Journal: bioRxiv

Article Title: Fgf9-Nolz-1-Wnt2 Signaling Axis Regulates Morphogenesis of the Lung

doi: 10.1101/2022.08.10.503529

Figure Lengend Snippet: (A) Treatment with rFgf10 (200 n /ml) of wild type explant lung culture for 48 hr. qRT-PCR shows that Nolz-1 and Wnt2 are not changed in the rFgf10 treated group compared to the vehicle control. Student’s t -test, P > 0.05, n = 4. Scale bar, 500 μm. (B) Treatment with rFgf9 (200 ng/ml) results in enlarged epithelia in wild-type explant lungs cultured for 48 hr. The qRT-PCR shows that Nolz-1 , Wnt2 and Lef1 mRNAs are increased in rFgf9 treated group in wild type lungs. Student’s t -test, * P < 0.05, ** P < 0.01, n = 4. Western blotting showed that rFgf9 treatment increases Nolz-1 protein by 99% in wild type lung culture compared to the vehicle-treated group. Student’s t -test, * P < 0.05, n = 3. (C) Working hypothesis. Nolz-1 controls the proliferation of mesenchymal cells and the growth of epithelial branches through the regulation of Wnt2 signaling in the early stages of the development of the lungs. In the late stages of development, Nolz-1 acts non-cell autonomously to regulate the development of epithelial cells through Wnt2 signaling. Fgf9 acts upstream to regulate Nolz-1 expression in developing lungs.

Article Snippet: The lung explants were cultivated with recombinant mouse Fgf9 protein (200 ng/ml, R & D Systems, Inc.) or Fgf10 protein (200 ng/ml, R & D Systems, Inc.).

Techniques: Quantitative RT-PCR, Control, Cell Culture, Western Blot, Expressing

Journal: PLoS Biology

Article Title: Hierarchical patterning modes orchestrate hair follicle morphogenesis

doi: 10.1371/journal.pbio.2002117

Figure Lengend Snippet: (A) Schematic illustrating the 2 general theoretical models explaining the emergence of repeating patterns: Turing reaction–diffusion-driven systems and mesenchymal self-organisation. (B) Schematic of the process of hair follicle formation depicting acquisition of epidermal foci of Dkk4 in placodes coupled with the underlying mesenchymal cell accumulation. (C) Pattern responses of skin to stimulation and repression of bone morphogenetic protein (BMP), fibroblast growth factor (FGF), and wingless-related integration site (WNT) pathways. Detection of the epidermal placode marker Dkk4 and cell arrangement through green fluorescent protein (GFP) signal in the TCF/Lef::H2B-GFP line in E13.5 mouse dorsal skin explant cultures treated for 27 h with BMP4 (500 ng/ml), LDN193189 (BMP receptor inhibitor) (10 μM), FGF9 (1 μg/ml), SU5402 (FGFR inhibitor) (25 μM), CHIR99021 (GSK3 inhibitor to stimulate WNT/β-catenin signalling) (10 μM), or IWR-1 (WNT/β-catenin signalling inhibitor) (50 μM). Scale bars: 250 μm. (D) Heatmap depicting quantitative reverse transcription polymerase chain reaction (qRT-PCR) fold changes of candidate genes in response to 6 h stimulation or inhibition of the BMP, FGF, or WNT signalling pathways. Pie charts show the fraction of dermal (yellow) to epidermal (purple) expression of each gene detected in unstimulated skin (see ). Statistical significance from control skins was calculated using a Student t test (* p < 0.05 and > ± 1.8-fold change). The raw numerical data for the heatmap can be found in . (E) Gene regulatory network derived from the transcriptional responses to BMP, FGF, or WNT pathway stimulation shown in (D). (F) Dispersion curves calculated from matrix of interactions show that Turing instability (curve breaking the x-axis) can be achieved by a regulatory network with this structure when components of each pathway can diffuse (see for details).

