Journal: Molecular medicine reports

Article Title: MicroRNA‑214 targets Wnt3a to suppress liver cancer cell proliferation.

doi: 10.3892/mmr.2017.7483

Figure Lengend Snippet: Figure 1. miR‑214 is downregulated in liver cancer and targets Wnt3a. (A) Reverse transcription‑quantitative polymerase chain reaction was performed to examine the expression of miR‑214 in 24 paired human hepatocellular carcinoma and non‑tumor tissues. (B) Relative expression of miR‑214 in liver cancer cell lines and a normal liver cell line. **P<0.01; *P<0.05. (C) miR‑214 seed region sequence in the 3'UTR of Wnt3a. (D) Wnt3a protein expression as detected by immunohistochemistry. (E) Protein expression levels of Wnt3a were measured by western blot analysis in HepG2 cells transfected with miR‑214 or miR‑ctrl. (F) miR‑214 was co‑transfected with pmirGLO, pmirGLO‑Wnt3a‑3'‑UTR‑wt or pmirGLO‑Wnt3a‑3'‑UTR‑mut in HepG2 cells. Relative luciferase activity was measured after 48 h. *P<0.05 vs. control. miR, microRNA; mut/M, mutant; UTR, untranslated region; wt/W, wild‑type.

Article Snippet: Membranes were then incubated with rabbit anti-human Wnt3a (cat no. bs-1700R; 1:100; Beijing Biosynthesis Biotechnology Co., Ltd.) and rabbit anti-human GAPDH (cat no. 10494-1-AP; 1:2,000; ProteinTech Group, Inc., Chicago, IL, USA) at 4 ̊C overnight.

Techniques: Polymerase Chain Reaction, Expressing, Sequencing, Immunohistochemistry, Western Blot, Transfection, Luciferase, Activity Assay, Control, Mutagenesis

Journal: Molecular medicine reports

Article Title: MicroRNA‑214 targets Wnt3a to suppress liver cancer cell proliferation.

doi: 10.3892/mmr.2017.7483

Figure Lengend Snippet: Figure 2. miR‑214 inhibits the proliferation of liver cancer cells. CCK8 assay was performed to detect the effects of miR‑214 on cell proliferation at 24, 48, and 72 h in (A) HepG2 and (B) Hep3B cells. CCK8 assay was performed to detect the effects of siWnt3a on cell proliferation at 24, 48 and 72 h in (C) HepG2 and (D) Hep3B cells. Wnt3a overexpression vector was co‑transfected with miR‑ctrl or miR‑214 into (E) HepG2 and (F) Hep3B cells, and cell proliferation was detected by CCK8 assay. *P<0.05; **P<0.01 vs. miR‑ctrl + Wnt3a‑ctrl. CCK8, Cell Counting kit‑8; ctrl, control; miR, microRNA; OD, optical density; si, small interfering RNA.

Article Snippet: Membranes were then incubated with rabbit anti-human Wnt3a (cat no. bs-1700R; 1:100; Beijing Biosynthesis Biotechnology Co., Ltd.) and rabbit anti-human GAPDH (cat no. 10494-1-AP; 1:2,000; ProteinTech Group, Inc., Chicago, IL, USA) at 4 ̊C overnight.

Techniques: CCK-8 Assay, Over Expression, Plasmid Preparation, Control, Small Interfering RNA

Journal: Molecular medicine reports

Article Title: MicroRNA‑214 targets Wnt3a to suppress liver cancer cell proliferation.

doi: 10.3892/mmr.2017.7483

Figure Lengend Snippet: Figure 3. Overexpression of miR‑214 or Wnt3a silencing affects cell cycle progression. Cell cycle analysis of (A) HepG2 and (B) Hep3B cells following transfection with miR‑214 or miR‑ctrl for 48 h. Cell cycle analysis of (C) HepG2 and (D) Hep3B cells following transfection with siWnt3a or si‑ctrl for 48 h. *P<0.05. ctrl, control; miR, microRNA; si, small interfering RNA.

