Journal: Nucleic Acids Research

Article Title: Polo-like kinase 4 transcription is activated via CRE and NRF1 elements, repressed by DREAM through CDE/CHR sites and deregulated by HPV E7 protein

doi: 10.1093/nar/gkt849

Figure Lengend Snippet: The Plk4 promoter is activated through NRF1 and CRE sites. B-Myb contributes to maximal expression and is bound through the CHR in proliferating cells. ( A ) Protein binding to the wt and mutant Plk4 promoter was analyzed with nuclear extracts from asynchronous growing NIH3T3 cells by DNA affinity purification and western blot. Band intensities were quantified by densitometric analyses. The intensities relative to the control are given below the bands. ( B ) Double thymidine- or nocodazole-blocked NIH3T3 cells were cotransfected with a vector expressing shRNA targeting Bmyb (sh-Bmyb) or an shRNA construct expressing a nontargeting luciferase shRNA (sh-Luc) as a negative control and with EGFP. Cells were sorted for green fluorescence before mRNA preparation. Relative expression of Bmyb, Plk4 and Ccnb2 mRNA was quantified by RT-PCR and normalized to U6 RNA levels. ( C ) Luciferase reporter assays were performed with wt and CHR mutant Plk4 promoter reporter constructs from double thymidine- or nocodazole-blocked NIH3T3 cells. Cells were cotransfected with sh-Bmyb or a shRNA construct expressing a nontargeting GFP-shRNA as a negative control. ( D ) Asynchronous growing HCT116 cells were transfected with siRNA targeting NRF1 or with siControl. Relative expression of NRF1 and PLK4 mRNA was quantified by RT-PCR and normalized to U6 RNA levels. ( E ) Luciferase reporter assays with wt or NRF1-mutant Plk4 promoter constructs. HCT116 cells were cotransfected with siRNA targeting NRF1 or with siControl. ( F ) Luciferase reporter assays from NIH3T3 cells transfected with wt or mutant Plk4 promoter constructs. NIH3T3 cells were arrested in G0 by serum starvation and stimulated to reenter the cell cycle by addition of FCS to the medium. After 24 h of serum addition, cells were enriched in G2/M phases. Twenty four hours after addition of serum, NIH3T3 cells were enriched in G2/M cell cycle phases. RLU, relative light units. ( G ) Bioinformatic identification of CCAAT, CRE and NRF1 elements in DREAM/CHR promoters.

Article Snippet: HCT116 wt, HCT116 p53 − / − and HCT116 p21 −/− cells, kindly provided by Bert Vogelstein , and NIH3T3, T98G and HeLa cells (DSMZ, Braunschweig, Germany) were grown in Dulbecco's modified Eagle's medium (DMEM; Lonza, Basel, Switzerland) supplemented with 10% fetal calf serum (FCS) (Biochrom, Berlin, Germany) and penicillin/streptomycin (PAA Laboratories, Pasching, Austria) and maintained at 37°C and 10% CO 2 .

Techniques: Expressing, Protein Binding, Mutagenesis, Affinity Purification, Western Blot, Control, Plasmid Preparation, shRNA, Construct, Luciferase, Negative Control, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Transfection

Journal: Nucleic Acids Research

Article Title: Polo-like kinase 4 transcription is activated via CRE and NRF1 elements, repressed by DREAM through CDE/CHR sites and deregulated by HPV E7 protein

doi: 10.1093/nar/gkt849

Figure Lengend Snippet: p53-controlled downregulation of Plk4 expression depends on p21 WAF1/CIP1 and binding of DREAM to the CDE and CHR sites. ( A ) Expression of Plk4 mRNA in NIH3T3 and in ( B ) HCT116 wt, p53 −/− or p21 −/− cells treated with doxorubicin and roscovitine for 24 h. Cells without treatment served as control. Relative expression of Plk4 mRNA was quantified by RT-PCR and normalized to U6 RNA levels. ( C ) Luciferase reporter assays with wt or mutant Plk4 promoter constructs were cotransfected in HCT116 p53 −/− or HCT116 p21 −/− cells with p53mut, p53wt, p21mut or p21wt expression vectors. Ratios of values from wt/mut transfections with p53 or p21 are shown as fold-repression relative to the vector control.

Article Snippet: HCT116 wt, HCT116 p53 − / − and HCT116 p21 −/− cells, kindly provided by Bert Vogelstein , and NIH3T3, T98G and HeLa cells (DSMZ, Braunschweig, Germany) were grown in Dulbecco's modified Eagle's medium (DMEM; Lonza, Basel, Switzerland) supplemented with 10% fetal calf serum (FCS) (Biochrom, Berlin, Germany) and penicillin/streptomycin (PAA Laboratories, Pasching, Austria) and maintained at 37°C and 10% CO 2 .

Techniques: Expressing, Binding Assay, Control, Reverse Transcription Polymerase Chain Reaction, Luciferase, Mutagenesis, Construct, Transfection, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Polo-like kinase 4 transcription is activated via CRE and NRF1 elements, repressed by DREAM through CDE/CHR sites and deregulated by HPV E7 protein

doi: 10.1093/nar/gkt849

Figure Lengend Snippet: In vivo protein binding to the Plk4 promoter in untreated or 48-h doxorubicin-treated HCT116 wt, p53 −/− or p21 −/− cells was tested by ChIP. Protein binding to the GAPDHS promoter served as a negative control.

Article Snippet: HCT116 wt, HCT116 p53 − / − and HCT116 p21 −/− cells, kindly provided by Bert Vogelstein , and NIH3T3, T98G and HeLa cells (DSMZ, Braunschweig, Germany) were grown in Dulbecco's modified Eagle's medium (DMEM; Lonza, Basel, Switzerland) supplemented with 10% fetal calf serum (FCS) (Biochrom, Berlin, Germany) and penicillin/streptomycin (PAA Laboratories, Pasching, Austria) and maintained at 37°C and 10% CO 2 .

Techniques: In Vivo, Protein Binding, Negative Control

Journal: Nucleic Acids Research

Article Title: Polo-like kinase 4 transcription is activated via CRE and NRF1 elements, repressed by DREAM through CDE/CHR sites and deregulated by HPV E7 protein

doi: 10.1093/nar/gkt849

Figure Lengend Snippet: Deregulation of PLK4 expression after expression of HPV-16 E7 depends on loss of DREAM binding to the CDE and CHR elements. ( A ) HCT116 wt and HCT116 stably transfected with a plasmid encoding HPV-16 E7 wt or its ΔDLYC mutant. Relative expression of PLK4 mRNA was quantified by RT-PCR and normalized to U6 RNA levels. ( B ) Luciferase reporter assays from lysates of HCT116 wt; p53 −/− or p21 −/− cells transfected with plasmids expressing wt or CDE/CHR mutant Plk4 promoter constructs. Plasmids expressing HPV-16 E7 wt or its ΔDLYC mutant were cotransfected. Results are given as relative light units. ( C ) Nuclear extracts from HCT116 E7 wt and E7 ΔDLYC cells were analyzed by DNA affinity purification with wt and CDE/CHR mutant Plk4 promoter probes followed by western blot. Band intensities were quantified by densitometric analyses. Relative intensities are given below the bands. Intensities of input bands were normalized to E7 ΔDLYC. Binding intensities to Plk4 promoters were normalized to CDE/CHR mutant probes from E7 ΔDLYC extracts. ( D ) Protein binding to the PLK4 promoter in HeLa cells was assessed by ChIP. Protein binding to the GAPDHS promoter served as a negative control. ( E ) HCT116 E7 wt and HeLa cells were treated with doxorubicin and roscovitine for 24 h. Cells without treatment served as control. Relative expression of PLK4 mRNA was quantified by RT-PCR and normalized to U6 RNA levels. ( F ) Luciferase reporter assays with wt and CDE/CHR mutant Plk4 promoter reporter constructs in HCT116 p53 −/− cells. Cells were cotransfected with p53mut, p53wt, p21mut or p21wt expression vectors and HPV-16 E7 wt or ΔDLYC mutant. Results are given as relative light units. ( G ) Venn diagram from DREAM-bound genes ( , ), genes upregulated by HPV E7 and genes downregulated by HPV E2 proteins repressing E7 ( , ).