Article Snippet: Recombinant FGF7 (mouse), FGF9 (mouse and human), BMP4 (mouse), and TGFβ2 (human) were from R&D Systems.

Techniques: Diffusion-based Assay, Marker, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Inhibition, Expressing, Control, Derivative Assay, Dispersion

Journal: PLoS Biology

Article Title: Hierarchical patterning modes orchestrate hair follicle morphogenesis

doi: 10.1371/journal.pbio.2002117

Figure Lengend Snippet: (A) Single frames from time-lapse sequences of E13.5 TCF/Lef::H2B-green fluorescent protein (GFP) skin explant culture captured by confocal microscopy. Dashed circles indicate ultimate condensate location. Scale bar: 50 μm. (B) Analysis of tracked cells showing the probability of joining the dermal condensate based upon initial location relative to its centre. Two hundred and forty individual cells were tracked across 8 condensates from 4 independent skins. (C) Protractor plot showing the distribution of Euclidean angles and Euclidean distances of individual cell movements in 6-h windows for cell tracks that start outside of, but ultimately terminate in, a follicle (condensate = red) and those that remain outside (intercondensate = blue). Tracking was halted on cell entry. (D) Plots showing the mean Euclidean angle (top) and mean level of persistence (bottom) of condensate-entering and intercondensate cells for 6-h windows relative to time of entry into the condensate. Error bars represent SEM (condensate cells n = 9, 14, and 20 and intercondensate n = 263, 245, and 197 for 12, 6, and 0 h before entry, respectively). Statistical significance was calculated using a Kruskal–Wallis test ( p < 0.0001 and p < 0.001 for angle and persistence, respectively) followed by Mann–Whitney U tests with Bonferroni’s correction (** p < 0.01). The raw numerical tracking data (for B, C, and D) can be found in . (E) Detection of a molecular prepattern prior to dermal condensate formation. TCF/Lef::H2B-GFP skin explants were fixed at intermediate stages of pattern formation, imaged to detect GFP, and Dkk4 expression determined in the same skin sample. Asterisk represents an area where Dkk4 -positive foci are present but corresponding dermal condensates are absent. Scale bar: 500 μm. (F) Time-lapse images of E12.75 TCF/Lef::H2B-GFP dorsal skin explants cultured with recombinant fibroblast growth factor (FGF) 9- or bovine serum albumin (BSA)-loaded beads. Cells accumulate around FGF9-loaded beads. Scale bar: 250 μm.

Article Snippet: Recombinant FGF7 (mouse), FGF9 (mouse and human), BMP4 (mouse), and TGFβ2 (human) were from R&D Systems.

Techniques: Confocal Microscopy, MANN-WHITNEY, Expressing, Cell Culture, Recombinant

Journal: PLoS Biology

Article Title: Hierarchical patterning modes orchestrate hair follicle morphogenesis

doi: 10.1371/journal.pbio.2002117

Figure Lengend Snippet: (A) Detection of green fluorescent protein (GFP) signal, Dkk4 expression, and histological appearance of TCF/Lef::H2B-GFP skin explants cultured with fibroblast growth factor (FGF) 9 (1 μg/ml), LDN193189 (LDN) (10 μM), or both agents. Large, periodically spaced dermal condensates form in the absence of epidermal placodes when FGF and LDN are administered. Scale bars: H2B-GFP & Dkk4 : 250 μm, haemotoxylin and eosin (HE): 50 μm. (B) Frozen sections of TCF/Lef::H2B-GFP dorsal skin explants treated as indicated and stained for neural cell adhesion molecule (NCAM) expression. Arrows indicate epidermal placode. Scale bar: 20 μm. (C) Expression of epidermal placode ( Shh , Edar , Bmp2 ) and dermal condensate ( Bmp4 , Sox2 ) marker genes in dorsal skin explants cultured with FGF and LDN. Scale bar: 250 μm. (D) Epidermis and dermis isolated from E13.5 TCF/Lef::H2B-GFP skin explants cultured with FGF9 and LDN, counterstained with propidium iodide (PI) and imaged using confocal microscopy. Epidermis is unpatterned while the large dermal condensates are accompanied by cell depletion from the intervening spaces. Scale bar: 100 μm.

