Journal: Oncotarget

Article Title: Modulation of estrogen related receptor alpha activity by the kinesin KIF17

doi: 10.18632/oncotarget.18104

Figure Lengend Snippet: A. Sequence of the KIF17-Tail. The 12 amino acid NR box peptide containing the LXXLL motif is underlined. B. Co-immunoprecipitation of GFP-ERR1 with myc-EV control, myc-KIF17-T or myc-KIF17T ΔNR expressed in HEK293 cells. Immunoprecipitates and total lysates were analyzed by immunoblot using anti-GFP and anti-myc IgG. C. , D. Luciferase reporter assays showing transcriptional activity of endogenous ERR1 in MCF7 cells expressing ERRE-Luc and myc-EV, myc-KIF17-T, myc-KIF17 ΔNR (panel C) or myc-KIF17 NR (panel D). E. Luciferase reporter assays showing transcriptional activity of endogenous ERR1 in parental LM2 and LM2 NR cells transfected with ERRE-Luc. F. Same as in panel E, but comparing cells transfected with control or ERR1 siRNAs. Graphs show normalized luminescence values pooled from ≥ 3 experiments performed in triplicate. Error = SEM. *** p < 0.05.

Article Snippet: Lysates were cleared using Protein-G sepharose beads (GE Healthcare) and incubated overnight with 6-8μg rabbit anti-GFP antibody (Novus Biologicals, NB 600-303) or mouse anti-myc antibody (Sigma M4439).

Techniques: Sequencing, Immunoprecipitation, Control, Western Blot, Luciferase, Activity Assay, Expressing, Transfection

Journal: PLoS ONE

Article Title: Connective Tissue Fibroblast Properties Are Position-Dependent during Mouse Digit Tip Regeneration

doi: 10.1371/journal.pone.0054764

Figure Lengend Snippet: A) LacZ positive P3 cells were injected into the digit tip of SCID mice 1 day prior to amputation and collected at 10 DPA when the regenerate at the blastema stage. LacZ positive cells are present at the injection site in the dorsal connective tissue (*) and are scattered throughout the blastema. B) During the differentiation stage (16 DPA) LacZ positive P3 cells are primarily found in the regenerating connective tissue with small clusters of cells present within the trabeculae of the regenerating bone (arrows). C–E) GFP+ human breast cancer cells injected into P3 connective tissue prior to amputation remained aggregated in the regeneration stump and did not enter the blastema. GFP positive cells (C) shown aggregated in the stump of a 16 DPA regenerate indicating that they survive engraftment. Immunohistochemical co-staining for the endothelial marker vWF (D, E) indicates that these cells differentiate in situ without participating in the regenerative response. A and B, scale bar = 200 µm; C–E, scale bar = 100 µm.

Article Snippet: Labeled cells were identified based on immunohistochemical staining with a GFP antibody (Novus biologicals).

Techniques: Injection, Immunohistochemical staining, Staining, Marker, In Situ

Journal: PLoS ONE

Article Title: Connective Tissue Fibroblast Properties Are Position-Dependent during Mouse Digit Tip Regeneration

doi: 10.1371/journal.pone.0054764

Figure Lengend Snippet: A–C) GFP and Ki67 co-immunohistochemical reveal P3 isolated cells engraft and proliferate. A) GFP labeled P3 cells are identified within the digit. B) Ki-67, a marker for proliferation, is expressed in engrafted and endogenous P3 cells. C) Merged GFP and Ki67 expression identifies a fraction of engrafted P3 cells that are proliferating. Double labeled cells shown in C are indicated with arrows in the image set. GFP- red, Ki67- green, DAPI nuclear stain- blue. Scale bar = 50 µm. D) Quantification of the proliferation indices of P2 and P3 engrafted cells compared to endogenous neighboring cells. Left: In unamputated digits 17 days post engraftment, P3 cells proliferated at a rate similar to neighboring control cells, whereas P2 cells are non-proliferative. Right: P3 cells participating in blastema formation at 10 DPA display a proliferation index that was lower than neighboring endogenous cells but significantly higher than P2 cells within the blastema. All chart values are expressed as means ± SEM. The proliferative index of P3 cells is significantly greater than P2 cells in both studies (**, p-value <0.005).

Article Snippet: Labeled cells were identified based on immunohistochemical staining with a GFP antibody (Novus biologicals).

