hek293 tet off advanced cell line  (TaKaRa)

Journal: Oncogene

Article Title: Wnt target IQGAP3 promotes Wnt signaling via disrupting Axin1-CK1α interaction

doi: 10.1038/s41388-025-03512-y

Figure Lengend Snippet: A Workflow for proximity-dependent identification of IQGAP3-associated proteins in HEK293 cells treated with either L-cells condition media (−Wnt3a) or L-Wnt3a cells condition media (+Wnt3a) treatment for 4 h, created with BioRender.com. B Venn diagram of biotinylated proteins detected in HEK293 cells treated with L-cells condition media (−Wnt3a) and L-Wnt3a cells condition media (+Wnt3a) (Fold change ≥ 4, p -value ≤ 0.01). C Interactome of −Wnt3a ∩ +Wnt3a (Fold change ≥ 4, p -value ≤ 0.01), node color denote degree distribution, created with Cytoscape. D Gene ontology: Biological Process of shared proximity partners in −Wnt3a and +Wnt3a treated cells. E Gene ontology: Molecular function of shared proximity partners in −Wnt3a and +Wnt3a treated cells. F Gene ontology: Cellular compartment of shared proximity partners in −Wnt3a and +Wnt3a treated cells.

Article Snippet: HEK293 Tet-On® 3G Cell Line #631182 was from Takara bio.

Techniques:

Journal: Oncogene

Article Title: Wnt target IQGAP3 promotes Wnt signaling via disrupting Axin1-CK1α interaction

doi: 10.1038/s41388-025-03512-y

Figure Lengend Snippet: A IQGAP3-biotinylated proteins exclusively found in cells treated with L-cells conditioned media (−Wnt3a). Shown are proximity partners with Fold change ≥ 4, p -value ≤ 0.01, node color denote degree distribution, created with Cytoscape. B IQGAP3-biotinylated proteins exclusively found in cells treated with L-Wnt3a cells conditioned media (+Wnt3a). Shown are proximity partners with Fold change ≥ 4, p -value ≤ 0.01, node color denote degree distribution, created with Cytoscape. C ClueGO analysis for Biological Process enrichment of biotinylated protein in HEK293 cells treated with −Wnt3a and +Wnt3a conditioned media. Size denote number of mapped genes and node color denote p -values. D ClueGO analysis for Cellular Compartment enrichment of biotinylated protein in HEK293 cells treated with −Wnt3a and +Wnt3a conditioned media. Size denote number of mapped genes and node color denote p -values.

Article Snippet: HEK293 Tet-On® 3G Cell Line #631182 was from Takara bio.

Techniques:

Journal: Oncogene

Article Title: Wnt target IQGAP3 promotes Wnt signaling via disrupting Axin1-CK1α interaction

doi: 10.1038/s41388-025-03512-y

Figure Lengend Snippet: A Immunoblot of endogenous IQGAP3 immunoprecipitation using an IQGAP3-specific antibody (1:100) and Mouse IgG as a control, with 1 mg of HEK293 cell lysate. Samples were subjected to SDS-PAGE (7.5%) followed by immunoblotting using the indicated antibodies. B HEK293T cells were exposed to Wnt3a conditioned medium at different time points as indicated. Stimulated lysates were subjected to immunoprecipitation using an Axin1-specific antibody and Rabbit IgG as control and were subjected to SDS-PAGE (7.5%) followed by immunoblotting using the indicated antibodies. C HEK293T cells were exposed to Wnt3a conditioned medium at different time points as indicated. Stimulated lysates were subjected to IQGAP3 immunoprecipitation using Myc-Tag antibody and Mouse IgG as control and were subjected to SDS-PAGE (7.5%) followed by immunoblotting using the indicated antibodies. D Immunoblot of Flag-Immunoprecipitated protein expressing HA-Axin1 C-terminal truncations with Flag-IQGAP3 to map the IQGAP3 binding site within Axin1. Samples were subjected to SDS-PAGE (7.5%) followed by immunoblotting using the indicated antibodies. E IQGAP3 reduced exogenous Axin1-CK1 interaction. HEK293T cells expressing Flag-Axin1 were transfected with limiting amounts of HA-CK1α and increasing amounts of Myc-IQGAP3 (5 µg/10 µg/15 µg) as indicated, and the lysates were subjected to anti-Flag immunoprecipitation. Samples were subjected to SDS-PAGE (7.5%) followed by immunoblotting using the indicated antibodies. The relative immunoblot bands of HA and Flag (HA/Flag) from the immunoprecipitation membrane were quantified by densitometry. F IQGAP3 reduced endogenous Axin1-CK1 interaction. HEK293T cells were transfected with increasing amounts of Myc-IQGAP3 (5 µg/10 µg/15 µg) as indicated, and the lysates were subjected to anti-Axin1 immunoprecipitation. Samples were subjected to SDS-PAGE (10%) followed by immunoblotting using the indicated antibodies. The relative immunoblot bands of CK1α and Axin1 (CK1α/Axin1) from the immunoprecipitation membrane were quantified by densitometry.