Article Snippet: Recombinant FGF7 (mouse), FGF9 (mouse and human), BMP4 (mouse), and TGFβ2 (human) were from R&D Systems.

Techniques: Expressing, Cell Culture, Staining, Marker, Isolation, Confocal Microscopy

Journal: PLoS Biology

Article Title: Hierarchical patterning modes orchestrate hair follicle morphogenesis

doi: 10.1371/journal.pbio.2002117

Figure Lengend Snippet: (A, B) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) of E13.5 or E13.75 (with condensates) skins treated with transforming growth factor (TGF) β2, fibroblast growth factor (FGF) 9, or bone morphogenetic protein (BMP) 4 for 8 or 24 h, respectively, followed by assessment of transcript abundance. TGFβ2 upregulates expression of genes associated with cell movement and the extracellular matrix. Statistical significance from control was calculated using a Student t test (* p < 0.05, ** p < 0.01, *** p < 0.001). Error bars represent SEM from at least 3 independent experiments. (C) Cell aggregation at FGF9 beads in E12.5 TCF/Lef::H2B-green fluorescent protein (GFP) skin explants. TGFβ2 (100 ng/ml) or LY2109761 (25 μM) is present in the culture medium as indicated. TGFβ2 enhances aggregation at FGF9 beads, while LY2109761 suppresses cell accumulation. (D) FGF9 presence in culture medium does not detectably increase cell recruitment to TGFβ2 beads. (E, F) Quantification of areas of high cell density around FGF9- or TGFβ2-coated beads under conditions as indicated. Statistical significance was calculated using Student t tests (* p < 0.05, ** p < 0.01, *** p < 0.001). Error bars represent SEM of at least 3 independent experiments. Scale bars: 250 μm. The raw numerical values (for A, B, E, and F) can be found in .

Article Snippet: Recombinant FGF7 (mouse), FGF9 (mouse and human), BMP4 (mouse), and TGFβ2 (human) were from R&D Systems.

Techniques: Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Control

Journal: Oncotarget

Article Title: Frequently rearranged and overexpressed δ-catenin is responsible for low sensitivity of prostate cancer cells to androgen receptor and β-catenin antagonists

doi: 10.18632/oncotarget.25319

Figure Lengend Snippet: ( A ) Graphical representation of CTNND2 expression in PCa cases based on microarray analysis . Green corresponds to normal prostate epithelial cells, light blue is prostatic intraepithelial neoplasia (PIN), dark blue is Gleason pattern (GP) 3, magenta is GP4, orange is GP5, red corresponds to metastases, cell lines are shown in purple. Grey and black are bulk tissue (without laser capture microdissection) with good and bad (systemic progression) outcome respectively. Tumors are grouped into lacking TMPRSS-ERG fusion gene (ERG-), harboring TMPRSS-ERG fusion gene (ERG+) and harboring ETV fusion gene (ETV+). ( B ) Comparison of catenin binding complexes between different PCa cell lines. Cell lysates (50 μg) from BPH1, Du145, 22RV1, LNCaP and VCaP were subjected to nondenaturing gel electrophoresis and probed with indicated antibodies. ( C and D ). Characterization of binding partners of δ-catenin and β-catenin in LNCaP cells. Protein (300 μg) isolated from LNCaP cells was subjected to immunoprecipitation using IgG, anti-α-catenin, anti-β-catenin, anti-E-cadherin, anti-p120 or anti-δ-catenin antibodies immobilized on beads. The immunoprecipitates were resolved on SDS-PAGE electrophoresis and blotted with the indicated antibodies.

