Journal: Development (Cambridge, England)

Article Title: Developmental vascular regression is regulated by a Wnt/β-catenin, MYC and CDKN1A pathway that controls cell proliferation and cell death

doi: 10.1242/dev.154898

Figure Lengend Snippet: Myc is required for cell proliferation and apoptosis in hyaloid VECs. Flat-mounted P8 hyaloid vessels from VEC conditional Myc heterozygote and homozygote mice as labeled. There is dramatic hyaloid persistence in homozygous Mycflox/flox; Pdgfb-icreERT2 mice (C) and mild persistence in heterozygous Myc (Mycflox/+; Pdgfb-icreERT2) mice (B) compared with wild-type littermate controls (Myc+/+; Pdgfb-icreERT2) (A). (D) Quantification of hyaloid vessel numbers in mice of the labeled genotypes. (E,F) Flat-mounted hyaloid vessels from P5 animals were labeled for BrdU (n≥6) (E,F) or TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) (H,I) along with Hoechst. Quantification of BrdU-positive nuclei (G) and TUNEL-positive apoptotic vascular segments (J). Data are mean±s.e.m. Statistical significance was tested using Student's t-test, n≥4. (K) Immunoblot showing active β-catenin (CTNNB1), MYC and β-tubulin (TUBB) in Lrp5/6 control and CKO BMVECs stimulated with Wnt3a.

Article Snippet: For induction of Wnt or the VEGF signaling pathway, Wnt3a (R&D Systems, 1324-WNP-010, 100 ng/ml) and hrVEGFA (Goldbio, #1350, 100 ng/ml) were used, respectively.

Techniques: Labeling, TUNEL Assay, Western Blot

Journal: bioRxiv

Article Title: The Amyloid Precursor Protein is a conserved Wnt receptor

doi: 10.1101/2021.01.18.426557

Figure Lengend Snippet: (A) APPL extracellular region contains a conserved CRD. The figure shows a CLUSTAL alignment of the CRD of different APP homologs. The 12 cysteine residues (as indicated by the red asterisks) are highly conserved across species. (B-B’) Wnt5a binds APPL and APP in a CRD dependent manner. (B) Co-immunoprecipitation (co-IP) of the full-length proteins APP-flag and APPL-flag but not their truncated forms APP ΔCRD -flag and APPL ΔCRD -flag with Wnt5a-myc. (B’) Reciprocal co-IP showing that Wnt5a-myc is co-IPed with flAPP-flag and APPL-flag can but not when the CRDs are deleted.

Article Snippet: Wnt5a(400ng/ml)(645-WN-010, R&D Systems), Wnt3a(150ng/ml)(1324-WNP-010, R&D Systems) and PBS/BSA(control) addition performed at Div 7.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: The Amyloid Precursor Protein is a conserved Wnt receptor

doi: 10.1101/2021.01.18.426557

Figure Lengend Snippet: (A) Co-immunoprecipitation (co-IP) of Wnt5a-Myc with full-length proteins mAPP-flag or mAPP-delatCRD-flag. The tagged proteins were co-expressed in HEK293T cells and immunoprecipiteted with ant-flag or anti-Myc antibody, wild type mAPP pulled down Wnt5a and vice versa, while mAPP lacking the CRD showed impaired ability to pull down Wnt5a even with higher protein levels compared to wild type mAPP in the input. (B) mAPP localization after 4 hours PBS/BSA or Wnt5a treatment. Immunofluorescence using antibodies to APP, Rab5 (early endosome marker) Golgin97 (TGN marker) or Lamp1 (lysosome marker) to reveal mAPP localization in different intracellular compartments, the inset shows a high magnification image of the area in the white box and arrows indicate the overlap of mAPP with respective cellular compartment marker. (C-E) Quantification of the overlap between mAPP and early endosome TGN or lysosome respectively after Wnt5a treatment. (F) mAPP protein expression is altered after Wnt5a treatment, Western blotting for mAPP and Actin was done on lysates from DIV7 primary cortical neurons. (G) Quantification of the Western blot result for fig F. (H) mAPP mRNA is not affected after Wnt5a treatment, qPCR for mAPP and actin was done in mRNA sample from DIV7 primary cortical neurons, APP−/− mice derived primary neurons were used as a negative control. (I-J) The lysosome inhibitor Bafilomycin rescues Wnt5a-induced mAPP reduction in mAPP protein levels. (I) untreated controls. (J) Cells treated with the Bafilomycin. (K-L) Quantification of the western blot result for fig(I-J). Bars represent the mean±s.e.m. for at least three independent experiments. 40-50 cells from at least two independent experiments were analyzed for each group. *P<0.05, **P<0.01. Scale bar = 10um.

