Journal: Molecular Cancer Research

Article Title: Demethylation of the SFRP4 Promoter Drives Gastric Cancer Progression via the Wnt Pathway

doi: 10.1158/1541-7786.mcr-20-0933

Figure Lengend Snippet: Figure 5. SFRP4 plays an antagonistic role on biology function of SFRP1. A, The expression of Wnt ligands (Wnt2, Wnt3a, Wnt4, Wnt5A) in different gas- tric cancer cells was examined by Western blotting. B, The binding activ- ity of Wnt5A to its receptor was exam- ined by flow cytometry in the absence or presence of recombinant SFRP4. C, Co-IP experiments in MGC80–3cells were performed using anti-SFRP4 or anti-b-catenin antibodies in the absence or presence of recombinant Wnt5a. IgG was used as a negative control. D, The Wnt pathway markers (GSK-3b, b-catenin, c-Jun, LEF1, CD44, and MMP7) were examined in MGC80–3 cell after treating with recombinant SFRP1 (100 ng/mL), recombinant SFRP4 (100 ng/mL), or both for 48 hours. E, Afterward, com- bination of recombinant SFRP1 and recombinant SFRP4 were addedto the MGC80–3 cell in a concentration gra- dient (1:0, 1:0.5, 1:1, 1:2, 1:3) for 48 hours before harvesting for Western blotting analysis. F, The effects of recombinant SFRP1 and SFRP4 on the proliferation of MGC80–3 and AGS cellswere exam- ined by CCK-8 assay. , P < 0.001. r, recombinant; NC, negative control. In (A–E), the images were representa- tive of three independent experi- ments. In F, the experiments were repeated three times. Statistical sig- nificance was determined by one-way ANOVA test (F).

Article Snippet: The recombinant human SFRP1 (1384-SF), SFRP4 (1827-SF) and Wnt5a(645-WN) protein were purchased from R&D Systems.

Techniques: Expressing, Western Blot, Binding Assay, Cytometry, Recombinant, Co-Immunoprecipitation Assay, Negative Control, Concentration Assay, CCK-8 Assay

Journal: Scientific Reports

Article Title: Secreted frizzled-related protein 1 overexpression in gastric cancer: Relationship with radiological findings of dual-energy spectral CT and PET-CT

doi: 10.1038/srep42020

Figure Lengend Snippet: ( a ) In vitro angiogenesis assays were performed by treating HUVEC cells with culture supernatants of SGC-7901/vector or SGC-7901/sFRP1 cells. Representative pictures are shown (original magnification,x40). Numbers of tube formation per field are plotted. The data are shown as mean ± SD of three independent experiments ( p < 0.05). ( b ) Migration of HUVECs was conducted using Boydon chamber assays. Same amount of SGC-7901/vector and SGC-7901/sFRP1 cells were plated in 24-well plate for 24 h, and the culture supernatants were collected and used as chemoattractants to HUVECs in the migration assays. Representative pictures are shown (left, original magnification,x100) and migrated cell numbers are plotted (right). The data are shown as mean ± SD of three independent experiments ( p < 0.05).

Article Snippet: Plasmid containing Myc-DDK-tagged ORF clone of Homo sFRP1 was purchased from Origene (Origene Technologies, Rockville, MD).

Techniques: In Vitro, Plasmid Preparation, Migration

Journal: Scientific Reports

Article Title: Secreted frizzled-related protein 1 overexpression in gastric cancer: Relationship with radiological findings of dual-energy spectral CT and PET-CT

doi: 10.1038/srep42020

Figure Lengend Snippet: ( a ) In vitro angiogenesis assays were performed by treating HUVEC cells with culture supernatants of SGC-7901/vector or SGC-7901/sFRP1 cells. Representative pictures are shown (original magnification,x40). Number of tube formation per field are plotted. ( b ) Migration of HUVECs was conducted using Boydon chamber assays. Same amount of SGC-7901/vector and SGC-7901/sFRP1 cells were plated in 24-well plate for 24 h, and the culture supernatants were collected and used as chemoattractants to HUVECs in the migration assays. Representative pictures are shown (left, original magnification,x100) and migrated cell numbers are plotted (right). ( c ) VEGF levels in culture supernatants supernatants of SGC-7901/vector and SGC-7901/sFRP1 cells were examined using ELISA analysis.

Article Snippet: Plasmid containing Myc-DDK-tagged ORF clone of Homo sFRP1 was purchased from Origene (Origene Technologies, Rockville, MD).

Techniques: In Vitro, Plasmid Preparation, Migration, Enzyme-linked Immunosorbent Assay

Journal: Experimental Physiology

Article Title: Tumour necrosis factor α regulates the miR‐27a‐3p–Sfrp1 axis in a mouse model of osteoporosis

doi: 10.1113/EP090311

Figure Lengend Snippet: Sfrp1 is a direct target of miR‐27a‐3p. (a) Bioinformatics analysis was performed using the online tools microT and miRDB to predict the potential targets of miR‐27a‐3p. (b) BMSCs from wild‐type (WT) mice were transfected with miR‐NC or miR‐27a‐3p mimics. The relative expressions of potential target genes ( Pkia , Gfpt2 , Gmps , Car10 and Sfrp1 ) were analysed by qPCR. (c) The complementary binding sequences between miR‐27a‐3p and WT or mutated (MUT) 3ʹ‐UTR of Sfrp1 mRNA are shown. (d) HEK293T cells and BMSCs were transfected with miR‐NC or miR‐27a‐3p mimics, together with luciferase reporter vector containing WT or Mut 3ʹ‐UTR of Sfrp1 mRNA. The relative luciferase activity was analysed 48 h later. (e) BMSCs were isolated from wild‐type mice and RNA immunoprecipitation assay was performed using an isotype control IgG or anti‐AGO2 antibody. The relative enrichment of Sfrp1 and miR‐27a‐3p was analysed by qPCR. (f) BMSCs were isolated from the Sham or OVX mice. The mRNA and protein expression of Sfrp1 was analysed by qPCR or western blot. (g, h) BMSCs isolated from WT mice were transfected with miR‐NC, miR‐27a‐3p mimics, inhibitor NC, or miR‐27a‐3p inhibitor. The expression of Sfrp1 mRNA (g) or protein (h) 7 days later was analysed by qPCR or western blot assays, respectively. * P < 0.05, ** P < 0.011, *** P < 0.001, between the indicated groups.

Article Snippet: The membranes were probed with the following primary antibodies: Runx2 (cat. no. 12556, Cell Signaling Technology, Danvers, MA, USA), Osterix (DF7731, Affinity Biosciences, Changzhou, China), Sfrp1 (cat. no. 3534, Cell Signaling Technology), β‐catenin (cat. no. 8480, Cell Signaling Technology), C‐Myc (cat. no. 18583, Cell Signaling Technology), Cyclin D1 (cat. no. 55506, Cell Signaling Technology), and the loading control, GAPDH (cat. no. 5174, Cell Signaling Technology).

Techniques: Transfection, Binding Assay, Luciferase, Plasmid Preparation, Activity Assay, Isolation, RNA Immunoprecipitation, Control, Expressing, Western Blot

Journal: Experimental Physiology

Article Title: Tumour necrosis factor α regulates the miR‐27a‐3p–Sfrp1 axis in a mouse model of osteoporosis

doi: 10.1113/EP090311

Figure Lengend Snippet: Knockdown of Sfrp1 promotes osteogenic differentiation of BMSCs in vitro. (a) BMSCs were isolated from wild‐type (WT) mice and cultured in vitro. The expression of Sfrp1 was analysed by qPCR at the indicated time points. (b–e) BMSCs were transfected with empty vector pcDNA3.1, or Sfp1 ‐expressing vector pcDNA3.1‐Sfrp1 alone or followed by TNF‐α treatment (10 ng/mL). (b) The expression of Sfrp1 was analysed by qPCR 48 h later. (c–e) Transfected BMSCs were cultured in vitro for 7 days. (c) The expression of ALP and formation of mineralized nodule were subsequently analysed by ALP staining and Alizarin Red‐S staining, respectively. (d) The mRNA expression of Runx2 and Osterix was analysed by qPCR. (e) The protein expression of Runx2 and Osterix was analysed by western blot. * P < 0.05, ** P < 0.01, *** P < 0.001, between the indicated groups.

