18 research outputs found
PI3 Kinase activation regulates Zscan4 metastate induction.
<p>(A) Western Blot analysis on ESCs treated with either RA or RA with 5.0 μM LY294002 for 12h. NANOG was considered the positive control for treatment evaluation. (B) The mRNA expression levels were assessed by qRT-PCR and normalized to RM condition. The average and SD of duplicate samples from each of three independent biological replicates are shown: **, p < .01; ***, p < .001, in a Student’s <i>t</i> test. (C) Western Blot analysis on ESCs were treated with either RA or RA with 20 μM PD0325901 for 12h. ESCs treatment with RA increased pERK1/ERK2, whereas PD treatment hampered pERK1/ERK2. (D) The mRNA expression levels are assessed by qRT-PCR and normalized to RM condition. The average and SD of duplicate samples from each of three independent biological replicates are shown: *, p < .05, in a Student’s <i>t</i> test.</p
Alterations of GSK3β / β-catenin are associated with the development of the glomerulocystic phenotype.
<p>Representative pictures of renal cortex from 30 (A, B) and 50 days old (C, D) mice. Ctr (A, C) and Dicer cKO (B, D) sections were stained with anti β-catenin antibody. In Dicer cKO, β-catenin expression is limited to the basolateral domain in both tubules and parietal cells of the Bowman’s capsule, at both P30 and P50 (B-D). (A-D) Magnification 63X, zoom1.5. Panel E and F show immunoblotting of Cortex/OSOM samples of 30 (E) and 50 (F) days old mice. GSK3β expression is already upregulated at the pre-cystic stage (P30) in Dicer cKO mice (E) and this is persistent at P50 (F). Glomerular cyst formation is associated with loss of cytosolic β-catenin expression and general downregulation of cortical protein level (F). No significant changes were observed in the fraction of their main regulating phosphorylated forms. Panel G represent qPCR analysis of mRNA of β-catenin in 30 (right) and 50 days old mice (left). mRNA level of β-catenin is downregulated in Dicer-cKO mice at P50. Data are expressed as mean ± sem; n power is 5 vs 5. * is for p value < 0.05; ** is for p-value <0.01.</p
Dicer suppression is associated with downregulation of AQP2, NKCC2 and lower density of collecting ducts.
<p>Representative pictures of the inner stripe of the outer medulla of control (A, C) and Dicer cKO (B, D) mice double labeled with anti-AQP2 (green) and anti-H<sup>+</sup>ATPase (red) antibodies. At P50, Dicer cKO mice (D) showed no alteration in the distribution pattern of principal (AQP2 +) and intercalated cells (H<sup>+</sup>ATPase +), but a lower density of collecting ducts compared to the controls (C). (A-D) Magnification 40X. (E) Western blot analysis for AQP2 and NKCC2 expression at P30 (upper) and P50 (lower) of samples of IM and ISOM, respectively. AQP2 expression is significantly downregulated in Dicer cKO mice compared to Ctr at P50. NKCC2 expression is significantly downregulated in Dicer cKO mice compared to Ctr at P30 and P50. Data are expressed as mean ± sem; n power is 5 vs 5. *** is for p value < 0.001.</p
Selective Dicer Suppression in the Kidney Alters GSK3β/β-Catenin Pathways Promoting a Glomerulocystic Disease - Table 1
<p>Data are expressed as mean ± sem; n is expressed in brackets.</p><p>** is for p <0.01;</p><p>*** is for <0.001</p><p>Selective Dicer Suppression in the Kidney Alters GSK3β/β-Catenin Pathways Promoting a Glomerulocystic Disease - Table 1 </p
Tubular dilatation mainly involves the distal segments of the nephron.
<p>Representative pictures of renal cortex from Ctr mice (A, D) and Dicer cKO at P30 (B, E) and at P50 (C, F) mice stained with LAH (Lectin from Arachis Hypogaea) (A- C) and AQP2 (D- F). Dilated tubules in P50 Dicer cKO mice are positive for LAH (C) and AQP2 (F), reflecting their origin from distal convolute tubules, connecting and collecting ducts. (A-F) Magnification 63X.</p
Dicer inactivation in mutant mice.
