l wnt-3a  (ATCC)

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: HEK293T (CRL-3216), L-Cells (CRL-2648), and L-Wnt3a cells (CRL-2647) were from ATCC.

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: HEK293T (CRL-3216), L-Cells (CRL-2648), and L-Wnt3a cells (CRL-2647) were from ATCC.

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: HEK293T (CRL-3216), L-Cells (CRL-2648), and L-Wnt3a cells (CRL-2647) were from ATCC.

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: HEK293T (CRL-3216), L-Cells (CRL-2648), and L-Wnt3a cells (CRL-2647) were from ATCC.

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: HEK293T (CRL-3216), L-Cells (CRL-2648), and L-Wnt3a cells (CRL-2647) were from ATCC.

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: HEK293T (CRL-3216), L-Cells (CRL-2648), and L-Wnt3a cells (CRL-2647) were from ATCC.

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

Journal: The Journal of Pathology

Article Title: Human colonic organoids for understanding early events of familial adenomatous polyposis pathogenesis

doi: 10.1002/path.6366

Figure Lengend Snippet: Characterization of FAP organoids. (A and B) In contrast to organoids from healthy controls (HCs), FAP‐organoid growth (NA, nonadenomatous; A, adenomatous) was independent of WNT3a‐conditioned medium or recombinant WNT3a. (A) n patients: HC = 6, NA/A = 7 (with WNT3a‐conditioned medium), NA/A = 4 (without WNT3a‐conditioned medium), organoid size mean ± SEM (diameter, around 200–300 organoids per condition), ANOVA comparison of A to HC: *** p < 0.001 **** p < 0.0001, A to NA ## p < 0.01 #### p < 0.0001, NA to HC § p < 0.05 §§ p < 0.01 §§§§ p < 0.0001. (B) n patients: HC = 3, NA/A = 3, percentage variation of organoid size from D2 mean ± SEM (diameter at D2, mean ± SEM: HC 144 ± 18 μm, NA 117 ± 16 μm, A 76 ± 17 μm; around 10 organoids per condition), ANOVA comparison: * p < 0.05 ** p < 0.01 **** p < 0.0001. (C) From D2 to D9 of culture, HC organoids develop as immature forms with a thin monolayer boarding a central lumen (cysts) to mature forms as colonospheres with a larger and polarized monolayer and then as colonoids with buds of neo‐crypts in formation. NA organoids show a delayed maturation with very large cysts. A organoid culture contains unique structures as cysts with buds. Scale bar, 100 μm. (D) Expression of proliferation marker Ki67 (immunolabeling) is increased in FAP organoids compared to HC. Labeling of nuclei by DAPI is shown. CD44 labeling is high in NA organoids and present in buds of A organoids. Representative of two independent experiments. Scale bar, 50 μm. (E) n patients: HC = 3, NA/A = 3, percentage cysts of total organoids mean ± SEM, ANOVA comparison: * p < 0.05 ** p < 0.01. (F) n patients: NA/A = 3, percentage budding structures of total organoids mean ± SEM, paired (NA to A) t ‐test comparison: * p < 0.05. A typical cyst with buds, structure unique to A organoid culture, is shown. (G) At D7 of culture, mRNA from A organoids compared to HC and/or NA organoids is enriched in markers of CBC stem cells ( EPHB2, LGR5 ), β‐catenin transcriptional targets ( AXIN2, CCND1 ), marker of cell proliferation ( MKI67 ), and β‐catenin interacting partners ( NOTCH1 , TROP2 ). Compared to HC, NA organoids are characterized by both an increase of MKI67 and a decrease of LGR4 and SPINT1 mRNA, two guardians of the crypt integrity. n patients: HC = 3–6, NA/A = 4–7, ANOVA comparison between HC and NA/A: * p < 0.05 ** p < 0.01 *** p < 0.001, paired t ‐test comparison between NA and A: * p < 0.05 ** p < 0.01.

Article Snippet: Recombinant WNT3a was added to HC organoid culture only (supplementary material, Table ), and in some experiments, WNT3a‐conditioned medium from subcutaneous connective tissue transfected with a WNT3a expression vector (ATCC®, L WNT‐3a, n°CRL‐2648) was added for both FAP and HC organoid cultures.

Techniques: Recombinant, Comparison, Expressing, Marker, Immunolabeling, Labeling