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

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

<p>A: Western blot with lysates of MEPC5 after apical stimulation with TGFβ1, TGFβ2 or TGFβ3 compared to untreated cells. TGFβ1 and TGFβ3 decreased the level of claudin-1 slightly 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 modest reduction of expression by TGFβ1 and TGFβ3 treatment which is not significant (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 with SEM indicated. p-values compared to untreated controls according to Students t-test.</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

Effects of alkannin on IκB-α activity in UVB-exposed cells.

<p>(A) Cells exposed to alkannin or UVB light (40 mJ/cm²) alone or both. Western blot analysis was performed with IκB-α protein (30 µg protein in each group). (B) Immunofluorescent assay for the detection of NFκB p65 in: untreated cells (a), cells pre-treated with 1 µM alkannin for 24 h (b), cells exposed to UVB light (c), cells pre-treated with 1 µM alkannin for 24 h and then exposed to 40 mJ/cm² UVB light (d). Results are representative of five independent experiments (original magnification a–d: 200×). (C) NFκB from the nuclear extracts was analyzed by Western blotting, and histone H1 was used as a loading control for nuclear proteins. These data are representative of 3 independent experiments.</p

Genes up- or down-regulated in HaCaT cells.

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

Effects of HSP70-inducing agents on caspase-3 activity in UVB-exposed cells.

<p>Cells pre-treated with HSP70-inducing agents at 1 µM for 24 h were exposed to UVB light (40 mJ/cm²). CaspGlowTM Fluorescein Active Caspase-3 Staining Kit was utilized to determine caspase-3 activity. Data are presented as mean ± S.D (n = 3). *p<0.001.</p

Induction of HSP70 protein by UVB radiation.

<p>Cells were exposed to UVB light (40 mJ/cm²) and harvested at various time intervals. Western blot analysis of HSP70 protein expression was then performed. Bands were quantified densitometrically and normalized to β-actin (n = 5).</p