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Metabolic and physiological parameters of UUO mice treated with Telmisartan or PXS64.

<p>Metabolic and physiological parameters of UUO mice treated with Telmisartan or PXS64.</p

PXS64 inhibited TGF-β1-induced fibrotic and inflammatory markers in HK2 cells.

<p>HK2 cells exposed to 100ng/ml latent TGF-β1 showed a significant increase in fibronectin and collagen IV mRNA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116888#pone.0116888.g002" target="_blank">Fig. 2A and B</a>) and protein (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116888#pone.0116888.g002" target="_blank">Fig. 2D and E</a>) expression, which were suppressed when co-exposed to PXS-64. These cells had increased mRNA expression of MCP-1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116888#pone.0116888.g002" target="_blank">Fig. 2C</a>), which was translated to increased secretory MCP-1 in the supernatant (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116888#pone.0116888.g002" target="_blank">Fig. 2F</a>). There were no difference mRNA and protein expression of the fibrotic and inflammatory markers s in HK2 cells under basal conditions or when cells were exposed to PXS64 alone (*<i>P</i> < 0.05, ** p<0.01 vs. appropriate control) Results are expressed as mean ± SEM, n = 4</p

PXS64 reduced extracellular matrix mRNA and protein expressions in the UUO.

<p>Real time PCR showing increased fibronectin and collagen IV mRNA levels in UUO mice vs. sham and reduced levels by telmisartan and PXS64 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116888#pone.0116888.g005" target="_blank">Fig. 5A and B</a>). Western blot and Hydroxy-proline showing fibronectin protein expression and collagen release in the UUO kidney, telmisartan and PXS64 treated mice (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116888#pone.0116888.g005" target="_blank">Fig. 5C and Fig. 5D</a> respectively). Western blot shows three lanes of each group representing three independent experiments of sham, UUO control, UUO+telmisartan and UUO+PXS64. Immunostaining of kidney tissue showing fibronectin and collagen IV protein expression in UUO mice in the absence and presence of telimisartan or PXS64 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116888#pone.0116888.g005" target="_blank">Fig. 5E and 5F</a>). Isotype specific IgG1 was used to confirm specificity of the staining (data not shown). (n = 8, *P < 0.05, ** P < 0.01).</p

Reduction in cellular infiltration and inflammatory markers in UUO kidneys exposed to telmisartan or PXS64.

<p>Untreated UUO kidneys showed increased F4/80, CD68 and CD45 positively stained cells as compared to the sham operated control animals. PXS64 significantly reduced F4/80 and CD45 positive stained cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116888#pone.0116888.g006" target="_blank">Fig. 6C and E</a>) with a trend to a reduction in CD68 cells, although this was not statistically significant (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116888#pone.0116888.g006" target="_blank">Fig. 6D</a>). Telmisartan treated kidneys showed a reduction in F4/80 positive cells but no difference in CD45 or CD68 stained cells, suggesting a differential action of PXS64 and telmisartan in modifying cellular infiltration. Results are presented as mean showed (n = 8, *P < 0.05 vs. UUO, ** P < 0.01 vs. UUO). Magnification x 400.</p

PXS64 suppressed conversion of latent to active TGFβ1 in HK2 cells.

<p>Active transforming growth factor TGF-β1 was determined in the concentrated supernatant after 48 hours exposure to 100ng/ml latent TGF-β1 with or without 10μM PXS64. There was increased active TGF-β1 concentration when cells were exposed to excess latent TGF-β1, which was markedly suppressed on co-exposure with PXS64. This data suggest that conversion of latent to active TGF-β1 can be effectively inhibited by a cationic independent mannose 6-phosphate receptor inhibitor (**<i>P</i> < 0.01 vs. control). Results are expressed as mean ± SEM, n = 3.</p

Details of primers used for pro-fibrotic and pro-inflammatory markers.

<p>Details of primers used for pro-fibrotic and pro-inflammatory markers.</p

PXS64 suppressed TGF-β1-Smad signaling pathways in HK2 cells.

<p>Phosphorylated Smad2 expression was markedly increased when HK2 cells were exposed to 100ng/ml latent TGF-β1 for 48 hours. This was suppressed when PXS64 was added to the media, which strongly suggests that PXS64 suppresses TGF-β1-Smad2 rather than AKT or ERK signaling pathways (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116888#pone.0116888.g003" target="_blank">Fig. 3A, B and C</a>). Results are expressed as mean ± SEM (n = 3, *P < 0.05, ** p<0.01 vs. appropriate control).</p

PXS64 reduced pSmad2 staining in UUO kidney cortex.

<p>Unilateral ureteric ligation increased tubular pSmad2 expression in the kidney cortex, which was markedly reduced when treated with PXS64 and telmisartan (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116888#pone.0116888.g007" target="_blank">Fig. 7</a>). Results are presented as Mean +/-SEM. (n = 8).</p