<p>Panel A shows the genotyping of Dicer, Cre and Pax8 genes in mice either homozygote (cKO) or heterozygote (Het) for Dicer and control mice (Ctr). PCR of renal cortical tissues at P50 shows the Dicer allele floxed, wild type and excised forms. Cre is detected only in homozygous and heterozygous cKO mice as expected, while Pax8 is present constitutively in all the groups. Panel B shows western blot analysis of renal zones Cortex/OSOM, ISOM and IM samples from 50 day old mice. A reduced abundance of Dicer in cKO mice can be observed in all the zones. Normalization was carried out with GAPDH. Panel C shows qPCR analysis of Dicer mRNA. A substantial reduction of Dicer can be seen in cKO mice compared to Ctr. Data are expressed as mean ± sem.</p
RA-Zscan4<sup>+</sup> cells molecular and cellular characterization.
<p>(A) The global gene expression profiles between RA-Zscan4<sup>+</sup> and RA-Zscan4<sup>-</sup> cell populations by DNA chip microarray hybridization. List of RA-Zscan4<sup>+</sup> probes upregulated more than 20-fold compared to RA-Zscan4<sup>-</sup>. (B) RA-Zscan4<sup>+</sup> cells expression versus RM-Zscan4<sup>+</sup>. (C) RA-Zscan4<sup>+</sup> and RA-Zscan4<sup>-</sup> cells were collected, separated by FACS and plated in RM. The mean % RA-Zscan4<sup>+</sup> or RA-Zscan4<sup>-</sup> cells ± SD of three independent experiments is presented with statistical analysis performed using Student’s <i>t</i> test (**, p < .01; ***, p < .001). (D) RA-Zscan4<sup>+</sup> and RA-Zscan4<sup>-</sup> cells were collected and separated by FACS, plated in RM and were characterized based on AP-positive colonies after 5 days in RM (<i>n</i> = 3).</p
Suppression of Dicer leads to a glomerulocystic disease.
<p>Representative pictures of hematoxylin and eosin (A-F) and Masson’s Trichrome (G,I). Staining of Ctr (A, D, G), Dicer cKO kidneys at P30 (B, E, H) and Dicer cKO kidneys at P50 (C, F, I). Dicer induced cyst formation is limited to the cortex (C). Dicer cKO mice present a glomerulocystic phenotype and tubular dilatation at P50 (F). These alterations are not so prominent at P30, where only sites of dilatation in Bowman capsule can be sporadically seen (E). Interstitial fibrosis is evident in Dicer cKO mice at P50 (I). (A, B, C) Magnification 5X. (D- I) Magnification 40X.</p
High cellular turnover precedes the development of cysts.
<p>Representative pictures of renal cortex from 30 (A, B, E, F) and 50 day old mice (C, D, G, H). Ctr (A, C, E, G) and Dicer cKO (B, D, F, H) renal sections were stained with anti-ki67 (marker of proliferation) (A-D) and anti-Cleaved Caspase 3 (marker of apoptosis) (E-H). Before developing the Dicer-induced glomerulocystic phenotype, the epithelium lining the dilating structure is already immunoreactive to the proliferation marker ki67 (B). Higher proliferation rate is evident also in dilated structure (D). Glomerular and tubular cells of Dicer cKO mice at both P30 and P50 (F, H) were positive for the apoptosis marker, Cleaved Caspase 3. (A-H) Magnification 40X.</p
The development of the glomerulocystic phenotype is progressively associated with loss of primary cilium.
<p>Representative pictures of renal cortex from 50 days old Ctr (B) and Dicer cKO mice (A, C, D) stained for α-Acetylated Tubulin (green) and counterstained with Dapi (blue). Primary cilium is normally found in non-dilating tubules of Dicer cKO mice (A) and in the parietal cells of Ctr mice (B) at P50. However, presence of cilia is decreased progressively in dilating glomeruli (C) until disappearing in frank cysts (D). Bars = 5 μm (A); 10 μm (B-D).</p