative real-time PCR of regulated genes. Pooled PBMC RNAs from HIV-1 viremic and aviremic individuals ( n = 5) were amplified using gene-specific primers as described in Methods. Expression pattern of up-regulated genes CCL2, CXCL10, IFN-γ, GCH1 and CCR1 are shown in (A). Individual patient's inflammatory gene array verification showed similar expression pattern in two HIV-1 viremic and aviremic individuals (B). Av and V stand for aviremic and viremic individuals, respectively. Fold change in mRNA was quantified in relation to internal housekeeper β-actin/GAPDH mRNA as described in Methods.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Expressing

    17) Product Images from "Alternative splicing is a developmental switch for hTERT expression"

    Article Title: Alternative splicing is a developmental switch for hTERT expression

    Journal: bioRxiv

    doi: 10.1101/2020.04.02.022087

    SON is a key regulator of hTERT alternative splicing. A) Schematic of minigenes designed to measure the efficiency of hTERT mRNA splicing using Nano- and Firefly luciferase activity as a readout. Retention of exon-2 leads to expression of Firefly luciferase, but not Nano luciferase. Conversely, exclusion of exon 2 leads to a (−1) frameshift in exon-3 prompting the expression of Nano-luciferase while shifting Firefly luciferase out-of-frame. B) The minigenes were integrated into HeLa cells and a small-scale RNAi screen using a curated list of splicing factors and RNA-binding proteins was performed. Graph depicts the average ratio of Nano/Firefly luciferase of 3 biological replicates for 442 genes. Data presented as a log-ratio, colors highlight genes with p-value
    Figure Legend Snippet: SON is a key regulator of hTERT alternative splicing. A) Schematic of minigenes designed to measure the efficiency of hTERT mRNA splicing using Nano- and Firefly luciferase activity as a readout. Retention of exon-2 leads to expression of Firefly luciferase, but not Nano luciferase. Conversely, exclusion of exon 2 leads to a (−1) frameshift in exon-3 prompting the expression of Nano-luciferase while shifting Firefly luciferase out-of-frame. B) The minigenes were integrated into HeLa cells and a small-scale RNAi screen using a curated list of splicing factors and RNA-binding proteins was performed. Graph depicts the average ratio of Nano/Firefly luciferase of 3 biological replicates for 442 genes. Data presented as a log-ratio, colors highlight genes with p-value

    Techniques Used: Luciferase, Activity Assay, Expressing, RNA Binding Assay

    Inclusion of exon-2 correlates with the abundance of telomerase mRNA. A) Sashimi plot representing RNA Capture-Seq for hTERT locus in H7 ESCs and ARPE cells. Reads from exons are depicted as pileups and exon-exon junctions denoted with arcs. Asterisks indicate ΔEx2 splice variant transcripts detected in differentiated cells. B) Schematic of full-length and hTERT-ΔEx2 transcripts, demonstrating the position of the premature termination codons (PTC, asterisks) generated upon exon-2 skipping. Two tandem PTC’s would likely target ΔEx2 transcript for degradation by nonsense-mediated decay (NMD). C) Schematic illustration of junction-spanning PCR strategy used to assay hTERT ΔEx2 abundance by quantitative RT-PCR. RNA was reverse-transcribed using an hTERT gene-specific primer (GSP) and cDNA was purified and equalized between samples prior to PCR amplification with the indicated primers. D) Quantification of the ratio of hTERT ΔEx2 relative to full-length as determined by qRT-PCR in mortal cell lines (ARPE BJ), ARPE and BJ cell lines immortalized with hTERT cDNA, iPSCs derived from BJ cells, and human ESCs (n=3, p
    Figure Legend Snippet: Inclusion of exon-2 correlates with the abundance of telomerase mRNA. A) Sashimi plot representing RNA Capture-Seq for hTERT locus in H7 ESCs and ARPE cells. Reads from exons are depicted as pileups and exon-exon junctions denoted with arcs. Asterisks indicate ΔEx2 splice variant transcripts detected in differentiated cells. B) Schematic of full-length and hTERT-ΔEx2 transcripts, demonstrating the position of the premature termination codons (PTC, asterisks) generated upon exon-2 skipping. Two tandem PTC’s would likely target ΔEx2 transcript for degradation by nonsense-mediated decay (NMD). C) Schematic illustration of junction-spanning PCR strategy used to assay hTERT ΔEx2 abundance by quantitative RT-PCR. RNA was reverse-transcribed using an hTERT gene-specific primer (GSP) and cDNA was purified and equalized between samples prior to PCR amplification with the indicated primers. D) Quantification of the ratio of hTERT ΔEx2 relative to full-length as determined by qRT-PCR in mortal cell lines (ARPE BJ), ARPE and BJ cell lines immortalized with hTERT cDNA, iPSCs derived from BJ cells, and human ESCs (n=3, p

    Techniques Used: Variant Assay, Generated, Polymerase Chain Reaction, Quantitative RT-PCR, Purification, Amplification, Derivative Assay

    18) Product Images from "Alternative splicing is a developmental switch for hTERT expression"

    Article Title: Alternative splicing is a developmental switch for hTERT expression

    Journal: bioRxiv

    doi: 10.1101/2020.04.02.022087

    SON is a key regulator of hTERT alternative splicing. A) Schematic of minigenes designed to measure the efficiency of hTERT mRNA splicing using Nano- and Firefly luciferase activity as a readout. Retention of exon-2 leads to expression of Firefly luciferase, but not Nano luciferase. Conversely, exclusion of exon 2 leads to a (−1) frameshift in exon-3 prompting the expression of Nano-luciferase while shifting Firefly luciferase out-of-frame. B) The minigenes were integrated into HeLa cells and a small-scale RNAi screen using a curated list of splicing factors and RNA-binding proteins was performed. Graph depicts the average ratio of Nano/Firefly luciferase of 3 biological replicates for 442 genes. Data presented as a log-ratio, colors highlight genes with p-value
    Figure Legend Snippet: SON is a key regulator of hTERT alternative splicing. A) Schematic of minigenes designed to measure the efficiency of hTERT mRNA splicing using Nano- and Firefly luciferase activity as a readout. Retention of exon-2 leads to expression of Firefly luciferase, but not Nano luciferase. Conversely, exclusion of exon 2 leads to a (−1) frameshift in exon-3 prompting the expression of Nano-luciferase while shifting Firefly luciferase out-of-frame. B) The minigenes were integrated into HeLa cells and a small-scale RNAi screen using a curated list of splicing factors and RNA-binding proteins was performed. Graph depicts the average ratio of Nano/Firefly luciferase of 3 biological replicates for 442 genes. Data presented as a log-ratio, colors highlight genes with p-value

    Techniques Used: Luciferase, Activity Assay, Expressing, RNA Binding Assay

    Forced retention of exon-2 abolishes silencing of hTERT upon differentiation. A) Schematic illustration of intron 1 deletion by CRISPR/Cas9 gene editing and the predicted splicing pattern. Cells were co-transfected with two sgRNA that cleave within hTERT intron-1 and a 200bp single-stranded (ss) DNA donor containing 100bp sequence from exon-1 and exon-2 directly concatenated. B) Genotyping PCR from cells with the indicated genotype. PCR products: wild type, 584bp; Δintron1, 480bp C) Quantitative RT-PCR for hTERT mRNA in cells with the indicated genotype. hTERT expression is silenced in fibroblasts derived from wildtype ESC clones, whereas hTERT levels remain elevated upon differentiation of hTERT Δin1/Δin1 ESCs. Values are normalized to hTERT +/+ H7 ESC (n=3, **: p
    Figure Legend Snippet: Forced retention of exon-2 abolishes silencing of hTERT upon differentiation. A) Schematic illustration of intron 1 deletion by CRISPR/Cas9 gene editing and the predicted splicing pattern. Cells were co-transfected with two sgRNA that cleave within hTERT intron-1 and a 200bp single-stranded (ss) DNA donor containing 100bp sequence from exon-1 and exon-2 directly concatenated. B) Genotyping PCR from cells with the indicated genotype. PCR products: wild type, 584bp; Δintron1, 480bp C) Quantitative RT-PCR for hTERT mRNA in cells with the indicated genotype. hTERT expression is silenced in fibroblasts derived from wildtype ESC clones, whereas hTERT levels remain elevated upon differentiation of hTERT Δin1/Δin1 ESCs. Values are normalized to hTERT +/+ H7 ESC (n=3, **: p

    Techniques Used: CRISPR, Transfection, Sequencing, Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Derivative Assay, Clone Assay

    Inclusion of exon-2 correlates with the abundance of telomerase mRNA. A) Sashimi plot representing RNA Capture-Seq for hTERT locus in H7 ESCs and ARPE cells. Reads from exons are depicted as pileups and exon-exon junctions denoted with arcs. Asterisks indicate ΔEx2 splice variant transcripts detected in differentiated cells. B) Schematic of full-length and hTERT-ΔEx2 transcripts, demonstrating the position of the premature termination codons (PTC, asterisks) generated upon exon-2 skipping. Two tandem PTC’s would likely target ΔEx2 transcript for degradation by nonsense-mediated decay (NMD). C) Schematic illustration of junction-spanning PCR strategy used to assay hTERT ΔEx2 abundance by quantitative RT-PCR. RNA was reverse-transcribed using an hTERT gene-specific primer (GSP) and cDNA was purified and equalized between samples prior to PCR amplification with the indicated primers. D) Quantification of the ratio of hTERT ΔEx2 relative to full-length as determined by qRT-PCR in mortal cell lines (ARPE BJ), ARPE and BJ cell lines immortalized with hTERT cDNA, iPSCs derived from BJ cells, and human ESCs (n=3, p
    Figure Legend Snippet: Inclusion of exon-2 correlates with the abundance of telomerase mRNA. A) Sashimi plot representing RNA Capture-Seq for hTERT locus in H7 ESCs and ARPE cells. Reads from exons are depicted as pileups and exon-exon junctions denoted with arcs. Asterisks indicate ΔEx2 splice variant transcripts detected in differentiated cells. B) Schematic of full-length and hTERT-ΔEx2 transcripts, demonstrating the position of the premature termination codons (PTC, asterisks) generated upon exon-2 skipping. Two tandem PTC’s would likely target ΔEx2 transcript for degradation by nonsense-mediated decay (NMD). C) Schematic illustration of junction-spanning PCR strategy used to assay hTERT ΔEx2 abundance by quantitative RT-PCR. RNA was reverse-transcribed using an hTERT gene-specific primer (GSP) and cDNA was purified and equalized between samples prior to PCR amplification with the indicated primers. D) Quantification of the ratio of hTERT ΔEx2 relative to full-length as determined by qRT-PCR in mortal cell lines (ARPE BJ), ARPE and BJ cell lines immortalized with hTERT cDNA, iPSCs derived from BJ cells, and human ESCs (n=3, p

    Techniques Used: Variant Assay, Generated, Polymerase Chain Reaction, Quantitative RT-PCR, Purification, Amplification, Derivative Assay

    19) Product Images from "Expression of Innate Immunity Genes and Damage of Primary Human Pancreatic Islets by Epidemic Strains of Echovirus: Implication for Post-Virus Islet Autoimmunity"

    Article Title: Expression of Innate Immunity Genes and Damage of Primary Human Pancreatic Islets by Epidemic Strains of Echovirus: Implication for Post-Virus Islet Autoimmunity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0077850

    Virus-induced cytopathic effect in primary human pancreatic islets cells. A. Uninfected islet. B. Islets infected with the E4 isolate 5 days post infection. C. Islets infected with E16 isolates 3 days post infection. D. Islets infected with E30 isolates 3 days post infection. The figure is representative of seven islet donors.
    Figure Legend Snippet: Virus-induced cytopathic effect in primary human pancreatic islets cells. A. Uninfected islet. B. Islets infected with the E4 isolate 5 days post infection. C. Islets infected with E16 isolates 3 days post infection. D. Islets infected with E30 isolates 3 days post infection. The figure is representative of seven islet donors.

    Techniques Used: Infection

    Viral titers of the clinical strains of E16, E30 strains and E4 in the culture medium of infected primary human islets during 3 days post-infection. Aliquots of the culture medium were withdrawn day 0 and day 3. Virus titers were obtained using the cell culture infectious dose 50 (CCID50) titration methods. The results are shown as the means ± SD from experiments performed in triplicate.
    Figure Legend Snippet: Viral titers of the clinical strains of E16, E30 strains and E4 in the culture medium of infected primary human islets during 3 days post-infection. Aliquots of the culture medium were withdrawn day 0 and day 3. Virus titers were obtained using the cell culture infectious dose 50 (CCID50) titration methods. The results are shown as the means ± SD from experiments performed in triplicate.

    Techniques Used: Infection, Cell Culture, Titration

    20) Product Images from "ZBTB48 is both a vertebrate telomere‐binding protein and a transcriptional activator"

    Article Title: ZBTB48 is both a vertebrate telomere‐binding protein and a transcriptional activator

    Journal: EMBO Reports

    doi: 10.15252/embr.201744095

    ZBTB48 and HOT1 KO clones show depleted expression Depiction of the ZBTB48 TALEN binding sites located in exon 2. Genotypes of HeLa and U2OS WT and ZBTB48 KO clones. The grey boxes represent the TALEN binding sites; insertions (blue) and deletions (red) are marked. The numbers in brackets represent the numbers of clones found with the specific genotype. Gel image of T7E1 assay showing ZBTB48 TALEN activity in HeLa cells. Western blot confirmation of depleted ZBTB48 expression in each five HeLa and U2OS ZBTB48 KO clones compared to parental cells. IF confirmation of depleted ZBTB48 expression (green) with DAPI (blue) as nuclear counterstain in U2OS ZBTB48 KO cells. ZBTB48 signals are reduced to background levels. Scale bars represent 5 μm. Depiction of the HOT1 TALEN binding sites located in exon 3. Genotypes of HeLa and U2OS WT and HOT1 KO clones. The grey boxes represent the TALEN binding sites; insertions (blue) and deletions (red) are marked. The numbers in brackets represent the numbers of clones found with the specific genotype. Gel image of T7E1 assay showing HOT1 TALEN activity in HeLa cells. IF confirmation of depleted HOT1 expression (green) with DAPI (blue) as nuclear counterstain in a representative HeLa HOT1 KO clone. Scale bars represent 5 μm. Source data are available online for this figure.
    Figure Legend Snippet: ZBTB48 and HOT1 KO clones show depleted expression Depiction of the ZBTB48 TALEN binding sites located in exon 2. Genotypes of HeLa and U2OS WT and ZBTB48 KO clones. The grey boxes represent the TALEN binding sites; insertions (blue) and deletions (red) are marked. The numbers in brackets represent the numbers of clones found with the specific genotype. Gel image of T7E1 assay showing ZBTB48 TALEN activity in HeLa cells. Western blot confirmation of depleted ZBTB48 expression in each five HeLa and U2OS ZBTB48 KO clones compared to parental cells. IF confirmation of depleted ZBTB48 expression (green) with DAPI (blue) as nuclear counterstain in U2OS ZBTB48 KO cells. ZBTB48 signals are reduced to background levels. Scale bars represent 5 μm. Depiction of the HOT1 TALEN binding sites located in exon 3. Genotypes of HeLa and U2OS WT and HOT1 KO clones. The grey boxes represent the TALEN binding sites; insertions (blue) and deletions (red) are marked. The numbers in brackets represent the numbers of clones found with the specific genotype. Gel image of T7E1 assay showing HOT1 TALEN activity in HeLa cells. IF confirmation of depleted HOT1 expression (green) with DAPI (blue) as nuclear counterstain in a representative HeLa HOT1 KO clone. Scale bars represent 5 μm. Source data are available online for this figure.

    Techniques Used: Clone Assay, Expressing, Binding Assay, Activity Assay, Western Blot

    ZBTB48 is a direct telomere‐binding protein but TRAP activity is not affected in ZBTB48 KO clones TTAGGG content of telomeric reads in ChIPseq samples from U2OS WT, U2OS ZBTB48 KO and U2OS HOT1 KO clones using IgG, TRF2, ZBTB48 Atlas, ZBTB48 GeneTex and HOT1 antibodies as well as FLAG antibody for FLAG‐ZBTB48 WT and point mutants for ZnF10 and ZnF11. The percentage contribution to all reads containing 1–8× TTAGGG repeats is shown. All reactions were performed in technical replicates on two independent WT and KO clones each. These reactions are biological replicates of Fig 2 A, performed with independent U2OS ZBTB48 WT and KO clones for ZBTB48 and with a second HOT1 antibody for HOT1. Telomerase activity was determined based on a quantitative TRAP assay. Heat‐inactivated HeLa extracts were used as a threshold to determine non‐specific background signal. Differences in C t values (∆∆ C t ) from the quantitative PCR measurements are displayed relative to the heat‐inactivation control. Error bars represent standard deviations ( n = 3). The P ‐value is based on Student's t ‐test.
    Figure Legend Snippet: ZBTB48 is a direct telomere‐binding protein but TRAP activity is not affected in ZBTB48 KO clones TTAGGG content of telomeric reads in ChIPseq samples from U2OS WT, U2OS ZBTB48 KO and U2OS HOT1 KO clones using IgG, TRF2, ZBTB48 Atlas, ZBTB48 GeneTex and HOT1 antibodies as well as FLAG antibody for FLAG‐ZBTB48 WT and point mutants for ZnF10 and ZnF11. The percentage contribution to all reads containing 1–8× TTAGGG repeats is shown. All reactions were performed in technical replicates on two independent WT and KO clones each. These reactions are biological replicates of Fig 2 A, performed with independent U2OS ZBTB48 WT and KO clones for ZBTB48 and with a second HOT1 antibody for HOT1. Telomerase activity was determined based on a quantitative TRAP assay. Heat‐inactivated HeLa extracts were used as a threshold to determine non‐specific background signal. Differences in C t values (∆∆ C t ) from the quantitative PCR measurements are displayed relative to the heat‐inactivation control. Error bars represent standard deviations ( n = 3). The P ‐value is based on Student's t ‐test.

