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Results: 5

1.
Figure 3

Figure 3. From: Alterations in NF-?B function in transgenic epithelial tissue demonstrate a growth inhibitory role for NF-?B.

Effect of a pharmacologic inhibitor of NF-κB function on normal murine skin. Histologic appearance of adult C57BL/6J mouse skin is shown after 7 days of twice daily topical application of (a) 10 mM PDTC in PBS versus (b) 0.1% SDS in PBS and (c) PBS alone (control). Brackets define the thickness of the epidermis (Bars = 100 μM.)

Cornelia S. Seitz, et al. Proc Natl Acad Sci U S A. 1998 March 3;95(5):2307-2312.
2.
Figure 1

Figure 1. From: Alterations in NF-?B function in transgenic epithelial tissue demonstrate a growth inhibitory role for NF-?B.

Expression of NF-κB in stratified epithelium in vivo. Frozen tissue sections of non-sun-exposed adult human abdominal skin were immunostained with antibody to p50. Immunoperoxidase staining for (a and c) p50 and (b and d) secondary antibody alone control. Immunofluorescence staining for (e) p50 and (f) secondary antibody alone control. E, epidermis; D, dermis; SC, stratum corneum; SG, stratum granulosum; S, squamous layer; B, basal layer; BMZ, basement membrane zone. Arrows denote suprabasal nuclei. [Bars = 75 μM (a and b) and 25 μM (cf).]

Cornelia S. Seitz, et al. Proc Natl Acad Sci U S A. 1998 March 3;95(5):2307-2312.
3.
Figure 4

Figure 4. From: Alterations in NF-?B function in transgenic epithelial tissue demonstrate a growth inhibitory role for NF-?B.

Growth characteristics of murine epidermis transgenic for gain and loss of NF-κB function. (ac) Clinical appearance of IκBαM[+], littermate control, and p50[+] skin harvested from 3-day-old transgenic mice and grafted to CB.17 scid/scid recipients, shown at 14 days postgrafting. (df) Histologic appearance of grafted skin from IκBαM[+], control, and p50[+] mice (scale bars = 150 μM). (gi) The proportion of epithelial cells actively synthesizing DNA in vivo as a function of altered NF-κB activity. CB.17 scid/scid mice bearing IκBαM[+], p50[+], and nontransgenic control skin were injected with BrdU (250 mg/kg of body weight) i.p. Skin biopsy specimens were obtained 2 h later, and tissue sections were stained with antibody to BrdU. Shown are representative immunofluorescence micrographs. Note the increased labeling activity in IκBαM[+] skin that extends into cells of the suprabasal layers and the marked decrease in labeling activity in p50[+] skin compared with control. (Bars = 150 μM.)

Cornelia S. Seitz, et al. Proc Natl Acad Sci U S A. 1998 March 3;95(5):2307-2312.
4.
Figure 2

Figure 2. From: Alterations in NF-?B function in transgenic epithelial tissue demonstrate a growth inhibitory role for NF-?B.

Transgenic mice engineered for gain and loss of epidermal NF-κB function. (a) Schematic of the K14-IκBαM and K14-p50 transgenes for targeted expression to murine epidermis. (b) Confirmation of transgene integration. Genomic DNA extracted from tail specimens of IκBαM[+] (lanes 1 and 2) and p50[+] (lanes 4 and 5) mice was subjected to PCR by using transgene-specific primers. Data obtained from unaffected littermates (lanes 3 and 6) with each primer set are also shown. (c) Western blot analysis of tissue protein extracts prepared from p50[+], IκBαM[+], and control mice. Lanes 1–4 are tissue extracts blotted with antibodies to IκBα. Lanes: 1, normal littermate skin; 2, IκBαM[+] skin; 3, normal littermate liver; 4, IκBαM[+] liver. Lanes 5–8 are blotted with antibodies to p50. Lanes: 5, normal littermate skin; 6, p50[+] skin; 7, normal littermate liver; 8, p50[+] liver. (d) Clinical appearance of K14-IκBαM transgenic mice. Five-day-old IκBαM[+] transgenic animals displayed clinical evidence of epidermal hyperplasia with markedly enhanced skin markings and a lack of visible hair growth compared with nontransgenic littermates. (e and f) Clinical appearance of 5-day-old p50[+] transgenic mice. p50[+] mice displayed clinical evidence of thin, slack skin compared with nontransgenic control. (gi) Histology of IκBαM[+], age and site-matched control, and p50[+] mice; brackets define the thickness of the epidermis. Note increased epidermal thickness relative to normal in IκBαM[+] transgenic tissue compared with decreased epidermal thickness in p50[+] transgenic skin. (Bars = 75 μM.) (jm) Epidermal expression pattern of p50 in (j) IκBαM[+] and (l) p50[+] transgenic mice along with (k) littermate and (m) secondary antibody alone controls.