Article Snippet: HCT116 wt, HCT116 p53 − / − and HCT116 p21 −/− cells, kindly provided by Bert Vogelstein , and NIH3T3, T98G and HeLa cells (DSMZ, Braunschweig, Germany) were grown in Dulbecco's modified Eagle's medium (DMEM; Lonza, Basel, Switzerland) supplemented with 10% fetal calf serum (FCS) (Biochrom, Berlin, Germany) and penicillin/streptomycin (PAA Laboratories, Pasching, Austria) and maintained at 37°C and 10% CO 2 .

Techniques: Expressing, Binding Assay, Stable Transfection, Transfection, Plasmid Preparation, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Luciferase, Construct, Affinity Purification, Western Blot, Protein Binding, Negative Control, Control

Journal: Nucleic Acids Research

Article Title: Polo-like kinase 4 transcription is activated via CRE and NRF1 elements, repressed by DREAM through CDE/CHR sites and deregulated by HPV E7 protein

doi: 10.1093/nar/gkt849

Figure Lengend Snippet: p53 controls transcriptional repression of target genes by an indirect mechanism through CDE and CHR elements. High levels of p53 lead to transcriptional activation of its target p21 WAF1/CIP1 . Inhibition of cyclin/CDK complex activity by p21 WAF1/CIP1 leads to hypophosphorylation of p130. Hypophosphorylated p130, together with E2F4/DP1, can then form a repressive complex with the MuvB core replacing B-Myb in the MMB complex. MMB only requires the CHR element. The complex of p107/p130 and E2F4/DP1 with the MuvB core is named DREAM. DREAM requires both elements, the CDE and the CHR, for binding and repression. This resembles the protein complex binding to promoters during quiescence. Expression of the viral oncoprotein HPV E7 impairs p21 WAF1/CIP1 function and blocks the pocket proteins p107/p130. Disruption of the DREAM complex then leads to derepression of cell cycle genes.

Article Snippet: HCT116 wt, HCT116 p53 − / − and HCT116 p21 −/− cells, kindly provided by Bert Vogelstein , and NIH3T3, T98G and HeLa cells (DSMZ, Braunschweig, Germany) were grown in Dulbecco's modified Eagle's medium (DMEM; Lonza, Basel, Switzerland) supplemented with 10% fetal calf serum (FCS) (Biochrom, Berlin, Germany) and penicillin/streptomycin (PAA Laboratories, Pasching, Austria) and maintained at 37°C and 10% CO 2 .

Techniques: Activation Assay, Inhibition, Activity Assay, Binding Assay, Expressing, Disruption

Journal: iScience

Article Title: The TRIP12’s intrinsically disordered region induces chromatin condensates and interferes with nuclear processes