    Techniques Used: Binding Assay, Activity Assay, Clone Assay, TRAP Assay, Real-time Polymerase Chain Reaction

    ZBTB 48 associates with short and long telomeres in vivo TTAGGG content of telomeric reads in ChIPseq samples from U2OS WT, U2OS ZBTB48 KO and U2OS HOT1 KO clones using IgG, TRF2, ZBTB48 Atlas, ZBTB48 GeneTex and HOT1 antibodies as well as FLAG antibody for FLAG‐ZBTB48 WT and point mutants for ZnF10 and ZnF11. The percentage contribution to all reads containing 1–8× TTAGGG repeats is shown. All reactions were performed in technical replicates on two independent WT and KO clones each. MEME sequence logo and bit score for the top 500 extratelomeric ChIPseq peaks for ZBTB48 Atlas, ZBTB48 GeneTex, TRF2 and HOT1 antibodies compared to IgG samples in U2OS. For each antibody, the most frequent motif is shown. TTAGGG enrichment in ChIPseq samples with reads with 7× or 8× TTAGGG repeats. Absolute read counts from ChIPseq reactions from each two HeLa and U2OS WT and KO clones with two independent antibodies for ZBTB48 and HOT1 are shown (left). The corresponding fold enrichments are calculated (right) and compared to TRF2 ChIPseq reactions for which fold enrichments are calculated relative to IgG samples. Error bars represent standard deviations ( n = 4), and P ‐values are based on Student's t ‐test with ** indicating P
    Figure Legend Snippet: ZBTB 48 associates with short and long telomeres in vivo TTAGGG content of telomeric reads in ChIPseq samples from U2OS WT, U2OS ZBTB48 KO and U2OS HOT1 KO clones using IgG, TRF2, ZBTB48 Atlas, ZBTB48 GeneTex and HOT1 antibodies as well as FLAG antibody for FLAG‐ZBTB48 WT and point mutants for ZnF10 and ZnF11. The percentage contribution to all reads containing 1–8× TTAGGG repeats is shown. All reactions were performed in technical replicates on two independent WT and KO clones each. MEME sequence logo and bit score for the top 500 extratelomeric ChIPseq peaks for ZBTB48 Atlas, ZBTB48 GeneTex, TRF2 and HOT1 antibodies compared to IgG samples in U2OS. For each antibody, the most frequent motif is shown. TTAGGG enrichment in ChIPseq samples with reads with 7× or 8× TTAGGG repeats. Absolute read counts from ChIPseq reactions from each two HeLa and U2OS WT and KO clones with two independent antibodies for ZBTB48 and HOT1 are shown (left). The corresponding fold enrichments are calculated (right) and compared to TRF2 ChIPseq reactions for which fold enrichments are calculated relative to IgG samples. Error bars represent standard deviations ( n = 4), and P ‐values are based on Student's t ‐test with ** indicating P

    Techniques Used: In Vivo, Clone Assay, Sequencing

    ZBTB48 ZnF11 is necessary to bind to telomeres Sequence‐specific DNA pull‐downs with either telomeric (TTAGGG) or a control sequence (GTGAGT) for FLAG‐ZBTB48 WT, domain deletion constructs for different zinc finger and combinations of deletion constructs with ZnF10 or 11 point mutants. Domain structures are indicated on the right. Sequence‐specific DNA pull‐downs for FLAG‐ZBTB48 WT and ZnF11 point mutant for telomeric repeat sequences of different phyla (green) and their respective scrambled controls (blue). Protein expression analysis of ZBTB48 by Western blot for the cell lines used in this study. GAPDH serves as a loading control. IF stainings for exogenous FLAG‐ZBTB48 WT and point mutants for ZnF10 and ZnF11 in U2OS cells. The same analysis as in Fig 1 E was performed and average co‐localization frequencies are shown ( n = 24–37 cells). Co‐localization analysis of endogenous ZBTB48 or exogenous FLAG‐ZBTB48 WT with TRF2 in HeLa cells by immunofluorescence (IF) staining. A representative image illustrating the co‐localization between ZBTB48 or FLAG‐ZBTB48 WT (green) and TRF2 (red) as a marker for telomeres is shown with DAPI (blue) used as a nuclear counterstain. Co‐localization events are indicated by white arrows. The quantification of frequency of co‐localization events (right) was done after 3D reconstruction of the acquired z ‐stacks ( n = 30 cells). Co‐localization analysis of endogenous ZBTB48 or exogenous FLAG‐ZBTB48 WT with TRF2 in HeLa 1.3 cells by immunofluorescence (IF) staining analogous to (E) ( n = 30 cells). Co‐localization analysis of endogenous ZBTB48 or exogenous FLAG‐ZBTB48 WT with TRF2 in HT1080 super‐telomerase cells by immunofluorescence (IF) staining analogous to (E) ( n = 30 cells). Data information: (D–G) Scale bars represent 5 μm. Error bars indicate standard deviations, and P ‐values are based on Student's t ‐test. Source data are available online for this figure.
    Figure Legend Snippet: ZBTB48 ZnF11 is necessary to bind to telomeres Sequence‐specific DNA pull‐downs with either telomeric (TTAGGG) or a control sequence (GTGAGT) for FLAG‐ZBTB48 WT, domain deletion constructs for different zinc finger and combinations of deletion constructs with ZnF10 or 11 point mutants. Domain structures are indicated on the right. Sequence‐specific DNA pull‐downs for FLAG‐ZBTB48 WT and ZnF11 point mutant for telomeric repeat sequences of different phyla (green) and their respective scrambled controls (blue). Protein expression analysis of ZBTB48 by Western blot for the cell lines used in this study. GAPDH serves as a loading control. IF stainings for exogenous FLAG‐ZBTB48 WT and point mutants for ZnF10 and ZnF11 in U2OS cells. The same analysis as in Fig 1 E was performed and average co‐localization frequencies are shown ( n = 24–37 cells). Co‐localization analysis of endogenous ZBTB48 or exogenous FLAG‐ZBTB48 WT with TRF2 in HeLa cells by immunofluorescence (IF) staining. A representative image illustrating the co‐localization between ZBTB48 or FLAG‐ZBTB48 WT (green) and TRF2 (red) as a marker for telomeres is shown with DAPI (blue) used as a nuclear counterstain. Co‐localization events are indicated by white arrows. The quantification of frequency of co‐localization events (right) was done after 3D reconstruction of the acquired z ‐stacks ( n = 30 cells). Co‐localization analysis of endogenous ZBTB48 or exogenous FLAG‐ZBTB48 WT with TRF2 in HeLa 1.3 cells by immunofluorescence (IF) staining analogous to (E) ( n = 30 cells). Co‐localization analysis of endogenous ZBTB48 or exogenous FLAG‐ZBTB48 WT with TRF2 in HT1080 super‐telomerase cells by immunofluorescence (IF) staining analogous to (E) ( n = 30 cells). Data information: (D–G) Scale bars represent 5 μm. Error bars indicate standard deviations, and P ‐values are based on Student's t ‐test. Source data are available online for this figure.

    Techniques Used: Sequencing, Construct, Mutagenesis, Expressing, Western Blot, Immunofluorescence, Staining, Marker

    ZBTB48 is a transcriptional activator Differential expression analysis of the RNA sequencing (RNAseq) gene quantitation, comparing each five WT and ZBTB48 KO clones for U2OS (left) and HeLa (right). Cut‐offs for significant differential expression were set to log 2 (fold change) > |1| and −log 10 (adjusted P ‐value) > 2 (FDR
    Figure Legend Snippet: ZBTB48 is a transcriptional activator Differential expression analysis of the RNA sequencing (RNAseq) gene quantitation, comparing each five WT and ZBTB48 KO clones for U2OS (left) and HeLa (right). Cut‐offs for significant differential expression were set to log 2 (fold change) > |1| and −log 10 (adjusted P ‐value) > 2 (FDR

    Techniques Used: Expressing, RNA Sequencing Assay, Quantitation Assay, Clone Assay

    ZBTB 48 is a negative regulator of telomere length Terminal restriction fragment (TRF) analysis for each five independent HeLa WT and HeLa ZBTB48 KO clones at passage 20. The average telomere length was determined from the telomeric distribution (left) and used for average quantification of WT vs. KO clones (right). Average telomere length is indicated for all WT samples (dotted line) and individually for all samples (red dots). Error bars indicate standard deviations, and the P ‐value is based on Student's t ‐test. TRF analysis using pulsed‐field gel electrophoresis (PFGE) of parental U2OS WT cells compared to five independent U2OS ZBTB48 KO clones at passage 37. Average telomere length is indicated for all WT samples (dotted line) and individually for all samples (red dots). Quantification of C‐circles in U2OS WT cells compared to five independent U2OS ZBTB48 KO clones. C‐circle reactions were carried out and spotted in triplicate (top), and average quantifications are displayed (bottom). No Φ indicates negative control reactions without the ΦDNA polymerase. 15 ng DNA was used as input material per reaction. The dashed line indicates cropping of the membrane between KO clones 4 and 5. Error bars indicate standard deviations, and P ‐values are based on Student's t ‐test with * indicating P
    Figure Legend Snippet: ZBTB 48 is a negative regulator of telomere length Terminal restriction fragment (TRF) analysis for each five independent HeLa WT and HeLa ZBTB48 KO clones at passage 20. The average telomere length was determined from the telomeric distribution (left) and used for average quantification of WT vs. KO clones (right). Average telomere length is indicated for all WT samples (dotted line) and individually for all samples (red dots). Error bars indicate standard deviations, and the P ‐value is based on Student's t ‐test. TRF analysis using pulsed‐field gel electrophoresis (PFGE) of parental U2OS WT cells compared to five independent U2OS ZBTB48 KO clones at passage 37. Average telomere length is indicated for all WT samples (dotted line) and individually for all samples (red dots). Quantification of C‐circles in U2OS WT cells compared to five independent U2OS ZBTB48 KO clones. C‐circle reactions were carried out and spotted in triplicate (top), and average quantifications are displayed (bottom). No Φ indicates negative control reactions without the ΦDNA polymerase. 15 ng DNA was used as input material per reaction. The dashed line indicates cropping of the membrane between KO clones 4 and 5. Error bars indicate standard deviations, and P ‐values are based on Student's t ‐test with * indicating P

    Techniques Used: Clone Assay, Pulsed-Field Gel, Electrophoresis, Negative Control

    ZBTB48 acts as a transcription factor in contrast to HOT1 Differential expression analysis of the RNA sequencing (RNAseq) gene quantitation, comparing each five WT and ZBTB48 KO clones for U2OS (left) and HeLa (right). Cut‐offs for significant differential expression were set to log 2 (fold change) > |1| and −log 10 (adjusted P ‐value) > 2 (FDR
    Figure Legend Snippet: ZBTB48 acts as a transcription factor in contrast to HOT1 Differential expression analysis of the RNA sequencing (RNAseq) gene quantitation, comparing each five WT and ZBTB48 KO clones for U2OS (left) and HeLa (right). Cut‐offs for significant differential expression were set to log 2 (fold change) > |1| and −log 10 (adjusted P ‐value) > 2 (FDR

    Techniques Used: Expressing, RNA Sequencing Assay, Quantitation Assay, Clone Assay

    ZBTB48‐dependent loss of MTFP1 phenocopies MTFP1 depletion Fluorescence microscopy analysis of the structure and localization of the mitochondrial network in HeLa WT and ZBTB48 KO clones. Mitochondria are marked with the MitoTracker dye (red), and nuclei are counterstained with DAPI (blue). Scale bars represent 20 μm. The same analysis as in (A) for U2OS WT and ZBTB48 KO clones.
    Figure Legend Snippet: ZBTB48‐dependent loss of MTFP1 phenocopies MTFP1 depletion Fluorescence microscopy analysis of the structure and localization of the mitochondrial network in HeLa WT and ZBTB48 KO clones. Mitochondria are marked with the MitoTracker dye (red), and nuclei are counterstained with DAPI (blue). Scale bars represent 20 μm. The same analysis as in (A) for U2OS WT and ZBTB48 KO clones.

    Techniques Used: Fluorescence, Microscopy, Clone Assay

    ZBTB48 is required for MTFP1 expression Western blot confirmation of reduced MTFP1 expression in each five HeLa and U2OS ZBTB48 KO clones compared to five WT clones each. Similar to ZBTB48, no detectable MTFP1 protein is found in the KO cells. Super‐resolution fluorescence microscopy analysis of the structure and localization of the mitochondrial network in HeLa WT and ZBTB48 KO clones. Mitochondria are marked with the MitoTracker dye (red), and nuclei are counterstained with DAPI (blue). Scale bars represent 5 μm. mtDNA level quantification comparing five HeLa ZBTB48 WT and KO clones. mtDNA levels were quantified based on three mtDNA loci and normalized to two genomic regions. Error bars represent standard deviations ( n = 3). The P ‐value is based on Student's t ‐test.
    Figure Legend Snippet: ZBTB48 is required for MTFP1 expression Western blot confirmation of reduced MTFP1 expression in each five HeLa and U2OS ZBTB48 KO clones compared to five WT clones each. Similar to ZBTB48, no detectable MTFP1 protein is found in the KO cells. Super‐resolution fluorescence microscopy analysis of the structure and localization of the mitochondrial network in HeLa WT and ZBTB48 KO clones. Mitochondria are marked with the MitoTracker dye (red), and nuclei are counterstained with DAPI (blue). Scale bars represent 5 μm. mtDNA level quantification comparing five HeLa ZBTB48 WT and KO clones. mtDNA levels were quantified based on three mtDNA loci and normalized to two genomic regions. Error bars represent standard deviations ( n = 3). The P ‐value is based on Student's t ‐test.

    Techniques Used: Expressing, Western Blot, Clone Assay, Fluorescence, Microscopy

    21) Product Images from "MGA, L3MBTL2 and E2F6 determine genomic binding of the non-canonical Polycomb repressive complex PRC1.6"

    Article Title: MGA, L3MBTL2 and E2F6 determine genomic binding of the non-canonical Polycomb repressive complex PRC1.6

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007193

    The role of PRC1.6 in HEK293 cell function. (A) Reduced proliferation of MGA ko , L3MBTL2 ko and E2F6 ko cells. Shown are growth curves of wildtype, MGA ko , L3MBTL2 ko , E2F6 ko and PCGF6 ko HEK293 cells. Cells were seed at 3x10 5 , and counted and replated at the indicated time points. Cumulative cell numbers were calculated by multiplying the initial cell number with the fold-increase in cell numbers in each interval. (B) Venn diagrams illustrating the overlap of MGA-bound genes and genes down- or up-regulated in MGA ko cells. Left circle, genes with ≥2-fold reduced transcript levels in MGA ko cells; right circle, genes with ≥2-fold increased transcript levels in MGA ko cells. (C) Representative genome browser screenshots of ChIP-seq and RNA-seq tracks illustrating binding of MGA, L3MBTL2, E2F6 and PCGF6 (top tracks) to the CNTD1 and SMC1B promoters, and RNA expression (bottom tracks) of the corresponding genes in three wild type samples (MGA_wt1, MGA_wt2 and MGA_wt3), and in three different MGA ko cell clones (MGA ko _cl26, MGA ko _cl27 and MGA ko _cl30). (D) RT-qPCR-based analysis of expression changes of selected genes in MGA ko , E2F6 ko , L3MBTL2 ko and PCGF6 ko cells. Transcript levels were normalized to B2M transcript levels, and are depicted relative to transcript levels in wild type cells.
    Figure Legend Snippet: The role of PRC1.6 in HEK293 cell function. (A) Reduced proliferation of MGA ko , L3MBTL2 ko and E2F6 ko cells. Shown are growth curves of wildtype, MGA ko , L3MBTL2 ko , E2F6 ko and PCGF6 ko HEK293 cells. Cells were seed at 3x10 5 , and counted and replated at the indicated time points. Cumulative cell numbers were calculated by multiplying the initial cell number with the fold-increase in cell numbers in each interval. (B) Venn diagrams illustrating the overlap of MGA-bound genes and genes down- or up-regulated in MGA ko cells. Left circle, genes with ≥2-fold reduced transcript levels in MGA ko cells; right circle, genes with ≥2-fold increased transcript levels in MGA ko cells. (C) Representative genome browser screenshots of ChIP-seq and RNA-seq tracks illustrating binding of MGA, L3MBTL2, E2F6 and PCGF6 (top tracks) to the CNTD1 and SMC1B promoters, and RNA expression (bottom tracks) of the corresponding genes in three wild type samples (MGA_wt1, MGA_wt2 and MGA_wt3), and in three different MGA ko cell clones (MGA ko _cl26, MGA ko _cl27 and MGA ko _cl30). (D) RT-qPCR-based analysis of expression changes of selected genes in MGA ko , E2F6 ko , L3MBTL2 ko and PCGF6 ko cells. Transcript levels were normalized to B2M transcript levels, and are depicted relative to transcript levels in wild type cells.

    Techniques Used: Cell Function Assay, Chromatin Immunoprecipitation, RNA Sequencing Assay, Binding Assay, RNA Expression, Clone Assay, Quantitative RT-PCR, Expressing

    MGA is essential for genomic binding of PRC1.6. (A) Heat map view of the distribution of union MGA, L3MBTL2 and E2F6 peaks in wild type cells (n = 8342) and in MGA-depleted cells at +/- 2 kb regions centred over the MGA peaks. (B) Representative genome browser screenshots showing binding of MGA, L3MBTL2, E2F6 and PCGF6 to the AEBP2 , RPA2 , RFC1 and SPOP promoters in wild type cells. MGA-depleted cells lack binding of L3MBTL2 and E2F6. (C) Western blot analysis of L3MBTL2, E2F6, PCGF6 and RING2 in wild type HEK293 cells and in two different MGA-depleted clones (cl26 and cl27). The anti-Tubulin blot served as a loading control. (D) L3MBTL2 -, E2F6 - and PCGF6 transcripts were determined in wild type cells and in MGA-depleted cell clones by RT-qPCR analysis. B2M transcript levels were used to normalize the data across samples, and transcript levels in wild type cells were arbitrarily set to 1. Data represent the average of technical replicates ± SD. (E) ChIP-qPCR data showing lack of L3MBTL2, E2F6, PCGF6, MAX, RING2, RYBP and HP1γ binding to representative PRC1.6 target promoters in MGA ko cells, and diminished deposition of H2AK119ub1. The CDC7 -2kb region served as a negative control region. Percent of input values represent the mean of at least three independent experiments +/- SD. (F) PRC1.6 target promoters are not bound by PRC2 and lack H3K27me3. Local levels of EZH2 and H3K27me3 at selected PRC1.6 target promoters in wild type (WT) and in MGA ko cells (clones cl26 and cl27) were determined by ChIP-qPCR analysis. Genomic regions known to be bound by canonical PRC1 ( FUT9 , MYT1 and TSH2B ) served as positive control regions. These regions were not bound by MGA (right panel). Percent of input values represent the mean of at least three independent experiments +/- SD.
    Figure Legend Snippet: MGA is essential for genomic binding of PRC1.6. (A) Heat map view of the distribution of union MGA, L3MBTL2 and E2F6 peaks in wild type cells (n = 8342) and in MGA-depleted cells at +/- 2 kb regions centred over the MGA peaks. (B) Representative genome browser screenshots showing binding of MGA, L3MBTL2, E2F6 and PCGF6 to the AEBP2 , RPA2 , RFC1 and SPOP promoters in wild type cells. MGA-depleted cells lack binding of L3MBTL2 and E2F6. (C) Western blot analysis of L3MBTL2, E2F6, PCGF6 and RING2 in wild type HEK293 cells and in two different MGA-depleted clones (cl26 and cl27). The anti-Tubulin blot served as a loading control. (D) L3MBTL2 -, E2F6 - and PCGF6 transcripts were determined in wild type cells and in MGA-depleted cell clones by RT-qPCR analysis. B2M transcript levels were used to normalize the data across samples, and transcript levels in wild type cells were arbitrarily set to 1. Data represent the average of technical replicates ± SD. (E) ChIP-qPCR data showing lack of L3MBTL2, E2F6, PCGF6, MAX, RING2, RYBP and HP1γ binding to representative PRC1.6 target promoters in MGA ko cells, and diminished deposition of H2AK119ub1. The CDC7 -2kb region served as a negative control region. Percent of input values represent the mean of at least three independent experiments +/- SD. (F) PRC1.6 target promoters are not bound by PRC2 and lack H3K27me3. Local levels of EZH2 and H3K27me3 at selected PRC1.6 target promoters in wild type (WT) and in MGA ko cells (clones cl26 and cl27) were determined by ChIP-qPCR analysis. Genomic regions known to be bound by canonical PRC1 ( FUT9 , MYT1 and TSH2B ) served as positive control regions. These regions were not bound by MGA (right panel). Percent of input values represent the mean of at least three independent experiments +/- SD.

