Regulation of Skin Microvasculature Angiogenesis, Cell Migration, and Permeability by a Specific Inhibitor of PKCα  Sirosh M. Bokhari, Lisa Zhou, Marvin A. Karasek, Sarita G. Paturi, Vaishali Chaudhuri  Journal of Investigative Dermatology  Volume 126, Issue 2, Pages 460-467 (February 2006) DOI: 10.1038/sj.jid.5700071 Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Phase-contrast micrographs of the effect of inhibitors Gö6976 and hispidin on IL-1β- and PMA-activated HDMEC at 24hours. (a) DMSO (control), (b) hispidin, and (c) Gö6976. Effect of PMA on cells treated with: (d) DMSO, (e) hispidin, and (f) Gö6976. Effect of IL-1β on cells treated with: (g) DMSO, (h) hispidin, and (i) Gö6976. Note the loss of contact inhibition in IL-1β- and PMA-treated HDMEC. Gö6976 completely inhibits epithelial to mesenchymal transformation (EMT) in IL-1β- and PMA-activated HDMEC. Morphological analysis was made on representative areas 24hours after activation. Photomicrographs are from representative areas of three repeat experiments. Bar: 250μm. Journal of Investigative Dermatology 2006 126, 460-467DOI: (10.1038/sj.jid.5700071) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Immunofluorescence studies showing the translocation pattern of PKCα on activation. Double staining of HDMEC membrane (green) and PKCα (red) following preincubation with Gö6976 and hispidin and activated with PMA for 24hours. (a) DMSO; (b) hispidin; (c) Gö6976; (d) DMSO and PMA; (e) hispidin with PMA; and (f) Gö6976 with PMA. After PMA activation, the membrane and perinuclear membrane are stained and they appear intense yellow due to translocation of PKCα (depicted by arrows). Note the complete inhibition of PKCα transfer to the HDMEC membranes in the presence of Gö6976. Bar: 250μm. Journal of Investigative Dermatology 2006 126, 460-467DOI: (10.1038/sj.jid.5700071) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 RT-PCR analysis shows the effect of Gö6976 and hispidin on mRNA synthesis following activation with (a) PMA or (b) IL-1β. HDMECs pre-treated with Gö6976, hispidin, or DMSO (control) at 0minute, 30minutes, 4hours, and 6hours time points. PKCα (top) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (bottom) for each panel. Gö6976 inhibits mRNA synthesis by 6hours. (c) RT-PCR analysis of other PKC isozymes apart from PKCα in Gö6976-pre-treated and PMA-activated HDMECs at 0minute, 30minutes, 4hours and 6hours time points. Results with IL-1β are not shown as they are similar to PMA activation. The results shown are representative of three repeat experiments. Gö6976 inhibits mRNA synthesis by 6hours. Journal of Investigative Dermatology 2006 126, 460-467DOI: (10.1038/sj.jid.5700071) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Western blot analysis of phosphorylated and nonphosphorylated PKCα in the cytoplasm and membrane fractions of HDMECs activated by PMA. (a) Distribution of PKCα in resting HDMECs and (b) distribution of PKCα in HDMECs on activation with PMA. PMA increases phosphorylated PKCα in the membranes in both control and hispidin-treated cells and this increase is inhibited on pre-treatment with Gö6976. Journal of Investigative Dermatology 2006 126, 460-467DOI: (10.1038/sj.jid.5700071) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Effect of Gö6976 and hispidin on initial migration of endothelial cells 24hours after monolayer disruption. The endothelial cell monolayer was pre-treated with inhibitors, or DMSO as control and followed by activation with IL-1β or PMA. (a) Control, (b) hispidin, (c) Gö6976, (d) IL-1β, (e) IL-1β and hispidin, (f) IL-1β and Gö6976, (g) PMA, (h) PMA and hispidin, and PMA and (i) Gö6976. Arrows show loss of contact inhibition in IL-1β-treated HDMECs. Note the conversion to spindle-shaped cells at the wound edge in PMA- and IL-1β-treated cultures. Wounds were made in confluent cultures of HDMECs pre-treated with inhibitors as described under Materials and Methods. Cultures were fixed with ethanol and stained with crystal violet at 24hours following wounding. Bar: 250μm. Journal of Investigative Dermatology 2006 126, 460-467DOI: (10.1038/sj.jid.5700071) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Quantitative comparison illustrating the percent of wound closure after 24hours in DMSO in control, hispidin, and Gö6976, pre-treated HDMEC, with and without activation by IL-1β or PMA. Data are expressed as the percent of wound closure and are the mean±standard deviation of three separate experiments. Levels of significance: P<0.001 compared with DMSO and hispidin (Student's t-test). Journal of Investigative Dermatology 2006 126, 460-467DOI: (10.1038/sj.jid.5700071) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Effect of collagen induction on endothelial cell vascular channel formation pre-treated (a) with DMSO as control and inhibitors, (b) Gö6976, and (c) hispidin. Confluent cultures of HDMECs were pre-treated with either Gö6976 or hispidin and induced with a collagen gel as described under Materials and Methods. Morphological analysis was made by phase-contrast microscopy of representative areas from three repeat experiments 24hours after induction. Note the complete inhibition of vessel formation in Gö6976-treated HDMECs. Bar: 250μm. Journal of Investigative Dermatology 2006 126, 460-467DOI: (10.1038/sj.jid.5700071) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Timeline changes on the effect of pre-treatment of DMSO, hispidin, and Gö6976 on changes in permeability following activation with IL-1β (a) or PMA (b). The shaded bars depict 3hours; 6hours; 24hours; and 48h. Values are adjusted with baseline values in the absence of IL-1β or PMA. Journal of Investigative Dermatology 2006 126, 460-467DOI: (10.1038/sj.jid.5700071) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions