Clustering of the altered genes in response to TLR1/2 or TLR4 activation in SZ95 sebocytes.

<p>Genes that were regulated both by PAM3CSK4 and LPS were functionally categorized using the Cytoscape classification system. The clusters clearly defined an immunocompetence for the activated sebocytes and pointed on so far unrevealed functions such as a possible involvement in wounding and leukocyte migration. Note that in black are the genes/clusters present in both the early (6 hours) as well as in the late responder (24 hours) group, while the genes in bold falling to the functional category of cholesterol metabolic process were only detected at 24 hours.</p

Meta-analysis using gene expression profiles from acne samples, TLR1/2- and TLR4-activated SZ95 sebocytes reveals a possible contribution of sebocytes to the inflammatory environment in acne.

<p>(A) Genes significantly up-regulated in acne samples (red circle) when compared to control samples and genes up-regulated in LPS (green circle) or PAM3CSK4 (blue circles) treated SZ95 sebocytes when compared to untreated cells at 24 hours are visualized as a Venn diagram. Gene expression data of acne samples were obtained from available gene expression profiles (2). (B) Overlapping genes presented in a heat map were functionally categorized using the Cytoscape classification system. (C) Biological process analysis by Cytoscape Analysis confirmed that sebocytes are possible candidates for contributing to the inflammatory environment in acne samples.</p

Immunohistochemical detection of Serum Amyloid A 1/2 marks activated sebocytes and reveals a structurally defined immune-competence in sebaceous glands.

<p>Immunohistochemical detection of SAA1/2 in human sebaceous glands in control skin as well as in papulopustular acne and papulopustular rosacea. FFPE tissue samples were stained with rabbit monoclonal SAA1/2 antibody as described in the Materials and Methods. Note that besides the increased staining intensities observed in papulopustular acne and papulopustular rosacea samples, SAA1/2 positivity had a characteristic distribution within the sebaceous glands, localizing exclusively to the basal cell layers. Images are representative of at least 5 samples from each disease and each staining. Staining intensities were independent of age and gender. Sections were counterstained with methylene green. Original magnification: 50X, 200X.</p

TLR1/2 and TLR4 pathways induce similar changes in the gene expression profile of SZ95 sebocytes.

<p>(A)Venn diagram visualizing genes that are regulated both by PAM3CSK4 and LPS in SZ95 sebocytes at 6 (orange) and 24 hours (dark blue). Note the large number of genes that are regulated by both stimuli at 6 hours and an even greater number at 24 hours. (B) Differentially expressed genes in untreated, LPS or PAM3CSK4 treated SZ95 sebocytes at 6 and 24 hours as observed by our RNAseq analysis shown as a heat map. Color intensities reflect the ratios of signal intensities as shown. Note that the detected changes were due to a sustained up-regulation in the vast majority of genes, while no genes with a significant down-regulation at both time-points were detected.</p

Identifying Serum amyloid A 1/2 as a marker for inflamed sebocytes.

<p>(A)Venn diagram visualizing the number of genes showing significantly different expression levels between treatment conditions (LPS and PAM3CSK4 treated SZ95 sebocytes at 6 and 24 hours when compared to untreated SZ95 sebocytes) as observed by our RNAseq analysis. Note that 207 genes were identified as differentially regulated in any of the examined conditions and time points, marked by yellow. (B) Venn diagram visualizing the overlap of the 207 genes and the differentially regulated genes in acne samples from available gene expression profiles (2). Note that 56 out of the 207 genes were significantly regulated also in acne samples. (C) Hierarchical clustering of the 56 genes that are differentially expressed in any of the conditions of LPS and PAM3CSK4 treated SZ95 sebocytes when compared to untreated cells both at 6 and at 24 hours and are also significantly altered in acne samples when compared to control ones as observed in the gene expression profiles of acne samples from the available work of Kelhala HL et al. (NCBI GEO accession number: GSE5379) (2). Out of the 56 genes presented on the heat map, Serum amyloid A 1/2 is highlighted, fulfilling our criteria (up-regulated in acne samples as well as by both treatments, its expression levels increased from 6 to 24 hours, detectable also at the level of protein) to define a possible marker for inflamed sebocytes. (D) Western blot analysis of SAA1/2 in control, LPS and PAM3CSK4 treated SZ95 sebocytes 24 h after treatment.</p

Identification of Biomarkers of Human Skin Ageing in Both Genders. Wnt Signalling - A Label of Skin Ageing?

<div><p>The goal of our work has been to investigate the mechanisms of gender-independent human skin ageing and examine the hypothesis of skin being an adequate model of global ageing. For this purpose, whole genome gene profiling was employed in sun-protected skin obtained from European Caucasian young and elderly females (mean age 26.7±4 years [n1 = 7] and 70.75±3.3 years [n2 = 4], respectively) and males (mean age 25.8±5.2 years [n3 = 6] and 76±3.8 years [n4 = 7], respectively) using the Illumina array platform. Confirmation of gene regulation was performed by real-time RT-PCR and immunohistochemistry. 523 genes were significantly regulated in female skin and 401 genes in male skin for the chosen criteria. Of these, 183 genes exhibited increased and 340 decreased expression in females whereas 210 genes showed increased and 191 decreased expression in males with age. In total, 39 genes were common in the target lists of significant regulated genes in males and females. 35 of these genes showed increased (16) or decreased (19) expression independent of gender. Only 4 overlapping genes (<em>OR52N2</em>, <em>F6FR1OP2</em>, <em>TUBAL3</em> and <em>STK40</em>) showed differential regulation with age. Interestingly, Wnt signalling pathway showed to be significantly downregulated in aged skin with decreased gene and protein expression for males and females, accordingly. In addition, several genes involved in central nervous system (CNS) ageing (f.i. <em>APP</em>, <em>TAU</em>) showed to be expressed in human skin and were significanlty regulated with age. In conclusion, our study provides biomarkers of endogenous human skin ageing in both genders and highlight the role of Wnt signalling in this process. Furthermore, our data give evidence that skin could be used as a good alternative to understand ageing of different tissues such as CNS.</p> </div