Article Snippet: Membranes were then incubated with rabbit anti-human Wnt3a (cat no. bs-1700R; 1:100; Beijing Biosynthesis Biotechnology Co., Ltd.) and rabbit anti-human GAPDH (cat no. 10494-1-AP; 1:2,000; ProteinTech Group, Inc., Chicago, IL, USA) at 4 ̊C overnight.

Techniques: Over Expression, Cell Cycle Assay, Transfection, Control, Small Interfering RNA

Journal: Cancer Immunology Research

Article Title: Wnt3a/β-Catenin Signaling Conditions Differentiation of Partially Exhausted T-effector Cells in Human Cancers

doi: 10.1158/2326-6066.cir-17-0712

Figure Lengend Snippet: Figure 4. Distribution of Wnt3a in serum nontumor- or tumor-derived conditioned media and expression of the Wnt3a/b-catenin axis in nontumor and tumor-infiltrating CD4þ

Article Snippet: ELISA was performed according to the manufacture's instruction of Cloud Clone Corp kit or to the manufacturer's instruction of Human Protein Wnt3a ELISA kit (Cat. CSB-EL026136HU, Cusabio Technology).

Techniques: Derivative Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Examining Crosstalk among Transforming Growth Factor β, Bone Morphogenetic Protein, and Wnt Pathways *

doi: 10.1074/jbc.M116.759654

Figure Lengend Snippet: Establishment of our system for investigating crosstalk. A , overview of experimental approach. Cells were treated with combinations of purified ligands, and nuclear transcription factor responses were measured by single-cell immunofluorescence imaging. Sample images of HCECs after 2 h with or without ligand show that Wnt3A globally increases β-catenin levels, TGFβ3 causes bulk nuclear translocation of Smad2/3, and BMP4 increases nuclear phospho-Smad1/5/8. Nuclei are outlined using the same segmentation approach as in all experiments (see “Experimental Procedures”). B , graphic of image-based nuclear transcription factor quantification. Total nuclear intensity was measured for each cell (see “Experimental Procedures”), and the population medians of these values ( filled circles ) from each distribution ( bottom ) were obtained for each of three replicate experiments. The means and standard deviations (S.D.) of these median values were then normalized so that the control mean was 0 and the canonical ligand-only mean was 1 ( top , open circles ). C and D , time-course ( C ) and dose-response curves ( D ) at 1 h for SKMEL2 ( solid lines ) and HCEC ( dashed lines ) in response to Wnt3A ( red ), TGFβ3 ( blue ), or BMP4 ( green ). Values were measured as in B and normalized to have the same minimum ( min ) and maximum ( max ) values, n = 3 per time point. Concentrations were as follows: Wnt3A (4.8 n m ), TGFβ3 (180 p m ), and BMP4 (2.6 n m ). E , the input/output relationships can be blocked by co-treatment with specific antagonists. Left , Dickkopf-1 (38 n m ) blocks Wnt3A (4.8 n m ) → β-catenin in SKMEL2s (2 h). Middle , a pan-TGFβ-blocking antibody (αTGFβ, 5 μg/ml) blocks TGFβ3 (450 p m ) → Smad2/3 in HCECs (2 h). Right , Noggin (4.3 n m ) blocks BMP4 (1.9 n m ) → pSmad1/5/8 in SKMEL2s (1.5 h). F , low-purity Wnt3A (used only in this panel) causes dose-dependent accumulation of Smad2/3. The Wnt → Smad2/3 response is likely due to trace contamination by TGFβ ligands. This response is completely blocked by a pan-TGFβ-blocking antibody (αTGFβ, 5 μg/ml) but not blocked by Wnt antagonist (Dkk1, 38 n m ) or observed for high-purity/carrier-free ( HP/CF ) Wnt ligands. Concentrations were as follows: Wnt3A (4.8 n m HP/CF, 4 n m low purity ( LP )). ctrl , control. G , cells show transcriptional changes to canonical ligands after 2-h treatments. Concentrations were as follows: Wnt3A (4.8 n m ), TGFβ3 (450 p m ), and BMP4 (1.9 n m ). Open circles show reference values used for scaling. n = 3 for all points, * indicates p value <0.05 compared with control (two-sided t test).