Article Snippet: HEK293 Tet-On® 3G Cell Line #631182 was from Takara bio.

Techniques: Western Blot, Immunoprecipitation, Control, SDS Page, Expressing, Binding Assay, Transfection, Membrane

Journal: Oncogene

Article Title: Wnt target IQGAP3 promotes Wnt signaling via disrupting Axin1-CK1α interaction

doi: 10.1038/s41388-025-03512-y

Figure Lengend Snippet: A TOPFlash assay of β-catenin co-overexpression with Axin1 or increasing IQGAP3 (300 µg, 600 µg) in HEK293T cells. The data is representative of three independent experiments. Student’s t test was performed, with ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, and ∗∗∗ p ≤ 0.001. B TOPFlash assay of β-catenin co-overexpression with siControl or increasing siIQGAP3 (20 nM, 50 nM) in HEK293T cells. The data is representative of three independent experiments. Student’s t test was performed, with ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, and ∗∗∗ p ≤ 0.001. C Immunoblot of IQGAP3 overexpression with and without Wnt3a conditioned media treatment. The data is representative of three independent experiments. D TOPFlash and Immunoblot assays of β-catenin overexpression with a series of IQGAP3 domain deletions. The data is representative of three independent experiments. HeLa cells transfected with E EGFP-empty, F EGFP-Axin1, and G EGFP-CC. Scale bars, 10 µm. H HeLa cells were transfected with EGFP-CC, subjected to FRAP bleaching, and observed for recovery and fusion events. Scale bars, 2 µm. I Mean normalized standard deviation of FRAP recovery of 10 bleached EGFP-CC condensates, half time of recovery (τ1/2) = 7.82 s. HeLa cells transfected with ( J – L ) EGFP-IQ domain and ( M – O ) mCherry2-IQGAP3ΔIQ. Scale bars, 10 µm. P Percentile of cell count for protein distribution, cells with higher nuclear intensity were counted as Nuclear > Cytosol (Nuc > Cyto), vice versa. Cell count for protein distribution, 50 cells per sample. Representative data were collected and are expressed as the mean ± SD from three independent experiments ( n = 3).

Article Snippet: HEK293 Tet-On® 3G Cell Line #631182 was from Takara bio.

Techniques: TOPFlash assay, Over Expression, Western Blot, Transfection, Standard Deviation, Cell Counting

Journal: Oncogene

Article Title: Wnt target IQGAP3 promotes Wnt signaling via disrupting Axin1-CK1α interaction

doi: 10.1038/s41388-025-03512-y

Figure Lengend Snippet: A Immunoblot of non-cancer/immortalized (HEK293T and HFE145) and gastric cancer cell lines (MKN1, MKN28, AGS, and NUGC3). Samples were subjected to SDS-PAGE (7.5%) followed by immunoblotting using the indicated antibodies. B Immunoblot of MKN28 cells expressing doxycycline-inducible 3×Flag-IQGAP3. Samples were subjected to SDS-PAGE (7.5%) followed by immunoblotting using the indicated antibodies. C GSEA data from RNA-seq of IQGAP3 overexpression in MKN28 cells, showing top 4 pathways. D Gene ontology: Biological Process Enrichment histogram of RNA-seq data from IQGAP3 overexpression in MKN28 cells. Wnt Signaling pathway represented with red bar. E Gene ontology: Molecular function Enrichment histogram of RNA-seq data from IQGAP3 overexpression in MKN28 cells. Wnt-protein and beta-catenin binding are represented with red bars. F KEGG pathway Enrichment histogram of RNA-seq data from IQGAP3 overexpression in MKN28 cells. Wnt Signaling pathway represented with red bar. G IQGAP3 interactome mapped from MKN28 cells stably expressing doxycycline-inducible TurboID-IQGAP3 (Fold change ≥ 2, p -value ≤ 0.05), node color denotes degree distribution, created with Cytoscape.

Article Snippet: HEK293 Tet-On® 3G Cell Line #631182 was from Takara bio.

Techniques: Western Blot, SDS Page, Expressing, RNA Sequencing, Over Expression, Binding Assay, Stable Transfection

Journal: Oncogene

Article Title: Wnt target IQGAP3 promotes Wnt signaling via disrupting Axin1-CK1α interaction

doi: 10.1038/s41388-025-03512-y

Figure Lengend Snippet: A HEK293T cells were treated with wnt3a conditioned media supplemented with 100 ng/ml of rhWnt3a. Cells were harvested, and the RNA collected were converted to cDNA and subjected to RT-PCR to quantify the amount of AXIN2, CCND1, MYC, LEF1, TCF7, TCF7L2 and IQGAP3 transcripts levels. B Time-course of HEK293T cells treated with Wnt3a conditioned media supplemented with 100 ng/ml of rhWnt3a. Image is best representative of three independent experiments. Samples were subjected to SDS-PAGE (7.5%) followed by immunoblotting using the indicated antibodies. The relative immunoblot bands of IQGAP3 and β-actin (IQ3/βact) were quantified by densitometry. C Luciferase assay of 5’ truncation of IQGAP3 promoter region upon treatment with L-cells conditioned or wnt3a conditioned media. D Luciferase assay of Site1 and Site2 within the IQGAP3 promoter region upon treatment with L-cells conditioned or wnt3a conditioned media. E Luciferase assay of Site1, Site1a mutant, Site1b mutant and Site1ab mutant upon treatment with L-cells conditioned or wnt3a conditioned media. F Time-course of HeLa cells treated with 5 µM of XAV939. Samples were subjected to SDS-PAGE (7.5%) followed by immunoblotting using the indicated antibodies. All the above data are representative of three independent experiments.

Article Snippet: HEK293 Tet-On® 3G Cell Line #631182 was from Takara bio.

Techniques: Reverse Transcription Polymerase Chain Reaction, SDS Page, Western Blot, Luciferase, Mutagenesis

Journal: bioRxiv

Article Title: Global cis-regulatory landscape of double-stranded DNA viruses

doi: 10.1101/2025.07.20.665756

Figure Lengend Snippet: (A) Distribution of MPRA activity ranks for HHV-1, HHV-3, HHV-4, and HHV-8 tiles that contain experimentally determined TSSs. Violin plots are shown together with box plots (median = solid black line, quartiles = box). Statistical significance determined by Kolmogorov-Smirnov test. (B) Observed versus expected distance of CREs active in HEK293T cells to the closest annotated transcription start site (TSS). Scatter plot of the observed versus expected fraction of CREs whose midpoint is located within 250 bp of an annotated TSS for each virus tested. (C) Observed versus expected distance of annotated TSSs to the closest active CRE in HEK293T cells. Scatter plot of the observed versus expected fraction of annotated TSSs located within 250 bp of the midpoint of an active CRE for each virus tested. (D) Fraction of active tiles per virus classified by CRE-class as promoter-like, proximal, distal, CTCF-only, or undetermined. Human DHSs active in K562, HepG2, or SK-N-SH cells are shown for comparison. (E) MPRA activity of tiles from each tile class active in K562 cells. For each tile, the maximum activity between the positive and negative strand is considered. The dashed red lines indicate the threshold to consider a tile active in the corresponding cell line. Violin plots are shown together with box plots (median = solid black line, quartiles = box) for each class of element in each cell line. (F) Schematic of saturation mutagenesis experiments and analyses. Each base pair of an active tile is substituted for the alternative 3 bases and activity is determined using MPRAs. Base contributions to activity are used to derive activity contributing motifs. Similarly, base contribution can be predicted using Malinois and TFmodisco-lite can be used to determine motifs that contribute to activity. (G) Example of an HPV-11:25+ tile showing the base contributions predicted using Malinois trained in K562 cells, and the best matches derived from TFmodisco-lite. Below are base contributions from the MPRA saturation mutagenesis experiments in HEK293T cells. (H) Distribution of number of motifs per active CRE tile across different viruses and human DHSs predicted to be active in K562 cells. (I) Average number of motifs per active tile derived from TFmodisco-lite and collapsed into single TF motifs. Activating and repressing motifs are shown in red and blue, respectively. (J) Average number of motifs per active tile relative to their frequency in human DHSs active in K562 cells.

Article Snippet: The assembled genome was transfected into HEK293 Tet-On® 3G cells (Takara Cat. #631185) using Lipofectamine 3000 (Invitrogen Cat. #L3000-015), following the manufacturer’s instructions.