    Techniques Used: Binding Assay, Western Blot, Clone Assay, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Positive Control

    MGA, L3MBTL2, E2F6 and PCGF6 colocalize in 293 cells. (A) Schematic representation of PRC1.6 core components. (B) Western blot analysis of MGA, L3MBTL2, E2F6 and PCGF6 expression in wild type 293 cells (wt) and in corresponding MGA-, L3MBTL2-, E2F6- and PCGF6-depleted cell clones (MGA ko , L3MBTL2 ko , E2F6 ko and PCGF6 ko ). Re-probing for tubulin (TUB) controlled loading of extracts. (C) Venn diagrams showing the overlap of MGA, L3MBTL2, E2F6 and PCGF6 binding regions in HEK293 cells. The total number of high-confidence MGA, L3MBTL2, E2F6 and PCGF6 ChIP-seq peaks (≥30 tags, ≥3-fold enrichment over knockout control) and their overlap is shown. (D) A heat map view of the distribution of union MGA, L3MBTL2, E2F6 and PCGF6 peaks in HEK293 cells (n = 8342) at +/- 2 kb regions centred over the MGA peaks. (E) Representative genome browser screenshots of a 0.7 Mb region of chromosome 19 showing co-localization of MGA, L3MBTL2, E2F6 and PCGF6 at the CTC-232P5 . 1 , RFX2 , MLLT1 and KHSRP promoters. (F) Distribution of MGA, L3MBTL2, E2F6 and PCGF6 peaks relative to positions -2000 bp upstream to +2000 bp downstream of gene bodies. TSS, transcription start site; TES, transcription end site. (G) ChIP-qPCR analysis of MGA, L3MBTL2, E2F6 and PCGF6 binding to selected promoters. The region -2 kb upstream of the CDC7 promoter served as a negative control. Percent of input values represent the mean of at least three independent experiments +/- SD. (H) Sequence motifs enriched in PRC1.6 binding regions. Logos were obtained by running MEME-ChIP with 300 bp summits of the top 600 union MGA-L3MBTL2-E2F6-PCGF6 ChIP-seq peaks. The numbers next to the logos indicate the occurrence of the motifs, the statistical significance (E-value) and the transcription factors that bind to the motif. Right panel, local motif enrichment analysis (CentriMo) showing central enrichment of the MGA/MAX bHLH and the E2F6/DP1 binding motifs within the 300 bp peak regions. The NRF1 binding motif was not centrally enriched.
    Figure Legend Snippet: MGA, L3MBTL2, E2F6 and PCGF6 colocalize in 293 cells. (A) Schematic representation of PRC1.6 core components. (B) Western blot analysis of MGA, L3MBTL2, E2F6 and PCGF6 expression in wild type 293 cells (wt) and in corresponding MGA-, L3MBTL2-, E2F6- and PCGF6-depleted cell clones (MGA ko , L3MBTL2 ko , E2F6 ko and PCGF6 ko ). Re-probing for tubulin (TUB) controlled loading of extracts. (C) Venn diagrams showing the overlap of MGA, L3MBTL2, E2F6 and PCGF6 binding regions in HEK293 cells. The total number of high-confidence MGA, L3MBTL2, E2F6 and PCGF6 ChIP-seq peaks (≥30 tags, ≥3-fold enrichment over knockout control) and their overlap is shown. (D) A heat map view of the distribution of union MGA, L3MBTL2, E2F6 and PCGF6 peaks in HEK293 cells (n = 8342) at +/- 2 kb regions centred over the MGA peaks. (E) Representative genome browser screenshots of a 0.7 Mb region of chromosome 19 showing co-localization of MGA, L3MBTL2, E2F6 and PCGF6 at the CTC-232P5 . 1 , RFX2 , MLLT1 and KHSRP promoters. (F) Distribution of MGA, L3MBTL2, E2F6 and PCGF6 peaks relative to positions -2000 bp upstream to +2000 bp downstream of gene bodies. TSS, transcription start site; TES, transcription end site. (G) ChIP-qPCR analysis of MGA, L3MBTL2, E2F6 and PCGF6 binding to selected promoters. The region -2 kb upstream of the CDC7 promoter served as a negative control. Percent of input values represent the mean of at least three independent experiments +/- SD. (H) Sequence motifs enriched in PRC1.6 binding regions. Logos were obtained by running MEME-ChIP with 300 bp summits of the top 600 union MGA-L3MBTL2-E2F6-PCGF6 ChIP-seq peaks. The numbers next to the logos indicate the occurrence of the motifs, the statistical significance (E-value) and the transcription factors that bind to the motif. Right panel, local motif enrichment analysis (CentriMo) showing central enrichment of the MGA/MAX bHLH and the E2F6/DP1 binding motifs within the 300 bp peak regions. The NRF1 binding motif was not centrally enriched.

    Techniques Used: Western Blot, Expressing, Clone Assay, Binding Assay, Chromatin Immunoprecipitation, Knock-Out, Real-time Polymerase Chain Reaction, Negative Control, Sequencing

    22) Product Images from "Targeting Multiple Effector Pathways in Pancreatic Ductal Adenocarcinoma with a G-Quadruplex-Binding Small Molecule"

    Article Title: Targeting Multiple Effector Pathways in Pancreatic Ductal Adenocarcinoma with a G-Quadruplex-Binding Small Molecule

    Journal: Journal of Medicinal Chemistry

    doi: 10.1021/acs.jmedchem.7b01781

    CM03 treatment induces DNA damage and increases the presence of nuclear G4. (a) Following 6 and 24 h treatment with 400 nM CM03, PANC-1 cells were fixed with paraformaldehyde and stained with antibodies against G4s (BG4, green) and the DNA damage marker 53BP1 (red). Z -Stack images (11 μm × 0.3 μm spacing) were captured using a Nikon wide-field microscope and deconvolved using Hugyens Professional software. The central slice of the Z -stack is shown in the representative images. White scale bar is 10 μm. Zoom panel represents increased magnification of the highlighted section of the nucleus (dotted square) in the Merge panel. Yellow scale bar is 2.5 μm. Detection and co-localization between BG4 and 53BP1 foci (white arrows) were performed using a custom protocol in the ICY software, which utilizes the spot detector and co-localization studio plugins with the wavelet and Ripley’s K functions (see Methods ). (b–e) The graphs show the mean number of (b) BG4 foci, (c) 53BP1 foci, (d) BG4-53BP1 co-localizations, or (e) percentage of total BG4 co-localizing with 53BP1 per nucleus with the standard deviation from three biological replicates. A two-tailed Student’s t test was used to determine statistical significance across each condition ( p
    Figure Legend Snippet: CM03 treatment induces DNA damage and increases the presence of nuclear G4. (a) Following 6 and 24 h treatment with 400 nM CM03, PANC-1 cells were fixed with paraformaldehyde and stained with antibodies against G4s (BG4, green) and the DNA damage marker 53BP1 (red). Z -Stack images (11 μm × 0.3 μm spacing) were captured using a Nikon wide-field microscope and deconvolved using Hugyens Professional software. The central slice of the Z -stack is shown in the representative images. White scale bar is 10 μm. Zoom panel represents increased magnification of the highlighted section of the nucleus (dotted square) in the Merge panel. Yellow scale bar is 2.5 μm. Detection and co-localization between BG4 and 53BP1 foci (white arrows) were performed using a custom protocol in the ICY software, which utilizes the spot detector and co-localization studio plugins with the wavelet and Ripley’s K functions (see Methods ). (b–e) The graphs show the mean number of (b) BG4 foci, (c) 53BP1 foci, (d) BG4-53BP1 co-localizations, or (e) percentage of total BG4 co-localizing with 53BP1 per nucleus with the standard deviation from three biological replicates. A two-tailed Student’s t test was used to determine statistical significance across each condition ( p

    Techniques Used: Staining, Marker, Microscopy, Software, Standard Deviation, Two Tailed Test

    CM03 treatment reduces tumor volume in a MIA PaCa-2 xenograft model of PDAC. (a) Plot showing the tumor volume of MIA PaCa-2 xenografts treated with CM03, MM41, gemcitabine, or saline (control) over 62 days. There are eight CD-1 mice per condition and dosing for all cohorts was stopped on day 28, shown by the red arrow. Standard error of the mean (SEM) is indicated for all growth curves for each tumor volume. * p
    Figure Legend Snippet: CM03 treatment reduces tumor volume in a MIA PaCa-2 xenograft model of PDAC. (a) Plot showing the tumor volume of MIA PaCa-2 xenografts treated with CM03, MM41, gemcitabine, or saline (control) over 62 days. There are eight CD-1 mice per condition and dosing for all cohorts was stopped on day 28, shown by the red arrow. Standard error of the mean (SEM) is indicated for all growth curves for each tumor volume. * p

    Techniques Used: Mouse Assay

    Synthesis of Compound CM03 ( 4 ) (i) 5,5-Dimethyl-1,3-dibromohydantoin, H 2 SO 4 , 80° C, 72 h; (ii) 3-morpholinopropylamine, acetic acid, microwave, 130 °C, 25 min (The intermediate 3b was not isolated); (iii) amine, NMP, microwave, 125 °C, 30 min. For CM03, n = 2, R = pyrrolidino. The overall yield of the synthesis for steps i to iii (compound 4 : CM03) was 26%. The yield for steps i and ii (from compound 1 to compound 3a ) was 35%; the yield for final step (iii) (compound 3a to compound 4 (CM03)) was 75%.
    Figure Legend Snippet: Synthesis of Compound CM03 ( 4 ) (i) 5,5-Dimethyl-1,3-dibromohydantoin, H 2 SO 4 , 80° C, 72 h; (ii) 3-morpholinopropylamine, acetic acid, microwave, 130 °C, 25 min (The intermediate 3b was not isolated); (iii) amine, NMP, microwave, 125 °C, 30 min. For CM03, n = 2, R = pyrrolidino. The overall yield of the synthesis for steps i to iii (compound 4 : CM03) was 26%. The yield for steps i and ii (from compound 1 to compound 3a ) was 35%; the yield for final step (iii) (compound 3a to compound 4 (CM03)) was 75%.

    Techniques Used: Isolation

    Differentially down-regulated genes common to both PANC-1 and MIA PaCa-2 are enriched in PQs after treatment with 400 nM CM03. (a,b) MIA PaCa-2 and PANC-1 cells were treated with 400 nM CM03 for 6 and 24 h and mRNA extracted for analysis by RNA-Seq. Genes were split into four subgroups according to their fold change upon CM03 treatment versus untreated: Down (Log 2 FC
    Figure Legend Snippet: Differentially down-regulated genes common to both PANC-1 and MIA PaCa-2 are enriched in PQs after treatment with 400 nM CM03. (a,b) MIA PaCa-2 and PANC-1 cells were treated with 400 nM CM03 for 6 and 24 h and mRNA extracted for analysis by RNA-Seq. Genes were split into four subgroups according to their fold change upon CM03 treatment versus untreated: Down (Log 2 FC

    Techniques Used: RNA Sequencing Assay

    G4 modeling, FRET, and cell line growth inhibition studies of the trisubstituted naphthalene diimide derivatives MM41 and CM03. (a) MM41 structure and (b) molecular model of MM41 bound to a human telomeric G4, following docking and minimization and using the co-crystal structure (PDB 3UYH) 32a as a starting point. The red dotted circle highlights the fourth pyrrolidino side chain substituent of MM41, which is not buried in a G4 groove, by contrast with the other three side chains. (c) CM03 structure and (d) molecular model of CM03 bound to the native parallel human telomeric G4 structure (PDB 1KF1) 32b following docking and minimization. MM41 and CM03 are shown in ball-and-stick representation and the G4 in surface representation, with electrostatic interaction regions colored yellow. (e) Table showing melting temperature changes (Δ T m ) in °C, for CM03 and MM41 with a panel of G4s, determined using a FRET procedure performed in solution containing 50 mM K + ion. Ligands were used at 1 μM concentration. Standard deviations are from triplicate measurements. (f) Growth inhibition assays. Short-term 96 h IC 50 values (in nM) for the two compounds in cancer and normal fibroblast cell line panel. Average growth inhibition is shown with standard deviations from > 3 individual determinations.
    Figure Legend Snippet: G4 modeling, FRET, and cell line growth inhibition studies of the trisubstituted naphthalene diimide derivatives MM41 and CM03. (a) MM41 structure and (b) molecular model of MM41 bound to a human telomeric G4, following docking and minimization and using the co-crystal structure (PDB 3UYH) 32a as a starting point. The red dotted circle highlights the fourth pyrrolidino side chain substituent of MM41, which is not buried in a G4 groove, by contrast with the other three side chains. (c) CM03 structure and (d) molecular model of CM03 bound to the native parallel human telomeric G4 structure (PDB 1KF1) 32b following docking and minimization. MM41 and CM03 are shown in ball-and-stick representation and the G4 in surface representation, with electrostatic interaction regions colored yellow. (e) Table showing melting temperature changes (Δ T m ) in °C, for CM03 and MM41 with a panel of G4s, determined using a FRET procedure performed in solution containing 50 mM K + ion. Ligands were used at 1 μM concentration. Standard deviations are from triplicate measurements. (f) Growth inhibition assays. Short-term 96 h IC 50 values (in nM) for the two compounds in cancer and normal fibroblast cell line panel. Average growth inhibition is shown with standard deviations from > 3 individual determinations.

    Techniques Used: Inhibition, Concentration Assay

    Validation of mRNA down regulation by qRT-PCR for a subset of down-regulated genes, selected from RNA-Seq experiments. (a–d) MIA PaCa-2 and PANC-1 cells were treated (a and b) with 400 nM CM03 and (c and d) with 400 nM gemcitabine, all for 6 and 24 h. Total mRNA was extracted, reverse transcribed into cDNA, and then qRT-PCR was performed. The C t values were normalized to the genomic mean of three housekeeping genes ( ACTB , GAPDH , and TUBB ), and the relative gene expression was determined using the Livak method, 2 –ΔΔ C t . The log-fold expression changes (Log 2 FC) for each gene are shown relative to vehicle-treated controls (PBS for CM03 and DMSO for gemcitabine). Student’s t test along with 2 –Δ C t values were used to determine the statistical significance of the observed changes, which are the mean of in each case at least three determinations. Those genes with changes in expression with p
    Figure Legend Snippet: Validation of mRNA down regulation by qRT-PCR for a subset of down-regulated genes, selected from RNA-Seq experiments. (a–d) MIA PaCa-2 and PANC-1 cells were treated (a and b) with 400 nM CM03 and (c and d) with 400 nM gemcitabine, all for 6 and 24 h. Total mRNA was extracted, reverse transcribed into cDNA, and then qRT-PCR was performed. The C t values were normalized to the genomic mean of three housekeeping genes ( ACTB , GAPDH , and TUBB ), and the relative gene expression was determined using the Livak method, 2 –ΔΔ C t . The log-fold expression changes (Log 2 FC) for each gene are shown relative to vehicle-treated controls (PBS for CM03 and DMSO for gemcitabine). Student’s t test along with 2 –Δ C t values were used to determine the statistical significance of the observed changes, which are the mean of in each case at least three determinations. Those genes with changes in expression with p

    Techniques Used: Quantitative RT-PCR, RNA Sequencing Assay, Expressing

    23) Product Images from "Limitations of a Murine Transgenic Breast Cancer Model for Studies of Erythropoietin-Induced Tumor Progression 1"

    Article Title: Limitations of a Murine Transgenic Breast Cancer Model for Studies of Erythropoietin-Induced Tumor Progression 1

    Journal: Translational Oncology

    doi:

    EPO does not increase the rate of tumor formation in MMTV-PyMT-FVB mice. Heterozygous MMTV-PyMT-FVB mice without palpable tumors were randomly assigned to EPO ( n = 27) or PBS ( n = 27) when they reached 48 to 51 days old (week 0) continuing weekly for a total of six injections. The number of mice without visible mammary tumors was recorded each week.
    Figure Legend Snippet: EPO does not increase the rate of tumor formation in MMTV-PyMT-FVB mice. Heterozygous MMTV-PyMT-FVB mice without palpable tumors were randomly assigned to EPO ( n = 27) or PBS ( n = 27) when they reached 48 to 51 days old (week 0) continuing weekly for a total of six injections. The number of mice without visible mammary tumors was recorded each week.

    Techniques Used: Mouse Assay

    24) Product Images from "Lineage-specific enhancers activate self-renewal genes in macrophages and embryonic stem cells"

    Article Title: Lineage-specific enhancers activate self-renewal genes in macrophages and embryonic stem cells

    Journal: Science (New York, N.Y.)

    doi: 10.1126/science.aad5510

    The self-renewal gene network is activated in alveolar macrophages expressing naturally low levels of MafB and cMaf A) Expression of MafB and cMaf relative to HPRT1, measured by RT-QPCR, in short-term cultures of bone marrow macrophages (BMM), peritoneal macrophages (PM) and alveolar macrophages (AM). Data are representative of three independent experiments using biological replicates. B) Growth curve showing number of AM over time in liquid culture. Data are representative of two independent experiments. C) Number of colony forming units (CFU) counted at day 21 per 10 4 AM and PM plated in methocult medium after first plating, or after replating 10 4 cells washed out from first plating (2 nd plating), or second plating (3 rd plating). Data are representative of three independent experiments. D) Box plots showing average, inter-quartile and 5–95 percentile relative expression levels of all self-renewal genes in Maf-DKO, WT BMM and AM, measured by nano-fluidic real-time PCR on Fluidigm array. * p-value
    Figure Legend Snippet: The self-renewal gene network is activated in alveolar macrophages expressing naturally low levels of MafB and cMaf A) Expression of MafB and cMaf relative to HPRT1, measured by RT-QPCR, in short-term cultures of bone marrow macrophages (BMM), peritoneal macrophages (PM) and alveolar macrophages (AM). Data are representative of three independent experiments using biological replicates. B) Growth curve showing number of AM over time in liquid culture. Data are representative of two independent experiments. C) Number of colony forming units (CFU) counted at day 21 per 10 4 AM and PM plated in methocult medium after first plating, or after replating 10 4 cells washed out from first plating (2 nd plating), or second plating (3 rd plating). Data are representative of three independent experiments. D) Box plots showing average, inter-quartile and 5–95 percentile relative expression levels of all self-renewal genes in Maf-DKO, WT BMM and AM, measured by nano-fluidic real-time PCR on Fluidigm array. * p-value

    Techniques Used: Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    A subset of lineage specific enhancers are enriched for activation marks in self-renewing Maf-DKO macrophages A) Representative predicted enhancer regions (red shading) with greater enrichment for enhancer activation marks p300 and H3K27ac in Maf-DKO versus WT BMM (red boxes). B) Direct alignment of p300, H3K27ac and PU.1 ChIP-Seq signals for enhancer regions differentially enriched for p300 marks in Maf-DKO (7323, light grey) and WT BMM (305, dark grey), centered and ranked on p300 signal. C) Aggregation plots showing average ChIP-seq signals for PU.1, H3K27ac, p300, H3K4m1 and H3K4m3 marks in Maf-DKO and WT BMM for p300 regions specifically enriched in Maf-DKO macrophages (depicted light grey in (B)). For each protein target, Chip-seq analysis was performed on at least two biological replicates, and results were reproducible in all cases. D) Microarray gene expression ratios of Maf-DKO versus WT BMM 2h after M-CSF stimulation, for total genes (white) or genes associated with Maf-DKO-only enhancers (blue). The box extends from the first to the third quartile with the whiskers denoting 1.5 times the interquartile range. Data were derived from three biological replicates.
    Figure Legend Snippet: A subset of lineage specific enhancers are enriched for activation marks in self-renewing Maf-DKO macrophages A) Representative predicted enhancer regions (red shading) with greater enrichment for enhancer activation marks p300 and H3K27ac in Maf-DKO versus WT BMM (red boxes). B) Direct alignment of p300, H3K27ac and PU.1 ChIP-Seq signals for enhancer regions differentially enriched for p300 marks in Maf-DKO (7323, light grey) and WT BMM (305, dark grey), centered and ranked on p300 signal. C) Aggregation plots showing average ChIP-seq signals for PU.1, H3K27ac, p300, H3K4m1 and H3K4m3 marks in Maf-DKO and WT BMM for p300 regions specifically enriched in Maf-DKO macrophages (depicted light grey in (B)). For each protein target, Chip-seq analysis was performed on at least two biological replicates, and results were reproducible in all cases. D) Microarray gene expression ratios of Maf-DKO versus WT BMM 2h after M-CSF stimulation, for total genes (white) or genes associated with Maf-DKO-only enhancers (blue). The box extends from the first to the third quartile with the whiskers denoting 1.5 times the interquartile range. Data were derived from three biological replicates.

