Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: A. Quantification of change in cell density (number of DAPI positive nuclei per cm 2 imaged area) upon FOXM1 overexpression or knockdown, 72h post transfection. Data is normalized to appropriate controls (Empty vector for pFOXM1 and non-targeting siRNA for siFOXM1). Bars represent Mean + SD (n = 4). P<0.0001 (Student’s t-test). B. Heatmap showing changes in gene expression of a panel of representative markers over a timecourse of RPE culture where cells are seeded at high (100000 cells/cm 2 ) or low (8000 cells/cm 2 ) density. C. Plot showing differential expression of BMP7 and Wnt5B transcripts extrapolated from the microarray data. The shaded area represents 95% confidence intervals around the point estimates (circles) of the difference between the mean high density expression vs the mean low density expression.

Article Snippet: Where required, media was supplemented with recombinant human Wnt5B (500ng/ml; R&D Systems), BMP-4/7 (75ng/ml; R&D Systems), Thiostrepton (Sigma), LDN-193189 (10μM; Stemgent), WAY-262611 (10μM; Enzo Lifesciences).

Techniques: Over Expression, Knockdown, Transfection, Plasmid Preparation, Gene Expression, Quantitative Proteomics, Microarray, Expressing

Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: A. Immunocytochemistry for PMEL17 where cells are seeded at either low density (16000 cells/cm 2 ) in the presence or absence of BMP4/7 (top left) or at high density (25000 cells/cm 2 ) in the presence or absence of Wnt5B (bottom left) and cultured for a period of 14 days. Also shown is the expression of BEST1 under the same conditions (top and bottom right). ACTB and B2M are used as housekeeping genes. Bars represent Mean + SD (n = 3). B. Quantification of immunocytochemistry for % CRALBP at Day 21 where cells are either treated with media alone (Control) or media supplemented with 10μM LDN-193189 added at Day 2,4,6,8,11,14 or 18. * indicates significant difference between control and compound treatment (One way ANOVA with Dunnett’s multiple comparisons). C. Quantification of immunocytochemistry for % CRALBP at Day 28 where cells are either treated with media alone (Control) or media supplemented with 10μM WAY-262611 added at Day 2,7,14 or 21. * indicates significant difference between control and compound treatment (One way ANOVA with Dunnett’s multiple comparisons). D. qPCR based measurement of BMP7 and Wnt5B transcript expression at Day 10 post siFOXM1 transfection (relative to transfection with non-targeting siRNA used as a control). GAPDH , HPRT1 and IPO8 were used as housekeeping genes. Bars represent Mean + SD (n = 3). P<0.05 (Student’s t-test).

Article Snippet: Where required, media was supplemented with recombinant human Wnt5B (500ng/ml; R&D Systems), BMP-4/7 (75ng/ml; R&D Systems), Thiostrepton (Sigma), LDN-193189 (10μM; Stemgent), WAY-262611 (10μM; Enzo Lifesciences).

Techniques: Immunocytochemistry, Cell Culture, Expressing, Control, Transfection

Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: RPE first acquire a mesenchymal morphology upon dissociation and culture followed by proliferation and mesenchymal-epithelial transition to re-uptake an epithelial phenotype. Proliferation of RPE is directly regulated by FOXM1 which also affects expression of BMP7 and Wnt5B by an unknown mechanism. Both these activities are required for successful MET and epithelialization.

Article Snippet: Where required, media was supplemented with recombinant human Wnt5B (500ng/ml; R&D Systems), BMP-4/7 (75ng/ml; R&D Systems), Thiostrepton (Sigma), LDN-193189 (10μM; Stemgent), WAY-262611 (10μM; Enzo Lifesciences).

Techniques: Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: WNT5B governs the phenotype of basal-like breast cancer by activating WNT signaling

doi: 10.1186/s12964-019-0419-2

Figure Lengend Snippet: WNT5B is a specific marker of BLBC with poor prognosis. a mRNA expression of WNT ligands and their receptors Frizzleds between luminal and BLBC cell lines are shown in the heatmap (data are extracted from CCLE and displayed by using GraphPad Prism 7 software). b mRNA and protein expression levels of WNT3A, WNT5A, and WNT5B in two normal breast cell lines, eight luminal, and seven BLBC cell lines by semi-q RT-PCR and western blot (S: Short exposure; L: Long exposure). c Expression of Wnt5a and Wnt5b in representative Her-2 positive, luminal A, luminal B and BLBC tissues by IHC (Red scale bar = 200 μm; Purple scale bar = 40 μm). d The association of Wnt5a or Wnt5b expression between basal-like and non-BLBC tissue subtypes were assessed using Pearson’s χ 2 test (*** p = 0.0003, **** p < 0.0001). Prognostic value of WNT5A ( e ) and WNT5B ( f ) mRNA levels in human breast cancer, data obtained from the KM-plotter

