human recombinant protein wnt5a  (R&D Systems)

Journal: Cancer Research Communications

Article Title: ROME, an Ancient Gene with a Novel Function in Vertebrates, Is a Key Modulator of Embryonal Development and Cancer Metastasis

doi: 10.1158/2767-9764.CRC-26-0068

Figure Lengend Snippet: ROME protein negatively regulates the Wnt pathway and calcium signaling in human cancer cells. A and B, Compared with EV control cells, TC-71 ( A ) and TC-32 ( B ) cells stably overexpressing ROME were less responsive to recombinant human Wnt3a protein as measured by TOPFlash β-catenin–responsive luciferase reporter. C, TC-71 cells with ROME knockdown by siRNA were more responsive to Wnt3a. D, Endogenous Wnt pathway activity in the colorectal cancer cell line HCT116 was significantly decreased by ROME expression (Western blots confirming ROME-HA expression on the right). Statistical significance was calculated by an unpaired t test (two-tailed) for A–D . E, ROME co-immunoprecipitates with β-catenin in β-catenin pulldown experiments. F, β-Catenin co-immunoprecipitates with ROME in ROME pulldown experiments. G, SPR sensorgrams showing direct binding between the recombinant full-length ROME protein and β-catenin protein. β-Catenin was immobilized on the surface and full-length ROME protein was injected in duplicate at concentrations of 1,250, 416.7, 138.9, 46.3, 15.4, and 5.12 nmol/L. The red lines are the actual data, and the black lines indicate the curve fit. H, ROME expression decreases β-catenin and TCF4 interaction (measured by co-IP). I–K, The Ca 2+ response was decreased in A4573, STA-ET-7.2, and TC-71 cells overexpressing ROME compared with that in EV-transfected cells when the cells were stimulated with ATP or FBS after overnight serum starvation. L, The Ca 2+ response was greater in the TC-71 cells with ROME KO than in the WT cells. Statistical significance was calculated by an unpaired t test (two-tailed) for I–L .

Article Snippet: On the next day, cells were stimulated with recombinant Wnt3a (R&D Systems, #645-WN-010/CF) for 4.5 hours, and then luciferase assay was conducted with the Dual Luciferase Reporter Assay System Kit (Promega, #E1960) according to the manufacturer’s protocol.

Techniques: Control, Stable Transfection, Recombinant, Luciferase, Knockdown, Activity Assay, Expressing, Western Blot, Two Tailed Test, Binding Assay, Injection, Co-Immunoprecipitation Assay, Transfection

Journal: Respiratory Research

Article Title: Downregulation of SLC40A1 leads to iron accumulation in fibrotic lung fibroblasts

doi: 10.1186/s12931-025-03479-0

Figure Lengend Snippet: Transcriptional regulation of SLC40A1 by TGF-β1. a HPFs were treated for 48 h with different cytokines [TGF-β1 (5 ng/ml)​​, WNT-1(50 ng/ml), WNT-3a (50 ng/ml), WNT-5a (50 ng/ml), TNFα (10 ng/ml)​, LPS (1,000 ng/ml), IL-1β (10 ng/ml), IL-4 (20 ng/ml), IL-5 (20 ng/ml), IL-13 (25 ng/ml), IL-21 (25 ng/ml), IL-25 (25 ng/ml), IL-33 (30 ng/ml), IFNβ (1 ng/mL), or IFNγ (20 ng/ml)]. SLC40A1 mRNA levels were determined by real-time PCR and expressed a ratio of the control (CON). b HPFs were treated with 50 ng/ml IL-6 for 48 h or serum-starved for 4 h or 24 h and then treated with 10 nM thrombin or 100 nM factor Xa for 48 h and SLC40A1 mRNA levels were determined by real-time PCR. c HPFs were co-transfected with the pGL3-Basic vector containing the SLC40A1 promoter fragment and firefly luciferase gene (180 ng) along with pRL-TK plasmid containing Renilla luciferase gene (20 ng) and then treated with 5 ng/ml TGF-β1 for 48 h. Firefly and Renilla luciferase activities in cell lysates were measured. The relative firefly luciferase activities were normalized to the respective Renilla luciferase activities. The results are presented as a ratio CON. d HPFs were treated with shSMAD2, shSMAD3, or shCON lentivirus at a MOI of 100 for 48 h. SMAD2 and SMAD3 protein levels were determined by western blot and quantitated. e HPFs were treated with shSMAD2, shSMAD3, or shCON lentivirus at a MOI of 100 for 48 h and then treated with 5 ng/ml TGF-β1 for 48 h. SLC40A1 mRNA levels were determined by real-time PCR. f HPFs were treated with SIS3 (10 µM, Milipore Sigma, Cat# 566405) for 2 h and then with TGF-β1 (5 ng/ml) for 48 h. SLC40A1 mRNA levels were determined by real-time PCR. g HPFs were treated with TGF-β1 (5 ng/ml) for 48 h. The cell lysates were subjected to chromatin immunoprecipitation (ChIP) using anti-SMAD3 antibodies. The SMAD3 binding site on the SLC40A1 promoter was detected by real-time PCR. n =3, * p < 0.05, ** p < 0.01, *** p < 0.001 v.s CON or shCON. ## p < 0.01 vs ShCon/TGF-β1. Student t-test ( c ), one-way ANOVA ( a and d ) or two-way ANOVA ( b , e , f and g ). followed by uncorrected Fisher’s least significant difference (LSD) test ( e , f and g ) or Turkey’s multiple comparison test ( b )

Article Snippet: HPFs (1x10 5 /well) were seeded into 12-well plates and treated with different cytokines and other compounds for 48 h: TNF-α (10 ng/ml, #300-01A, PeproTech, Cranbury, NJ, USA), lipopolysaccharides (LPS) (1,000 ng/ml, #00–4976-93, Invitrogen), TGF-β1 (5 ng/ml, #240-B, R&D Systems, Minneapolis, MN, USA) , IL-4 (20 ng/ml, #200-04, PeproTech), IFN-γ (20 ng/ml, #300-02, PeproTech) , IL-21 (25 ng/ml, #200-21, PeproTech), IL-13 (25 ng/ml, #200-13, PeproTech), IFN-β (1 ng/mL, #300-02BC, PeproTech), IL-25 (25 ng/ml, #1258-IL, R&D Systems), IL-33 (30 ng/ml, #200-33, PeproTech), WNT-1 (50 ng/ml, #120-17, PeproTech), IL-1β (10 ng/ml, #200-01B, PeproTech), IL-5 (20 ng/ml, #200-05, PeproTech), WNT-3a (50 ng/mL, #5036-WN, R&D Systems), WNT-5a (50 g/mL, #645-WN, R&D Systems), IL-6 (50 ng/ml, #200-06, Gibco), thrombin (10 nM, #RP-43100, Invitrogen), factor Xa (100 nM, #RP-43114, Invitrogen), ferric ammonium citrate (FAC) (200 μM, # RES20400 -A7, Sigma), deferoxamine (DFO) (10 μM #D9533, Sigma).

Techniques: Real-time Polymerase Chain Reaction, Control, Transfection, Plasmid Preparation, Luciferase, Western Blot, Chromatin Immunoprecipitation, Binding Assay, Comparison