Article Snippet: The membranes were probed with the following primary antibodies: Runx2 (cat. no. 12556, Cell Signaling Technology, Danvers, MA, USA), Osterix (DF7731, Affinity Biosciences, Changzhou, China), Sfrp1 (cat. no. 3534, Cell Signaling Technology), β‐catenin (cat. no. 8480, Cell Signaling Technology), C‐Myc (cat. no. 18583, Cell Signaling Technology), Cyclin D1 (cat. no. 55506, Cell Signaling Technology), and the loading control, GAPDH (cat. no. 5174, Cell Signaling Technology).

Techniques: Knockdown, In Vitro, Isolation, Cell Culture, Expressing, Transfection, Plasmid Preparation, Staining, Western Blot

Journal: Experimental Physiology

Article Title: Tumour necrosis factor α regulates the miR‐27a‐3p–Sfrp1 axis in a mouse model of osteoporosis

doi: 10.1113/EP090311

Figure Lengend Snippet: Overexpression of Sfrp1 abrogates the promotive effect of miR‐27a‐3p on osteogenic differentiation of BMSCs. (a–f) BMSCs from wild‐type (WT) mice were transfected with pcDNA 3.1, or pcDNA3.1‐Sfrp1, or (pcDNA3.1‐Sfrp1+miR‐NC), or (pcDNA3.1‐Sfrp1+miR‐27a‐3p mimics) alone or followed by TNF‐α treatment (10 ng/mL). (a) The relative expression of miR‐27a‐3p and Sfrp1 was examined by qPCR 48 h later. (b–f) Transfected cells were cultured for 7 days. (b, c) Formation of mineralized nodule was analysed by Alizarin Red‐S staining. Scale bar, 100 μm. (d) The expression of ALP was analysed by ALP staining. (e) The mRNA expression of Runx2 and Osterix was analysed by qPCR. (f) The protein expression of Sfrp1, Runx2 and Osterix was analysed by western blot. * P < 0.05, ** P < 0.01, *** P < 0.001, between the indicated groups.

Article Snippet: The membranes were probed with the following primary antibodies: Runx2 (cat. no. 12556, Cell Signaling Technology, Danvers, MA, USA), Osterix (DF7731, Affinity Biosciences, Changzhou, China), Sfrp1 (cat. no. 3534, Cell Signaling Technology), β‐catenin (cat. no. 8480, Cell Signaling Technology), C‐Myc (cat. no. 18583, Cell Signaling Technology), Cyclin D1 (cat. no. 55506, Cell Signaling Technology), and the loading control, GAPDH (cat. no. 5174, Cell Signaling Technology).

Techniques: Over Expression, Transfection, Expressing, Cell Culture, Staining, Western Blot

Journal: Experimental Physiology

Article Title: Tumour necrosis factor α regulates the miR‐27a‐3p–Sfrp1 axis in a mouse model of osteoporosis

doi: 10.1113/EP090311

Figure Lengend Snippet: MiR‐27a‐3p–Sfrp1 axis regulates the Wnt3a–β‐catenin signalling in osteogenic differentiation of BMSCs. (a–c) BMSCs from the Sham or OVX mice were isolated. The mRNA (a) and protein (b) expression of Wnt3a and β‐catenin were analysed by qPCR and western blot, respectively. (c) The expression levels of β‐catenin in nucleus and cytoplasm were determined by western blot. (d–g) BMSCs from wild‐type mice were transfected with pcDNA3.1, or pcDNA3.1‐Sfrp1, or (pcDNA3.1‐Sfrp1 + miR‐NC), or (pcDNA3.1‐Sfrp1 + miR‐27a‐3p mimics) alone or followed by TNF‐α treatment (10 ng/mL). The mRNA (d) and protein (e, f) expression of Wnt3a and β‐catenin were analysed by qPCR and western blot, respectively. (g) The expression levels of β‐catenin in nucleus and cytoplasm were determined by western blot. * P < 0.05, ** P < 0.01, *** P < 0.001, between the indicated groups.