Article Snippet: Breast cancer cells were treated with or without recombinant human Wnt3a (200 ng/mL, R&D, 5036-WN/CF), Wnt5a (500 ng/mL, R&D, 645-WN/CF), or Wnt5b (500 ng/mL, R&D, 7347-WN/CF) protein for 24 h before IF staining.

Techniques: Marker, Expressing, Software, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: WNT5B governs the phenotype of basal-like breast cancer by activating WNT signaling

doi: 10.1186/s12964-019-0419-2

Figure Lengend Snippet: WNT5B is positively correlated with WNT signaling targets, EMT and basal-like markers, but inversely correlated with luminal markers. a WNT3A, WNT5A, WNT5B, WNT signaling targets, EMT markers, basal-like markers, and luminal markers mRNA expression between luminal and BLBC cell lines are shown in the heatmap (data are extracted from CCLE and displayed by using GraphPad Prism 7 software). b WNT5B is positively correlated with most of WNT signaling targets. c WNT5B is positively correlated with most of EMT markers. d WNT5B is inversely correlated with luminal markers. e WNT5B is positively correlated with basal-like markers. b - e are based on GEPIA (breast invasive carcinoma; n = 1085; Red: positive correlation; Green: negative correlation; Black: no correlation)

Article Snippet: Breast cancer cells were treated with or without recombinant human Wnt3a (200 ng/mL, R&D, 5036-WN/CF), Wnt5a (500 ng/mL, R&D, 645-WN/CF), or Wnt5b (500 ng/mL, R&D, 7347-WN/CF) protein for 24 h before IF staining.

Techniques: Expressing, Software

Journal: Cell Communication and Signaling : CCS

Article Title: WNT5B governs the phenotype of basal-like breast cancer by activating WNT signaling

doi: 10.1186/s12964-019-0419-2

Figure Lengend Snippet: WNT5B controls the phenotype of BLBC by activating canonical and non-canonical WNT signaling. a Western blot showing Wnt5b upon knockdown of WNT5B by lentivirus-mediated shRNA in Hs-578 T, MDA-MB-231, and Bcap-37 cells. b Migration and invasion of sh-control with or without Wnt5b ligand and Wnt5b-KD Bcap-37 cells and MDA-MB-231 cells were measured using transwell chamber assays (scale bar = 100 μm). c Data represent the average cell number from 5 viewing fields (B-37: Bcap-37; M-231: MDA-MB-231; ** p < 0.005, *** p < 0.001, **** p < 0.0001). d Active β-Catenin (Non-phospho-ser45 and Non-phospho-Ser33/37/Thr41 and total β-Catenin were analyzed by western blot (Hs-578 T and MDA-MB-231 cells were pretreated with Wnt3a, Wnt5a, or Wnt5b; S: Short exposure; L: Long exposure). e Protein level of phospho-JNK (Thr183/Tyr185), JNK, phospho-Erk1/2 (Thr202/Tyr204), Erk1/2, phospho-STAT3 (Tyr705), and STAT3 were analyzed by western blot (Hs-578 T and MDA-MB-231 cells were pretreated with Wnt3a, Wnt5a, or Wnt5b). f Protein level of canonical and non-canonical markers in WNT5B-KD Hs-578 T and MDA-MB-231 cells was analyzed by western blot. g Protein level of EMT markers in WNT5B-KD Hs-578 T and MDA-MB-231 cells was analyzed by western blot. h Protein level of luminal and basal-like markers in WNT5B-KD Hs-578 T and MDA-MB-231 cells was analyzed by western blot. i Cytoskeleton F-actin proteins were stained with phalloidin and viewed under a confocal microscope (scale bar = 20 μm). j Protein expression of phospho-STAT3 (Tyr705) in representative Her-2 positive, luminal A, luminal B and BLBC tissues by IHC (Red scale bar = 200 μm; Purple scale bar = 40 μm). k The association of phospho-STAT3 (Tyr705) expression between basal-like and non-BLBC tissue subtypes were assessed using Pearson’s χ 2 test (*** p = 0.0003)

Article Snippet: Breast cancer cells were treated with or without recombinant human Wnt3a (200 ng/mL, R&D, 5036-WN/CF), Wnt5a (500 ng/mL, R&D, 645-WN/CF), or Wnt5b (500 ng/mL, R&D, 7347-WN/CF) protein for 24 h before IF staining.

Techniques: Western Blot, shRNA, Migration, Staining, Microscopy, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: WNT5B governs the phenotype of basal-like breast cancer by activating WNT signaling

doi: 10.1186/s12964-019-0419-2

Figure Lengend Snippet: Knockdown of WNT5B inhibits the tumorigenicity of basal-like cancer cells in vivo. RFP-expressed MDA-MB-231 ( a ) and Bcap-37 ( b ) cell pellets under visible light. c Fluorescence images of BLBC cells bearing mice under 530 nm laser irradiation at day 14, 22, and 30 for MDA-MB-231 or at day 6, 12, and 18 for Bcap-37 with/without WNT5B-KD. d Ultrasound images of BLBC cells bearing mice under B-type ultrasonic image system at day 14, 22, and 30 for MDA-MB-231 or at day 6, 12, and 18 for Bcap-37 with/without WNT5B-KD. Subcutaneous tumor of MDA-MB-231 ( e ) and Bcap-37 ( f ) with/without WNT5B-KD at the experimental endpoint. Tumor volume of MDA-MB-231 ( g ) and Bcap-37 ( h ) at the experimental endpoint (* p < 0.05; ** p < 0.005). Tumor growth curve of MDA-MB-231 ( i ) and Bcap-37 ( j ) (** p < 0.005; **** p < 0.0001). Tumor weight of MDA-MB-231 ( k ) and Bcap-37 ( l ) at the experimental endpoint (* p < 0.05; ** p < 0.005)

Article Snippet: Breast cancer cells were treated with or without recombinant human Wnt3a (200 ng/mL, R&D, 5036-WN/CF), Wnt5a (500 ng/mL, R&D, 645-WN/CF), or Wnt5b (500 ng/mL, R&D, 7347-WN/CF) protein for 24 h before IF staining.

Techniques: In Vivo, Fluorescence, Irradiation

Journal: Cell Communication and Signaling : CCS

Article Title: WNT5B governs the phenotype of basal-like breast cancer by activating WNT signaling

doi: 10.1186/s12964-019-0419-2

Figure Lengend Snippet: Small-molecule compounds inhibit tumor growth by targeting Wnt5b-mediated non-canonical WNT signaling and CK1α-mediated canonical WNT signaling in vitro and in vivo. a Chemical structure of LGK-974 and pyrvinium pamoate. Cell viability was determined in MDA-MB-231 and Bcap-37 cells following treatment with LGK-974 ( b ) and pyrvinium pamoate ( c ) for 72 h, mean ± SEM is shown (assays performed in quadruplicate). d LGK-974 and pyrvinium pamoate inhibited cell lines with a high expression of Wnt5b and a low expression of CK1α by foci formation assay. e Graphs represent LGK-974-treated or pyrvinium pamoate-treated Hs-578 T and Bcap-37 with or without Wnt5b (500 ng/ml), mean ± SD ( n = 3 per treatment group), (n.s.: no significant, * p < 0.05, ** p < 0.005, *** p < 0.001, **** p < 0.0001). f Images of Bcap-37 cells bearing mice under 530 nm laser irradiation at day 5, 10, and 15 following treatment with LGK-974 (5 mg/kg) or pyrvinium pamoate (1 mg/kg). Tumor weight ( g ), Tumor volume ( h ), Tumor growth curve ( i ), and subcutaneous tumor ( j ) of Bcap-37 at the experimental endpoint. k Representative images of Ki-67 staining by IHC after 14 days of treatment

Article Snippet: Breast cancer cells were treated with or without recombinant human Wnt3a (200 ng/mL, R&D, 5036-WN/CF), Wnt5a (500 ng/mL, R&D, 645-WN/CF), or Wnt5b (500 ng/mL, R&D, 7347-WN/CF) protein for 24 h before IF staining.

Techniques: In Vitro, In Vivo, Expressing, Tube Formation Assay, Irradiation, Staining