Techniques: Immunohistochemical staining, Isolation, Labeling, Marker, Expressing, Staining, Control

Journal: bioRxiv

Article Title: Axin1 and Axin2 regulate the WNT-signaling landscape to promote distinct mesoderm programs

doi: 10.1101/2024.09.11.612342

Figure Lengend Snippet: ( A ) UMAP generated using integrated scRNA-seq expression data, split by genotype, and colored by normalized gene expression of Kdr (top), Cdx2 (middle) and Foxf1 (bottom) in E6.5 wildtype and Axin1 ΔEpi ;Axin2 -/- embryos. ( B ) IF staining of longitudinal cryosections of E6.5 wild-type, Axin1 ΔEpi ;Axin2 -/- and Smad2 -/- embryos detecting KDR, CDX2 and FOXF1. ( C ) UMAP generated using integrated scRNA-seq expression data, split by genotype, and colored by normalized gene expression of Bmp4 (top), Bmp5 (middle) and Bmp7 (bottom) in E6.5 wildtype and Axin1 ΔEpi ;Axin2 -/- embryos. ( D ) Whole-mount in situ hybridization detecting Bmp4 expression in E6.5 control, Axin1 ΔEpi ;Axin2 -/- and Smad2 -/- embryos. Scale bar is 100μm. ( E ) IF in longitudinal sections detecting pSMAD1/5/9 and GFP on E6.5 wild-type, Axin1 ΔEpi ;Axin2 and Smad2 mutant. Scale bars on IFs are 50μm. ( B,E) ExE (Extraembryonic Ectoderm) and Em (Embryo). ( F ) Graph showing ratio of GFP (TCF/Lef:H2B); pSMAD1/5/9 and nuclei positive for both GFP and pSMAD1/5/9 relative to the total of nuclei in the E6.5 wild-type, Axin1 ΔEpi ;Axin2 -/- and Smad2 -/- embryos. Bars represent the mean± SEM. Statistically significant differences were determined by t-test *p<0.02, **p<0.004, ***p<0.0002.

Article Snippet: The following primary antibodies were incubated overnight in blocking buffer: CDH1 (1:300 Sigma-Aldrich), CDH2 (1:300, Cell Signaling Technology), T (1:400, Cell Signaling), KDR (1:200, BD Pharmigen), CDX2 (1:400, Cell Signaling), FOXF1 (1:300, R&D Systems), TBX6 (1:300, R&D Systems), pSMAD1/5/9 (1:100 Cell Signaling), FOXA2 1:500 (Abcam), GFP (1:500, R&D Systems), SOX2 (1:400 Cell Signaling), POUF5F1 (1:500, SantaCruz), NANOG (1:300 BD Biosciences).

Techniques: Generated, Expressing, Gene Expression, Staining, In Situ Hybridization, Control, Mutagenesis

Journal: bioRxiv

Article Title: Axin1 and Axin2 regulate the WNT-signaling landscape to promote distinct mesoderm programs

doi: 10.1101/2024.09.11.612342

Figure Lengend Snippet: ( A ) Cartoon describing the mice mating strategy to generate the triple mutants. “Created with BioRender.com ”. ( B ) Bright field images of E7.75 wild-type, Axin1 ΔEpi; Axin2 -/- and Axin1 ΔEpi ;Axin2 -/- ;Bmpr1a ΔEpi triple mutants. Scale bar is 100μm. ( C ) Immunostainings for TBX6 on E7.75 wild-type and Axin1 ΔEpi ;Axin2 -/- ;Bmpr1a ΔEpi triple mutants. ( D ) Quantitation of TBX6, KDR and pSMAD1/5/9-positive cells in wild-type and Axin1 ΔEpi ;Axin2 -/- ;Bmpr1a ΔEpi triple mutants embryos. Bars represent the mean± SEM. Statistically significant differences were determined by t-test. **p<0.005. ( E ) IFs for pSMAD1/5/9, GFP (TCF/Lef:H2B-GFP) and KDR in wild-type and Axin1 ΔEpi ;Axin2 -/- ;Bmpr1a ΔEpi E7.75 longitudinal sections. Scale bars on IFs are 50μm.

Article Snippet: The following primary antibodies were incubated overnight in blocking buffer: CDH1 (1:300 Sigma-Aldrich), CDH2 (1:300, Cell Signaling Technology), T (1:400, Cell Signaling), KDR (1:200, BD Pharmigen), CDX2 (1:400, Cell Signaling), FOXF1 (1:300, R&D Systems), TBX6 (1:300, R&D Systems), pSMAD1/5/9 (1:100 Cell Signaling), FOXA2 1:500 (Abcam), GFP (1:500, R&D Systems), SOX2 (1:400 Cell Signaling), POUF5F1 (1:500, SantaCruz), NANOG (1:300 BD Biosciences).

Techniques: Quantitation Assay