Cornelia S. Seitz, et al. Proc Natl Acad Sci U S A. 1998 March 3;95(5):2307-2312.
5.
Figure 5

Figure 5. From: Alterations in NF-?B function in transgenic epithelial tissue demonstrate a growth inhibitory role for NF-?B.

Impact of NF-κB on human epithelial growth in vivo. (a) Retroviral expression vectors for proteins exerting inhibitory and activating effects on NF-κB function. Diagrammed are vectors for dominant-negative mutant IκBαM, constitutively nuclear p50 (p50), and p65. The LZRS lacZ vector (35) served as a control for these studies. (b) Impact of inhibitory and activating NF-κB proteins on NF-κB-driven gene expression in epithelial cells in vitro. The panel of vectors above was expressed in human keratinocytes along with a reporter construct containing three copies of NF-κB DNA consensus binding sites driving expression of the luciferase reporter gene. All transfections were performed in triplicate. Twenty-four hours following gene transfer, cell extracts were prepared and analyzed for reporter gene activity. Data are reported as fold induction in reporter gene activity, normalized for transfection efficiency by using a cotransfected Rous sarcoma virus–chloramphenicol acetyltransferase internal control. C, lacZ control. For assessment of IκBαM dominant-negative effects, NF-κB activity was induced for 4 h with 30 ng/ml of the phorbol ester phorbol 12-myristate 13-acetate prior to performance of reporter gene analysis. (c) Tissue architecture of human skin expressing either activating or inhibitory NF-κB subunits and lacZ control. Retroviral expression vectors for proteins activating or inhibiting NF-κB function were utilized to transduce primary human keratinocytes in vitro. These cells were then used to regenerate human skin on CB.17 scid/scid mice. Histologic appearance is shown; immunostaining of each tissue section with species-specific antibodies to human involucrin was used to confirm human tissue origin prior to hematoxylin/eosin staining. Brackets define epidermal thickness; arrows in the low power (×10) field of IκBαM skin denote focal areas of deep hyperplasia. (d) Frequency of histologic abnormalities in human epidermis in vivo. Multiple 5-μM sections were obtained in a stepwise fashion through tissue biopsies that spanned the full 1.5 cm thickness of each regenerated human graft. Atrophic changes were defined as less than 30% thickness of viable epidermis as compared with normal average value of 0.1 mm found in controls. Deep hyperplasia was defined as epithelial tissue penetrating underlying dermis to a depth of at least 0.3 mm. For p50, a total of 9 representative tissue sections were analyzed from all grafted mice; for IκBαM, n =12; and for lacZ and unengineered control, n = 6. The data are expressed as the percent of individual tissue sections displaying the given histologic abnormality. (e) Expression of involucrin in human skin expressing either activating or inhibitory NF-κB subunits and lacZ control. Double immunostaining was performed with human species-specific antibodies to involucrin and laminin 5, a basement membrane zone protein used to highlight the inferior boundary of the basal epidermal layer; involucrin (rhodamine), and laminin 5 (fluorescein isothiocyanate). Low power field of laminin 5 immunostained IκBαM[+] skin is also shown to highlight the boundaries of deep human epithelium. E, epidermis; D, dermis.

Cornelia S. Seitz, et al. Proc Natl Acad Sci U S A. 1998 March 3;95(5):2307-2312.

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