Proliferating Keratinocytes Are Putative Sources of the Psoriasis Susceptibility-Related EDA+(Extra Domain A of Fibronectin) Oncofetal Fibronectin  Márta Széll, Zsuzsanna Bata-Csörgő, Andrea Koreck, Andor Pivarcsi, Hilda Polyánka, Csilla Szeg, Magdolna Gaál, Attila Dobozy, Lajos Kemény  Journal of Investigative Dermatology  Volume 123, Issue 3, Pages 537-546 (September 2004) DOI: 10.1111/j.0022-202X.2004.23224.x Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 The primers were designed around the EDA motive of fibronectin. With the EDA motive, the PCR product was 1221 bp long, without it was 847 bp long. Journal of Investigative Dermatology 2004 123, 537-546DOI: (10.1111/j.0022-202X.2004.23224.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Fibroblasts, normal cultured keratinocytes, and HaCaT cells express different amounts of total fibronectin mRNA and EDA+:EDA− isoforms. Since fibroblasts, normal human keratinocytes, and HaCaT cells express considerably different levels of fibronectin, different amounts of cDNAs were used for fibronectin-specific PCR reactions in order to obtain good-quality presentation for the comparison of isoform ratios. The resulting PCR products were run on 1% agarose gel (A) and after densitometry the amount of total fibronectin as well as the ratio of EDA+ and EDA− isoforms were calculated (B). The gel photo (A) shows a representative experiment; data (B) (mean (±SE)) were obtained from two independent fibroblast, three independent normal keratinocyte, and four independent HaCaT cell cultures. Journal of Investigative Dermatology 2004 123, 537-546DOI: (10.1111/j.0022-202X.2004.23224.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Immunocytochemistry on cultured human fibroblasts, keratinocytes, and HaCaT cells. Subconfluent cell cultures were stained with a mouse monoclonal antibody specific for human extra domain (EDA sequence) of cellular fibronectin (A, C, E) and mouse IgG1 as isotypic control (B, D, F). Fibroblasts showed a uniform intracytoplasmic staining (A), whereas among keratinocytes (C) and HaCaT cells (E), mostly mitotic cells revealed positive intracytoplasmic staining. Journal of Investigative Dermatology 2004 123, 537-546DOI: (10.1111/j.0022-202X.2004.23224.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 The amount of total fibronectin mRNA as well as the ratio of EDA+/EDA− mRNA splice variants depend on the proliferation/differentiation stage of HaCaT cells. HaCaT cells were synchronized by contact inhibition and serum-starvation (0 h), and then released from cell quiescence by serum re-addition and passage. Samples for RT-PCR analysis were taken at the indicated time points. After running the resulted PCR products on 1% agarose gel (A), both the EDA+ and EDA− bands were measured by densitometry. Changes of total EDA+ and EDA− mRNAs at different times, relative to the amounts measured in the 0 h samples, are shown (B). The calculated ratios of EDA+ and EDA− mRNA splice variants are also shown at different times (C). Values represent the mean of two independent experiments (±SE). Journal of Investigative Dermatology 2004 123, 537-546DOI: (10.1111/j.0022-202X.2004.23224.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 The amount of total fibronectin changes with the proliferation/differentiation stage of HaCaT cells. HaCaT cells were synchronized by contact inhibition and serum-starvation (0 h), and then released from cell quiescence by serum re-addition and passage. Samples for analysis were taken at the indicated time points, run on SDS-PAGE, and stained with Coomassie Brilliant Blue (CBB). The staining indicates equal loading (A). The same amounts of proteins were blotted on nylon membrane, and western blot analysis was performed for the detection of total fibronectin by colorimetric method (B). Three relatively steady bands from the CBB gel were chosen, their average calculated, and used to normalize the total fibronectin bands of the western blot. Normalized band intensities (±SE) of two independent experiments are shown (C). Journal of Investigative Dermatology 2004 123, 537-546DOI: (10.1111/j.0022-202X.2004.23224.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 The EDA+ oncofetal form of fibronectin is expressed in highly proliferating HaCaT cells. HaCaT cells were synchronized by contact inhibition and serum-starvation (0 h), and then released from cell quiescence by serum re-addition and passage. Flow cytometry demonstrated that in the 0 h, contact-inhibited and serum-starved HaCaT cells, EDA+ fibronectin (continuous line), were indistinguishable from isotype staining (dashed line) (A). 72 h after re-entering the cell cycle, the EDA+ fibronectin staining exceeded isotype staining in HaCaT cells (B, left panel). 12.56% of the cells fall above the background level in this representative experiment. Double staining with PCNA demonstrated that the EDA+ fibronectin-expressing cells (B, right panel) were all positive for PCNA (upper right quadrant). Journal of Investigative Dermatology 2004 123, 537-546DOI: (10.1111/j.0022-202X.2004.23224.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 A small population of uninvolved psoriatic, but not normal epidermal cells, express EDA+ fibronectin after a short-term culture. Normal (A and B) and psoriatic non-lesional (C and D) epidermal cells were cultured for 72 h, and then stained with an mAb for EDA+ fibronectin and with mouse IgG1 as isotype control. Although in the normal cultured epidermal cells the EDA+ FITC fluorescence level did not exceed the level of isotype fluorescence, among the uninvolved epidermal cells a small population (3.06%) of cells fall above the isotype level in the EDA+ FITC samples. Journal of Investigative Dermatology 2004 123, 537-546DOI: (10.1111/j.0022-202X.2004.23224.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Culture conditions substantially affect the expression of fibronectin mRNA and the EDA+/EDA− spice variant ratio. The effects of different culture surfaces were tested on HaCaT cells. A pre-confluent culture of HaCaT cells (0 h) was passaged to tissue-culture dish (TC), fibronectin-coated tissue culture dish (F), Teflon (TF), and bacterial plate (B). Cells were harvested, and the expression of fibronectin was studied by RT-PCR. Cells grown on tissue culture dish and fibronectin-coated tissue culture dish did not show drastic changes in the total fibronectin mRNA expression, but showed a slight shift in the EDA+/EDA− mRNA splice variants. HaCaT cells that were seeded onto teflon and bacterial plates expressed substantially lower levels of fibronectin mRNA (A). The most pronounced decrease in EDA+/EDA− mRNA splice variant ratio was detected in HaCaT cells cultured on bacterial plates (B). The gel photo (A) shows a representative experiment; data (B) (mean (±SE)) were obtained from two independent experiments. Journal of Investigative Dermatology 2004 123, 537-546DOI: (10.1111/j.0022-202X.2004.23224.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions