c-Myc accumulation in outlet obstruction.

<p>(A) Fold change (vs. sham) of c-Myc (Myc), Hdac3 and Ezh2 mRNAs in outlet obstruction and de-obstruction (n=6-8). (B) Inverse correlation between miR-29b and Myc mRNA level (both expressed relative to sham) in outlet obstruction and its reversal (n=6-8). The leftmost symbol represents 10 d obstruction, the rightmost symbol represents de-obstruction, and the symbols close to x=1 represent sham and 6 weeks of obstruction, respectively. Western blots and bar graph summaries showing increased c-Myc (C) and Ezh2 (D) levels in outlet obstruction (n=6 for both). (E) Western blots and bar graph summary showing increased phospho-NF-κB p105 in outlet obstruction (n=6). Blots for phospho-NF-κB p65 and IκB-α are shown without bar graph summaries as no significant differences were seen. </p

Rat bladder outlet obstruction leads to SMAD and extracellular-signal-regulated kinase (ERK) phosphorylation and to increased transcript levels of established transforming growth factor-β (TGF-β) targets.

<p>Phosphorylation of SMAD proteins (A) SMAD2/3 and (B) SMAD1/5/8 was measured by western blotting using phosphorylation-sensitive antibodies at various times following outlet obstruction. Gapdh was used as loading and normalization control. Protein expression was normalized first to Gapdh and then to the mean for the sham-operated controls. (C) Transcript levels for three established TGF-β targets are shown at 6 weeks of obstruction. The fold changes were large so the log₂ expression is shown on the y-axis. Data is from the mRNA microarray described in further detail in the text. (D) Time-course of ERK1/2 activation. All samples: n=6-8. * <i>p</i> < 0.05 versus sham. *** <i>p</i> < 0.001 versus sham.</p

MiR-29 depletion by genetic deletion of Dicer in smooth muscle increases detrusor stiffness in Ca²⁺-free conditions.

<p>Real-time quantitative polymerase chain reaction (<i>n</i>=5-7) for miR-29b (A), miR-29c (B) and elastin (Eln, C) in control (Ctrl) and Dicer knockout (KO) mouse bladders. Electron micrographs from control (D) and Dicer KO (E) detrusor. SMC: smooth muscle cell; N: neural varicosity. White arrowheads point to elastin fibrils. Scale bars represent 1 µm. Quantitative morphometry of collagen fibril diameters (F) and basal membrane thickness (G). (H) Western blots for Eln, Gapdh and Cav1 in detrusors from control and smooth muscle-specific Dicer knockout (KO) mice. Summarized data on Eln expression in control and Dicer KO bladders (I, <i>n</i>=12). (J) Passive circumference-stress curves in Dicer KO and control bladder strips (<i>n</i>=12). (K) Passive circumference-stress relations in control and elastase-treated rat detrusor strips (n=4).</p

Repression of miR-29 after outlet obstruction is associated with increased levels of miR-29 target proteins.

<p>(A) Western blots for eight miR-29 targets in sham-operated control bladders and at 6 weeks of obstruction. Gapdh and Cav1 were used as loading controls (<i>n</i>=6 throughout). (B) Relative fold change at 6 weeks of obstruction (vs. sham) of miR-29 target messenger RNAs (mRNA; black circles) and proteins (white circles). The fold change of mRNA compared to the fold change of the protein was significantly different throughout, except for Eln and Mybl2. MRNA expression is from the microarrays (<i>n</i>=6−8), and protein expression is from the western blots in A. Protein expression was normalized first to Gapdh and then to the mean for the sham-operated controls. (C) Immunofluorescence staining for collagen IV (col4a1, green) in sham-operated control and 6-wk obstructed detrusor. Scale bars represent 100 µm. The fluorescent pear-shaped profile in the center of the 6 weeks specimen represents the outline of a muscle bundle. Individual cell profiles are visible within the bundles. Nuclei are stained blue and the outer bladder surface is facing upward in both micrographs. </p