Article Snippet: Treatments (supplier, product number, approximate molecular mass, approximate purity) are as follows: Wnt3A (high-purity, R&D Systems 5036-WNP/CF, 37 kDa, 90%); Wnt3A (low-purity, R&D Systems 5036-WN, Lot RSK311102B, 37 kDa, 75%); TGFβ3 (Cell Signaling Technology 8425, 22 kDa (dimer), 98%); BMP4 (Cell Signaling Technology 4697, 26 kDa (dimer), 95%); Dickkopf-1 (R&D Systems 5439-DK, 26 kDa, 95%); Noggin (R&D Systems 6057-NG, 23 kDa (monomer), 95%); and αTGFβ blocking antibody (R&D Systems MAB1835).

Techniques: Purification, Immunofluorescence, Imaging, Translocation Assay, Control, Blocking Assay

Journal: The Journal of Biological Chemistry

Article Title: Examining Crosstalk among Transforming Growth Factor β, Bone Morphogenetic Protein, and Wnt Pathways *

doi: 10.1074/jbc.M116.759654

Figure Lengend Snippet: Wnt3A and TGFβ3 are insulated during signaling but show cell type-dependent transcriptional crosstalk. A , at 2 h, Wnt3A and TGFβ3 show little to no cross-pathway modulation of nuclear transcription factor ( Nuclear TF ) accumulation in HCECs or SKMEL2s. Ligand concentrations were as follows: Wnt3A (2.4 n m ) and TGFβ3 (9 p m ). B , red arrowheads indicate HCEC-specific 2-fold reduction of Axin2 expression caused by TGFβ3 at the same 2-h time point (measured by qPCR, see “Experimental Procedures”). C , by 18 h, dramatic HCEC-specific activation of β-catenin by TGFβ3 is observed ( arrowheads ). A and C , data as in B , with open circles showing reference values used for scaling. B and C , ligand concentrations were as follows: Wnt3A (4.8 n m ) and TGFβ3 (450 p m ). A–C , n = 3 for all points. The no-treatment and canonical ligand-only treatment are significantly different in all cases ( p value <0.05 in two-sided t test).

Article Snippet: Treatments (supplier, product number, approximate molecular mass, approximate purity) are as follows: Wnt3A (high-purity, R&D Systems 5036-WNP/CF, 37 kDa, 90%); Wnt3A (low-purity, R&D Systems 5036-WN, Lot RSK311102B, 37 kDa, 75%); TGFβ3 (Cell Signaling Technology 8425, 22 kDa (dimer), 98%); BMP4 (Cell Signaling Technology 4697, 26 kDa (dimer), 95%); Dickkopf-1 (R&D Systems 5439-DK, 26 kDa, 95%); Noggin (R&D Systems 6057-NG, 23 kDa (monomer), 95%); and αTGFβ blocking antibody (R&D Systems MAB1835).

Techniques: Expressing, Activation Assay

Journal: Blood

Article Title: Inhibition of WNT signaling in the bone marrow niche prevents the development of MDS in the Apc del/ + MDS mouse model