Article Snippet: Wnt5a(400ng/ml)(645-WN-010, R&D Systems), Wnt3a(150ng/ml)(1324-WNP-010, R&D Systems) and PBS/BSA(control) addition performed at Div 7.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Immunofluorescence, Marker, Expressing, Western Blot, Derivative Assay, Negative Control

Journal: bioRxiv

Article Title: The Amyloid Precursor Protein is a conserved Wnt receptor

doi: 10.1101/2021.01.18.426557

Figure Lengend Snippet: (A) Co-immunoprecipitation (co-IP) of Wnt3a-V5 with full-length proteins mAPP-flag or mAPPL ΔCRD. The tagged proteins were co-expressed in HEK293T cells and immunoprecipiteted with ant-flag or anti-v5 antibody, wild type mAPP pulled down Wnt3a and vice versa, while mAPP lacking the CRD showed impaired ability to pull down Wnt3a even with higher protein levels compared to wild type mAPP in the input. (B) mAPP localization after 4 hours PBS/BSA or Wnt3a treatment. Immunofluorescence using antibodies to APP, Rab5, Golgin97 or Lamp1 to reveal mAPP localization in different intracellular compartments, the inset shows a high magnification image of the area in the white box and arrows indicate the overlap of mAPP with respective cellular compartment marker. (C-E) Quantification of the overlap between mAPP and early endosome, TGN or lysosome, respectively, after Wnt3a treatment. (F) mAPP protein expression after Wnt3a treatment. Western blotting for mAPP and Actin was done on lysates from DIV7 primary cortical neurons. (G) Quantification of the Western blot results for fig F. (H) mAPP mRNA is not affected after Wnt3a treatment, qPCR for mAPP and actin was done in mRNA sample from DIV7 primary cortical neurons, APP−/− mice derived primary neurons were used as a negative control. (I-J) The Retromer inhibitor Ly294002 rescues Wnt3a-induced increase in mAPP expression levels (I) untreated controls. (J) Cells treated with Ly294002. (K-L) Quantification of the Western blot result for fig(I-J). (M-O) Wnt5a and Wnt3a working in a competing way on affecting mAPP protein expression. (M) Western blot performed with cell lysate from the DIV7 primary cortical neuron treated with Wnt3a and Wnt5a at the same time for 4 hours, PBS/BSA group act as control group. (N) Quantification of the Western blot result for fig M. (O) qPCR results of mAPP knockout neurons (negative control) and PBS/BSA or Wnt3a+Wnt5a treated neurons. Bars represent the mean±s.e.m. for at least three independent experiments. 40-50 cells from at least two independent experiments were analyzed for each group. *P<0.05, **P<0.01. Scale bar = 10um.

Article Snippet: Wnt5a(400ng/ml)(645-WN-010, R&D Systems), Wnt3a(150ng/ml)(1324-WNP-010, R&D Systems) and PBS/BSA(control) addition performed at Div 7.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Immunofluorescence, Marker, Expressing, Western Blot, Derivative Assay, Negative Control, Knock-Out

Journal: bioRxiv

Article Title: The Amyloid Precursor Protein is a conserved Wnt receptor

doi: 10.1101/2021.01.18.426557

Figure Lengend Snippet: (A-D) CRD is critical for Wnt3a/5a regulation of mAPP protein expression. (A-B) APP knock-out primary cortical neuron expressing exogenous wild type or CRD mutant mouse APP via lenti-virus transduction were treated with Wnt3a or Wnt5a at DIV7, 4 hours later protein samples were collected for Western blots. Wnt3a upregulated fl-mAPP and Wnt5a downregulated mAPP (A), in contrast both Wnt3a or Wnt5a failed to affect mAPPΔCRD expression (B). (C-D) quantification of the Western blots results for figure A and B respectively. (E-H) Routing of mAPP trafficking by Wnt3a/5a requires the CRD. (E) Localization of exogenous mAPP in APP knock-out primary cortical neurons after 4 hours of Wnt3a or Wnt5a treatment. Immunofluorescence using antibodies to APP, Rab5 (early endosome marker) Golgin97 (TGN marker) or Lamp1 (lysosome marker) to reveal mAPP localization in different intracellular compartments, the inset shows a high magnification image of the area in the white box and arrows indicate the overlap of mAPP with respective cellular compartment marker. (F-H) Quantification of the overlap between mAPP and early endosome, TGN, or lysosome, respectively, after Wnt3a or Wnt5a treatment. Bars represent the mean±s.e.m. for at least three independent experiments. 40-50 cells from at least two independent experiments were analyzed for each group. *P<0.05, Scale bar = 10um.

Article Snippet: Wnt5a(400ng/ml)(645-WN-010, R&D Systems), Wnt3a(150ng/ml)(1324-WNP-010, R&D Systems) and PBS/BSA(control) addition performed at Div 7.

Techniques: Expressing, Knock-Out, Mutagenesis, Transduction, Western Blot, Immunofluorescence, Marker

Journal: bioRxiv

Article Title: The Amyloid Precursor Protein is a conserved Wnt receptor

doi: 10.1101/2021.01.18.426557

Figure Lengend Snippet: (A-D) Interaction between exogenous wild type mAPP with Wnts (A) location of exogenous mAPP in APP knock-out primary cortical neurons After 4 hour Wnt3a or Wnt5a treatment. Immunofluorescence using antibody app with rab5(early endosome marker) golgin97(TGN marker) or lamp1(lysosome marker) to reveal mAPP location in different intracellular compartment, the inset fig with White arrow is a high zoom in of the area in white box, arrow indicate the overlap of mAPP with relative cellular compartment. (B-D) quantification of the overlap between mAPP and early endosome TGN or lysosome respectively after Wnt3a or Wnt5a treatment. *P<0.05. scale bar = 10um.

Article Snippet: Wnt5a(400ng/ml)(645-WN-010, R&D Systems), Wnt3a(150ng/ml)(1324-WNP-010, R&D Systems) and PBS/BSA(control) addition performed at Div 7.

Techniques: Knock-Out, Immunofluorescence, Marker