Journal: Kidney Diseases

Article Title: Clinical and Experimental Insights into the Role of NETosis in IgA Nephropathy Pathogenesis

doi: 10.1159/000546343

Figure Lengend Snippet: Elevated serum NETosis markers in IgAN patients correlate with CitH3 and Gd-IgA1 levels, and increased NETosis markers in renal tissue. a–c Comparison of serum levels of Citrullinated Histone H3 (CitH3), myeloperoxidase (MPO), and neutrophil elastase (NE) between IgAN patients ( n = 16) and healthy controls (HC, n = 13). d–f Pearson correlation analysis between serum CitH3, MPO, NE, and Gd-IgA1 levels. g Immunofluorescence staining of CitH3 and MPO in kidney tissues from IgAN ( n = 5) and minimal change disease (MCD, n = 5) patients. h–i Quantitative analysis of immunofluorescence staining intensity in the kidneys. Unpaired t tests were used for group comparisons, and Pearson correlation was employed for correlation analysis. Data are presented as mean ± SEM.

Article Snippet: Bone marrow was harvested from healthy C57BL/6J mice, and neutrophils were isolated utilizing a mouse bone marrow neutrophil isolation kit (Solarbio, P8550).

Techniques: Comparison, Immunofluorescence, Staining

Journal: Blood

Article Title: MicroRNA-130a-mediated down-regulation of Smad4 contributes to reduced sensitivity to TGF-β1 stimulation in granulocytic precursors.

doi: 10.1182/blood-2011-03-339978

Figure Lengend Snippet: Figure 3. Biologic effect of increased miR-130a expression. Stable transfection of 32Dcl3 cells with the pEGP-miR-130a, pEGP-miR-223, or pEGP-miR-Null vectors was performed, generating 3 pEGP-miR-130a (Cl 3, Cl 6, and Cl 7) clones, 1 pEGP-miR223 (miR-223) clone, and 1 pEGP-miR-null (null) clone. (A) Relative miR-130a expression in Cl 3, Cl 6, Cl 7, null, and miR-223 was determined by real-time PCR and normalized to Sno234 expression. (B) Relative miR-223 expres- sion in clone miR-223 and null was determined by real-time PCR and normalized to Sno234 expression. (C) Immunoblot showing Smad4 protein expression in the Cl 3, Cl 6, Cl 7, null, and miR-223 clones. The graph shows the relative expression of Smad4 protein relative to the expression of -actin. The highest value among those being compared was assigned the value 1, and the remaining values were recalculated accordingly. (D) The relative expression level of Smad4 mRNA in the Cl 3, Cl 6, Cl 7, null, and miR-223 clones was measured by real-time PCR and normalized to -actin expression. Data are shown as the mean SD from triplicate measurements. (E) Cell proliferation assays were performed with the clones Cl 3, Cl 6, Cl 7, null, and miR-223 in the presence or absence of human recombinant TGF-1 (20nM). The graph shows the relative number of cells after 4 days when TGF-1– stimulated cells are compared with unstimulated cells for each clone. The mean reduction in cell number (Red. in cell no.) for TGF-1–stimulated cells for each clone was 8% for Cl 3, 31% for Cl 6, 8% for Cl 7, 43% for null, and 52% for miR-223 (the values are based on 3 independent growth experiments). The cell number for the unstimulated cells was assigned the value 1, and the relative number of TGF-1–

Article Snippet: 32Dcl3 cells (ATCC CRL-11 346) were cultured in RPMI 1640 medium (Invitrogen), 10% non–heat-inactivated FBS (Gibco BRL), 1% P/S, and 1 ng/mL murine IL-3 (Sigma-Aldrich).

Techniques: Expressing, Stable Transfection, Clone Assay, Real-time Polymerase Chain Reaction, Western Blot, Recombinant

Journal: Blood

Article Title: MicroRNA-130a-mediated down-regulation of Smad4 contributes to reduced sensitivity to TGF-β1 stimulation in granulocytic precursors.