doi: 10.1182/blood-2016-08-736454

Figure Lengend Snippet: Loss of 1 copy of Ctnnb1 in an Apc-haploinsufficient microenvironment prevents or delays the development of MDS by 8 to 10 months. (A) Kaplan-Meier survival curves for Apcdel/+ (A; n = 4), Apcdel/+, Ctnnb1del/+ (AC; n = 11), Ctnnb1del/+ (C; n = 3), and Apcfl/+, Ctnnb1fl/+ (Cre−; n = 9) recipient mice. Median survival of Apcdel/+ and Apcdel/+, Ctnnb1del/+ recipient mice was significantly different (115 vs 413 days; P < .0001). All control mice (C and Cre−) survived until the end of the study, with the exception of 1 Apcfl/+, Ctnnb1fl/+ recipient that died at 343 days, likely due to a hemorrhagic renal cyst. (B) Percentage of CD71+Ter119+ erythroid cells, Gr1+CD11b+ myeloid cells, and CD19+IgM+ B cells in spleen isolated from Cre− (∼400 days), A (70-115 days), and AC (303-413 days) recipients that eventually displayed a fatal anemia. At sacrifice, the AC cell populations were more similar to A than Cre− recipients. (C) RBC and Hb counts in all 4 cohorts over time. The development of anemia is delayed in AC recipients after 35 weeks (a point in time when all A recipients have already been sacrificed due to severe anemia). In Cre− control recipients, 2 mice developed moderate anemia at 57 weeks. However, 1 mouse had an apparent colorectal tumor, and the other had a hemorrhagic renal cyst; neither had developed MDS. (D) MSCs were isolated from Cre− (control), A, and AC littermates 2 months posttreatment with pIpC to induce Cre-mediated deletion, and before development of disease. Following in vitro Wnt3a stimulation for 6 hours, nuclear and cytoplasmic fractions were isolated and immunoblotted with Ctnnb1, β-actin (cytoplasmic), and Hdac1 (nuclear) antibodies. Quantification of 3 independent experiments shows increased nuclear and cytoplasmic Ctnnb1 protein expression in Apcdel/+ MSCs that is reduced by ∼50% upon haploinsufficient loss of Ctnnb1. (E) RNA was isolated from Cre−, A, and AC MSCs (no Wnt3a stimulation), transcribed to complementary DNA (cDNA), and PCRs (run in triplicate) were quantified using Fast-SYBR Green. Gene expression was normalized to Gapdh and data are presented as mean ± standard error of the mean (SEM) of 3 independent experiments. ***P < .0001, **P < .001, *P < .05. NS, not significant.

Article Snippet: MSCs were stimulated with 50 ng/mL recombinant mouse Wnt3a (Pepro Tech, Rocky Hill, NJ) or human WNT3A (R&D Systems, Minneapolis, MN) with or without 50 nM PT for times indicated.

Techniques: Isolation, In Vitro, Expressing, SYBR Green Assay

Journal: Blood

Article Title: Inhibition of WNT signaling in the bone marrow niche prevents the development of MDS in the Apc del/ + MDS mouse model

