Pemphigus Vulgaris-IgG Causes a Rapid Depletion of Desmoglein 3 (Dsg3) from the Triton X-100 Soluble Pools, Leading to the Formation of Dsg3-Depleted Desmosomes in a Human Squamous Carcinoma Cell Line, DJM-1 Cells  Yurni Aoyama, Yasuo Kitajima  Journal of Investigative Dermatology  Volume 112, Issue 1, Pages 67-71 (January 1999) DOI: 10.1046/j.1523-1747.1999.00463.x Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Immunoblots of membrane (PBS insoluble-Triton X-100 soluble) fractions obtained after 20 min or 30 h stimulation with PV-IgG (PV) or normal human IgG (Nor) in DJM-1 cells, using anti-Dsg1, Dsg3, and plakoglobin (PG) antibodies. Dsg 3 was depleted almost completely from membrane fractions 20 min and also 30 h after PV-IgG stimulation, whereas normal IgG stimulation caused no depletion of Dsg3. In contrast, Dsg1 and PG were not depleted at any time after PV-IgG stimulation as well as normal IgG stimulation. Journal of Investigative Dermatology 1999 112, 67-71DOI: (10.1046/j.1523-1747.1999.00463.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Immunoblots of cytoskeleton (Triton X-100 insoluble) fractions obtained after 20 min or 30 h stimulation with PV-IgG (PV) or normal human IgG (Nor) in DJM-1 cells, using anti-Dsg1, Dsg3, Dpk1, PG, and keratin 8 (K8). Stimulation with PV-IgG for 30 h caused almost complete depletion of Dsg 3 from cytoskeleton fractions, whereas Dsg1, Dpk1, and PG were not deleted by this stimulation. Note that stimulation with PV-IgG for 20 min is not enough to cause depletion of Dsg3, as is clearly demonstrated by comparing the densities of bands of keratin 8 and Dsg3. Keratin 8 (K8) is used for a quantitative control. Journal of Investigative Dermatology 1999 112, 67-71DOI: (10.1046/j.1523-1747.1999.00463.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Double-staining immunofluorescence microscopy of DJM-1 cells stimulated with PV-IgG for 20 min using anti-keratin 8 (a), anti-Dsg1 (c), anti-Dpk1 (e), anti-PG (g), and Dsg3 (b, d, f, h) antibodies. Cells were fixed with cold methanol (–20°C) for 10 min. Dsg3 are colocalized with any of Dsg1, Dpk1, and PG. Journal of Investigative Dermatology 1999 112, 67-71DOI: (10.1046/j.1523-1747.1999.00463.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Double-staining immunofluorescence microscopy of DJM-1 cells stimulated with PV-IgG (c, d, g, h, k, l, o, p) or normal IgG (a, b, e, f, i, j, m, n) for 30 h using anti-keratin 8 (a, c), anti-Dsg1 (e, g), anti-Dpk1 (i, k), anti-PG (m, o), and Dsg3 (b, d, f, h, i, l, n, p) antibodies. Cells were fixed with cold methanol (–20°C) for 10 min. Note that the number of immunofluorescent points of Dsg3 was dramatically reduced from the majority of points (desmosomes) where keratin intermediate filaments are connected point-to-point with each other 30 h after stimulation with PV-IgG (c, d), whereas Dsg3 is clearly detected at cell–cell contacts, forming a linear fluorescence 30 h after normal IgG stimulation (a, b). Small arrows (d) indicate remnants of Dsg3 at desmosornes, whereas arrowheads indicate the points where no Dsg3 is detected at joints of bundles of keratin intermediate filaments (c, d). Dsg3 (h, l, p;arrowheads) is dramatically deleted from desmosomes where Dsg1 (g;arrowheads), Dpk1 (k;arrowheads), and PG (p;arrowheads) are clearly detected in samples obtained 30 h after PV-IgG stimulation. A deletion or a marked reduction in the fluorescence intensity of Dsg3 (d, h, l, p) was also clearly demonstrated 30 h after PV-IgG stimulation, as compared with cells treated with normal IgG (b, f, j, n). Journal of Investigative Dermatology 1999 112, 67-71DOI: (10.1046/j.1523-1747.1999.00463.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions