Journal: Oncology reports

Article Title: Supervillin promotes tumor angiogenesis in liver cancer.

doi: 10.3892/or.2020.7621

Figure Lengend Snippet: Figure 1. SVIL is highly expressed in liver cancer and localized to new tumor vessels. (A) Analysis of microarray data from The Cancer Genome Atlas. (B) Immunohistochemistry was performed to detect the expression of SVIL in normal liver tissue, grade I, grade II and grade III liver cancer tissue samples (DAB staining; magnification, x100). (C) Liver cancer tissue was serially sectioned and analyzed for SVIL, CD31, CD34, CD146 and CD248 expression (DAB staining; magnification, x100). (D) CD34/PAS staining and SVIL/PAS staining were performed on the same liver cancer tissue area (DAB staining; magnification, x100 and x200). Statistics of the proportion of CD34‑labeled endothelial blood vessels in SVIL‑labeled tumor blood vessels. Data are presented as the mean ± standard deviation. SVIL, supervillin; TCGA, The Cancer Genome Atlas; PAS, periodic acid‑Schiff.

Article Snippet: A liver cancer tissue microarray consisting of 173 pathological samples was purchased from US Biomax, Inc. Liver cancer tissue microarrays were immunohistochemically treated with antibodies against SVIL (dilution 1:1,000; cat. no. S8695; Sigma-Aldrich, Inc.) (24), cluster of differentiation (CD) 31 (dilution 1:1,500; product no. 3528; Cell Signaling Technology, Inc.), CD34 (dilution 1:50; product no. 3569; Cell Signaling Technology, Inc.), CD146 (dilution 1:500; cat. no. GTX60775; GeneTex, Inc.) and CD248 (dilution 1:500; cat. no. 564993; BD Biosciences).

Techniques: Microarray, Immunohistochemistry, Expressing, Staining, Standard Deviation

Journal: Cellular & molecular biology letters

Article Title: Reciprocal negative feedback between Prrx1 and miR-140-3p regulates rapid chondrogenesis in the regenerating antler.

doi: 10.1186/s11658-024-00573-x

Figure Lengend Snippet: Fig. 4 Characteristics of tissue layers of the antler growth center and isolated RM cells. A Immunofluorescence assay of CD73, CD90, Nestin, CD34, and Prrx1 (red color) in the RM, PC, and CA layers, respectively. B Third passage of cultured cells from the RM layer were identified using mesenchymal stem cell markers: CD73 and CD90 (red), Nestin and CD34 (green), respectively; nuclei were stained blue with DAPI. C Chondrogenic differentiation of RM cells, Alcian blue, and Col II immunofluorescence staining. D Osteogenic differentiation of RM cells with Alizarin red staining. E Adipogenic differentiation of RM cells with Oil Red O staining (scale bars, 125 μm and magnification ×200)

Article Snippet: The cells and tissues were fixed, permeabilized, blocked, and incubated overnight with primary antibodies: CD90 (1:300, proteintech, USA, 66766-1-lg), CD73 (1:1000, proteintech, USA, 12231- 1-AP), Nestin (1:300, BIOSS, China, bs-0008R), CD34 (1:300, proteintech, USA, 14486- 1-AP), and Prrx1 (1:200, Absin, China, abs134576).

