wnt3a conditioned media  (R&D Systems)

Journal: bioRxiv

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

doi: 10.1101/2024.12.10.626710

Figure Lengend Snippet: (A) Workflow for proximity-dependent entification of IQGAP3-associated proteins in HEK293 cells treated with either L-cells ndition media (-Wnt3a) or L-Wnt3a cells condition media (+Wnt3a) treatment for 4 ours, Created with BioRender.com. (B) Venn diagram of biotinylated proteins detected HEK293 cells treated with L-cells condition media (-Wnt3a) and L-Wnt3a cells ndition 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, eated with Cytoscape. (D) Gene ontology: Biological Process of shared proximity rtners in -Wnt3a and +Wnt3a treated cells. (E) Gene ontology: Molecular function of ared proximity partners in -Wnt3a and +Wnt3a treated cells. (F) Gene ontology: llular compartment of shared proximity partners in -Wnt3a and +Wnt3a treated cells.

Article Snippet: All Wnt3a conditioned media were supplemented with 100 ng/ml rhWnt3a #5036-WN (RnDSystems).

Techniques:

Journal: bioRxiv

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

doi: 10.1101/2024.12.10.626710

Figure Lengend Snippet: (A) IQGAP3-biotinylated proteins exclusively found in cells treated with L-cells ditioned media (-Wnt3a). Shown are proximity partners with Fold change ≥ 4, p-value ≤ , node color denote degree distribution, Created with Cytoscape. (B) IQGAP3- inylated proteins exclusively found in cells treated with L-Wnt3a cells conditioned media nt3a). Shown are proximity partners with Fold change ≥ 4, p-value ≤ 0.01, node color ote degree distribution, Created with Cytoscape. (C) ClueGO analysis for Biological cess enrichment of biotinylated protein in HEK293 cells treated with -Wnt3a and +Wnt3a ditioned media. Size denote number of mapped genes and node color denote p-values. ClueGO analysis for Cellular Compartment enrichment of biotinylated protein in HEK293 s treated with -Wnt3a and +Wnt3a conditioned media. Size denote number of mapped es and node color denote p-values.

Article Snippet: All Wnt3a conditioned media were supplemented with 100 ng/ml rhWnt3a #5036-WN (RnDSystems).

Techniques:

Journal: bioRxiv

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

doi: 10.1101/2024.12.10.626710

Figure Lengend Snippet: (A) Immunoblot analysis of Doxycycline ction in HeLa Tet-On cells. Cells were induced with Doxycycline for 48 hours prior to cell . (B) Live images of HeLa Tet-On cells expressing EGFP-empty and EGFP-IQGAP3 without with Wnt3a treatment after 30 minutes. Cells were induced with Doxycycline for 48 rs prior to imaging. Images of untreated and post-treated cells are not the same cells but the best representative examples. Scale bars, 10 µm. (C) Timelapse images of HeLa Tet- cells expressing EGFP-IQGAP3 0 - 50 minutes post-Wnt3a treatment. Cells were induced Doxycycline for 48 hours prior to imaging. Images of untreated and post-treated cells the same cells (refer to timelapse video in supplementary). Scale bars, 10 µm. (D) elapse images of HeLa Tet-On cells expressing EGFP-IQGAP3 treated with either 1% 2,5- anediol (control) or 1% 1,6-Hexanediol for 0-5 minutes. Images of untreated and post- ted cells are the same cells (refer to timelapse video in supplementary). Scale bars, 10 (E) HeLa Tet-On cells stably expressing EGFP-IQGAP3 were subjected to FRAP bleaching observed for recovery and fusion events. (For FRAP video refer to supplementary). Scale , 2 µm. (F) Mean normalized standard deviation of FRAP recovery of 5 bleached EGFP- AP3 condensates, half time of recovery (τ1/2) = 30.87s.

Article Snippet: All Wnt3a conditioned media were supplemented with 100 ng/ml rhWnt3a #5036-WN (RnDSystems).

Techniques: Western Blot, Expressing, Imaging, Control, Stable Transfection, Standard Deviation

Journal: bioRxiv

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

doi: 10.1101/2024.12.10.626710

Figure Lengend Snippet: (A) Immunoblot of endogenous AP3 immunoprecipitation using an IQGAP3-specific antibody (1:100) and Mouse IgG as a trol, with 1 mg of HEK293 cell lysate. Samples were subjected to SDS-PAGE (7.5%) wed by immunoblotting using the indicated antibodies. (B) HEK293T cells were exposed Wnt3a conditioned medium at different time points as indicated. Stimulated lysates were jected to immunoprecipitation using an Axin1-specific antibody and Rabbit IgG as control were subjected to SDS-PAGE (7.5%) followed by immunoblotting using the indicated bodies. (C) HEK293T cells were exposed to Wnt3a conditioned medium at different time ts as indicated. Stimulated lysates were subjected to IQGAP3 immunoprecipitation using-Tag antibody and Mouse IgG as control and were subjected to SDS-PAGE (7.5%) wed by immunoblotting using the indicated antibodies. (D) Immunoblot of Flag- unoprecipitated protein expressing HA-Axin1 C-terminal truncations with Flag-IQGAP3 map the IQGAP3 binding site within Axin1. Samples were subjected to SDS-PAGE (7.5%) wed by immunoblotting using the indicated antibodies. (E) IQGAP3 reduced exogenous 1-CK1 interaction. HEK293T cells expressing Flag-Axin1 were transfected with limiting unts of HA-CK1α and increasing amounts of Myc-IQGAP3 (5µg/10µg/15µg) as indicated, the lysates were subjected to anti-Flag immunoprecipitation. Samples were subjected to -PAGE (7.5%) followed by immunoblotting using the indicated antibodies. The relative unoblot bands of HA and Flag (HA/Flag) from the immunoprecipitation membrane were ntified by densitometry. (F) IQGAP3 reduced endogenous Axin1-CK1 interaction. 293T cells were transfected with increasing amounts of Myc-IQGAP3 (5µg/10µg/15µg) ndicated, and the lysates were subjected to anti-Axin1 immunoprecipitation. Samples e subjected to SDS-PAGE (10%) followed by immunoblotting using the indicated bodies. The relative immunoblot bands of CK1α and Axin1 (CK1α/Axin1) from the unoprecipitation membrane were quantified by densitometry.

Article Snippet: All Wnt3a conditioned media were supplemented with 100 ng/ml rhWnt3a #5036-WN (RnDSystems).

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

Journal: bioRxiv

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

doi: 10.1101/2024.12.10.626710

Figure Lengend Snippet: (A) TOPFlash y of β-catenin co-overexpression with Axin1 or increasing IQGAP3 (300µg, 600µg) in 293T cells. The data is representative of three independent experiments. Student’s t test performed, with ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. (B) TOPFlash assay of β- nin co-overexpression with siControl or increasing siIQGAP3 (20nM, 50nM) in HEK293T s. The data is representative of three independent experiments. Student’s t test was ormed, with ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. (C) Immunoblot of IQGAP3 rexpression with and without Wnt3a conditioned media treatment. The data is esentative of three independent experiments. (D) TOPFlash and Immunoblot assays of tenin overexpression with a series of IQGAP3 domain deletions. The data is esentative of three independent experiments. HeLa cells transfected with (E) EGFP- pty, (F) EGFP-Axin1, and (G) EGFP-CC. Scale bars, 10 µm. (H) HeLa cells were transfected EGFP-CC, subjected to FRAP bleaching, and observed for recovery and fusion events. e bars, 2µm. (I) Mean normalized standard deviation of FRAP recovery of 10 bleached P-CC condensates, half time of recovery (τ1/2) = 7.82s. HeLa cells transfected with (J-L) P-IQ domain and (M-O) mCherry2-IQGAP3ΔIQ. Scale bars, 10 µm. (P) Percentile of cell nt for protein distribution, cells with higher nuclear intensity were counted as Nuclear > osol (Nuc > Cyto), vice versa. Cell count for protein distribution, 50 cells per sample. resentative data were collected and are expressed as the mean ± SD from three pendent experiments (n=3).

Article Snippet: All Wnt3a conditioned media were supplemented with 100 ng/ml rhWnt3a #5036-WN (RnDSystems).

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

Journal: bioRxiv

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

doi: 10.1101/2024.12.10.626710

Figure Lengend Snippet: p. (A) HEK293T cells were treated with wnt3a conditioned media supplemented with ng/ml of rhWnt3a. Cells were harvested, and the RNA collected were converted to cDNA subjected to RT-PCR to quantify the amount of AXIN2, CCND1, MYC, LEF1, TCF7, TCF7L2 IQGAP3 transcripts levels. (B) Time-course of HEK293T cells treated with Wnt3a ditioned media supplemented with 100ng/ml of rhWnt3a. Image is best representative hree independent experiments. Samples were subjected to SDS-PAGE (7.5%) followed by unoblotting using the indicated antibodies. The relative immunoblot bands of IQGAP3 β-actin (IQ3/βact) were quantified by densitometry. (C) Luciferase assay of 5’ truncation QGAP3 promoter region upon treatment with L-cells conditioned or wnt3a conditioned ia. (D) Luciferase assay of Site1 and Site2 within the IQGAP3 promoter region upon tment with L-cells conditioned or wnt3a conditioned media. (E) Luciferase assay of Site1, 1a mutant, Site1b mutant and Site1ab mutant upon treatment with L-cells conditioned wnt3a conditioned media. All the above data are representative of three independent eriments.

Article Snippet: All Wnt3a conditioned media were supplemented with 100 ng/ml rhWnt3a #5036-WN (RnDSystems).

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