Article Snippet: The membranes were probed with the following primary antibodies: Runx2 (cat. no. 12556, Cell Signaling Technology, Danvers, MA, USA), Osterix (DF7731, Affinity Biosciences, Changzhou, China), Sfrp1 (cat. no. 3534, Cell Signaling Technology), β‐catenin (cat. no. 8480, Cell Signaling Technology), C‐Myc (cat. no. 18583, Cell Signaling Technology), Cyclin D1 (cat. no. 55506, Cell Signaling Technology), and the loading control, GAPDH (cat. no. 5174, Cell Signaling Technology).

Techniques: Isolation, Expressing, Western Blot, Transfection

Journal: BIOCELL

Article Title: Identification of tumorigenic risk genes in human placenta-derived mesenchymal stem cells treated with 3-methylcholanthrene

doi: 10.32604/biocell.2022.015390

Figure Lengend Snippet: FIGURE 4. The verification of tumor transformation-related and pluripotency-related DEGs. (A) Detection of mRNA of transformation and pluripotency related DEGs in 3-MC treated hPTMSCs. (B) mRNA of pluripotent markers sox2, cmyc, oct4, and nanog measured by qPCR and normalized against gapdh or actb. MOCK was arbitrarily set to 1.0. (C and D) western blotting of OCT4 and CMYC in hPTMSCs and A549 cells with sfrp1 and ptgs2 overexpression and knockdown. *P < 0.05, **P < 0.01 denote significant differences compared with the MOCK or sh-NC group.

Article Snippet: Equal protein from all samples was separated by 10% or 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA), then probed with rabbit anti SFRP1, PTGS2, SOX2, CMYC, OCT4, NANOG, and ACTB antibody (Proteintech, Wuhan, China) at 4°C overnight followed by appropriate secondary antibodies (BOSTER, Wuhan, China).

Techniques: Transformation Assay, Western Blot, Over Expression, Knockdown

Journal: BIOCELL

Article Title: Identification of tumorigenic risk genes in human placenta-derived mesenchymal stem cells treated with 3-methylcholanthrene

doi: 10.32604/biocell.2022.015390

Figure Lengend Snippet: FIGURE 5. The inhibitory or promotion effects of SFRP1 and PTGS2 overexpression on the cell viability, colony formation, and migration of A549 cells. (A) Detections of mRNA of sfrp1 and ptgs2 overexpression and knockdown efficiencies in stable A549 subcell lines constructed with indicated viruses. (B) Cell viability of A549 cells was analyzed by CCK-8 assay at 24 h. sfrp1 silence significantly facilitated the cell viability compared with the sh-NC group. ptgs2 silence significantly suppressed the cell viability compared with the sh-NC group. (C) Representative images of the indicated A549 subcell lines assessed by colony formation assay. sfrp1 overexpression significantly suppressed the colony formation compared with the MOCK or sh-NC group. ptgs2 overexpression significantly facilitated the colony formation compared with the MOCK or sh-NC group. (D) Representative images and quantitative data of migration of the indicated A549 subcell lines assessed by wound-healing assay. sfrp1 silence significantly facilitated the migration compared with the MOCK or sh- NC group. ptgs2 overexpression significantly facilitated the migration compared with the MOCK or sh-NC group. All the experiments were performed in triplicate, and the data are presented as the mean ± SD. *P < 0.05, **P < 0.01 denote significant differences compared with the MOCK or sh-NC group.

Article Snippet: Equal protein from all samples was separated by 10% or 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA), then probed with rabbit anti SFRP1, PTGS2, SOX2, CMYC, OCT4, NANOG, and ACTB antibody (Proteintech, Wuhan, China) at 4°C overnight followed by appropriate secondary antibodies (BOSTER, Wuhan, China).

Techniques: Over Expression, Migration, Knockdown, Construct, CCK-8 Assay, Colony Assay, Wound Healing Assay