Techniques: Isolation, Immunofluorescence, Cell Culture, Staining

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: GATA-1 was located on the upstream of miR-23a∼27a∼24-2 cluster and activated its expression during erythropoiesis. ( A ) Q-PCR analysis of the primary transcript of the miR-23a∼27a∼24-2 cluster in K562s undergoing erythroid differentiation caused by hemin treatment for 0, 24, 48 and 72 h. ( B ) Q-PCR analysis of the mature transcripts of miR-27a, miR-24 and miR-23a in K562s undergoing erythroid differentiation caused by hemin treatment for 0, 24, 48 and 72 h. ( C ) Northern blot analysis of miR-27a, miR-24 and miR-23a in K562s undergoing erythroid differentiation. U6 snRNA was used as a loading control. ( D ) Q-PCR analysis of mature miR-27a and miR-24 expression in CD34+ HPCs undergoing day 4, 7, 11, 15 and 18 of E culture. ( E ) Representation of the human −603 bp miR-23a∼27a∼24 cluster promoter fragments. ( F ) ChIP-PCR and ChIP-qPCR analysis of the GATA-1 hit on the −557 site at the miR-23a∼27a∼24 cluster promoter in K562s. ChIP-q-PCR results are shown as fold enrichment compared with input. Error bars represent the standard deviation obtained from three independent experiments. ** P < 0.01. ( G ) Immunoblot analysis of GATA-1 expression in K562s treated with siRNAs specific to GATA-1 (si_GATA-1) for 48 h or K562s transfected with a construct overexpressing GATA-1 (over_GATA-1) for 48 h, respectively. ( H ) Q-PCR analysis of Pri-27a∼24, mature miR-27a and miR-24 expression in K562s treated as described in (G). ( I ) Immunoblot analysis of GATA-1 expression in K562s undergoing erythroid differentiation. ( J ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1 and pol II occupancy at the −557 site in hemin-treated K562s at 0 and 48 h. ChIP-q-PCR results are shown as fold enrichment compared with input. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( K ) Functional activity of GATA-1 on the wild-type GATA site (WT) or mutant site (MUT) of the miR-23a∼27a∼24 promoter in a luciferase reporter analysis. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Expressing, Northern Blot, Control, ChIP-qPCR, Standard Deviation, Western Blot, Transfection, Construct, Functional Assay, Activity Assay, Mutagenesis, Luciferase

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: MiR-27a and miR-24 promoted erythroid differentiation in CD34+ HPCs. ( A ) Monitoring of the GFP + population (left panel) and the CD235a stained GFP + fraction (medium panel) of Lenti-miRNA-transduced CD34+ HPCs on day 15 of E culture. The morphology (May-Grunwald Giemsa staining) of CD34+ HPCs derivatives on day 15 is shown in the right panel. A 400X magnification of a representative field is shown. ( B ) Detection of the erythroid differentiation degree of CD34+ HPCs transduced with Lenti-27a, Lenti-24 or Lenti-GFP in E culture at the indicated time. Percentages of basophilic (Bas), polychromatophilic (Pol), orthochromatic (Ort) erythroblasts and erythrocytes (Ery) were determined by May-Grunwald/Giemsa staining of cytospin preparations. ( C ) Q-PCR analysis of gamma-globin mRNA expression in CD34+ HPCs transduced with Lenti-27a, Lenti-24 or Lenti-GFP in E culture at the indicated time. ( D ) A comparison of the erythroid colony-forming capacity (CFU-E, BFU-E) of CD34+ HPCs transduced with Lenti-miRNAs. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( E ) FACS monitoring of CD34+ HPCs transduced with Zip-miRNA or Zip-GFP as described in (A). ( F ) Detection of the erythroid differentiation degree of CD34+ HPCs transduced with Zip-27a, Zip-24 or Zip-GFP as described in (B). ( G ) Detection of gamma-globin mRNA level in CD34+ HPCs transduced with Zip-miRNA. ( H ) Colony-forming assay of CD34+ HPCs transduced with Zip-miRNA as described in (D).

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Staining, Transduction, Expressing, Comparison, Standard Deviation