doi: 10.1182/blood-2011-03-339978

Figure Lengend Snippet: Figure 4. The effect of knocking-down Smad4 expression with a shRNA against Smad4 mRNA. (A) Immunoblot showing Smad4 protein expression in 32Dcl3 stably transfected with a plasmid encoding a short hairpin against Smad4 (sh-Smad4) or a nonsilencing short hairpin (sh-neg), respectively. The graph shows the relative expression of Smad4 normalized to -actin expression. (B) The relative expression level of Smad4 mRNA in the 32Dcl3 clones sh-Smad4 and sh-neg, respectively. Smad4 mRNA expression was measured by real-time PCR and normalized to -actin expression. Data are shown as the mean SD from triplicate measurements. (C) Cell proliferation assays were performed with the 32Dcl3 clones sh-Smad4 and sh-neg in the presence or absence of human recombinant TGF-1 (20nM). The graph shows the relative number of cells after 4 days when TGF-1–stimulated cells are compared with unstimulated cells for each clone. The reduction in cell number (Red. in cell no.) for TGF-1–stimulated cells for each clone was 19% for sh-Smad4 and 47% for sh-neg. The cell number for the unstimulated cells was assigned the value 1, and the relative number of TGF-1–stimulated cells was recalculated accordingly.

Article Snippet: 32Dcl3 cells (ATCC CRL-11 346) were cultured in RPMI 1640 medium (Invitrogen), 10% non–heat-inactivated FBS (Gibco BRL), 1% P/S, and 1 ng/mL murine IL-3 (Sigma-Aldrich).

Techniques: Expressing, shRNA, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Clone Assay, Real-time Polymerase Chain Reaction, Recombinant

Journal: Blood

Article Title: MicroRNA-130a-mediated down-regulation of Smad4 contributes to reduced sensitivity to TGF-β1 stimulation in granulocytic precursors.

doi: 10.1182/blood-2011-03-339978

Figure Lengend Snippet: Figure 5. TGF-1 sensitivity is restored by antisense RNA against miRNA-130a or expression of a Smad4 mRNA lacking miR-130a–binding sites. (A) 2 32Dcl3 clones stably transfected with pmiRZIP-130a (pZIP-130a–cl 1 and pZIP-130a–cl 2) or pmiRZIP-null (pZIP-null–cl 1and pZIP-null–cl 2) were tested for Smad4 expression by immunoblotting. The graph shows the relative expression of Smad4 normalized to -actin expression. (B) Cell proliferation assays were performed with pZIP-null–cl 1, pZIP-null–cl 2, pZIP-130a–cl 1, and pZIP-130a–cl 2 in the presence or absence of human recombinant TGF-1 (20nM). The mean reduction in cell number (Red. in cell no.) for TGF-1–stimulated cells for each clone was 36% for pZIP-null–cl 1, 39% for

Article Snippet: 32Dcl3 cells (ATCC CRL-11 346) were cultured in RPMI 1640 medium (Invitrogen), 10% non–heat-inactivated FBS (Gibco BRL), 1% P/S, and 1 ng/mL murine IL-3 (Sigma-Aldrich).

Techniques: Expressing, Binding Assay, Clone Assay, Stable Transfection, Transfection, Western Blot, Recombinant

Journal: Blood

Article Title: MicroRNA-130a-mediated down-regulation of Smad4 contributes to reduced sensitivity to TGF-β1 stimulation in granulocytic precursors.

doi: 10.1182/blood-2011-03-339978

Figure Lengend Snippet: Figure 6. Identification of miR-130a binding sites in the Smad4 mRNA. (A) Schematic drawing showing the cloning of the predicted miR-130a binding sites in the 3-UTR of pMIR-REPORT. (B-C) 32Dcl3 cells were transfected individually with the firefly expression vector pMIR-REPORT containing 1 of the 4 different constructs (wt1/wt2, mut1/wt2, wt1/mut2, and mut1/mut2) along with a renilla luciferase vector (for normalization) and pre–miR-130a/scrambled miRNA (B) or LNA-130a/LNA-neg (C), respectively. Relative firefly luciferase activity is shown after normalizing to the renilla luminescence. Data are shown as the mean SD from triplicate measure- ments. The expression from the transfections with the controls (miR-neg and LNA-neg) was assigned the value 1, and the relative expression measured from the constructs cotransfected with miR-130a or LNA-130a was recalculated accordingly.

Article Snippet: 32Dcl3 cells (ATCC CRL-11 346) were cultured in RPMI 1640 medium (Invitrogen), 10% non–heat-inactivated FBS (Gibco BRL), 1% P/S, and 1 ng/mL murine IL-3 (Sigma-Aldrich).

Techniques: Binding Assay, Cloning, Transfection, Expressing, Plasmid Preparation, Construct, Luciferase, Activity Assay