    Techniques Used: Activation Assay, Chromatin Immunoprecipitation, Microarray, Expressing, Derivative Assay

    Self-renewing Maf-DKO macrophages activate genes required for ES cell self-renewal A) ) for adult tissue stem cells and core embryonic stem cell modules (Broad Institute MSigDB M1999 and M7079). comparing the expression of genes associated with Maf-DKO-only associated enhancers in Maf-DKO versus WT BMM. NES, Normalized enrichment score; FDR, false-discovery rate. B) ). C) Gene expression by quantitative real-time PCR of self-renewal genes in Maf-DKO and WT BMM stimulated with M-CSF for the indicated times. Heatmap shows the average signal of technical replicates. Data are representative of four independent experiments.
    Figure Legend Snippet: Self-renewing Maf-DKO macrophages activate genes required for ES cell self-renewal A) ) for adult tissue stem cells and core embryonic stem cell modules (Broad Institute MSigDB M1999 and M7079). comparing the expression of genes associated with Maf-DKO-only associated enhancers in Maf-DKO versus WT BMM. NES, Normalized enrichment score; FDR, false-discovery rate. B) ). C) Gene expression by quantitative real-time PCR of self-renewal genes in Maf-DKO and WT BMM stimulated with M-CSF for the indicated times. Heatmap shows the average signal of technical replicates. Data are representative of four independent experiments.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Self-renewal genes are organized in a network and functionally important for Maf-DKO macrophage proliferation A) Gene expression analysis of Maf-DKO macrophages uninfected or infected with shLacZ control or shRNAs targeting self-renewal genes (rows) for self-renewal genes associated with Maf-DKO macrophage activated enhancers, not associated self-renewal genes (SR), house keeping (C) and macrophage specific (myeloid) genes (columns) using quadruplicate nano-fluidic real-time PCR on Fluidigm array. For each gene, the heatmap presents normalized values as percent change over average expression in non-infected and control lacZ shRNA infected cell samples. Data are representative of three independent experiments. B) A network model using an FDR-approach showing significant repression of an output target gene resulting from shRNA knockdown of a regulator gene, with darker lines denoting regulation in all replicates, arrows denoting repression and blue bars activation by shRNA. Circle size is a function of the number of times the target is affected by knockdown of other regulators. C) Number of colony forming units (CFU) obtained from equal numbers of Maf-DKO macrophages infected with shRNAs against control (shLacZ) or self-renewal gene targets after 12 days of culture in methocult medium containing M-CSF. The mean number of CFU for self-renewal gene shRNA infected populations is significantly different from the mean number of CFU for controls (One-way analysis of variance, P
    Figure Legend Snippet: Self-renewal genes are organized in a network and functionally important for Maf-DKO macrophage proliferation A) Gene expression analysis of Maf-DKO macrophages uninfected or infected with shLacZ control or shRNAs targeting self-renewal genes (rows) for self-renewal genes associated with Maf-DKO macrophage activated enhancers, not associated self-renewal genes (SR), house keeping (C) and macrophage specific (myeloid) genes (columns) using quadruplicate nano-fluidic real-time PCR on Fluidigm array. For each gene, the heatmap presents normalized values as percent change over average expression in non-infected and control lacZ shRNA infected cell samples. Data are representative of three independent experiments. B) A network model using an FDR-approach showing significant repression of an output target gene resulting from shRNA knockdown of a regulator gene, with darker lines denoting regulation in all replicates, arrows denoting repression and blue bars activation by shRNA. Circle size is a function of the number of times the target is affected by knockdown of other regulators. C) Number of colony forming units (CFU) obtained from equal numbers of Maf-DKO macrophages infected with shRNAs against control (shLacZ) or self-renewal gene targets after 12 days of culture in methocult medium containing M-CSF. The mean number of CFU for self-renewal gene shRNA infected populations is significantly different from the mean number of CFU for controls (One-way analysis of variance, P

    Techniques Used: Expressing, Infection, Real-time Polymerase Chain Reaction, shRNA, Activation Assay

    Self-renewal genes are associated with distinct enhancers in ES cells and macrophages A) Genomic regions surrounding MYC, KLF2 and KLF4 genes showing distinct ES cell (blue) and macrophage (red) specific predicted enhancer regions with differential H3K27ac and p300 enrichment in Maf-DKO over WT BMM (red boxes). B) Heatmaps and k-means clustering (k=2) of Chip-seq signal of all H3K27ac+ regions associated with self-renewal genes in ES cells, Maf-DKO and WT BMM, including both differentially regulated Maf-DKO-only and non-differentially regulated regions. Corresponding regions are shown for p300, H3K4m1 and PU.1 (ES, Maf-DKO and WT BMM). C) Model based on panels A) and B) to describe tissue-specific macrophage and ES cell enhancer platforms associated with individual self-renewal genes.
    Figure Legend Snippet: Self-renewal genes are associated with distinct enhancers in ES cells and macrophages A) Genomic regions surrounding MYC, KLF2 and KLF4 genes showing distinct ES cell (blue) and macrophage (red) specific predicted enhancer regions with differential H3K27ac and p300 enrichment in Maf-DKO over WT BMM (red boxes). B) Heatmaps and k-means clustering (k=2) of Chip-seq signal of all H3K27ac+ regions associated with self-renewal genes in ES cells, Maf-DKO and WT BMM, including both differentially regulated Maf-DKO-only and non-differentially regulated regions. Corresponding regions are shown for p300, H3K4m1 and PU.1 (ES, Maf-DKO and WT BMM). C) Model based on panels A) and B) to describe tissue-specific macrophage and ES cell enhancer platforms associated with individual self-renewal genes.

    Techniques Used: Chromatin Immunoprecipitation

    MafB inhibits macrophage self-renewal by direct repression of self-renewal gene enhancers A) Colony assays for Maf-DKO macrophages expressing empty vector (-MafB) or a doxycycline-inducible flag-tagged MafB allele (+MafB) counted after 14 days of culture in methocult medium containing M-CSF and doxycycline (DOX), showing culture dishes (0.63x), and number of colony-forming units (CFU). Error bars represent the SD of two technical replicates and data are representative of three independent experiments. B) Expression of self-renewal genes in Maf-DKO macrophages (-MafB) and Maf-DKO macrophages expressing a doxycycline-inducible, flag-tagged MafB allele (+MafB) after 2 hours stimulation with M-CSF determined by nano-fluidic real-time PCR on Fluidigm array. Data are representative of three independent experiments. C) Aggregation plots showing average ChIP-seq signals for P300 and H3K27ac in Maf-DKO, WT BMM and Maf-DKO macrophages expressing a doxycycline-inducible, flag-tagged MafB allele in the presence of doxycycline (Maf-DKO+MafB) for the self-renewal associated enhancers regions (total=88 regions). D) Direct alignment of regions proximal to Maf-DKO-only enhancers for flag-MafB binding in Maf-DKO+MafB and corresponding regions in Maf-DKO and Maf-DKO+MafB macrophages for P300 and H3K27ac binding. E) Histogram showing the percent of WT BMM-only, Maf-DKO-only and self-renewal gene-associated enhancers bound by MafB as determined by ChIP-seq for flag-MafB in Maf-DKO+MafB cells. F) Genomic regions surrounding MYC gene with Chip-seq tracks as labelled.
    Figure Legend Snippet: MafB inhibits macrophage self-renewal by direct repression of self-renewal gene enhancers A) Colony assays for Maf-DKO macrophages expressing empty vector (-MafB) or a doxycycline-inducible flag-tagged MafB allele (+MafB) counted after 14 days of culture in methocult medium containing M-CSF and doxycycline (DOX), showing culture dishes (0.63x), and number of colony-forming units (CFU). Error bars represent the SD of two technical replicates and data are representative of three independent experiments. B) Expression of self-renewal genes in Maf-DKO macrophages (-MafB) and Maf-DKO macrophages expressing a doxycycline-inducible, flag-tagged MafB allele (+MafB) after 2 hours stimulation with M-CSF determined by nano-fluidic real-time PCR on Fluidigm array. Data are representative of three independent experiments. C) Aggregation plots showing average ChIP-seq signals for P300 and H3K27ac in Maf-DKO, WT BMM and Maf-DKO macrophages expressing a doxycycline-inducible, flag-tagged MafB allele in the presence of doxycycline (Maf-DKO+MafB) for the self-renewal associated enhancers regions (total=88 regions). D) Direct alignment of regions proximal to Maf-DKO-only enhancers for flag-MafB binding in Maf-DKO+MafB and corresponding regions in Maf-DKO and Maf-DKO+MafB macrophages for P300 and H3K27ac binding. E) Histogram showing the percent of WT BMM-only, Maf-DKO-only and self-renewal gene-associated enhancers bound by MafB as determined by ChIP-seq for flag-MafB in Maf-DKO+MafB cells. F) Genomic regions surrounding MYC gene with Chip-seq tracks as labelled.

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

    25) Product Images from "Pseudoexfoliation syndrome-associated genetic variants affect transcription factor binding and alternative splicing of LOXL1"

    Article Title: Pseudoexfoliation syndrome-associated genetic variants affect transcription factor binding and alternative splicing of LOXL1

    Journal: Nature Communications

    doi: 10.1038/ncomms15466

    Effects of risk variants on LOXL1 transcriptional activity in vivo. ( a ) Scatter plot of TaqMan-based allelic discrimination of the LOXL1 SNP 13 (rs12441130). The genotypes of hTCF homoyzgous for the risk (C) or non-risk (T) alleles are shown in relation to genomic DNA and pre-mRNA containing cDNA of heterozygous hTCF cell lines ( n =15). Relative abundance of risk allele C over non-risk allele T in heterozygous hTCF cell lines ( n =15); expression level of T allele was set at 100%. ( b ) ChIP assay for RNA polymerase II (Pol II) binding at rs12441130 (SNP13)-containing region of LOXL1 in heterozygous hTCF cell lines ( n =2) using antibodies against Pol II, histone H3 and acetylated histone H3K27Ac (positive controls), and non-immune IgG (negative control); input represents total chromatin applied for immunoprecipitation. Allele-specific ChIP-qPCR analysis for Pol II chromatin binding and histone H3 is shown (left); expression levels of the non-risk allele T were set at 100%. DNA isolated from immunoprecipitated complexes was analysed on 2% agarose gel (top right) and by qPCR (bottom right) with primers specific for the SNP13 region producing a 121 bp PCR fragment (arrow). Data are expressed as per cent of input (Lane 1: hTCF 1, lane 2: hTCF 2, lane M: DNA marker, lane N: primer control without chromatin).
    Figure Legend Snippet: Effects of risk variants on LOXL1 transcriptional activity in vivo. ( a ) Scatter plot of TaqMan-based allelic discrimination of the LOXL1 SNP 13 (rs12441130). The genotypes of hTCF homoyzgous for the risk (C) or non-risk (T) alleles are shown in relation to genomic DNA and pre-mRNA containing cDNA of heterozygous hTCF cell lines ( n =15). Relative abundance of risk allele C over non-risk allele T in heterozygous hTCF cell lines ( n =15); expression level of T allele was set at 100%. ( b ) ChIP assay for RNA polymerase II (Pol II) binding at rs12441130 (SNP13)-containing region of LOXL1 in heterozygous hTCF cell lines ( n =2) using antibodies against Pol II, histone H3 and acetylated histone H3K27Ac (positive controls), and non-immune IgG (negative control); input represents total chromatin applied for immunoprecipitation. Allele-specific ChIP-qPCR analysis for Pol II chromatin binding and histone H3 is shown (left); expression levels of the non-risk allele T were set at 100%. DNA isolated from immunoprecipitated complexes was analysed on 2% agarose gel (top right) and by qPCR (bottom right) with primers specific for the SNP13 region producing a 121 bp PCR fragment (arrow). Data are expressed as per cent of input (Lane 1: hTCF 1, lane 2: hTCF 2, lane M: DNA marker, lane N: primer control without chromatin).

    Techniques Used: Activity Assay, In Vivo, Expressing, Chromatin Immunoprecipitation, Binding Assay, Negative Control, Immunoprecipitation, Real-time Polymerase Chain Reaction, Isolation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Marker

    26) Product Images from "Activation of the aryl hydrocarbon receptor by a component of cigarette smoke reduces germ cell proliferation in the human fetal ovary"

    Article Title: Activation of the aryl hydrocarbon receptor by a component of cigarette smoke reduces germ cell proliferation in the human fetal ovary

    Journal: Molecular Human Reproduction

    doi: 10.1093/molehr/gat059

    Expression of the aryl hydrocarbon receptor AHR ( A ) increases with gestation ( P = 0.008), but ARNT (aryl hydrocarbon translocator, an AhR co-factor) ( B ) was unchanged ( n = 5–6 ovaries per group).
    Figure Legend Snippet: Expression of the aryl hydrocarbon receptor AHR ( A ) increases with gestation ( P = 0.008), but ARNT (aryl hydrocarbon translocator, an AhR co-factor) ( B ) was unchanged ( n = 5–6 ovaries per group).

    Techniques Used: Expressing

    27) Product Images from "The lncRNA Firre anchors the inactive X chromosome to the nucleolus by binding CTCF and maintains H3K27me3 methylation"

    Article Title: The lncRNA Firre anchors the inactive X chromosome to the nucleolus by binding CTCF and maintains H3K27me3 methylation

    Journal: Genome Biology

    doi: 10.1186/s13059-015-0618-0

    CTCF binding and expression change during male and female development. (A) CTCF occupancy at Firre in mouse female ES cells PGK12.1 before (FES D0) and after 15-day differentiation (FES D15) and in female 12.5 dpc embryos (FE) is compared to occupancy in male mouse ES cells WD44 before (MES D0) and after 15-day differentiation (MES D15) and in male 12.5 dpc embryos (ME). ChIP-chip data are shown as log 2 ChIP/input. (B) Box plots of CTCF occupancy (log 2 ChIP/input) at Firre in male (MD0, MD15) and female (FD0, FD15) ES cells, 12.5 dpc embryos (ME, FE), and adult livers (ML, FL). Same data as in (A) and Figure 1 B. (C) Firre expression level is significantly higher in female than in male ES cells (undifferentiated, D0, differentiated, D15) and liver (box insert). Note that expression is much higher in ES cells than in liver. Expression in male ES cells (D0) is set as 1. Average expression levels were measured by qRT-PCR of biological replicates and normalized to 18S RNA. P values were determined by one sample t-test. Error bars, s.e.m. (D, E) Same analysis as in (A, C) for Dxz4 .
    Figure Legend Snippet: CTCF binding and expression change during male and female development. (A) CTCF occupancy at Firre in mouse female ES cells PGK12.1 before (FES D0) and after 15-day differentiation (FES D15) and in female 12.5 dpc embryos (FE) is compared to occupancy in male mouse ES cells WD44 before (MES D0) and after 15-day differentiation (MES D15) and in male 12.5 dpc embryos (ME). ChIP-chip data are shown as log 2 ChIP/input. (B) Box plots of CTCF occupancy (log 2 ChIP/input) at Firre in male (MD0, MD15) and female (FD0, FD15) ES cells, 12.5 dpc embryos (ME, FE), and adult livers (ML, FL). Same data as in (A) and Figure 1 B. (C) Firre expression level is significantly higher in female than in male ES cells (undifferentiated, D0, differentiated, D15) and liver (box insert). Note that expression is much higher in ES cells than in liver. Expression in male ES cells (D0) is set as 1. Average expression levels were measured by qRT-PCR of biological replicates and normalized to 18S RNA. P values were determined by one sample t-test. Error bars, s.e.m. (D, E) Same analysis as in (A, C) for Dxz4 .

    Techniques Used: Binding Assay, Expressing, Chromatin Immunoprecipitation, Quantitative RT-PCR

    28) Product Images from "ZBTB48 is both a vertebrate telomere‐binding protein and a transcriptional activator"

    Article Title: ZBTB48 is both a vertebrate telomere‐binding protein and a transcriptional activator

    Journal: EMBO Reports

    doi: 10.15252/embr.201744095

    ZBTB48 is a direct telomere‐binding protein but TRAP activity is not affected in ZBTB48 KO clones TTAGGG content of telomeric reads in ChIPseq samples from U2OS WT, U2OS ZBTB48 KO and U2OS HOT1 KO clones using IgG, TRF2, ZBTB48 Atlas, ZBTB48 GeneTex and HOT1 antibodies as well as FLAG antibody for FLAG‐ZBTB48 WT and point mutants for ZnF10 and ZnF11. The percentage contribution to all reads containing 1–8× TTAGGG repeats is shown. All reactions were performed in technical replicates on two independent WT and KO clones each. These reactions are biological replicates of Fig 2 A, performed with independent U2OS ZBTB48 WT and KO clones for ZBTB48 and with a second HOT1 antibody for HOT1. Telomerase activity was determined based on a quantitative TRAP assay. Heat‐inactivated HeLa extracts were used as a threshold to determine non‐specific background signal. Differences in C t values (∆∆ C t ) from the quantitative PCR measurements are displayed relative to the heat‐inactivation control. Error bars represent standard deviations ( n = 3). The P ‐value is based on Student's t ‐test.
    Figure Legend Snippet: ZBTB48 is a direct telomere‐binding protein but TRAP activity is not affected in ZBTB48 KO clones TTAGGG content of telomeric reads in ChIPseq samples from U2OS WT, U2OS ZBTB48 KO and U2OS HOT1 KO clones using IgG, TRF2, ZBTB48 Atlas, ZBTB48 GeneTex and HOT1 antibodies as well as FLAG antibody for FLAG‐ZBTB48 WT and point mutants for ZnF10 and ZnF11. The percentage contribution to all reads containing 1–8× TTAGGG repeats is shown. All reactions were performed in technical replicates on two independent WT and KO clones each. These reactions are biological replicates of Fig 2 A, performed with independent U2OS ZBTB48 WT and KO clones for ZBTB48 and with a second HOT1 antibody for HOT1. Telomerase activity was determined based on a quantitative TRAP assay. Heat‐inactivated HeLa extracts were used as a threshold to determine non‐specific background signal. Differences in C t values (∆∆ C t ) from the quantitative PCR measurements are displayed relative to the heat‐inactivation control. Error bars represent standard deviations ( n = 3). The P ‐value is based on Student's t ‐test.

    Techniques Used: Binding Assay, Activity Assay, Clone Assay, TRAP Assay, Real-time Polymerase Chain Reaction

    29) Product Images from "Limitations of a Murine Transgenic Breast Cancer Model for Studies of Erythropoietin-Induced Tumor Progression 1"

    Article Title: Limitations of a Murine Transgenic Breast Cancer Model for Studies of Erythropoietin-Induced Tumor Progression 1

    Journal: Translational Oncology

    doi:

    EPO does not increase the volume of subcutaneous B16F10 melanoma tumors. Mice were inoculated on day 0 with B16F10 cells, and EPO or PBS was administered at the time of tumor cell implantation continuing weekly for a total of two injections (Exp. 1) or three injections (Exp. 2–4). The tumor volumes at days 10 and 14 (Exp. 1) or at days 10, 14, 17, and 20 (Exp. 2–4) are plotted relative to the maximal tumor volume observed in each experiment. *Mice removed from the study before the end point owing to excessive tumor growth. † Mouse that died.
    Figure Legend Snippet: EPO does not increase the volume of subcutaneous B16F10 melanoma tumors. Mice were inoculated on day 0 with B16F10 cells, and EPO or PBS was administered at the time of tumor cell implantation continuing weekly for a total of two injections (Exp. 1) or three injections (Exp. 2–4). The tumor volumes at days 10 and 14 (Exp. 1) or at days 10, 14, 17, and 20 (Exp. 2–4) are plotted relative to the maximal tumor volume observed in each experiment. *Mice removed from the study before the end point owing to excessive tumor growth. † Mouse that died.

    Techniques Used: Mouse Assay

    30) Product Images from "MGA, L3MBTL2 and E2F6 determine genomic binding of the non-canonical Polycomb repressive complex PRC1.6"

    Article Title: MGA, L3MBTL2 and E2F6 determine genomic binding of the non-canonical Polycomb repressive complex PRC1.6

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007193

    The role of PRC1.6 in HEK293 cell function. (A) Reduced proliferation of MGA ko , L3MBTL2 ko and E2F6 ko cells. Shown are growth curves of wildtype, MGA ko , L3MBTL2 ko , E2F6 ko and PCGF6 ko HEK293 cells. Cells were seed at 3x10 5 , and counted and replated at the indicated time points. Cumulative cell numbers were calculated by multiplying the initial cell number with the fold-increase in cell numbers in each interval. (B) Venn diagrams illustrating the overlap of MGA-bound genes and genes down- or up-regulated in MGA ko cells. Left circle, genes with ≥2-fold reduced transcript levels in MGA ko cells; right circle, genes with ≥2-fold increased transcript levels in MGA ko cells. (C) Representative genome browser screenshots of ChIP-seq and RNA-seq tracks illustrating binding of MGA, L3MBTL2, E2F6 and PCGF6 (top tracks) to the CNTD1 and SMC1B promoters, and RNA expression (bottom tracks) of the corresponding genes in three wild type samples (MGA_wt1, MGA_wt2 and MGA_wt3), and in three different MGA ko cell clones (MGA ko _cl26, MGA ko _cl27 and MGA ko _cl30). (D) RT-qPCR-based analysis of expression changes of selected genes in MGA ko , E2F6 ko , L3MBTL2 ko and PCGF6 ko cells. Transcript levels were normalized to B2M transcript levels, and are depicted relative to transcript levels in wild type cells.
    Figure Legend Snippet: The role of PRC1.6 in HEK293 cell function. (A) Reduced proliferation of MGA ko , L3MBTL2 ko and E2F6 ko cells. Shown are growth curves of wildtype, MGA ko , L3MBTL2 ko , E2F6 ko and PCGF6 ko HEK293 cells. Cells were seed at 3x10 5 , and counted and replated at the indicated time points. Cumulative cell numbers were calculated by multiplying the initial cell number with the fold-increase in cell numbers in each interval. (B) Venn diagrams illustrating the overlap of MGA-bound genes and genes down- or up-regulated in MGA ko cells. Left circle, genes with ≥2-fold reduced transcript levels in MGA ko cells; right circle, genes with ≥2-fold increased transcript levels in MGA ko cells. (C) Representative genome browser screenshots of ChIP-seq and RNA-seq tracks illustrating binding of MGA, L3MBTL2, E2F6 and PCGF6 (top tracks) to the CNTD1 and SMC1B promoters, and RNA expression (bottom tracks) of the corresponding genes in three wild type samples (MGA_wt1, MGA_wt2 and MGA_wt3), and in three different MGA ko cell clones (MGA ko _cl26, MGA ko _cl27 and MGA ko _cl30). (D) RT-qPCR-based analysis of expression changes of selected genes in MGA ko , E2F6 ko , L3MBTL2 ko and PCGF6 ko cells. Transcript levels were normalized to B2M transcript levels, and are depicted relative to transcript levels in wild type cells.