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: GATA-2 was post-transcriptionally regulated by miR-27a and miR-24 during erythropoiesis. ( A ) A computer prediction of conserved and mutated binding sites within the 3′ UTR of GATA-2 mRNA for miR-27a and miR-24. ( B ) Relative luciferase activity of the indicated GATA-2 reporter constructs. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( C ) Immunoblot analysis of GATA-2 in K562s transfected with scramble or miRNA mimics (miR-27a, miR-24) or inhibitors (Anti-27a, Anti-24). ( D ) Immunoblot analysis of GATA-2 in CD34+ HPCs transduced with Lenti-GFP control and lentivirus expressing miR-27a or miR-24 (Lenti-27a or Lenti-24). ( E, F ) ‘Rescue’ assays for miRNAs and GATA-2 during erythroid differentiation. Immunoblot analysis of GATA-2 in K562s treated with scramble or Anti-27a/24 (E) for 24 h. These cells were subsequently treated for another 24 h with control siRNAs or siRNAs specific to GATA-2 and were then treated with hemin treatment for 0, 48 and 72 h. (F) FACS analysis of K562s stained for CD71 and CD235a expression after 48 h of hemin induction as described earlier in the text.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Binding Assay, Luciferase, Activity Assay, Construct, Standard Deviation, Western Blot, Transfection, Transduction, Control, Expressing, Staining

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: The GATA switch regulated miR-27a and miR-24 expression. ( A ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1, GATA-2 and Pol II occupancy at the −557 site in hemin-treated K562s at 0 and 48 h. ChIP-q-PCR results are shown as fold enrichment compared with input. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( B ) Immunoblot analysis of GATA-1 and GATA-2 expression in K562s undergoing erythroid differentiation for 0, 24, 48 and 72 h. ( C ) Functional activity of GATA-1 and GATA-2 on the wild-type GATA site (WT) or mutant site (MUT) of the miR-23a∼27a∼24 promoter in a luciferase reporter analysis. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( D ) Immunoblot analysis of GATA-2 in K562s treated with siRNAs specific to GATA-2 (si_GATA-2) for 48 h or K562s transfected with a construct overexpressing GATA-2 (over_GATA-2) for 48 h, respectively. ( E ) Q-PCR analysis of Pri-27a∼24, mature miR-27a and miR-24 expression in K562s treated as described in (D). ( F ) ChIP-PCR analysis of GATA-1 and GATA-2 occupancy at the −557 site in K562s transfected with control siRNAs or siRNAs specific to GATA-1/GATA-2 or K562s transfected with empty pcDNA3.1 vectors or pcDNA3.1_GATA-1/GATA-2. ( G ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1 and GATA-2 occupancy at the −557 site in CD34+ HPCs of E culture. ( H ) Q-PCR analysis of Pri-27a-24 abundance in CD34+ HPCs of E culture (Top panel). Error bars represent the standard deviation obtained from three independent experiments. An immunoblot analysis of GATA-1 and GATA-2 expression in CD34+ HPCs of E culture (Bottom panel). ( I ) Q-PCR (left panel) and immunoblot (right panel) analysis of GATA-1 and GATA-2 expression in CD34+ HPCs transduced with lentivirus-expressing siRNAs against GATA-1 or control on day 11 of E culture. Error bars represent standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( J ) Q-PCR analysis of Pri-27a∼24, mature miR-27a and miR-24 expression in CD34+ HPCs treated as described in (I). Error bars represent the standard deviation obtained from three independent experiments. ** P < 0.01.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Expressing, Standard Deviation, Western Blot, Functional Assay, Activity Assay, Mutagenesis, Luciferase, Transfection, Construct, Control, Transduction

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: A regulatory circuit involving GATA-1, GATA-2 and miR-27a/24 in erythropoiesis. ( A ) A schematic representation of the regulatory circuit comprised GATA-1, GATA-2, miR-27a and miR-24 in erythroid differentiation. ( B , C ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1 and GATA-2 occupancy at the −557 site in K562s transfected with scramble or miRNA mimics and inhibitors (B, r miR-27a and C, miR-24). ( D ) Q-PCR analysis of Pri-27a∼24 abundance in K562s treated as described in (B) and (C). Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( E ) Q-PCR analysis of Pri-27a∼24 abundance in CD34+ HPCs transduced with Lenti-miR-27a/24 or Lenti-GFP control for 11 days. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( F ) Q-PCR analysis of Pri-27a∼24 abundance in CD34+ HPCs transduced with Zip-miRNA or Zip-GFP for 11 days. Error bars represent the standard deviation obtained from three independent experiments. ** P < 0.01.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Transfection, Standard Deviation, Transduction, Control