doi: 10.1016/j.isci.2025.114592

Figure Lengend Snippet: The IDR of TRIP12 is responsible for the formation of chromatin condensates (A) Prediction of TRIP12 3D-structure by AlphaFold model. Predicted local distance difference test (pLDDT) indicates the confidence level of the predicted structure. Dark blue, light blue, orange, and yellow represent very high, confident, low, and very low model confidence, respectively. (B) Graphical representation of TRIP12 different domains fused to the GFP reporter protein. Dark blue rectangles locate endogenous or artificial NLSs. IDR, intrinsically disordered region; ARM, armadillo domain; WWE, tryptophan-tryptophan-glutamate-rich domain; HECT, homologous to E6-AP carboxyl terminus; and GFP, green fluorescent protein. (C) Representative images of TRIP12-domains fused to GFP expressing HeLa S3 cells by immunofluorescence. Nuclei were counterstained with DAPI. Cells with three different GFP intensities are represented. Insertion of an artificial NLS in ARM-WWE-GFP, ΔIDR/ARM-WWE/HECT-GFP, HECT-GFP, and ΔIDR-GFP constructs allowed for nuclear localization with a faint presence in the cytoplasm. Scale bars represent 2 μm. (D) Determination of DNA granularity relative to GFP expression level in TRIP12-domains fused to GFP expressing HeLa S3 cells. For each cell, DAPI granularity and GFP expression were determined as described in “ ” on over 50 cells. Blue and green filled circles correspond to individual non-transfected and transfected cells, respectively. The linear regression curve is indicated in black. A Spearman r coefficient test and a two tailed-p value are indicated. (E) Representative images of chromatin condensates induced by TRIP12-IDR overexpression in HCT-116, U2OS, and hTert-RPE1 cell lines by immunofluorescence. Nuclei were counterstained with DAPI. Scale bars represent 2 μm. (F) Comparison of isoelectric point (pI) and capacity to form chromatin condensates (slope) of the different TRIP12-GFP constructs. The pI of the different TRIP12 fragments was determined using ProtParam on Expasy website. The capacity to form chromatin condensates is indicated by the slope values obtained in D, 2F, and C. A Spearman r coefficient test and a two tailed-p value are indicated. (G) Representative images of DNA organization in IDR-GFP deletion constructs in high expressing HeLa S3 cells (left). The cytoplasmic expression of 325-445-GFP constructs is explained by the loss of NLS sequences. Determination of DNA granularity relative to GFP expression level for the different IDR-GFP deletion constructs (right). The DAPI granularity and GFP expression were determined as described in “ ” on more than 40 cells. Blue and green filled circles correspond to individual non-transfected and transfected cells, respectively. The linear regression curve is indicated in black. Scale bars represent 2 μm. A Spearman r coefficient test and a two tailed-p value are indicated. (H) Comparison of isoelectric point (pI), capacity to form chromatin condensates (slope), and the length (in aa) of the different IDR-GFP deletion constructs. The pI and the length of the different TRIP12 fragments were determined using ProtParam on Expasy website. The capacity to form chromatin condensates was assessed from the slope values obtained in G. A Spearman r coefficient test and a two tailed-p value are indicated. (I) Representative image of cellular fractionation of HeLa S3 cells expressing 1-107-GFP, 108-207-GFP, 208-324-GFP, and 325-445-GFP constructs. GFP expression in soluble and chromatin-bound fractions was determined by western blot analysis. The level of HSP90 and PanH3 protein expression were used as loading and enrichment controls. The graph represents the percentage of GFP expression in the different fraction. Results are expressed as mean ± SEM of four different experiments. (J) Electric net charge of [1–107], [108–207], [208–324], and [325–445] fragments of TRIP12 IDR at pH 7.4 determined by Prot pi|Protein Tool (left graph). Percentage of basic, acidic, and uncharged residues in [1–107], [108–207], [208–324], and [325–445] fragments of TRIP12 IDR determined by Prot pi|Protein Tool (right graph). (K) Graphical representation of MED1-IDR fused to GFP protein. The dark blue rectangle indicates artificial NLSs. Representative images of MED1-IDR-GFP expressing HeLa S3 cells obtained by immunofluorescence. Nuclei were counterstained with DAPI. Cells with three different GFP intensities are represented. For each cell, DAPI granularity and GFP expression were determined as described in “ ” on more than 50 cells. Blue and green filled circles correspond to individual non-transfected and transfected cells, respectively. The linear regression curve is represented in black. Scale bars represent 2 μm. A Spearman r coefficient test and a two tailed-p value are indicated.

Article Snippet: HeLa S3 (CCL-2TM, female), HEK-293T (CRL-3216TM, female), U2OS (HTB-96 TM , female), hTERT RPE-1 (CRL-4000 TM , female) and HCT-116 (CCL-247 TM , male) cell lines were procured from ATCC ( https://www.atcc.org ).

Techniques: Expressing, Immunofluorescence, Construct, Transfection, Two Tailed Test, Over Expression, Comparison, Cell Fractionation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Generation of antibodies to an extracellular region of the transporters Glut1/Glut4 by immunization with a designed antigen

doi: 10.1016/j.jbc.2024.105640

Figure Lengend Snippet: Analysis of the binding of VHHs to Glut1 on the cell membranes. FCM histograms for ( A and B ) Expi293 cells transfected with Glut1 in red or empty vector in black and ( C and D ) HCT116 cells. FCM, flow cytometry; Glut1, glucose transporter 1; VHH, variable region of heavy-chain antibodies.

Article Snippet: Cell lysate samples from Expi293 overexpressing Glut1 or HCT116 were prepared with radioimmunoprecipitation assay cell lysis buffer (Santa Cruz) supplemented with cOmplete Protease Inhibitor Cocktail (Roche Applied Science).

