miR-200 family members can ameliorate EMT and tubulointerstitial fibrosis in UUO model mouse kidneys.

<p><b>a</b>, Western blotting analysis confirms down-regulation of E-cadherin and up-regulation of N-cadherin during EMT. <b>b</b>,<b>c</b>, Western blotting of E-cadherin and N-cadherin in HK-2 cells treated with TGF-beta and transfected with control miR or miR-200 members precursors individually. In western blotting, each band has been quantitated and subjected to densitometry to determine if statistically significant difference exists between groups <b>d</b>, Changes in miR-200 family levels in UUO model mice kidneys, as measured by <i>TaqMan</i> qRT-PCR and normalized to U6 expression. The data are means from a representative time course experiment measured in triplicate and are presented as mean±S.E. <b>e</b>, Azan staining of intact Balbc mouse kidney, UUO day-6 kidneys after transfection of negative control pre-miR or miR-200b precursor. Fibrosis score; The tubulointerstitial fibrosis score in kidneys of miR-200b precursor injected group was significantly lower than of control group: <i>P</i><0.05, The data are means from experiment measured in triplicate and are presented as mean±S.E (n = 8) <b>f</b>, Immunofluorescence studies of vimentin and cytokeratin 18 in the kidneys of UUO day6 mice and UUO day6 mice injected by miR-200b precursor. miR-200b ameliorates up-regulation of vimentin and down-regulation of cytokeratin 18. <b>g</b>, real-time PCR confirms that miR-200b precursor reverses up-regulation of type I,III collagen and fibronectin of UUO day-6 kidneys. The data are normalized to beta-actin mRNA and shown as mean±S.E.(n = 8).</p

TH immunostaining in the substantia nigra pars compacta (SNc), and the ratio to the intact side.

<p>(A) TH immunostaining in the intact SNc. Severe loss of TH-positive neurons was seen in the lesioned side SNc of control group. Preservation of TH-positive neurons was seen at the lesioned side SNc of 50 Hz SCS group. Scale bar: 200 µm. (B) Significant preservation of TH-positive neurons in the lesioned-side SNc of 50 Hz SCS group, compared to those of control group (*p<0.05, n = 10, respectively).</p

Time course and SCS electrode, and the brain region punched out for protein assay.

<p>(A) Scheme showing overall experimental design. (B) Scheme showing experimental design for protein assay. (C) Photograph showing SCS electrode used in this study (diameter: 2 mm; wire length: 60 mm). (D) Scheme showing a rat during stimulation. (E) Brain tissue (diameter: 3 mm showing gray circle), corresponding to the striatum, was punched out from both the lesioned and the intact side.</p

Tyrosine hydroxylase (TH) immunostaining in the striatum and the ratio to the intact side.

<p>(A) TH immunostaining in the striatum. Severe loss of TH-positive fibers was seen in the lesioned striatum of control group. Preservation of TH-positive fibers was seen in the lesioned striatum of all SCS groups. Scale bar: 200 µm. (B) The all SCS groups showed significant preservation of TH-positive fibers in the lesioned striatum, compared to those in control group (*p<0.05, n = 10, respectively).</p

miR-200 family members target the transcriptional repressors ZEB1 and ZEB2.

<p>Normalized activity of luciferase reporter with the ZEB1 or ZEB2 3′UTR in HK-2 cells in the presence of co-transfected negative control pre-miR or miR-200 family individually. Luciferase activity was measured after 24 hours. The data are mean±S.E. of triplicates and are shown as the ratio of firefly to Renilla luciferase activity. <i>P</i><0.05: HK-2 cells in the presence of co-transfected miR-200 family compared with negative control pre-miR</p

Injected miR-200b precursor can be converted to mature miR-200b in kidneys.

<p><b>a</b>. Laser microscopy images of kidney, frozen sections at 24 hr after intravenously administration of Cy3 dye-labeled Pre-miR control. <b>b</b>. Changes in miR-200 family levels in Balbc mice kidneys 12, 24, 48 hours after intravenously administration of miR-200b precursor, as measured by <i>TaqMan</i> qRT-PCR and normalized to U6 expression. The data are means from experiment measured in triplicate and are presented as mean±S.E. <i>P</i><0.05: kidneys 12, 24 hours after intravenously administration of miR-200b precursor compared with 0 hour.</p

Results of ELISA analysis for VEGF and GDNF.

<p>(A, B) In the lesioned striatum, VEGF was significantly increased by SCS at 1 week after 6-OHDA lesion (*p<0.05). At 2 weeks after 6-OHDA lesion, VEGF level in the lesioned striatum also appeared elevated, but did not reach statistical significance. (C, D) GDNF in the striatum of both sides was not significantly increased by SCS at 1 and 2 weeks after 6-OHDA lesion. (C-lesion: control group lesioned side Striatum; C-intact: control group intact side Striatum; S-lesion: 50 Hz SCS group lesioned side Striatum; S-intact: 50 Hz SCS group intact side Striatum, n = 10, respectively).</p

The results of cylinder test and amphetamine-induced rotation test.

<p>(A) Rats receiving 2 Hz and 50 Hz SCS showed reduction of the contralateral bias at 2 weeks after 6-OHDA lesion, compared to that of rats in control group. (B) The number of amphetamine-induced rotations in all SCS groups decreased, compared to that of control group. There was a significant amelioration in 50 Hz SCS group, compared to control group (*p<0.05, n = 10, respectively).</p