    Techniques Used: Cell Function Assay, Chromatin Immunoprecipitation, RNA Sequencing Assay, Binding Assay, RNA Expression, Clone Assay, Quantitative RT-PCR, Expressing

    31) Product Images from "Targeting Multiple Effector Pathways in Pancreatic Ductal Adenocarcinoma with a G-Quadruplex-Binding Small Molecule"

    Article Title: Targeting Multiple Effector Pathways in Pancreatic Ductal Adenocarcinoma with a G-Quadruplex-Binding Small Molecule

    Journal: Journal of Medicinal Chemistry

    doi: 10.1021/acs.jmedchem.7b01781

    CM03 treatment reduces tumor volume in a MIA PaCa-2 xenograft model of PDAC. (a) Plot showing the tumor volume of MIA PaCa-2 xenografts treated with CM03, MM41, gemcitabine, or saline (control) over 62 days. There are eight CD-1 mice per condition and dosing for all cohorts was stopped on day 28, shown by the red arrow. Standard error of the mean (SEM) is indicated for all growth curves for each tumor volume. * p
    Figure Legend Snippet: CM03 treatment reduces tumor volume in a MIA PaCa-2 xenograft model of PDAC. (a) Plot showing the tumor volume of MIA PaCa-2 xenografts treated with CM03, MM41, gemcitabine, or saline (control) over 62 days. There are eight CD-1 mice per condition and dosing for all cohorts was stopped on day 28, shown by the red arrow. Standard error of the mean (SEM) is indicated for all growth curves for each tumor volume. * p

    Techniques Used: Mouse Assay

    32) Product Images from "Orientation-dependent Dxz4 contacts shape the 3D structure of the inactive X chromosome"

    Article Title: Orientation-dependent Dxz4 contacts shape the 3D structure of the inactive X chromosome

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03694-y

    Epigenetic features of the Xi in Dxz4 -edited cell lines. a Percentage of nuclei (`200 nuclei scored in each line) with 1 (light blue), 2 (orange), 3 (dark blue), or 4 (purple) X signals after DNA-FISH using an X-paint in WT, Patski2-4, Del-hinge, and Inv-Dxz4 cells. b Example of a z -stack image of a Del-hinge nucleus after H3K27me3 immunostaining (red), DNA-FISH using an X-paint (green), and Hoechst 33342 staining (blue). The Xa and Xi volumes were called based on the X-paint signals (lines show the microscope grid), using H3K27me immunostaining to identify the Xi. A 10 µm scale is shown. c – e Box plots of fluorescence intensities for the X-paint, Hoechst 33342, and H3K27me3 in wild-type Patski2-4 (blue), Del-hinge (red), and Inv-Dxz4 (gray) cells. No difference was detected between these lines by Wilcoxon rank sum test. Note that the Xi shows higher intensity of staining compared to the Xa. The boxes demarcate the interquartile range (IQR) with median. Whiskers are ±1.5 times the IQR. Outliers plotted as individual points. f Box plots of the Xi volume normalized to the Xa volume show a slight but significant (by Wilcoxon rank sum test) increase of the median value in the Xi volume in Del-hinge (7%; p -value = 0.01) and in Inv-Dxz4 (6%; p -value = 0.03). Boxes as described in c – e . g Left, examples of nuclei from WT, Patski2-4, Del-hinge, and Inv-Dxz4 cells after RNA-FISH for Xist (green; 10 µm scale shown). Right, bar plots of the percentage of nuclei (`200 nuclei scored in each line) with 0 (magenta), 1 (green), or 2 (yellow/green) Xist RNA clouds. The majority of nuclei have one Xist for Xist expression analyses. h Left, examples of nuclei stained with Hoechst 33342 (blue) and immunostained for H3K27me3 (red) to locate the Xi, and for nucleophosmin (green) to locate the nucleolus, show the Xi located either at the periphery (i), near the nucleolus (ii), sandwiched between periphery and nucleolus (iii), or at neither of these locations (iv). A 10 µm scale is shown. Right, the percentage of nuclei with the Xi near the periphery, the nucleolus, and at neither of these locations does not significantly differ between WT (blue), Del-hinge (red), and Inv-Dxz4 (gray) by Fisher’s exact test. A total of ~200 nuclei were scored per cell type by at least two different observers
    Figure Legend Snippet: Epigenetic features of the Xi in Dxz4 -edited cell lines. a Percentage of nuclei (`200 nuclei scored in each line) with 1 (light blue), 2 (orange), 3 (dark blue), or 4 (purple) X signals after DNA-FISH using an X-paint in WT, Patski2-4, Del-hinge, and Inv-Dxz4 cells. b Example of a z -stack image of a Del-hinge nucleus after H3K27me3 immunostaining (red), DNA-FISH using an X-paint (green), and Hoechst 33342 staining (blue). The Xa and Xi volumes were called based on the X-paint signals (lines show the microscope grid), using H3K27me immunostaining to identify the Xi. A 10 µm scale is shown. c – e Box plots of fluorescence intensities for the X-paint, Hoechst 33342, and H3K27me3 in wild-type Patski2-4 (blue), Del-hinge (red), and Inv-Dxz4 (gray) cells. No difference was detected between these lines by Wilcoxon rank sum test. Note that the Xi shows higher intensity of staining compared to the Xa. The boxes demarcate the interquartile range (IQR) with median. Whiskers are ±1.5 times the IQR. Outliers plotted as individual points. f Box plots of the Xi volume normalized to the Xa volume show a slight but significant (by Wilcoxon rank sum test) increase of the median value in the Xi volume in Del-hinge (7%; p -value = 0.01) and in Inv-Dxz4 (6%; p -value = 0.03). Boxes as described in c – e . g Left, examples of nuclei from WT, Patski2-4, Del-hinge, and Inv-Dxz4 cells after RNA-FISH for Xist (green; 10 µm scale shown). Right, bar plots of the percentage of nuclei (`200 nuclei scored in each line) with 0 (magenta), 1 (green), or 2 (yellow/green) Xist RNA clouds. The majority of nuclei have one Xist for Xist expression analyses. h Left, examples of nuclei stained with Hoechst 33342 (blue) and immunostained for H3K27me3 (red) to locate the Xi, and for nucleophosmin (green) to locate the nucleolus, show the Xi located either at the periphery (i), near the nucleolus (ii), sandwiched between periphery and nucleolus (iii), or at neither of these locations (iv). A 10 µm scale is shown. Right, the percentage of nuclei with the Xi near the periphery, the nucleolus, and at neither of these locations does not significantly differ between WT (blue), Del-hinge (red), and Inv-Dxz4 (gray) by Fisher’s exact test. A total of ~200 nuclei were scored per cell type by at least two different observers

    Techniques Used: Fluorescence In Situ Hybridization, Immunostaining, Staining, Microscopy, Fluorescence, Expressing

    33) Product Images from "Alternative splicing is a developmental switch for hTERT expression"

    Article Title: Alternative splicing is a developmental switch for hTERT expression

    Journal: bioRxiv

    doi: 10.1101/2020.04.02.022087

    SON is a key regulator of hTERT alternative splicing. A) Schematic of minigenes designed to measure the efficiency of hTERT mRNA splicing using Nano- and Firefly luciferase activity as a readout. Retention of exon-2 leads to expression of Firefly luciferase, but not Nano luciferase. Conversely, exclusion of exon 2 leads to a (−1) frameshift in exon-3 prompting the expression of Nano-luciferase while shifting Firefly luciferase out-of-frame. B) The minigenes were integrated into HeLa cells and a small-scale RNAi screen using a curated list of splicing factors and RNA-binding proteins was performed. Graph depicts the average ratio of Nano/Firefly luciferase of 3 biological replicates for 442 genes. Data presented as a log-ratio, colors highlight genes with p-value
    Figure Legend Snippet: SON is a key regulator of hTERT alternative splicing. A) Schematic of minigenes designed to measure the efficiency of hTERT mRNA splicing using Nano- and Firefly luciferase activity as a readout. Retention of exon-2 leads to expression of Firefly luciferase, but not Nano luciferase. Conversely, exclusion of exon 2 leads to a (−1) frameshift in exon-3 prompting the expression of Nano-luciferase while shifting Firefly luciferase out-of-frame. B) The minigenes were integrated into HeLa cells and a small-scale RNAi screen using a curated list of splicing factors and RNA-binding proteins was performed. Graph depicts the average ratio of Nano/Firefly luciferase of 3 biological replicates for 442 genes. Data presented as a log-ratio, colors highlight genes with p-value

    Techniques Used: Luciferase, Activity Assay, Expressing, RNA Binding Assay

    Inclusion of exon-2 correlates with the abundance of telomerase mRNA. A) Sashimi plot representing RNA Capture-Seq for hTERT locus in H7 ESCs and ARPE cells. Reads from exons are depicted as pileups and exon-exon junctions denoted with arcs. Asterisks indicate ΔEx2 splice variant transcripts detected in differentiated cells. B) Schematic of full-length and hTERT-ΔEx2 transcripts, demonstrating the position of the premature termination codons (PTC, asterisks) generated upon exon-2 skipping. Two tandem PTC’s would likely target ΔEx2 transcript for degradation by nonsense-mediated decay (NMD). C) Schematic illustration of junction-spanning PCR strategy used to assay hTERT ΔEx2 abundance by quantitative RT-PCR. RNA was reverse-transcribed using an hTERT gene-specific primer (GSP) and cDNA was purified and equalized between samples prior to PCR amplification with the indicated primers. D) Quantification of the ratio of hTERT ΔEx2 relative to full-length as determined by qRT-PCR in mortal cell lines (ARPE BJ), ARPE and BJ cell lines immortalized with hTERT cDNA, iPSCs derived from BJ cells, and human ESCs (n=3, p
    Figure Legend Snippet: Inclusion of exon-2 correlates with the abundance of telomerase mRNA. A) Sashimi plot representing RNA Capture-Seq for hTERT locus in H7 ESCs and ARPE cells. Reads from exons are depicted as pileups and exon-exon junctions denoted with arcs. Asterisks indicate ΔEx2 splice variant transcripts detected in differentiated cells. B) Schematic of full-length and hTERT-ΔEx2 transcripts, demonstrating the position of the premature termination codons (PTC, asterisks) generated upon exon-2 skipping. Two tandem PTC’s would likely target ΔEx2 transcript for degradation by nonsense-mediated decay (NMD). C) Schematic illustration of junction-spanning PCR strategy used to assay hTERT ΔEx2 abundance by quantitative RT-PCR. RNA was reverse-transcribed using an hTERT gene-specific primer (GSP) and cDNA was purified and equalized between samples prior to PCR amplification with the indicated primers. D) Quantification of the ratio of hTERT ΔEx2 relative to full-length as determined by qRT-PCR in mortal cell lines (ARPE BJ), ARPE and BJ cell lines immortalized with hTERT cDNA, iPSCs derived from BJ cells, and human ESCs (n=3, p

    Techniques Used: Variant Assay, Generated, Polymerase Chain Reaction, Quantitative RT-PCR, Purification, Amplification, Derivative Assay

    34) Product Images from "Orientation-dependent Dxz4 contacts shape the 3D structure of the inactive X chromosome"

    Article Title: Orientation-dependent Dxz4 contacts shape the 3D structure of the inactive X chromosome

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03694-y

    Model for the role of Dxz4 in unidirectional contacts with other loci on the Xi. Diagram of the Dxz4 locus (black) with adjacent centromeric (red) and telomeric (green) superdomains of the Xi. The orientation of CTCF-binding motifs is shown with black arrows, with 14 motifs being represented. Potential CTCF sites located in the telomeric superdomain are shown as green arrows, and on the centromeric domain as red arrows. In WT cells contacts between Dxz4 and loci telomeric to the locus would result in the formation of loops anchored at Dxz4 by the correct alignment of CTCF motifs, which would stall cohesin rings that continuously extrude loops (not depicted). Dxz4 would be pulled to the telomeric end of the hinge. After Dxz4 inversion contacts would shift to the centromeric superdomain, and be especially enhanced between Dxz4 and Firre . A new de-condensed hinge would form, with Dxz4 located at its centromeric end. Note that in WT and Inv-Dxz4, loops are depicted as anchored at each CTCF-binding site on Dxz4 . However, the process of loop formation is not static, but rather highly dynamic; thus, at a given time not all loops are likely to be anchored and larger or smaller loops may form.
    Figure Legend Snippet: Model for the role of Dxz4 in unidirectional contacts with other loci on the Xi. Diagram of the Dxz4 locus (black) with adjacent centromeric (red) and telomeric (green) superdomains of the Xi. The orientation of CTCF-binding motifs is shown with black arrows, with 14 motifs being represented. Potential CTCF sites located in the telomeric superdomain are shown as green arrows, and on the centromeric domain as red arrows. In WT cells contacts between Dxz4 and loci telomeric to the locus would result in the formation of loops anchored at Dxz4 by the correct alignment of CTCF motifs, which would stall cohesin rings that continuously extrude loops (not depicted). Dxz4 would be pulled to the telomeric end of the hinge. After Dxz4 inversion contacts would shift to the centromeric superdomain, and be especially enhanced between Dxz4 and Firre . A new de-condensed hinge would form, with Dxz4 located at its centromeric end. Note that in WT and Inv-Dxz4, loops are depicted as anchored at each CTCF-binding site on Dxz4 . However, the process of loop formation is not static, but rather highly dynamic; thus, at a given time not all loops are likely to be anchored and larger or smaller loops may form.

    Techniques Used: Binding Assay

    Local changes in TAD structure after Dxz4 deletion or inversion. a Comparisons of contacts on the Xa and Xi in WT* and Del-hinge/Dxz4 at individual loci. Changes in TAD configuration at 40 kb resolution are shown within 4 Mb regions, each centered at a specific gene, including two genes normally subject to XCI, Edar2 and Zfx , and a gene that escapes XCI, Ddx3x . b Scatter plots of WT* Xi, Del-hinge/Dxz4 Xi, Inv-Dxz4 Xi, and Del-hinge/Dxz4 Xa insulation scores at the TAD boundaries identified on WT* Xa. The dash red line shows the correlation between the samples as a linear fit, while the black line shows the expected fit for perfectly correlated data
    Figure Legend Snippet: Local changes in TAD structure after Dxz4 deletion or inversion. a Comparisons of contacts on the Xa and Xi in WT* and Del-hinge/Dxz4 at individual loci. Changes in TAD configuration at 40 kb resolution are shown within 4 Mb regions, each centered at a specific gene, including two genes normally subject to XCI, Edar2 and Zfx , and a gene that escapes XCI, Ddx3x . b Scatter plots of WT* Xi, Del-hinge/Dxz4 Xi, Inv-Dxz4 Xi, and Del-hinge/Dxz4 Xa insulation scores at the TAD boundaries identified on WT* Xa. The dash red line shows the correlation between the samples as a linear fit, while the black line shows the expected fit for perfectly correlated data

    Techniques Used:

    Epigenetic features of the Xi in Dxz4 -edited cell lines. a Percentage of nuclei (`200 nuclei scored in each line) with 1 (light blue), 2 (orange), 3 (dark blue), or 4 (purple) X signals after DNA-FISH using an X-paint in WT, Patski2-4, Del-hinge, and Inv-Dxz4 cells. b Example of a z -stack image of a Del-hinge nucleus after H3K27me3 immunostaining (red), DNA-FISH using an X-paint (green), and Hoechst 33342 staining (blue). The Xa and Xi volumes were called based on the X-paint signals (lines show the microscope grid), using H3K27me immunostaining to identify the Xi. A 10 µm scale is shown. c – e Box plots of fluorescence intensities for the X-paint, Hoechst 33342, and H3K27me3 in wild-type Patski2-4 (blue), Del-hinge (red), and Inv-Dxz4 (gray) cells. No difference was detected between these lines by Wilcoxon rank sum test. Note that the Xi shows higher intensity of staining compared to the Xa. The boxes demarcate the interquartile range (IQR) with median. Whiskers are ±1.5 times the IQR. Outliers plotted as individual points. f Box plots of the Xi volume normalized to the Xa volume show a slight but significant (by Wilcoxon rank sum test) increase of the median value in the Xi volume in Del-hinge (7%; p -value = 0.01) and in Inv-Dxz4 (6%; p -value = 0.03). Boxes as described in c – e . g Left, examples of nuclei from WT, Patski2-4, Del-hinge, and Inv-Dxz4 cells after RNA-FISH for Xist (green; 10 µm scale shown). Right, bar plots of the percentage of nuclei (`200 nuclei scored in each line) with 0 (magenta), 1 (green), or 2 (yellow/green) Xist RNA clouds. The majority of nuclei have one Xist for Xist expression analyses. h Left, examples of nuclei stained with Hoechst 33342 (blue) and immunostained for H3K27me3 (red) to locate the Xi, and for nucleophosmin (green) to locate the nucleolus, show the Xi located either at the periphery (i), near the nucleolus (ii), sandwiched between periphery and nucleolus (iii), or at neither of these locations (iv). A 10 µm scale is shown. Right, the percentage of nuclei with the Xi near the periphery, the nucleolus, and at neither of these locations does not significantly differ between WT (blue), Del-hinge (red), and Inv-Dxz4 (gray) by Fisher’s exact test. A total of ~200 nuclei were scored per cell type by at least two different observers
    Figure Legend Snippet: Epigenetic features of the Xi in Dxz4 -edited cell lines. a Percentage of nuclei (`200 nuclei scored in each line) with 1 (light blue), 2 (orange), 3 (dark blue), or 4 (purple) X signals after DNA-FISH using an X-paint in WT, Patski2-4, Del-hinge, and Inv-Dxz4 cells. b Example of a z -stack image of a Del-hinge nucleus after H3K27me3 immunostaining (red), DNA-FISH using an X-paint (green), and Hoechst 33342 staining (blue). The Xa and Xi volumes were called based on the X-paint signals (lines show the microscope grid), using H3K27me immunostaining to identify the Xi. A 10 µm scale is shown. c – e Box plots of fluorescence intensities for the X-paint, Hoechst 33342, and H3K27me3 in wild-type Patski2-4 (blue), Del-hinge (red), and Inv-Dxz4 (gray) cells. No difference was detected between these lines by Wilcoxon rank sum test. Note that the Xi shows higher intensity of staining compared to the Xa. The boxes demarcate the interquartile range (IQR) with median. Whiskers are ±1.5 times the IQR. Outliers plotted as individual points. f Box plots of the Xi volume normalized to the Xa volume show a slight but significant (by Wilcoxon rank sum test) increase of the median value in the Xi volume in Del-hinge (7%; p -value = 0.01) and in Inv-Dxz4 (6%; p -value = 0.03). Boxes as described in c – e . g Left, examples of nuclei from WT, Patski2-4, Del-hinge, and Inv-Dxz4 cells after RNA-FISH for Xist (green; 10 µm scale shown). Right, bar plots of the percentage of nuclei (`200 nuclei scored in each line) with 0 (magenta), 1 (green), or 2 (yellow/green) Xist RNA clouds. The majority of nuclei have one Xist for Xist expression analyses. h Left, examples of nuclei stained with Hoechst 33342 (blue) and immunostained for H3K27me3 (red) to locate the Xi, and for nucleophosmin (green) to locate the nucleolus, show the Xi located either at the periphery (i), near the nucleolus (ii), sandwiched between periphery and nucleolus (iii), or at neither of these locations (iv). A 10 µm scale is shown. Right, the percentage of nuclei with the Xi near the periphery, the nucleolus, and at neither of these locations does not significantly differ between WT (blue), Del-hinge (red), and Inv-Dxz4 (gray) by Fisher’s exact test. A total of ~200 nuclei were scored per cell type by at least two different observers

    Techniques Used: Fluorescence In Situ Hybridization, Immunostaining, Staining, Microscopy, Fluorescence, Expressing

    CTCF peak distribution on the Xi in Del-hinge and Inv-Dxz4. a Density histograms of the distribution of allelic proportions of CTCF peaks ( spretus /( spretus ). A shift in the distribution of allelic proportions due to an increase in CTCF binding on the Xi is evident for the X chromosome in Del-hinge and to a lesser extent Inv-Dxz4, compared to WT. b Percentages of CTCF peaks in WT (blue), Del-hinge (red), and Inv-Dxz4 along the autosomes and the X chromosomes classified as spretus -specific, BL6-specific, or common peaks. c Genome browser tracks of allelic CTCF ChIP-seq reads on the Xa and Xi, of CTCF peak d -scores ((Xa/(Xa + Xi) − 0.5), and of CTCF peaks assigned as Xa-specific, common, or Xi-specific for WT (blue), Del-hinge (red), and Inv-Dxz4 along a region of the X chromosome (pink background) that includes Zfx (a gene subject to XCI in WT, which reactivated in Del-hinge) and a region (green background) that includes Eif2s3x (a gene that escapes XCI). CTCF peaks that appear on the Xi in a region around Zfx
    Figure Legend Snippet: CTCF peak distribution on the Xi in Del-hinge and Inv-Dxz4. a Density histograms of the distribution of allelic proportions of CTCF peaks ( spretus /( spretus ). A shift in the distribution of allelic proportions due to an increase in CTCF binding on the Xi is evident for the X chromosome in Del-hinge and to a lesser extent Inv-Dxz4, compared to WT. b Percentages of CTCF peaks in WT (blue), Del-hinge (red), and Inv-Dxz4 along the autosomes and the X chromosomes classified as spretus -specific, BL6-specific, or common peaks. c Genome browser tracks of allelic CTCF ChIP-seq reads on the Xa and Xi, of CTCF peak d -scores ((Xa/(Xa + Xi) − 0.5), and of CTCF peaks assigned as Xa-specific, common, or Xi-specific for WT (blue), Del-hinge (red), and Inv-Dxz4 along a region of the X chromosome (pink background) that includes Zfx (a gene subject to XCI in WT, which reactivated in Del-hinge) and a region (green background) that includes Eif2s3x (a gene that escapes XCI). CTCF peaks that appear on the Xi in a region around Zfx

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation

    Dxz4 alone is necessary to maintain the bipartite structure of the Xi. a . The location of Dxz4 , Firre , and Xist for contact maps of the Xa in the same cell lines. b Relative position of the loci within the hinge region and location of the CRISPR/Cas9-induced alterations. Arrows indicate the orientation of CTCF motifs at Dxz4 and at the 5′end of Ds-TR for CTCF binding at Dxz4 on the Xi by ChIP-seq
    Figure Legend Snippet: Dxz4 alone is necessary to maintain the bipartite structure of the Xi. a . The location of Dxz4 , Firre , and Xist for contact maps of the Xa in the same cell lines. b Relative position of the loci within the hinge region and location of the CRISPR/Cas9-induced alterations. Arrows indicate the orientation of CTCF motifs at Dxz4 and at the 5′end of Ds-TR for CTCF binding at Dxz4 on the Xi by ChIP-seq

    Techniques Used: CRISPR, Binding Assay, Chromatin Immunoprecipitation

    Dxz4 deletion or inversion changes contact distribution on the Xi. a Contact maps (500 kb resolution) for the Xi in WT*, Del-hinge/Dxz4, and Inv-Dxz4 visualized using Pearson correlation to highlight contact probabilities. The color scale shows Pearson correlation values. b Pearson correlation-transformed contact maps (500 kb resolution) for 50 Mb around the Dxz4 locus to highlight the loss of superdomain structure in Del-hinge/Dxz4 and the shift in contacts in Inv-Dxz4, where a new contact domain forms between Firre and Dxz4 . The color scale shows Pearson correlation values. c b, c for analyses of autosomes. d Pair-wise Spearman correlation values and associated scatter plots between allelic PC1 scores for the Xa and Xi in WT*, Del-hinge/Dxz4, and Inv-Dxz4. Xi PC1 scores are less correlated between WT* and Del-hinge/Dxz4 (Spearman ρ = 0.35) compared to those of Xa ( ρ = 0.95) and autosomes ( ρ > 0.9). The Xi PC1 score in Inv-Dxz4 is less correlated with the WT* Xi PC1 profile ( ρ = 0.75) than Xa PC1 scores ( ρ for analysis of autosomes. e – h . Plots of the average Hi-C interaction frequencies (at 500 kb resolution) as a function of genomic distance along the Xa (dotted line) and Xi (line) for WT* (blue), Del-hinge/Dxz4 (red), and Inv-Dxz4 (black) for the entire length of the X chromosomes ( e ) and for three regions along the X chromosome: Firre-Xist (~53 Mb, f ); Firre-Dxz4 (~25 Mb, g ); Dxz4-Xist (~28 Mb, h ). Inset: plots of average contacts as a function of distance for autosomes
    Figure Legend Snippet: Dxz4 deletion or inversion changes contact distribution on the Xi. a Contact maps (500 kb resolution) for the Xi in WT*, Del-hinge/Dxz4, and Inv-Dxz4 visualized using Pearson correlation to highlight contact probabilities. The color scale shows Pearson correlation values. b Pearson correlation-transformed contact maps (500 kb resolution) for 50 Mb around the Dxz4 locus to highlight the loss of superdomain structure in Del-hinge/Dxz4 and the shift in contacts in Inv-Dxz4, where a new contact domain forms between Firre and Dxz4 . The color scale shows Pearson correlation values. c b, c for analyses of autosomes. d Pair-wise Spearman correlation values and associated scatter plots between allelic PC1 scores for the Xa and Xi in WT*, Del-hinge/Dxz4, and Inv-Dxz4. Xi PC1 scores are less correlated between WT* and Del-hinge/Dxz4 (Spearman ρ = 0.35) compared to those of Xa ( ρ = 0.95) and autosomes ( ρ > 0.9). The Xi PC1 score in Inv-Dxz4 is less correlated with the WT* Xi PC1 profile ( ρ = 0.75) than Xa PC1 scores ( ρ for analysis of autosomes. e – h . Plots of the average Hi-C interaction frequencies (at 500 kb resolution) as a function of genomic distance along the Xa (dotted line) and Xi (line) for WT* (blue), Del-hinge/Dxz4 (red), and Inv-Dxz4 (black) for the entire length of the X chromosomes ( e ) and for three regions along the X chromosome: Firre-Xist (~53 Mb, f ); Firre-Dxz4 (~25 Mb, g ); Dxz4-Xist (~28 Mb, h ). Inset: plots of average contacts as a function of distance for autosomes

    Techniques Used: Transformation Assay, Hi-C

    Changes in Xi TAD configuration after Dxz4 deletion or inversion. a Insulation score profiles at 500 kb resolution for the whole Xi in WT* (blue), Del-hinge/Dxz4 (red), and Inv-Dxz4 (black). The positions of Firre , Dxz4 , Zfx , Eda2r , and Xist for analysis of insulation scores of Xi and Xa in all cell lines. b As in a but based on 40 kb resolution data for a region from Firre to Xist along the X chromosome. c Differential insulation score profiles for Del-hinge/Dxz4 Xi (top), Inv-Dxz4 Xi (middle), and WT* Xa (bottom) relative to WT* Xi based on 40 kb resolution data for a region from Firre to Xist . d , e Hierarchical clustering based on the Euclidean distance ( d ) and Pearson correlation (using 1 − r as the distance measure; e for analysis of all cell lines. f Hierarchical clustering based on the adjusted Rand index to quantify the correspondence between TADs called using insulation scores at 40 kb resolution along the Xi and Xa in WT*, Del-hinge/Dxz4, and Inv-Dxz4. g Violin plots showing the distributions of TAD boundary strength scores (see Methods) for the Xa (left) and Xi (right) in WT* (blue), Del-hinge/Dxz4 (pink), and Inv-Dxz4 (gray) for the entire X chromosome (top) and for the region from Firre to Xist (bottom). The boxes demarcate the interquartile range (IQR) with median. Whiskers are ±1.5 times the IQR
    Figure Legend Snippet: Changes in Xi TAD configuration after Dxz4 deletion or inversion. a Insulation score profiles at 500 kb resolution for the whole Xi in WT* (blue), Del-hinge/Dxz4 (red), and Inv-Dxz4 (black). The positions of Firre , Dxz4 , Zfx , Eda2r , and Xist for analysis of insulation scores of Xi and Xa in all cell lines. b As in a but based on 40 kb resolution data for a region from Firre to Xist along the X chromosome. c Differential insulation score profiles for Del-hinge/Dxz4 Xi (top), Inv-Dxz4 Xi (middle), and WT* Xa (bottom) relative to WT* Xi based on 40 kb resolution data for a region from Firre to Xist . d , e Hierarchical clustering based on the Euclidean distance ( d ) and Pearson correlation (using 1 − r as the distance measure; e for analysis of all cell lines. f Hierarchical clustering based on the adjusted Rand index to quantify the correspondence between TADs called using insulation scores at 40 kb resolution along the Xi and Xa in WT*, Del-hinge/Dxz4, and Inv-Dxz4. g Violin plots showing the distributions of TAD boundary strength scores (see Methods) for the Xa (left) and Xi (right) in WT* (blue), Del-hinge/Dxz4 (pink), and Inv-Dxz4 (gray) for the entire X chromosome (top) and for the region from Firre to Xist (bottom). The boxes demarcate the interquartile range (IQR) with median. Whiskers are ±1.5 times the IQR

    Techniques Used:

    Unidirectional disruption of contacts on the Xi after Dxz4 deletion or inversion. a for comparison with differential contact maps based on untransformed count data. b As in a , h for comparison with differential contact maps based on untransformed count data. c Virtual 4C plots derived from Hi-C data at 500 kb resolution using a 500 kb region around Dxz4 as the viewpoint on the Xi in WT* (blue), Del-hinge/Dxz4 (red), and Inv-Dxz4 (black). Y -axis (contact counts) limited to 20% of maximum. The positions of Firre , Dxz4 , and Xist . d Standardized coverage score profiles at 500 kb resolution for the Xi in WT* (blue), Del-hinge/Dxz4 (red), and Inv-Dxz4 (black). The positions of Firre , Dxz4 , and Xist a-d. e Coverage scores (rescaled to [0; 1]) at 40 kb resolution within a 8 Mb region around Dxz4 for the Xi. The light blue background highlights the Xi boundary region of minimal interaction in WT* based on a threshold of 0.015 (horizontal red dashed line). The light gray background shows how this region is shifted to the right in Inv-Dxz4
    Figure Legend Snippet: Unidirectional disruption of contacts on the Xi after Dxz4 deletion or inversion. a for comparison with differential contact maps based on untransformed count data. b As in a , h for comparison with differential contact maps based on untransformed count data. c Virtual 4C plots derived from Hi-C data at 500 kb resolution using a 500 kb region around Dxz4 as the viewpoint on the Xi in WT* (blue), Del-hinge/Dxz4 (red), and Inv-Dxz4 (black). Y -axis (contact counts) limited to 20% of maximum. The positions of Firre , Dxz4 , and Xist . d Standardized coverage score profiles at 500 kb resolution for the Xi in WT* (blue), Del-hinge/Dxz4 (red), and Inv-Dxz4 (black). The positions of Firre , Dxz4 , and Xist a-d. e Coverage scores (rescaled to [0; 1]) at 40 kb resolution within a 8 Mb region around Dxz4 for the Xi. The light blue background highlights the Xi boundary region of minimal interaction in WT* based on a threshold of 0.015 (horizontal red dashed line). The light gray background shows how this region is shifted to the right in Inv-Dxz4

    Techniques Used: Derivative Assay, Hi-C

    35) Product Images from "Alternative splicing is a developmental switch for hTERT expression"

    Article Title: Alternative splicing is a developmental switch for hTERT expression

    Journal: bioRxiv

    doi: 10.1101/2020.04.02.022087

    SON is a key regulator of hTERT alternative splicing. A) Schematic of minigenes designed to measure the efficiency of hTERT mRNA splicing using Nano- and Firefly luciferase activity as a readout. Retention of exon-2 leads to expression of Firefly luciferase, but not Nano luciferase. Conversely, exclusion of exon 2 leads to a (−1) frameshift in exon-3 prompting the expression of Nano-luciferase while shifting Firefly luciferase out-of-frame. B) The minigenes were integrated into HeLa cells and a small-scale RNAi screen using a curated list of splicing factors and RNA-binding proteins was performed. Graph depicts the average ratio of Nano/Firefly luciferase of 3 biological replicates for 442 genes. Data presented as a log-ratio, colors highlight genes with p-value
    Figure Legend Snippet: SON is a key regulator of hTERT alternative splicing. A) Schematic of minigenes designed to measure the efficiency of hTERT mRNA splicing using Nano- and Firefly luciferase activity as a readout. Retention of exon-2 leads to expression of Firefly luciferase, but not Nano luciferase. Conversely, exclusion of exon 2 leads to a (−1) frameshift in exon-3 prompting the expression of Nano-luciferase while shifting Firefly luciferase out-of-frame. B) The minigenes were integrated into HeLa cells and a small-scale RNAi screen using a curated list of splicing factors and RNA-binding proteins was performed. Graph depicts the average ratio of Nano/Firefly luciferase of 3 biological replicates for 442 genes. Data presented as a log-ratio, colors highlight genes with p-value

    Techniques Used: Luciferase, Activity Assay, Expressing, RNA Binding Assay

    Inclusion of exon-2 correlates with the abundance of telomerase mRNA. A) Sashimi plot representing RNA Capture-Seq for hTERT locus in H7 ESCs and ARPE cells. Reads from exons are depicted as pileups and exon-exon junctions denoted with arcs. Asterisks indicate ΔEx2 splice variant transcripts detected in differentiated cells. B) Schematic of full-length and hTERT-ΔEx2 transcripts, demonstrating the position of the premature termination codons (PTC, asterisks) generated upon exon-2 skipping. Two tandem PTC’s would likely target ΔEx2 transcript for degradation by nonsense-mediated decay (NMD). C) Schematic illustration of junction-spanning PCR strategy used to assay hTERT ΔEx2 abundance by quantitative RT-PCR. RNA was reverse-transcribed using an hTERT gene-specific primer (GSP) and cDNA was purified and equalized between samples prior to PCR amplification with the indicated primers. D) Quantification of the ratio of hTERT ΔEx2 relative to full-length as determined by qRT-PCR in mortal cell lines (ARPE BJ), ARPE and BJ cell lines immortalized with hTERT cDNA, iPSCs derived from BJ cells, and human ESCs (n=3, p
    Figure Legend Snippet: Inclusion of exon-2 correlates with the abundance of telomerase mRNA. A) Sashimi plot representing RNA Capture-Seq for hTERT locus in H7 ESCs and ARPE cells. Reads from exons are depicted as pileups and exon-exon junctions denoted with arcs. Asterisks indicate ΔEx2 splice variant transcripts detected in differentiated cells. B) Schematic of full-length and hTERT-ΔEx2 transcripts, demonstrating the position of the premature termination codons (PTC, asterisks) generated upon exon-2 skipping. Two tandem PTC’s would likely target ΔEx2 transcript for degradation by nonsense-mediated decay (NMD). C) Schematic illustration of junction-spanning PCR strategy used to assay hTERT ΔEx2 abundance by quantitative RT-PCR. RNA was reverse-transcribed using an hTERT gene-specific primer (GSP) and cDNA was purified and equalized between samples prior to PCR amplification with the indicated primers. D) Quantification of the ratio of hTERT ΔEx2 relative to full-length as determined by qRT-PCR in mortal cell lines (ARPE BJ), ARPE and BJ cell lines immortalized with hTERT cDNA, iPSCs derived from BJ cells, and human ESCs (n=3, p

    Techniques Used: Variant Assay, Generated, Polymerase Chain Reaction, Quantitative RT-PCR, Purification, Amplification, Derivative Assay

    36) Product Images from "VEGF amplifies transcription through ETS1 acetylation to enable angiogenesis"

    Article Title: VEGF amplifies transcription through ETS1 acetylation to enable angiogenesis

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00405-x

    ETS1 promoter occupancy and gene expression. ETS1 occupied promoters of most expressed genes, and its promoter occupancy correlated with gene expression. a Overview of the experimental design used for in vitro studies. Samples were collected prior to stimulation (0 h) and at 1, 4, and 12 h of VEGF stimulation. b ETS1 chromatin occupancy at 0 h with respect to genome annotations. c Heatmap of indicated chromatin features at promoter regions at the 0 h time point. Regions are ordered by ETS1 binding strength at 0 h after VEGF stimulation. Features positively correlated with gene expression correlated with ETS1 binding strength. d ETS1 signal at TSS region and associated gene expression at the 0 h time point. ETS1 bound most expressed genes. Left panel (ETS1 signal): tag heatmap with high ChiP-seq signal shown in red . Right panel (mRNA): red lines indicate expressed genes, as determined by RNA-seq. e Correlation plot of promoter ETS1 and RNAPII occupancy at the 0 h time point. f Correlation plot of promoter ETS1 occupancy and RNA-seq gene expression at the 0 h time point
    Figure Legend Snippet: ETS1 promoter occupancy and gene expression. ETS1 occupied promoters of most expressed genes, and its promoter occupancy correlated with gene expression. a Overview of the experimental design used for in vitro studies. Samples were collected prior to stimulation (0 h) and at 1, 4, and 12 h of VEGF stimulation. b ETS1 chromatin occupancy at 0 h with respect to genome annotations. c Heatmap of indicated chromatin features at promoter regions at the 0 h time point. Regions are ordered by ETS1 binding strength at 0 h after VEGF stimulation. Features positively correlated with gene expression correlated with ETS1 binding strength. d ETS1 signal at TSS region and associated gene expression at the 0 h time point. ETS1 bound most expressed genes. Left panel (ETS1 signal): tag heatmap with high ChiP-seq signal shown in red . Right panel (mRNA): red lines indicate expressed genes, as determined by RNA-seq. e Correlation plot of promoter ETS1 and RNAPII occupancy at the 0 h time point. f Correlation plot of promoter ETS1 occupancy and RNA-seq gene expression at the 0 h time point

    Techniques Used: Expressing, In Vitro, Binding Assay, Chromatin Immunoprecipitation, RNA Sequencing Assay