Article Snippet: The following antibodies were used for western blotting and co-immunoprecipitation: δ-catenin (Abnova, cat. H00001501-A01), β-catenin (Sigma, cat. c2206), p120 (Santa Cruz, cat. SC-13957), α-catenin (Novus, cat. NB100-74356), cyclin D1 (Cell Signaling, cat. 2922S), NKX3.1 (R&D systems; cat. AF6080) and Slug (Cell signaling; cat. 9585).

Techniques: Expressing, Microarray, Laser Capture Microdissection, Binding Assay, Nucleic Acid Electrophoresis, Isolation, Immunoprecipitation, SDS Page, Electrophoresis

Journal: Oncotarget

Article Title: Frequently rearranged and overexpressed δ-catenin is responsible for low sensitivity of prostate cancer cells to androgen receptor and β-catenin antagonists

doi: 10.18632/oncotarget.25319

Figure Lengend Snippet: ( A ) SDA-PAGE and Western blot analysis of δ-catenin and β-catenin levels in clones (designated O8 and M1) with targeted CTNND2 gene. ( B ) Western blot analysis of indicated proteins in O8 and M1 clones. 30 μg of cell lysates were used in A and B, representative gels and corresponding quantification are shown. ( C ) Changes in cell migration were examined using Boyden chamber assay. Shown are images of stained cells with invasive phenotype after 36 hours. OD is optic density in arbitrary units measured at 36 hr. ( D ) Soft agar colony formation assay. Images of representative cells (left panel) and quantification (right panel) are shown. Data are presented as mean ± SD, based on 3 independent experiments. P values are as indicated. Quantification of expression of catenin proteins normalized to GAPDH expression is shown. Con is a vector only transfected control. ( E ) Proliferation of VCaP clones with knocked down expression of δ-catenin compared to parental cells at 48 hours after plating. Data presented as per cent of control parental VCaP cells. Mean and ±SD are shown based on 3 independent experiments, p values are as indicated. ( F ) SDA-PAGE and Western blot analysis of AR and NKX3.1 levels in VCaP clones and their corresponding quantification.

Article Snippet: The following antibodies were used for western blotting and co-immunoprecipitation: δ-catenin (Abnova, cat. H00001501-A01), β-catenin (Sigma, cat. c2206), p120 (Santa Cruz, cat. SC-13957), α-catenin (Novus, cat. NB100-74356), cyclin D1 (Cell Signaling, cat. 2922S), NKX3.1 (R&D systems; cat. AF6080) and Slug (Cell signaling; cat. 9585).

Techniques: Western Blot, Clone Assay, Migration, Boyden Chamber Assay, Staining, Soft Agar Assay, Expressing, Plasmid Preparation, Transfection

Journal: Oncotarget

Article Title: Frequently rearranged and overexpressed δ-catenin is responsible for low sensitivity of prostate cancer cells to androgen receptor and β-catenin antagonists

doi: 10.18632/oncotarget.25319

Figure Lengend Snippet: ( A ) Comparison of levels of δ-catenin in overexpressing LNCaP clones and other PCa cell lines. Full length protein is depicted by arrow. ( B ) Levels of catenin proteins in δ-catenin overexpressing LNCaP clone (designated as OE1b) are shown. Gradient loading of total protein for OE1b clone was used to illustrate an increase in expression of each protein (right panel). Quantification graphs showing protein levels normalized to GAPDH level (arbitrary units, a.u.) are at the bottom. ( C ) Characterization of nuclear (NE) and cytoplasmic (CE) levels of β-catenin. Nuclear and cytoplasmic protein was isolated, β-catenin was detected by Western blotting using specific antibody. TBP and GAPDH were used as loading control of nuclear and cytoplasmic protein respectively. Normalized level of β-catenin in each compartment is shown at the bottom. ( D and E ) Characterization of levels of proteins, downstream targets of Wnt/β-catenin pathway (D), and androgen regulated genes AR and NKX3.1 (E) by Western blotting. Corresponding quantification is shown. Three clones (OE3a, OR1b, OE1c) overexpressing δ-catenin at various levels were examined. Total amount of 30ug of protein was used in each experiment, unless otherwise specified (in B). SDS-PAGE and Western blot conditions as in Figure .