Journal: NPJ Biofilms and Microbiomes

Article Title: Disruption of bile acid homeostasis potentiates Paneth cell ablation by activating the intestinal Farnesoid X receptor in necrotizing enterocolitis

doi: 10.1038/s41522-025-00904-6

Figure Lengend Snippet: ( A ) UMAP of epithelial subtypes in control and NEC mice. ( B ) Representative immunofluorescence images for Lyz (red), Epacm (green) and DAPI (blue) on paraffin-embedded distal ileum from indicated group in experiment 3. White arrow indicates the Lyz stained PCs, Scale bar: 10 μm. ( C , D ) The number of Lyz + PCs in B ( C ) and mRNA levels of Lyz ( D ) were compared among different groups. ( E ) Violin plots showed the expression of Wnt/PCP pathway genes in mouse PCs subsets revealed by scRNA-seq analysis. ( F ) The mRNA level of Damm1 was compared in IECs isolated from different groups. ( G ) Organoids derived from mice were cultured in medium with DMSO, FEX (5 μm), or FEX (10 μm) from day 1. The organoids were collected and stained for Lyz on day 7. Arrowheads point at PCs (Lyz + , in pink), scale bars: 50 μm. ( H ) qRT-PCR analysis of Daam1 in organoids cultured with DMSO, FEX (5 μm), or FEX (10 μm). ( I ) Representative organoid formation images on day 7 in different groups supplemented with ENR, Wnt5a-containing ENR, and Wnt5a-containing ENR + FEX. Representative marker genes for PCs (Lyz, green) cells are highlighted by fluorescent reporters. Arrowheads point at PCs (Lyz + , in green), scale bars: 50 μm. All statistical data are shown as the means ± SEMs, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: For experiments involving Wnt5a (645-WN-010/CF, R&D Systems) ligands, the media was made by supplementing ENR media with 100 ng/ml Wnt5a.

Techniques: Control, Immunofluorescence, Staining, Expressing, Isolation, Derivative Assay, Cell Culture, Quantitative RT-PCR, Marker

Journal: Communications Biology

Article Title: PRICKLE3 protects VANGL proteins from CK1-mediated phosphorylation and RNF43-mediated degradation

doi: 10.1038/s42003-025-09422-9

Figure Lengend Snippet: a General schematic of the TetON inducible gene expression system used in this study. In the absence of doxycycline, the reverse tetracycline-controlled transactivator (rtTA) is inactive. Upon doxycycline addition, rtTA binds to the tetO promoter and activates transcription of the gene of interest, leading to protein expression. b Doxycycline-induced overexpression of HA-tagged PRICKLE3 in HEK T-REx 293 PRICKLE3 TetON cells and of PRICKLE1 in the corresponding inducible cell line. c Induction of HA-PRICKLE3 expression by doxycycline in HEK T-REx 293 PRICKLE3 TetON cells. HEK T-REx 293 wildtype (WT) cells served as a control for doxycycline effects. Arrowheads indicate phosphorylation-dependent shifts in the electrophoretic mobility of VANGL proteins. α-TUBULIN was used as a loading control. Representative result from n = 5. d , e Densitometric quantification of Western blot signals. Values were normalized to untreated cells. Statistical analysis was performed using an unpaired t -test; corresponding p -values are shown ( n = 5). f Effect of recombinant WNT stimulation on VANGL phosphorylation. HEK T-REx 293 PRICKLE3 TetON cells were pre-treated overnight with the porcupine inhibitor LGK-974 to block endogenous WNT ligand secretion and subsequently stimulated with 100 ng/ml human recombinant WNT5A or WNT3A for 3 h. Arrowheads indicate phosphorylation-dependent mobility shifts of ROR. α-TUBULIN served as a loading control. Representative result from n = 4. g , h Densitometric quantification of Western blot signals from panel f. Data were normalized to untreated controls. Statistical analysis was performed using an unpaired t -test; corresponding p -values are reported ( n = 4).

Article Snippet: Following this, non-canonical WNT signaling was activated by incubating the cells with recombinant human WNT5A (645-WN, R&D Systems) at a concentration of 100 ng/mL for 3 h. Similarly, canonical WNT signaling was activated by incubating the cells with 100 ng/mL of recombinant human WNT3A (5036-WN, R&D Systems) for 3 h. To assess protein degradation kinetics, cells were pretreated with doxycycline overnight.

Techniques: Gene Expression, Expressing, Over Expression, Control, Phospho-proteomics, Western Blot, Recombinant, Blocking Assay