Techniques: Activity Assay, Virus, Comparison, Mutagenesis, Derivative Assay

Journal: bioRxiv

Article Title: Global cis-regulatory landscape of double-stranded DNA viruses

doi: 10.1101/2025.07.20.665756

Figure Lengend Snippet: (A) MPRA activity maps for BK, JC, and Merkel Cell Polyomaviruses in HEK293T cells. Genome maps depicting the main genes and the noncoding control region (NCCR) are shown below. (B) Number of activating or repressing motifs for each TF in the NCCRs of the BK, JC, and Merkel Cell polyomaviruses obtained from MPRA saturation mutagenesis experiments in HEK293T cells. (C) Example of MPRA saturation mutagenesis in HEK293T cells for tiles 100+, 1+ and 3+ that cover the JC polyomavirus NCCR. Activating and repressive motifs are indicated in red and blue, respectively. (D) Violin plots of the activity measured by MPRAs in HEK293T cells for different isolates from BK, JC, and Merkel Cell polyomaviruses across tiles that cover the NCCR. The median and quartiles are indicated by bold solid and thin dashed lines, respectively. (E) Activity comparison between JC polyomavirus isolates that include the “a” region and those that do not, for tiles 100+ and 1+. Statistical significance determined by Mann-Whitney’s U test. (F) Violin plots of the activity measured by MPRAs in HEK293T cells for different isolates from BK, JC, and Merkel Cell polyomaviruses across different active tiles. The median and quartiles are indicated by bold solid and thin dashed lines, respectively.

Article Snippet: The assembled genome was transfected into HEK293 Tet-On® 3G cells (Takara Cat. #631185) using Lipofectamine 3000 (Invitrogen Cat. #L3000-015), following the manufacturer’s instructions.

Techniques: Activity Assay, Control, Mutagenesis, Comparison

Journal: bioRxiv

Article Title: Global cis-regulatory landscape of double-stranded DNA viruses

doi: 10.1101/2025.07.20.665756

Figure Lengend Snippet: (A) MPRA activity maps for different HPV strains in HEK293T cells. (B) Number of activating (pink-red) or repressing (blue) motifs for each TF in the tiles overlapping the long control region (LCR) for HPV-16 and -52. Motifs were obtained from MPRA saturation mutagenesis experiments in HEK293T cells. (C-D) Violin plots of the activity measured by MPRAs in HEK293T cells for different isolates from HPV-16 and -52 across tiles that cover the LCR. The median and quartiles are indicated by bold solid and thin dashed lines, respectively. The LCR maps and tiles that cover them are shown above. (E) Activity of 56 HPV-16 isolates for region 136+ of the LCR. The top plot shows the base contributions derived from MPRA saturation mutagenesis in HEK293T cells. The SREBF motif is indicated. The sequence alignment for region 136+ is shown across isolates. A = green, C = blue, G = yellow, T = red. The MPRA activity of each isolate is shown on the right. Purple and teal arrows indicate variants that reduce or increase activity, respectively. (F-H) MPRA saturation mutagenesis experiments in HEK293T cells for regions located in the E7 (F), E1 (G), and E2 (H) genes. TFs contributing to activity are indicated.

Article Snippet: The assembled genome was transfected into HEK293 Tet-On® 3G cells (Takara Cat. #631185) using Lipofectamine 3000 (Invitrogen Cat. #L3000-015), following the manufacturer’s instructions.

Techniques: Activity Assay, Control, Mutagenesis, Derivative Assay, Sequencing

Journal: bioRxiv

Article Title: Characterization of the BRAF interactome identifies BRAF V600E <=>TP53 interaction in melanoma

doi: 10.1101/2025.06.20.660711

Figure Lengend Snippet: A. Schematic depicting BRAF-TurboID fusion proteins and inherent ability to biotinylate proximal protein interactors, such as proteins 1 and 3, but not proteins 2 and 4. B. Representative images of endogenous BRAF and transiently transfected HA-BRAF-TurboID (wild-type and V600E) constructs in HEK293 cells as visualized by fluorescence microscopy with primary antibodies to BRAF and HA, respectively. (Scale bar = 25μM. Representative images shown from n=3 biological replicates). C. Representative images of TurboID constructs with biotinylation of endogenous proteins visualized by fluorescent neutravidin. (Scale bar = 25μM. Representative images shown from n=3 biological replicates). D. Immunoblot analyses demonstrate that all TurboID constructs are expressed following 1 μg/mL of doxycycline treatment, resulting in elevated protein biotinylation. E. Immunoblots depicting transient expression of a series of plasmids including empty vector, wild-type BRAF, and siRNA-resistant wild-type BRAF-TurboID with and without siBRAF treatment. F. Immunoblots depicting transient expression of a series of plasmids including empty vector, BRAF V600E , and siRNA-resistant BRAF V600E -TurboID with and without siBRAF treatment.

Article Snippet: HEK293 TetOn cells were purchased from Takara (631182).

Techniques: Transfection, Construct, Fluorescence, Microscopy, Western Blot, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: Characterization of the BRAF interactome identifies BRAF V600E <=>TP53 interaction in melanoma

doi: 10.1101/2025.06.20.660711

Figure Lengend Snippet: A. HEK293 cells (wild-type BRAF). B. Mel-9 cells (NRAS Q61R , wild-type BRAF). C. SKMEL-239 cells (heterozygous for BRAF V600E ). D. A375 cells (homozygous for BRAF V600E ). E. Quantification of colocalization of BRAF and TP53 in the cell lines listed above (quantification of n=4-5 fields of view from each of n=3 biological replicates; one-way ANOVA with multiple comparisons. **** = adjusted p-valued <0.0001). F. Quantification of TP53 localization in the nucleus by DAPI staining in the cell lines listed above (quantification of n=4-5 fields of view from each of n=3 biological replicates; one-way ANOVA with multiple comparisons. * = adjusted p-valued <0.03, *** = adjusted p-value <0.0009). Immunofluorescence microscopy depicting TP53 (magenta) and BRAF (green) localization in: G. Primary epidermal melanocytes expressing wild-type BRAF with no doxycycline. H. Primary epidermal melanocytes expressing BRAF V600E with the addition of doxycycline. I. Quantification of TP53 and BRAF colocalization in the epidermal melanocytes (quantification of n=4-5 fields of view from each of n=3 biological replicates; unpaired t-test p-value <0.0001). J. Quantification of TP53 localization in the nucleus by DAPI staining (quantification of n=4-5 fields of view from each of n=3 biological replicates; unpaired t-test p-value <0.0001). K. Immunoblots detecting BRAF and TP53 localization in the cytoplasm vs. nucleus after cell fractionation of SKMEL-239 cells, A375 cells, and HEK293 cells. L. Immunoblots detecting BRAF and TP53 localization in the cytoplasm vs. nucleus after cell fractionation of primary epidermal melanocytes with and without the addition of doxycycline to turn on BRAF V600E expression.

Article Snippet: HEK293 TetOn cells were purchased from Takara (631182).

Techniques: Staining, Immunofluorescence, Microscopy, Expressing, Western Blot, Cell Fractionation

Journal: bioRxiv

Article Title: Characterization of the BRAF interactome identifies BRAF V600E <=>TP53 interaction in melanoma

doi: 10.1101/2025.06.20.660711

Figure Lengend Snippet: A. PLA in human epidermal melanocytes with a doxycycline-inducible BRAF V600E with increasing doxycycline concentrations, stained with DAPI (blue) and cytoplasmic PLA signal (red) for BRAF:TP53 (Scale bar = 25μM. Representative images shown from n=3 biological replicates). B. Quantification of BRAF:TP53 PLA signal from primary epidermal melanocytes with and without increasing doses of doxycycline to induce BRAF V600E expression, reported as average PLA signals per nucleus (quantification of n=4-5 fields of view from each of n=3 biological replicates). C. Immunoblot analyses of increasing BRAF V600E expression and increased phosphorylated ERK with increasing doxycycline. D. Immunoblots of HEK293 cells treated with increasing doses of Nutlin-3a. E. Immunoblots of A375 cells treated with increasing doses of Nutlin-3a. F. Immunoblots of SKMEL-239 cells treated with increasing doses of Nutlin-3a. G. Immunoblots of primary epidermal melanocytes expressing wild-type BRAF treated with increasing doses of Nutlin-3a. H. Immunoblots of primary epidermal melanocytes with doxycycline expressing BRAF V600E treated with increasing doses of Nutlin-3a. I. Normalized RNA counts of TP53 and MDM2 from RNA-sequencing of epidermal melanocytes with doxycycline-inducible BRAF V600E . J. Relative TP53 activity in various human cell lines. The PG13-luc luciferase reporter for TP53 activity was co-transfected with a renilla control vector and dual luciferase activities were assessed. (Individual dots represent five technical replicates. One-way ANOVA for multiple comparisons, ** = adjusted P value = 0.0003). K. Relative TP53 activity in human cell lines exposed to increasing doses of UVB as indicated. The PG13-luc luciferase reporter for TP53 activity was co-transfected with a renilla control vector and dual luciferase activities were assessed. (Individual dots represent five technical replicates).

Article Snippet: HEK293 TetOn cells were purchased from Takara (631182).

Techniques: Staining, Expressing, Western Blot, RNA Sequencing, Activity Assay, Luciferase, Transfection, Control, Plasmid Preparation