Cell morphology and claudin-1 immunostaining after treatment with TGFβs.

<p>MEPC5 monolayers photographed in bright field (A–D), DAPI staining (E–H), and claudin-1 immunostaining (I–L). A: untreated MEPC5; B–D: MEPC5 cells treated with TGFβs for 24 h. No obvious changes in cell morphology are detectable. E: DAPI labeling of the nucleus in untreated MEPC5; F–H: MEPC5 cells treated with TGFβs for 24 h. No obvious changes are detectable. I: Immunostaining of claudin-1 in untreated MEPC5, DAPI labeling of the nucleus. The pattern of claudin-1 localization shows numerous cell clusters characterized by an intense immunofluorescence at the cell borders. J–L: MEPC5 cells treated with TGFβs for 24 h. After treatment with TGFβ3 (and to a lesser intent with TGFβ1) the staining was generally weaker, the striking immunofluorescence at cell borders was clearly reduced. Photos are examples from at least three independent experiments. Scale bar: 12.5 µm in A–D, 25 µm in E–L.</p

Time-dependent TGFβ effects on TER in the BEB <i>in vitro</i> model.

<p>TGFβs decreased epithelial barrier in a time-dependent manner. TGFβ3 decreased absolute values of TER (indicated by Ω x cm²) significantly within 4 h, 6 h and 24 h compared to untreated control each. TGFβ1 and TGFβ2 influenced the TER significantly after 6 h and 24 h. Data points represent mean values obtained from n = 6, generated by three independent repetitions performed in duplicate. SEM is indicated, p-values ≤0.05 (Mann-Whitney-test) were considered significant (_*), p≤0.005 highly significant (_**).</p

Time-dependent effects of TGFβ pathway inhibitors on TER in the <i>in vitro</i> model.

<p>In case of inhibitor application, cells were incubated with inhibitors alone for 3βs + inhibitors for 2 h, 4 h and 6 h. Values prior to any treatment were taken as baseline (100%). A: The inhibition of TGFβ-R1 kinase activity by Ly364947 (Ly36) compared to untreated and vehicle-treated cells. B-D: TGFβs-affected TER values in the presence and absence of Ly364947. Effects observed for TGFβs are significantly inhibited by TGFβ-R1 inhibition. E: The inhibition of Smad3 by SiS3 compared to untreated and vehicle-treated cells. F–H: TGFβs-affected TER values in the presence and absence of SiS3. Inhibition of Smad3 resulted in attenuation of TGFβ effects. Data points represent mean values obtained from n = 6, generated by three independent repetitions performed in duplicate. SEM is indicated, p-values ±0.05 (Mann-Whitney-test) were considered significant (_*), p±0.005 highly significant (_**).</p

Western blot analysis of claudin-1 and occludin expression after treatment with TGFβs.

<p>A: Western blot with lysates of MEPC5 after combined basolateral and apical stimulation with TGFβ1, TGFβ2 or TGFβ3 for 24 h compared to untreated cells. Lysates from three independent experiments (I–III) were used. TGFβ1 and especially TGFβ3 decreased the level of claudin-1 compared to the untreated sample. The levels of occludin were unaffected. Membrane (M) and cytosolic (C) protein fractions of mouse epididymis served as controls. B, C: Densitometric analysis of claudin-1 (B) and occludin (C) protein expression. Claudin-1 showed a significant reduction of expression by TGFβ3 by TGFβ1 treatment (p±0.05). All arbitrary units were normalized to the corresponding values of vinculin, serving as loading control. Columns represent mean values of three independent experiments (I–III) with SEM indicated. p-values compared to untreated controls according to Students t-test.</p

Verification of microarray results by qPCR assay.

<p>Cells exposed to alkannin or UVB light (40 mJ/cm²) or both were used for real-time qPCR assay of the selected genes BCOR and TBPL1. Each mRNA expression level was normalized with GADPH. Data are presented as mean ± SD (n = 3). *p<0.005, **p<0.05.</p

Genes up- or down-regulated in HaCaT cells.

<p>The fold change is the ratio between control and each experiment area.</p

Musashi-1 Post-Transcriptionally Enhances Phosphotyrosine-Binding Domain-Containing m-Numb Protein Expression in Regenerating Gastric Mucosa

<div><h3>Objective</h3><p>Upregulation of the RNA-binding protein Musashi-1 (Msi1) has been shown to occur in rat gastric corpus mucosa after ethanol-induced mucosal injury. However, there is no direct evidence linking Msi1 with gastric regeneration. We examined the process of tissue repair after acute gastric mucosal injury with Msi1-knock-out (KO) mice to clarify the role of Msi1 and Msi1-dependent regulation of m-Numb expression in regenerating gastric mucosa.</p> <h3>Methods</h3><p>Acute gastric injury was induced in Msi1-KO and wild-type ICR mice by administering absolute ethanol. Expression of the splicing variants of <em>m-Numb</em> mRNA and protein in the gastric mucosa were analyzed by quantitative RT-PCR and western blotting, respectively.</p> <h3>Results</h3><p>We demonstrated that phosphotyrosine-binding domain-containing m-Numb expression was significantly upregulated at both the mRNA and protein levels in wild-type mice at 3 h after ethanol-induced acute gastric injury. In contrast, in Msi1-KO mice, the m-Numb protein was expressed weakly, and was associated with delayed regeneration of the injured gastric mucosal epithelium. In the Msi1-KO mouse, the ratio of <em>m-Numb</em> mRNA to total <em>m-Numb</em> mRNA in the heavy polysome fractions was lower than that in the wild-type mouse. Further, we showed that m-Numb-enhancement in gastric mucous cells induced the expression of prostate stem cell antigen and metallothionein-2. Under the m-Numb enhancing condition, the gastric cells exhibited enhanced cell proliferation and were significantly more resistant to H₂O₂-induced cell death than control cells.</p> <h3>Conclusions</h3><p>Msi1-dependent post-transcriptional enhancement of m-Numb is crucial in gastric epithelial regeneration.</p> </div