Decrease of membrane potential and increase of ROS production upon wortmannin treatment in AD-iPSCs.

<p>(A) Decreased membrane potential of mitochondria was determined by flow cytometry after TMRM⁺ staining in AD-iPSCs. Cells were treated with different concentrations of wortmannin (1 μM, 2 μM, 4 μM) for different times (1 h, 2 h, 24 h). Treated cells (open graphs) were compared to untreated controls (gray). (B) The quantitative data represent mean values of triplicate experiments +/-SD. (C) The production of ROS was determined after H2DCFDA staining in AD-iPSCs treated with two different concentrations of wortmannin (2–4 μM) or 4 μM of MK-2206 at two different time points (2h, 6h), by flow cytometry. Treated cells (open graphs) were compared to untreated controls (gray). (D) Quantitative data represent the median values of treated cells compared to control cells. ROS production was depicted as fold-increase, the control was set to 1. Two independent experiments with triplicates revealed comparable results</p

Western blot analysis of key proteins of apoptosis in wortmannin treated AD-iPSCs.

<p>Protein lysates were analyzed in AD-iPSCs after treatment with 4 μM wortmannin for different times. On the last lane of the blot at the right we subjected protein lysate of dead cells (DC) raised after 24 h in the supernatant to follow the basal apoptosis. Fourty μg of protein each were separated by SDS-PAGE (12%). Western blot analysis was used to monitor the expression of phosphorylated AKT (p-AKT, at serine 473) and total AKT, phosphorylated BAD and total BAD and p53. The anti-human Caspase-3 antibody used recognizes only the cleaved active form of caspase-3. The anti-human LC3A antibody recognizes both isomers, LC3I and LC3II. Furthermore we used anti-human BAX, BAK, BCL-2, BCL-X_L and PUMA antibodies, against members of the Bcl-2 family. Beta-actin and Coomassie blue staining were used to confirm similar protein loading across samples.</p

Wortmannin significantly induced apoptosis in BIHi001-A and BIHi004-A iPSC lines.

<p>(A,E) Apoptosis (percentage of sub-G1 cells) was determined by cell cycle analysis in iPSCs treated for 24 h with 10ng/ml TRAIL, 4 μM L-779,450, 4 μM BMS, 50 nM MLN-8237 (AKA-I), 4 μM MK-2206, 4 μM wortmannin (Wort.). (B,F) Whole cell cycle analysis after treatment with small molecules mentioned in A with respect to the amount of different phases (sub-G1, G1, S, G2/M) in percent. (C,D) Histogram examples of cells treated with BMS, AKA-I or wortmannin as compared to controls (DMSO). Sub-G1 cell populations are indicated (sG1). (G) Apoptosis (percentage of sub-G1 cells) was determined by cell cycle analysis in orginal fibroblasts (HFF and NHDF) treated for 24 h with 10ng/ml TRAIL, 4 μM L-779,450, 4 μM BMS, 50 nM MLN-8237 (AKA-I), 4 μM MK-2206, 4 μM wortmannin (Wort.). Insets: Histogram examples of cells treated with AKA-I or wortmannin compared to controls (DMSO). Sub-G1 cell populations are indicated (sG1). (H,I) Apoptosis (percentage of sub-G1 cells) was determined by cell cycle analysis in two iPSC-derived neurons treated for 24 h with 10ng/ml TRAIL, 4 μM L-779,450, 4 μM BMS, 50 nM MLN-8237 (AKA-I), 4 μM MK-2206, 4 μM wortmannin (Wort.). Insets: Histogram examples of cells treated with, BMS, AKA-I or wortmannin compared to controls (DMSO). Means and SDs are shown of three independent experiments in triplicates. Statistical significance (*; p < 0.05) is indicated for comparison of control cells and wortmannin-treated cells.</p

Comparative analysis of proteins between AD-iPSCs and AD-iPSC-derived neurons after wortmannin treatment.

<p>Protein lysates of AD-iPSCs and iPSC-derived neurons (INC) were analyzed after treatment with 4μM wortmannin for different times. Fourty μg of protein each were separated by SDS-PAGE (12%). (A) Western blot analysis was used to monitor the expression of total and phosphorylated Akt (p-Akt, at serine 473). The anti-human LC3A recognizes both isoforms LC3I and LC3II and could also show phosphorylated LC3II. (B) iPSCs compared to iPSC-derived neurons and original fibroblasts. The anti-human Caspase-3 used recognizes only the cleaved active form of caspase-3. Beta-tubulin and Coomassie blue staining were used to confirm similar protein loading across samples.</p