Techniques: Binding Assay, Transfection, Plasmid Preparation, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Generation of antibodies to an extracellular region of the transporters Glut1/Glut4 by immunization with a designed antigen

doi: 10.1016/j.jbc.2024.105640

Figure Lengend Snippet: Interaction analysis of the obtained anti-Glut4 VHH antibody. A , ITC results. Binding of the VHH to Adhiron-Glut4 ( left ) and to Adhiron-WT ( right ). Table S3 lists the thermodynamic parameters. B , differences of hydrogen–deuterium exchange ratio between the data with the VHH and that without the VHH. The data including the peptide in the grafted region derived from Glut4 are shaded in purple . C , hydrogen–deuterium exchange ratio of one peptide 84 to 92 as a function of time. The region of the peptide is colored in cyan in the putative model structure of Adhiron-Glut4. The grafted region in the model structure is indicated by purple shading . D , SPR analysis of the binding of the VHH to full-length Glut4. Concentration of VHH ranged from 123 nM to 10 μM. E and F , analysis of the binding of the VHH to Glut4 on cell membranes. Flow cytometry histograms for ( E ) Expi293 cells transfected with Glut4 in red or empty vector in black and ( F ) HCT116 cells. Glut4, glucose transporter 4; ITC, isothermal titration calorimetry; SPR, surface plasmon resonance; VHH, variable region of heavy-chain antibodies.

Article Snippet: Cell lysate samples from Expi293 overexpressing Glut1 or HCT116 were prepared with radioimmunoprecipitation assay cell lysis buffer (Santa Cruz) supplemented with cOmplete Protease Inhibitor Cocktail (Roche Applied Science).

Techniques: Binding Assay, Derivative Assay, Concentration Assay, Flow Cytometry, Transfection, Plasmid Preparation, Isothermal Titration Calorimetry, SPR Assay

Journal: PLoS ONE

Article Title: Use of Fluorescence Lifetime Imaging Microscopy (FLIM) as a Timer of Cell Cycle S Phase

doi: 10.1371/journal.pone.0167385

Figure Lengend Snippet: (a,b) Images of live synchronized HCT116 cells released from APH block without (a) and with (b) BrdU labeling (100 μM, 4 h), stained with HXT (1 μM, 30 min). Scale bar is 50 μm. (c) Representative examples of HXT fluorescence decays (data trace) for individual pixels in selected nuclei (indicated by circles on (b)) showing mono- and double-exponential fittings. (d) Average τ m (left) and intensity (right) signals for no BrdU (red, n = 12) and +BrdU (blue, n = 15) nuclei. Asterisks indicate significant difference between groups (p < 0.05): *—p < 0.001, **—p < 0.00001. Error bars show the standard deviation.

Article Snippet: Tumor spheroids were formed by seeding HCT116 cells on a Lipidure-coat TM plate (Amsbio, UK) at concentration of 200 cells/ well, and by growing them for 4 days.

Techniques: Blocking Assay, Labeling, Staining, Fluorescence, Standard Deviation

Journal: PLoS ONE

Article Title: Use of Fluorescence Lifetime Imaging Microscopy (FLIM) as a Timer of Cell Cycle S Phase

doi: 10.1371/journal.pone.0167385

Figure Lengend Snippet: ( a) Asynchronous and synchronized live HCT116 cells were incubated with BrdU (100 μM, 4 h) and stained with HXT (1 μM, 30 min). Immediately after FLIM cells were fixed with 4% paraformaldehyde and stained with anti-BrdU antibody. Scale bar is 50 μm. (b) Average (n = 5) distributions of τ m . Black arrow indicates threshold τ m , which differentiates between S phase and non S-phase cells. (c) Cell proliferation rates calculated by the different methods. Bar chart shows fractions of total cell numbers and standard deviation for +BrdU cells (S-phase). The mean values were calculated from five different images of the asynchronous and synchronized cell cultures.

Article Snippet: Tumor spheroids were formed by seeding HCT116 cells on a Lipidure-coat TM plate (Amsbio, UK) at concentration of 200 cells/ well, and by growing them for 4 days.

Techniques: Incubation, Staining, Standard Deviation