doi: 10.1182/blood-2016-08-736454

Figure Lengend Snippet: Pyrvinium modulation of WNT signaling is more effective before the onset of moderate-severe anemia. (A) Two-month-old Mx1-Cre−Apcfl/+ (Cre−) or Mx1-Cre+Apcfl/+ (Cre+, also referred to as Apcdel/+) recipients were treated with pIpC (to induce Apc deletion) and vehicle (DMSO) or PT 2 weeks before lethal irradiation and transplantation with WT (CD45.1) BM cells. Mice were injected twice per week with 0.01, 0.1, or 0.5 mg/kg PT or DMSO until sacrifice. Kaplan-Meier curves for overall survival show that mice treated with 0.1 mg/kg or 0.5 mg/kg PT survived about 1 to 2 months longer than vehicle-treated mice (P = .0302 and P = .0064, respectively). (B) RBC and Hb counts of DMSO and PT-treated mice at ∼100 days posttransplant (for some mice in the DMSO and 0.01 mg/kg PT groups, counts from <100 days were plotted since they died before 100 days). The RBC and Hb counts of 0.5 mg/kg PT-treated mice were higher than 0.01 mg/kg PT-treated mice, indicating the administration of 0.5 mg/kg PT delays development of anemia (P = .0018 and P = .0320). The 2 mice in the 0.1 mg/kg PT-treated group that survived beyond 200 days had noticeably higher RBC and Hb counts at 100 days (circled). (C) Cre+ recipients were treated with 0.5 mg/kg PT once they developed mild (Hb, 12-13.5 g/dL), moderate (Hb, 10-11.5 g/dL), or severe anemia (Hb, <10 g/dL). A Kaplan-Meier survival curve of all PT-treated mice indicates survival is extended by almost 2 months (DMSO vs PT: 104 days vs 159 days; P < .0001). (D) The average median survival of recipients from the mild, moderate, and severe anemia group vs the DMSO-treated control group is shown. A longer survival is achieved if treatment is started before the onset of severe anemia. (E) MSCs were isolated from Apcdel/+mice ∼2 months post-pIpC-induced deletion and before development of disease. MSCs were treated with or without 50 ng/mL Wnt3a ± 50 nM PT, as indicated, for 16 hours. A representative immunoblot of nuclear and cytoplasmic fractions immunoblotted with Ctnnb1, β-actin (cytoplasmic), and Hdac1 (nuclear) is shown. In 3 independent experiments, PT treatment decreased Wnt3a-mediated elevation of Ctnnb1 by 56% ± 11.5% (P = .04). (F) Apcdel/+ MSCs were stimulated in vitro ± 50 ng/mL Wnt3a ± 50 nM PT for 16 hours. RNA was isolated and transcribed to cDNA, and PCRs (run in triplicate) were quantified using Fast-SYBR Green. Gene expression was normalized to Gapdh and data are presented as mean ± SEM of 3 independent experiments. **P < .001, *P < .05. NR, not reached.

Article Snippet: MSCs were stimulated with 50 ng/mL recombinant mouse Wnt3a (Pepro Tech, Rocky Hill, NJ) or human WNT3A (R&D Systems, Minneapolis, MN) with or without 50 nM PT for times indicated.

Techniques: Irradiation, Transplantation Assay, Injection, Isolation, Western Blot, In Vitro, SYBR Green Assay, Expressing

Journal: Blood

Article Title: Inhibition of WNT signaling in the bone marrow niche prevents the development of MDS in the Apc del/ + MDS mouse model

doi: 10.1182/blood-2016-08-736454

Figure Lengend Snippet: Pyrvinium suppresses WNT activation in MSCs isolated from patients with myeloid neoplasms with a del(5q). (A) MSCs were isolated from the BM of 2 del(5q) patients with primary MDS or t-MDS and were treated in vitro with or without 50 ng/mL WNT3A ± 50 nM PT, as indicated, for 16 hours. Nuclear and cytoplasmic fractions were isolated and immunoblotted with antibodies specific for CTNNB1, β-actin, and Lamin A/C. Quantitation of the immunoblots revealed a ∼50% reduction in CTNNB1 levels in samples treated with PT. *A smaller CTNNB1 degradation product. (B) Patient MSCs were treated with WNT3A ± 50 nM PT for 16 hours. RNA was isolated and transcribed to cDNA, and PCRs (run in triplicate) were quantified using Fast-SYBR Green. Gene expression was normalized to the ACTB gene and data are presented as mean ± SEM of 1 patient sample, run in triplicate. PT significantly decreased WNT3A-mediated transcription of WNT target genes, but not the control GAPDH gene. *P < .05. Increased GAPDH may reflect emerging evidence suggesting that GAPDH gene expression can be modulated by external factors.58

Article Snippet: MSCs were stimulated with 50 ng/mL recombinant mouse Wnt3a (Pepro Tech, Rocky Hill, NJ) or human WNT3A (R&D Systems, Minneapolis, MN) with or without 50 nM PT for times indicated.

Techniques: Activation Assay, Isolation, In Vitro, Quantitation Assay, Western Blot, SYBR Green Assay, Expressing