    ETS1 stimulated RNAPII pause release. a Immunoblot of ETS1 expression in HUVEC cells 12 h after transfection by the indicated dose of ETS1 modRNA. b ETS1 overexpression reduced RNAPII pausing at ETS1-bound promoters. HUVEC cells were treated with ETS1 or GFP modRNA. Pausing index (PI) of ETS1-bound genes, a measure of a gene’s RNAPII paused at its promoter, was calculated from RNAPII ChIP-seq performed 12 h after transfection. ETS1 shifted the distribution of ETS1-bound genes to lower PI in treatment compared to control. c ETS1 knockdown increased RNAPII pausing at ETS1-bound promoters. Experiment as in b , except that cells were treated with control or ETS1 siRNAs. d ETS1 overexpression using modRNA increased actively elongating RNAPII (RNAPII-pS2) but not total RNAPII. HUVEC cells treated with GFP or ETS1 modRNA were analyzed by immunoblot at 12 h. e ETS1 overexpression increased actively elongating RNAPII through BRD4 and P-TEFb. ETS1 modRNA-induced increase of RNAPII-pS2 was blocked by BRD4 inhibitor JQ1 or P-TEFb inhibitor flavopiridole (FP). f – i ETS1 overexpression broadly increased mRNA expression. Total RNA f or mRNA g content per cell were measured by Qubit assay. Alternatively, mRNA was converted to RNA-seq libraries, using external spike-in RNA for normalization to cell number. Relative RNA-seq library yield per cell was measured by quantitative RTPCR. Cumulative distribution plot of RNA abundance per cell. Cumulative distribution plot of gene expression i showed that that ETS1 modRNA broadly increased gene expression. P -values were calculated by Student’s t -test ( f – h ) or by Kolmogorov–Smirnov test b , c , i . Bar graphs show mean ± s.d
    Figure Legend Snippet: ETS1 stimulated RNAPII pause release. a Immunoblot of ETS1 expression in HUVEC cells 12 h after transfection by the indicated dose of ETS1 modRNA. b ETS1 overexpression reduced RNAPII pausing at ETS1-bound promoters. HUVEC cells were treated with ETS1 or GFP modRNA. Pausing index (PI) of ETS1-bound genes, a measure of a gene’s RNAPII paused at its promoter, was calculated from RNAPII ChIP-seq performed 12 h after transfection. ETS1 shifted the distribution of ETS1-bound genes to lower PI in treatment compared to control. c ETS1 knockdown increased RNAPII pausing at ETS1-bound promoters. Experiment as in b , except that cells were treated with control or ETS1 siRNAs. d ETS1 overexpression using modRNA increased actively elongating RNAPII (RNAPII-pS2) but not total RNAPII. HUVEC cells treated with GFP or ETS1 modRNA were analyzed by immunoblot at 12 h. e ETS1 overexpression increased actively elongating RNAPII through BRD4 and P-TEFb. ETS1 modRNA-induced increase of RNAPII-pS2 was blocked by BRD4 inhibitor JQ1 or P-TEFb inhibitor flavopiridole (FP). f – i ETS1 overexpression broadly increased mRNA expression. Total RNA f or mRNA g content per cell were measured by Qubit assay. Alternatively, mRNA was converted to RNA-seq libraries, using external spike-in RNA for normalization to cell number. Relative RNA-seq library yield per cell was measured by quantitative RTPCR. Cumulative distribution plot of RNA abundance per cell. Cumulative distribution plot of gene expression i showed that that ETS1 modRNA broadly increased gene expression. P -values were calculated by Student’s t -test ( f – h ) or by Kolmogorov–Smirnov test b , c , i . Bar graphs show mean ± s.d

    Techniques Used: Expressing, Transfection, Over Expression, Chromatin Immunoprecipitation, RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction

    37) Product Images from "ZBTB48 is both a vertebrate telomere‐binding protein and a transcriptional activator"

    Article Title: ZBTB48 is both a vertebrate telomere‐binding protein and a transcriptional activator

    Journal: EMBO Reports

    doi: 10.15252/embr.201744095

    ZBTB48 is a transcriptional activator Differential expression analysis of the RNA sequencing (RNAseq) gene quantitation, comparing each five WT and ZBTB48 KO clones for U2OS (left) and HeLa (right). Cut‐offs for significant differential expression were set to log 2 (fold change) > |1| and −log 10 (adjusted P ‐value) > 2 (FDR
    Figure Legend Snippet: ZBTB48 is a transcriptional activator Differential expression analysis of the RNA sequencing (RNAseq) gene quantitation, comparing each five WT and ZBTB48 KO clones for U2OS (left) and HeLa (right). Cut‐offs for significant differential expression were set to log 2 (fold change) > |1| and −log 10 (adjusted P ‐value) > 2 (FDR

    Techniques Used: Expressing, RNA Sequencing Assay, Quantitation Assay, Clone Assay

    ZBTB48 acts as a transcription factor in contrast to HOT1 Differential expression analysis of the RNA sequencing (RNAseq) gene quantitation, comparing each five WT and ZBTB48 KO clones for U2OS (left) and HeLa (right). Cut‐offs for significant differential expression were set to log 2 (fold change) > |1| and −log 10 (adjusted P ‐value) > 2 (FDR
    Figure Legend Snippet: ZBTB48 acts as a transcription factor in contrast to HOT1 Differential expression analysis of the RNA sequencing (RNAseq) gene quantitation, comparing each five WT and ZBTB48 KO clones for U2OS (left) and HeLa (right). Cut‐offs for significant differential expression were set to log 2 (fold change) > |1| and −log 10 (adjusted P ‐value) > 2 (FDR

    Techniques Used: Expressing, RNA Sequencing Assay, Quantitation Assay, Clone Assay

    38) Product Images from "ZBTB48 is both a vertebrate telomere‐binding protein and a transcriptional activator"

    Article Title: ZBTB48 is both a vertebrate telomere‐binding protein and a transcriptional activator

    Journal: EMBO Reports

    doi: 10.15252/embr.201744095

    ZBTB48 and HOT1 KO clones show depleted expression Depiction of the ZBTB48 TALEN binding sites located in exon 2. Genotypes of HeLa and U2OS WT and ZBTB48 KO clones. The grey boxes represent the TALEN binding sites; insertions (blue) and deletions (red) are marked. The numbers in brackets represent the numbers of clones found with the specific genotype. Gel image of T7E1 assay showing ZBTB48 TALEN activity in HeLa cells. Western blot confirmation of depleted ZBTB48 expression in each five HeLa and U2OS ZBTB48 KO clones compared to parental cells. IF confirmation of depleted ZBTB48 expression (green) with DAPI (blue) as nuclear counterstain in U2OS ZBTB48 KO cells. ZBTB48 signals are reduced to background levels. Scale bars represent 5 μm. Depiction of the HOT1 TALEN binding sites located in exon 3. Genotypes of HeLa and U2OS WT and HOT1 KO clones. The grey boxes represent the TALEN binding sites; insertions (blue) and deletions (red) are marked. The numbers in brackets represent the numbers of clones found with the specific genotype. Gel image of T7E1 assay showing HOT1 TALEN activity in HeLa cells. IF confirmation of depleted HOT1 expression (green) with DAPI (blue) as nuclear counterstain in a representative HeLa HOT1 KO clone. Scale bars represent 5 μm. Source data are available online for this figure.
    Figure Legend Snippet: ZBTB48 and HOT1 KO clones show depleted expression Depiction of the ZBTB48 TALEN binding sites located in exon 2. Genotypes of HeLa and U2OS WT and ZBTB48 KO clones. The grey boxes represent the TALEN binding sites; insertions (blue) and deletions (red) are marked. The numbers in brackets represent the numbers of clones found with the specific genotype. Gel image of T7E1 assay showing ZBTB48 TALEN activity in HeLa cells. Western blot confirmation of depleted ZBTB48 expression in each five HeLa and U2OS ZBTB48 KO clones compared to parental cells. IF confirmation of depleted ZBTB48 expression (green) with DAPI (blue) as nuclear counterstain in U2OS ZBTB48 KO cells. ZBTB48 signals are reduced to background levels. Scale bars represent 5 μm. Depiction of the HOT1 TALEN binding sites located in exon 3. Genotypes of HeLa and U2OS WT and HOT1 KO clones. The grey boxes represent the TALEN binding sites; insertions (blue) and deletions (red) are marked. The numbers in brackets represent the numbers of clones found with the specific genotype. Gel image of T7E1 assay showing HOT1 TALEN activity in HeLa cells. IF confirmation of depleted HOT1 expression (green) with DAPI (blue) as nuclear counterstain in a representative HeLa HOT1 KO clone. Scale bars represent 5 μm. Source data are available online for this figure.

    Techniques Used: Clone Assay, Expressing, Binding Assay, Activity Assay, Western Blot

    ZBTB48 is a direct telomere‐binding protein but TRAP activity is not affected in ZBTB48 KO clones TTAGGG content of telomeric reads in ChIPseq samples from U2OS WT, U2OS ZBTB48 KO and U2OS HOT1 KO clones using IgG, TRF2, ZBTB48 Atlas, ZBTB48 GeneTex and HOT1 antibodies as well as FLAG antibody for FLAG‐ZBTB48 WT and point mutants for ZnF10 and ZnF11. The percentage contribution to all reads containing 1–8× TTAGGG repeats is shown. All reactions were performed in technical replicates on two independent WT and KO clones each. These reactions are biological replicates of Fig 2 A, performed with independent U2OS ZBTB48 WT and KO clones for ZBTB48 and with a second HOT1 antibody for HOT1. Telomerase activity was determined based on a quantitative TRAP assay. Heat‐inactivated HeLa extracts were used as a threshold to determine non‐specific background signal. Differences in C t values (∆∆ C t ) from the quantitative PCR measurements are displayed relative to the heat‐inactivation control. Error bars represent standard deviations ( n = 3). The P ‐value is based on Student's t ‐test.
    Figure Legend Snippet: ZBTB48 is a direct telomere‐binding protein but TRAP activity is not affected in ZBTB48 KO clones TTAGGG content of telomeric reads in ChIPseq samples from U2OS WT, U2OS ZBTB48 KO and U2OS HOT1 KO clones using IgG, TRF2, ZBTB48 Atlas, ZBTB48 GeneTex and HOT1 antibodies as well as FLAG antibody for FLAG‐ZBTB48 WT and point mutants for ZnF10 and ZnF11. The percentage contribution to all reads containing 1–8× TTAGGG repeats is shown. All reactions were performed in technical replicates on two independent WT and KO clones each. These reactions are biological replicates of Fig 2 A, performed with independent U2OS ZBTB48 WT and KO clones for ZBTB48 and with a second HOT1 antibody for HOT1. Telomerase activity was determined based on a quantitative TRAP assay. Heat‐inactivated HeLa extracts were used as a threshold to determine non‐specific background signal. Differences in C t values (∆∆ C t ) from the quantitative PCR measurements are displayed relative to the heat‐inactivation control. Error bars represent standard deviations ( n = 3). The P ‐value is based on Student's t ‐test.

    Techniques Used: Binding Assay, Activity Assay, Clone Assay, TRAP Assay, Real-time Polymerase Chain Reaction

    ZBTB 48 associates with short and long telomeres in vivo TTAGGG content of telomeric reads in ChIPseq samples from U2OS WT, U2OS ZBTB48 KO and U2OS HOT1 KO clones using IgG, TRF2, ZBTB48 Atlas, ZBTB48 GeneTex and HOT1 antibodies as well as FLAG antibody for FLAG‐ZBTB48 WT and point mutants for ZnF10 and ZnF11. The percentage contribution to all reads containing 1–8× TTAGGG repeats is shown. All reactions were performed in technical replicates on two independent WT and KO clones each. MEME sequence logo and bit score for the top 500 extratelomeric ChIPseq peaks for ZBTB48 Atlas, ZBTB48 GeneTex, TRF2 and HOT1 antibodies compared to IgG samples in U2OS. For each antibody, the most frequent motif is shown. TTAGGG enrichment in ChIPseq samples with reads with 7× or 8× TTAGGG repeats. Absolute read counts from ChIPseq reactions from each two HeLa and U2OS WT and KO clones with two independent antibodies for ZBTB48 and HOT1 are shown (left). The corresponding fold enrichments are calculated (right) and compared to TRF2 ChIPseq reactions for which fold enrichments are calculated relative to IgG samples. Error bars represent standard deviations ( n = 4), and P ‐values are based on Student's t ‐test with ** indicating P
    Figure Legend Snippet: ZBTB 48 associates with short and long telomeres in vivo TTAGGG content of telomeric reads in ChIPseq samples from U2OS WT, U2OS ZBTB48 KO and U2OS HOT1 KO clones using IgG, TRF2, ZBTB48 Atlas, ZBTB48 GeneTex and HOT1 antibodies as well as FLAG antibody for FLAG‐ZBTB48 WT and point mutants for ZnF10 and ZnF11. The percentage contribution to all reads containing 1–8× TTAGGG repeats is shown. All reactions were performed in technical replicates on two independent WT and KO clones each. MEME sequence logo and bit score for the top 500 extratelomeric ChIPseq peaks for ZBTB48 Atlas, ZBTB48 GeneTex, TRF2 and HOT1 antibodies compared to IgG samples in U2OS. For each antibody, the most frequent motif is shown. TTAGGG enrichment in ChIPseq samples with reads with 7× or 8× TTAGGG repeats. Absolute read counts from ChIPseq reactions from each two HeLa and U2OS WT and KO clones with two independent antibodies for ZBTB48 and HOT1 are shown (left). The corresponding fold enrichments are calculated (right) and compared to TRF2 ChIPseq reactions for which fold enrichments are calculated relative to IgG samples. Error bars represent standard deviations ( n = 4), and P ‐values are based on Student's t ‐test with ** indicating P

    Techniques Used: In Vivo, Clone Assay, Sequencing

    ZBTB48 acts as a transcription factor in contrast to HOT1 Differential expression analysis of the RNA sequencing (RNAseq) gene quantitation, comparing each five WT and ZBTB48 KO clones for U2OS (left) and HeLa (right). Cut‐offs for significant differential expression were set to log 2 (fold change) > |1| and −log 10 (adjusted P ‐value) > 2 (FDR
    Figure Legend Snippet: ZBTB48 acts as a transcription factor in contrast to HOT1 Differential expression analysis of the RNA sequencing (RNAseq) gene quantitation, comparing each five WT and ZBTB48 KO clones for U2OS (left) and HeLa (right). Cut‐offs for significant differential expression were set to log 2 (fold change) > |1| and −log 10 (adjusted P ‐value) > 2 (FDR

    Techniques Used: Expressing, RNA Sequencing Assay, Quantitation Assay, Clone Assay

    39) Product Images from "ZBTB48 is both a vertebrate telomere‐binding protein and a transcriptional activator"

    Article Title: ZBTB48 is both a vertebrate telomere‐binding protein and a transcriptional activator

    Journal: EMBO Reports

    doi: 10.15252/embr.201744095

    ZBTB48 is required for MTFP1 expression Western blot confirmation of reduced MTFP1 expression in each five HeLa and U2OS ZBTB48 KO clones compared to five WT clones each. Similar to ZBTB48, no detectable MTFP1 protein is found in the KO cells. Super‐resolution fluorescence microscopy analysis of the structure and localization of the mitochondrial network in HeLa WT and ZBTB48 KO clones. Mitochondria are marked with the MitoTracker dye (red), and nuclei are counterstained with DAPI (blue). Scale bars represent 5 μm. mtDNA level quantification comparing five HeLa ZBTB48 WT and KO clones. mtDNA levels were quantified based on three mtDNA loci and normalized to two genomic regions. Error bars represent standard deviations ( n = 3). The P ‐value is based on Student's t ‐test.
    Figure Legend Snippet: ZBTB48 is required for MTFP1 expression Western blot confirmation of reduced MTFP1 expression in each five HeLa and U2OS ZBTB48 KO clones compared to five WT clones each. Similar to ZBTB48, no detectable MTFP1 protein is found in the KO cells. Super‐resolution fluorescence microscopy analysis of the structure and localization of the mitochondrial network in HeLa WT and ZBTB48 KO clones. Mitochondria are marked with the MitoTracker dye (red), and nuclei are counterstained with DAPI (blue). Scale bars represent 5 μm. mtDNA level quantification comparing five HeLa ZBTB48 WT and KO clones. mtDNA levels were quantified based on three mtDNA loci and normalized to two genomic regions. Error bars represent standard deviations ( n = 3). The P ‐value is based on Student's t ‐test.

    Techniques Used: Expressing, Western Blot, Clone Assay, Fluorescence, Microscopy

    40) Product Images from "MGA, L3MBTL2 and E2F6 determine genomic binding of the non-canonical Polycomb repressive complex PRC1.6"

    Article Title: MGA, L3MBTL2 and E2F6 determine genomic binding of the non-canonical Polycomb repressive complex PRC1.6

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007193

    The role of PRC1.6 in HEK293 cell function. (A) Reduced proliferation of MGA ko , L3MBTL2 ko and E2F6 ko cells. Shown are growth curves of wildtype, MGA ko , L3MBTL2 ko , E2F6 ko and PCGF6 ko HEK293 cells. Cells were seed at 3x10 5 , and counted and replated at the indicated time points. Cumulative cell numbers were calculated by multiplying the initial cell number with the fold-increase in cell numbers in each interval. (B) Venn diagrams illustrating the overlap of MGA-bound genes and genes down- or up-regulated in MGA ko cells. Left circle, genes with ≥2-fold reduced transcript levels in MGA ko cells; right circle, genes with ≥2-fold increased transcript levels in MGA ko cells. (C) Representative genome browser screenshots of ChIP-seq and RNA-seq tracks illustrating binding of MGA, L3MBTL2, E2F6 and PCGF6 (top tracks) to the CNTD1 and SMC1B promoters, and RNA expression (bottom tracks) of the corresponding genes in three wild type samples (MGA_wt1, MGA_wt2 and MGA_wt3), and in three different MGA ko cell clones (MGA ko _cl26, MGA ko _cl27 and MGA ko _cl30). (D) RT-qPCR-based analysis of expression changes of selected genes in MGA ko , E2F6 ko , L3MBTL2 ko and PCGF6 ko cells. Transcript levels were normalized to B2M transcript levels, and are depicted relative to transcript levels in wild type cells.
    Figure Legend Snippet: The role of PRC1.6 in HEK293 cell function. (A) Reduced proliferation of MGA ko , L3MBTL2 ko and E2F6 ko cells. Shown are growth curves of wildtype, MGA ko , L3MBTL2 ko , E2F6 ko and PCGF6 ko HEK293 cells. Cells were seed at 3x10 5 , and counted and replated at the indicated time points. Cumulative cell numbers were calculated by multiplying the initial cell number with the fold-increase in cell numbers in each interval. (B) Venn diagrams illustrating the overlap of MGA-bound genes and genes down- or up-regulated in MGA ko cells. Left circle, genes with ≥2-fold reduced transcript levels in MGA ko cells; right circle, genes with ≥2-fold increased transcript levels in MGA ko cells. (C) Representative genome browser screenshots of ChIP-seq and RNA-seq tracks illustrating binding of MGA, L3MBTL2, E2F6 and PCGF6 (top tracks) to the CNTD1 and SMC1B promoters, and RNA expression (bottom tracks) of the corresponding genes in three wild type samples (MGA_wt1, MGA_wt2 and MGA_wt3), and in three different MGA ko cell clones (MGA ko _cl26, MGA ko _cl27 and MGA ko _cl30). (D) RT-qPCR-based analysis of expression changes of selected genes in MGA ko , E2F6 ko , L3MBTL2 ko and PCGF6 ko cells. Transcript levels were normalized to B2M transcript levels, and are depicted relative to transcript levels in wild type cells.