Article Snippet: The following antibodies were used for western blotting and co-immunoprecipitation: δ-catenin (Abnova, cat. H00001501-A01), β-catenin (Sigma, cat. c2206), p120 (Santa Cruz, cat. SC-13957), α-catenin (Novus, cat. NB100-74356), cyclin D1 (Cell Signaling, cat. 2922S), NKX3.1 (R&D systems; cat. AF6080) and Slug (Cell signaling; cat. 9585).

Techniques: Clone Assay, Expressing, Isolation, Western Blot, SDS Page

Journal: Oncotarget

Article Title: Frequently rearranged and overexpressed δ-catenin is responsible for low sensitivity of prostate cancer cells to androgen receptor and β-catenin antagonists

doi: 10.18632/oncotarget.25319

Figure Lengend Snippet: ( A , B ) Comparison of binding partners of δ-catenin and β-catenin in LNCaP parental cells and overexpressing δ-catenin clone. Immunoprecipitation experiments were done as in Figure . ( C ) A cartoon depicts binding complex of catenin proteins and E-cadherin in LNCaP cells, proposed on pull down experiments.

Article Snippet: The following antibodies were used for western blotting and co-immunoprecipitation: δ-catenin (Abnova, cat. H00001501-A01), β-catenin (Sigma, cat. c2206), p120 (Santa Cruz, cat. SC-13957), α-catenin (Novus, cat. NB100-74356), cyclin D1 (Cell Signaling, cat. 2922S), NKX3.1 (R&D systems; cat. AF6080) and Slug (Cell signaling; cat. 9585).

Techniques: Binding Assay, Immunoprecipitation

Journal: Oncotarget

Article Title: Frequently rearranged and overexpressed δ-catenin is responsible for low sensitivity of prostate cancer cells to androgen receptor and β-catenin antagonists

doi: 10.18632/oncotarget.25319

Figure Lengend Snippet: ( A, B ) LNCaP, OE3a and OE1b cells were grown in RPMI 1640 supplemented with 10% charcoal stripped FBS for 48 hours and then treated with pyrvinium (Pyr, in A.) or casodex (CXD, in B). Concentrations and time are as indicated. Data are presented as mean ± SD, based on 3 independent experiments. ( C ) Western blotting showing changes in levels of δ-catenin and β-catenin upon indicated treatment; quantification is shown in right panel.

Article Snippet: The following antibodies were used for western blotting and co-immunoprecipitation: δ-catenin (Abnova, cat. H00001501-A01), β-catenin (Sigma, cat. c2206), p120 (Santa Cruz, cat. SC-13957), α-catenin (Novus, cat. NB100-74356), cyclin D1 (Cell Signaling, cat. 2922S), NKX3.1 (R&D systems; cat. AF6080) and Slug (Cell signaling; cat. 9585).

Techniques: Western Blot

Journal: Oncotarget

Article Title: Frequently rearranged and overexpressed δ-catenin is responsible for low sensitivity of prostate cancer cells to androgen receptor and β-catenin antagonists

doi: 10.18632/oncotarget.25319

Figure Lengend Snippet: ( A ) Survival of indicated clones is expressed as % of that of corresponding untreated clones. Data are presented as mean ± SD, based on 3 independent experiments. ( B ) Western blot showing changes in levels of AR and β-catenin upon indicated treatment, quantification is shown in ( C ).

Article Snippet: The following antibodies were used for western blotting and co-immunoprecipitation: δ-catenin (Abnova, cat. H00001501-A01), β-catenin (Sigma, cat. c2206), p120 (Santa Cruz, cat. SC-13957), α-catenin (Novus, cat. NB100-74356), cyclin D1 (Cell Signaling, cat. 2922S), NKX3.1 (R&D systems; cat. AF6080) and Slug (Cell signaling; cat. 9585).

Techniques: Clone Assay, Western Blot