    Techniques Used: Cell Function Assay, Chromatin Immunoprecipitation, RNA Sequencing Assay, Binding Assay, RNA Expression, Clone Assay, Quantitative RT-PCR, Expressing

    MGA is essential for genomic binding of PRC1.6. (A) Heat map view of the distribution of union MGA, L3MBTL2 and E2F6 peaks in wild type cells (n = 8342) and in MGA-depleted cells at +/- 2 kb regions centred over the MGA peaks. (B) Representative genome browser screenshots showing binding of MGA, L3MBTL2, E2F6 and PCGF6 to the AEBP2 , RPA2 , RFC1 and SPOP promoters in wild type cells. MGA-depleted cells lack binding of L3MBTL2 and E2F6. (C) Western blot analysis of L3MBTL2, E2F6, PCGF6 and RING2 in wild type HEK293 cells and in two different MGA-depleted clones (cl26 and cl27). The anti-Tubulin blot served as a loading control. (D) L3MBTL2 -, E2F6 - and PCGF6 transcripts were determined in wild type cells and in MGA-depleted cell clones by RT-qPCR analysis. B2M transcript levels were used to normalize the data across samples, and transcript levels in wild type cells were arbitrarily set to 1. Data represent the average of technical replicates ± SD. (E) ChIP-qPCR data showing lack of L3MBTL2, E2F6, PCGF6, MAX, RING2, RYBP and HP1γ binding to representative PRC1.6 target promoters in MGA ko cells, and diminished deposition of H2AK119ub1. The CDC7 -2kb region served as a negative control region. Percent of input values represent the mean of at least three independent experiments +/- SD. (F) PRC1.6 target promoters are not bound by PRC2 and lack H3K27me3. Local levels of EZH2 and H3K27me3 at selected PRC1.6 target promoters in wild type (WT) and in MGA ko cells (clones cl26 and cl27) were determined by ChIP-qPCR analysis. Genomic regions known to be bound by canonical PRC1 ( FUT9 , MYT1 and TSH2B ) served as positive control regions. These regions were not bound by MGA (right panel). Percent of input values represent the mean of at least three independent experiments +/- SD.
    Figure Legend Snippet: MGA is essential for genomic binding of PRC1.6. (A) Heat map view of the distribution of union MGA, L3MBTL2 and E2F6 peaks in wild type cells (n = 8342) and in MGA-depleted cells at +/- 2 kb regions centred over the MGA peaks. (B) Representative genome browser screenshots showing binding of MGA, L3MBTL2, E2F6 and PCGF6 to the AEBP2 , RPA2 , RFC1 and SPOP promoters in wild type cells. MGA-depleted cells lack binding of L3MBTL2 and E2F6. (C) Western blot analysis of L3MBTL2, E2F6, PCGF6 and RING2 in wild type HEK293 cells and in two different MGA-depleted clones (cl26 and cl27). The anti-Tubulin blot served as a loading control. (D) L3MBTL2 -, E2F6 - and PCGF6 transcripts were determined in wild type cells and in MGA-depleted cell clones by RT-qPCR analysis. B2M transcript levels were used to normalize the data across samples, and transcript levels in wild type cells were arbitrarily set to 1. Data represent the average of technical replicates ± SD. (E) ChIP-qPCR data showing lack of L3MBTL2, E2F6, PCGF6, MAX, RING2, RYBP and HP1γ binding to representative PRC1.6 target promoters in MGA ko cells, and diminished deposition of H2AK119ub1. The CDC7 -2kb region served as a negative control region. Percent of input values represent the mean of at least three independent experiments +/- SD. (F) PRC1.6 target promoters are not bound by PRC2 and lack H3K27me3. Local levels of EZH2 and H3K27me3 at selected PRC1.6 target promoters in wild type (WT) and in MGA ko cells (clones cl26 and cl27) were determined by ChIP-qPCR analysis. Genomic regions known to be bound by canonical PRC1 ( FUT9 , MYT1 and TSH2B ) served as positive control regions. These regions were not bound by MGA (right panel). Percent of input values represent the mean of at least three independent experiments +/- SD.

    Techniques Used: Binding Assay, Western Blot, Clone Assay, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Positive Control

    MGA, L3MBTL2, E2F6 and PCGF6 colocalize in 293 cells. (A) Schematic representation of PRC1.6 core components. (B) Western blot analysis of MGA, L3MBTL2, E2F6 and PCGF6 expression in wild type 293 cells (wt) and in corresponding MGA-, L3MBTL2-, E2F6- and PCGF6-depleted cell clones (MGA ko , L3MBTL2 ko , E2F6 ko and PCGF6 ko ). Re-probing for tubulin (TUB) controlled loading of extracts. (C) Venn diagrams showing the overlap of MGA, L3MBTL2, E2F6 and PCGF6 binding regions in HEK293 cells. The total number of high-confidence MGA, L3MBTL2, E2F6 and PCGF6 ChIP-seq peaks (≥30 tags, ≥3-fold enrichment over knockout control) and their overlap is shown. (D) A heat map view of the distribution of union MGA, L3MBTL2, E2F6 and PCGF6 peaks in HEK293 cells (n = 8342) at +/- 2 kb regions centred over the MGA peaks. (E) Representative genome browser screenshots of a 0.7 Mb region of chromosome 19 showing co-localization of MGA, L3MBTL2, E2F6 and PCGF6 at the CTC-232P5 . 1 , RFX2 , MLLT1 and KHSRP promoters. (F) Distribution of MGA, L3MBTL2, E2F6 and PCGF6 peaks relative to positions -2000 bp upstream to +2000 bp downstream of gene bodies. TSS, transcription start site; TES, transcription end site. (G) ChIP-qPCR analysis of MGA, L3MBTL2, E2F6 and PCGF6 binding to selected promoters. The region -2 kb upstream of the CDC7 promoter served as a negative control. Percent of input values represent the mean of at least three independent experiments +/- SD. (H) Sequence motifs enriched in PRC1.6 binding regions. Logos were obtained by running MEME-ChIP with 300 bp summits of the top 600 union MGA-L3MBTL2-E2F6-PCGF6 ChIP-seq peaks. The numbers next to the logos indicate the occurrence of the motifs, the statistical significance (E-value) and the transcription factors that bind to the motif. Right panel, local motif enrichment analysis (CentriMo) showing central enrichment of the MGA/MAX bHLH and the E2F6/DP1 binding motifs within the 300 bp peak regions. The NRF1 binding motif was not centrally enriched.
    Figure Legend Snippet: MGA, L3MBTL2, E2F6 and PCGF6 colocalize in 293 cells. (A) Schematic representation of PRC1.6 core components. (B) Western blot analysis of MGA, L3MBTL2, E2F6 and PCGF6 expression in wild type 293 cells (wt) and in corresponding MGA-, L3MBTL2-, E2F6- and PCGF6-depleted cell clones (MGA ko , L3MBTL2 ko , E2F6 ko and PCGF6 ko ). Re-probing for tubulin (TUB) controlled loading of extracts. (C) Venn diagrams showing the overlap of MGA, L3MBTL2, E2F6 and PCGF6 binding regions in HEK293 cells. The total number of high-confidence MGA, L3MBTL2, E2F6 and PCGF6 ChIP-seq peaks (≥30 tags, ≥3-fold enrichment over knockout control) and their overlap is shown. (D) A heat map view of the distribution of union MGA, L3MBTL2, E2F6 and PCGF6 peaks in HEK293 cells (n = 8342) at +/- 2 kb regions centred over the MGA peaks. (E) Representative genome browser screenshots of a 0.7 Mb region of chromosome 19 showing co-localization of MGA, L3MBTL2, E2F6 and PCGF6 at the CTC-232P5 . 1 , RFX2 , MLLT1 and KHSRP promoters. (F) Distribution of MGA, L3MBTL2, E2F6 and PCGF6 peaks relative to positions -2000 bp upstream to +2000 bp downstream of gene bodies. TSS, transcription start site; TES, transcription end site. (G) ChIP-qPCR analysis of MGA, L3MBTL2, E2F6 and PCGF6 binding to selected promoters. The region -2 kb upstream of the CDC7 promoter served as a negative control. Percent of input values represent the mean of at least three independent experiments +/- SD. (H) Sequence motifs enriched in PRC1.6 binding regions. Logos were obtained by running MEME-ChIP with 300 bp summits of the top 600 union MGA-L3MBTL2-E2F6-PCGF6 ChIP-seq peaks. The numbers next to the logos indicate the occurrence of the motifs, the statistical significance (E-value) and the transcription factors that bind to the motif. Right panel, local motif enrichment analysis (CentriMo) showing central enrichment of the MGA/MAX bHLH and the E2F6/DP1 binding motifs within the 300 bp peak regions. The NRF1 binding motif was not centrally enriched.

    Techniques Used: Western Blot, Expressing, Clone Assay, Binding Assay, Chromatin Immunoprecipitation, Knock-Out, Real-time Polymerase Chain Reaction, Negative Control, Sequencing

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    Qiagen on column dnase i digestion
    Summary of in vitro primer extension and <t>DNase</t> I and KMnO 4 footprinting. ( A ) Sequence of P vpsL from −60 to +30. Bold and underlined A with black arrow at +1 and bold G (+3) represent the TSS determined by primer extensions; the −10 element and the −35 region are labeled and boxed in green; sequences in bold and red denote the VpsR binding site. Protection sites from DNase I footprinting and hypersensitive sites are depicted as rectangular boxes and triangles, respectively, either above (non-template) or below (template) the sequences: gray, RNAP with or without c-di-GMP or VpsR; black, RNAP with VpsR and c-di-GMP; red, VpsR with or without c-di-GMP. The open transcription bubble detected using KMnO 4 footprinting is shown as separated ssDNA from position −11 to +2 with sites of KMnO 4 cleavage indicated as purple asterisks. ( B ) Summary of positions of protection and hypersensitive sites on non-template and template strand DNA.
    On Column Dnase I Digestion, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen on column dnase i digestion step
    <t>DNase</t> I footprinting assay of EsaR binding sites in the noncoding strand of P dkgA , coding strand of P glpF , and noncoding strand of P lrhA . (A to C) Capillary electrophoresis of FAM-labeled DNA fragments P dkgA (A), P glpF (B), and P lrhA (C) from DNase I
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    Summary of in vitro primer extension and DNase I and KMnO 4 footprinting. ( A ) Sequence of P vpsL from −60 to +30. Bold and underlined A with black arrow at +1 and bold G (+3) represent the TSS determined by primer extensions; the −10 element and the −35 region are labeled and boxed in green; sequences in bold and red denote the VpsR binding site. Protection sites from DNase I footprinting and hypersensitive sites are depicted as rectangular boxes and triangles, respectively, either above (non-template) or below (template) the sequences: gray, RNAP with or without c-di-GMP or VpsR; black, RNAP with VpsR and c-di-GMP; red, VpsR with or without c-di-GMP. The open transcription bubble detected using KMnO 4 footprinting is shown as separated ssDNA from position −11 to +2 with sites of KMnO 4 cleavage indicated as purple asterisks. ( B ) Summary of positions of protection and hypersensitive sites on non-template and template strand DNA.

    Journal: Nucleic Acids Research

    Article Title: VpsR and cyclic di-GMP together drive transcription initiation to activate biofilm formation in Vibrio cholerae

    doi: 10.1093/nar/gky606

    Figure Lengend Snippet: Summary of in vitro primer extension and DNase I and KMnO 4 footprinting. ( A ) Sequence of P vpsL from −60 to +30. Bold and underlined A with black arrow at +1 and bold G (+3) represent the TSS determined by primer extensions; the −10 element and the −35 region are labeled and boxed in green; sequences in bold and red denote the VpsR binding site. Protection sites from DNase I footprinting and hypersensitive sites are depicted as rectangular boxes and triangles, respectively, either above (non-template) or below (template) the sequences: gray, RNAP with or without c-di-GMP or VpsR; black, RNAP with VpsR and c-di-GMP; red, VpsR with or without c-di-GMP. The open transcription bubble detected using KMnO 4 footprinting is shown as separated ssDNA from position −11 to +2 with sites of KMnO 4 cleavage indicated as purple asterisks. ( B ) Summary of positions of protection and hypersensitive sites on non-template and template strand DNA.

    Article Snippet: Cells were harvested by centrifugation and RNA was extracted using the RNeasy kit (Qiagen), and an on-column DNase I digestion (Qiagen) was performed according to manufacturer's instructions.

    Techniques: In Vitro, Footprinting, Sequencing, Labeling, Binding Assay

    DNase I footprinting of P vpsL complexes on ( A ) nontemplate DNA and ( B ) template DNA. GA corresponds to G+A ladder. VpsR, c-di-GMP and/or RNAP are present as indicated. To the right of each gel image, a schematic indicates the −10 and −35 regions and the +1. The VpsR binding site is indicated as a dashed black line. DNase I protection regions and hypersensitive sites seen with the activated complex of RNAP, VpsR, c-di-GMP and DNA are depicted as black rectangles and horizontal arrows, respectively. The dashed red boxes indicate the regions of DNA where the protection/enhancement within and immediately adjacent to the VpsR binding site changes when comparing complexes containing RNAP with or without VpsR or c-di-GMP to the activated complex. (See text for details.)

    Journal: Nucleic Acids Research

    Article Title: VpsR and cyclic di-GMP together drive transcription initiation to activate biofilm formation in Vibrio cholerae

    doi: 10.1093/nar/gky606

    Figure Lengend Snippet: DNase I footprinting of P vpsL complexes on ( A ) nontemplate DNA and ( B ) template DNA. GA corresponds to G+A ladder. VpsR, c-di-GMP and/or RNAP are present as indicated. To the right of each gel image, a schematic indicates the −10 and −35 regions and the +1. The VpsR binding site is indicated as a dashed black line. DNase I protection regions and hypersensitive sites seen with the activated complex of RNAP, VpsR, c-di-GMP and DNA are depicted as black rectangles and horizontal arrows, respectively. The dashed red boxes indicate the regions of DNA where the protection/enhancement within and immediately adjacent to the VpsR binding site changes when comparing complexes containing RNAP with or without VpsR or c-di-GMP to the activated complex. (See text for details.)

    Article Snippet: Cells were harvested by centrifugation and RNA was extracted using the RNeasy kit (Qiagen), and an on-column DNase I digestion (Qiagen) was performed according to manufacturer's instructions.

    Techniques: Footprinting, Binding Assay

    Summary of in vitro primer extension and DNase I and KMnO 4 footprinting. ( A ) Sequence of P vpsL from −60 to +30. Bold and underlined A with black arrow at +1 and bold G (+3) represent the TSS determined by primer extensions; the −10 element and the −35 region are labeled and boxed in green; sequences in bold and red denote the VpsR binding site. Protection sites from DNase I footprinting and hypersensitive sites are depicted as rectangular boxes and triangles, respectively, either above (non-template) or below (template) the sequences: gray, RNAP with or without c-di-GMP or VpsR; black, RNAP with VpsR and c-di-GMP; red, VpsR with or without c-di-GMP. The open transcription bubble detected using KMnO 4 footprinting is shown as separated ssDNA from position −11 to +2 with sites of KMnO 4 cleavage indicated as purple asterisks. ( B ) Summary of positions of protection and hypersensitive sites on non-template and template strand DNA.

    Journal: Nucleic Acids Research

    Article Title: VpsR and cyclic di-GMP together drive transcription initiation to activate biofilm formation in Vibrio cholerae

    doi: 10.1093/nar/gky606

    Figure Lengend Snippet: Summary of in vitro primer extension and DNase I and KMnO 4 footprinting. ( A ) Sequence of P vpsL from −60 to +30. Bold and underlined A with black arrow at +1 and bold G (+3) represent the TSS determined by primer extensions; the −10 element and the −35 region are labeled and boxed in green; sequences in bold and red denote the VpsR binding site. Protection sites from DNase I footprinting and hypersensitive sites are depicted as rectangular boxes and triangles, respectively, either above (non-template) or below (template) the sequences: gray, RNAP with or without c-di-GMP or VpsR; black, RNAP with VpsR and c-di-GMP; red, VpsR with or without c-di-GMP. The open transcription bubble detected using KMnO 4 footprinting is shown as separated ssDNA from position −11 to +2 with sites of KMnO 4 cleavage indicated as purple asterisks. ( B ) Summary of positions of protection and hypersensitive sites on non-template and template strand DNA.

    Article Snippet: Cells were harvested by centrifugation and RNA was extracted using the RNeasy kit (Qiagen), and an on-column DNase I digestion (Qiagen) was performed according to manufacturer's instructions.

    Techniques: In Vitro, Footprinting, Sequencing, Labeling, Binding Assay

    DNase I footprinting of P vpsL complexes on ( A ) nontemplate DNA and ( B ) template DNA. GA corresponds to G+A ladder. VpsR, c-di-GMP and/or RNAP are present as indicated. To the right of each gel image, a schematic indicates the −10 and −35 regions and the +1. The VpsR binding site is indicated as a dashed black line. DNase I protection regions and hypersensitive sites seen with the activated complex of RNAP, VpsR, c-di-GMP and DNA are depicted as black rectangles and horizontal arrows, respectively. The dashed red boxes indicate the regions of DNA where the protection/enhancement within and immediately adjacent to the VpsR binding site changes when comparing complexes containing RNAP with or without VpsR or c-di-GMP to the activated complex. (See text for details.)

    Journal: Nucleic Acids Research

    Article Title: VpsR and cyclic di-GMP together drive transcription initiation to activate biofilm formation in Vibrio cholerae

    doi: 10.1093/nar/gky606

    Figure Lengend Snippet: DNase I footprinting of P vpsL complexes on ( A ) nontemplate DNA and ( B ) template DNA. GA corresponds to G+A ladder. VpsR, c-di-GMP and/or RNAP are present as indicated. To the right of each gel image, a schematic indicates the −10 and −35 regions and the +1. The VpsR binding site is indicated as a dashed black line. DNase I protection regions and hypersensitive sites seen with the activated complex of RNAP, VpsR, c-di-GMP and DNA are depicted as black rectangles and horizontal arrows, respectively. The dashed red boxes indicate the regions of DNA where the protection/enhancement within and immediately adjacent to the VpsR binding site changes when comparing complexes containing RNAP with or without VpsR or c-di-GMP to the activated complex. (See text for details.)

    Article Snippet: Cells were harvested by centrifugation and RNA was extracted using the RNeasy kit (Qiagen), and an on-column DNase I digestion (Qiagen) was performed according to manufacturer's instructions.

    Techniques: Footprinting, Binding Assay

    DNase I footprinting analysis of N. gonorrhoeae Fur protein with gonococcal fur and tonB promoter/operator regions. (A) DNase I footprint analysis of the fur promoter operator region. Radiolabeled DNA was incubated with increasing concentrations of Fur

    Journal: Journal of Bacteriology

    Article Title: Expression of the Gonococcal Global Regulatory Protein Fur and Genes Encompassing the Fur and Iron Regulon during In Vitro and In Vivo Infection in Women

    doi: 10.1128/JB.01830-07

    Figure Lengend Snippet: DNase I footprinting analysis of N. gonorrhoeae Fur protein with gonococcal fur and tonB promoter/operator regions. (A) DNase I footprint analysis of the fur promoter operator region. Radiolabeled DNA was incubated with increasing concentrations of Fur

    Article Snippet: On-column DNase I digestion was also performed for each sample according to the manufacturer's instructions (Qiagen).

    Techniques: Footprinting, Incubation

    DNase I footprinting assay of EsaR binding sites in the noncoding strand of P dkgA , coding strand of P glpF , and noncoding strand of P lrhA . (A to C) Capillary electrophoresis of FAM-labeled DNA fragments P dkgA (A), P glpF (B), and P lrhA (C) from DNase I

    Journal: Applied and Environmental Microbiology

    Article Title: Proteomic Analysis of the Quorum-Sensing Regulon in Pantoea stewartii and Identification of Direct Targets of EsaR

    doi: 10.1128/AEM.01744-13

    Figure Lengend Snippet: DNase I footprinting assay of EsaR binding sites in the noncoding strand of P dkgA , coding strand of P glpF , and noncoding strand of P lrhA . (A to C) Capillary electrophoresis of FAM-labeled DNA fragments P dkgA (A), P glpF (B), and P lrhA (C) from DNase I

    Article Snippet: The cell pellets were stored at −80°C prior to total RNA extraction using the RNeasy minikit (Qiagen) with an additional on-column DNase I digestion step using the RNase-free DNase set (Qiagen).

    Techniques: Footprinting, Binding Assay, Electrophoresis, Labeling

    Nested deletion EMSA analysis of EsaR direct targets. (A to C) The region protected by DNase I digestion in P dkgA (A), P glpF (B), and P lrhA (C) is the gray-shaded sequence (5′ to 3′), and the underlined bases are the 20-bp EsaR binding

    Journal: Applied and Environmental Microbiology

    Article Title: Proteomic Analysis of the Quorum-Sensing Regulon in Pantoea stewartii and Identification of Direct Targets of EsaR

    doi: 10.1128/AEM.01744-13

    Figure Lengend Snippet: Nested deletion EMSA analysis of EsaR direct targets. (A to C) The region protected by DNase I digestion in P dkgA (A), P glpF (B), and P lrhA (C) is the gray-shaded sequence (5′ to 3′), and the underlined bases are the 20-bp EsaR binding

    Article Snippet: The cell pellets were stored at −80°C prior to total RNA extraction using the RNeasy minikit (Qiagen) with an additional on-column DNase I digestion step using the RNase-free DNase set (Qiagen).

    Techniques: Sequencing, Binding Assay