Journal: Cancer Research

Article Title: Protein Kinase Cε, which Sensitizes Skin to Sun's UV Radiation–Induced Cutaneous Damage and Development of Squamous Cell Carcinomas, Associates with Stat3

doi: 10.1158/0008-5472.can-06-3350

Figure Lengend Snippet: Figure 1. UVR treatment leads to constitutive phosphorylation of Stat3 at both Tyr705 and Ser727 residues. PKCq transgenic mice (line 215) and their wild-type littermates (four mice per group) were exposed to a single UVR dose (4 kJ/m2). The mice were sacrificed at 1, 3, 6, 12, 18, 24, and 96 h after UVR exposure. In a parallel experiment, PKCq (line 215 and 224), PKCy transgenic mice (50), and their wild-type littermates (four mice per group) were exposed to chronic UVR exposure [2 kJ/m2, four times (Monday, Wednesday, Friday, and Monday)]. The mice were sacrificed at 1 and 3 h after the fourth treatment of UVR. The mouse epidermis was scraped off and total lysates were prepared. The epidermal extract (25 Ag protein) was subjected to SDS-PAGE followed by immunoblot analysis with pStat3Tyr705 (A), pStat3Ser727 (C), and whole Stat3 (A) antibodies from Cell Signaling Technology, Inc. (Beverly, MA). Equal loading was confirmed by stripping the blot and reprobing it for h-actin. The quantification of proteins (normalized to h-actin; B and D) was done by densitometry analysis using Total Lab Nonlinear Dynamics Image Analysis Software (Nonlinear USA). Y axis, relative numbers. Columns, mean of Western blots of epidermal extracts from four different mice; bars, SE. Shown is the Western blot of pooled epidermal extracts from four mice. Similar results were obtained in a repeat experiment.

Article Snippet: Subsequently, the slides were incubated overnight with a mixture of PKCq (goat polyclonal, 1:50 dilution) and Stat3 (rabbit polyclonal, 1:50 dilution) primary antibodies from Santa Cruz Biotechnology (Santa Cruz, CA) in a humidified chamber.

Techniques: Phospho-proteomics, Transgenic Assay, SDS Page, Western Blot, Stripping Membranes, Software

Journal: Cancer Research

Article Title: Protein Kinase Cε, which Sensitizes Skin to Sun's UV Radiation–Induced Cutaneous Damage and Development of Squamous Cell Carcinomas, Associates with Stat3

doi: 10.1158/0008-5472.can-06-3350

Figure Lengend Snippet: Figure 2. Immunostaining of Stat3. Mice were treated as described in the legend to Fig. 1. Mice were sacrificed at 3 h after the fourth UV exposure. Immunostaining for Stat3 was done as described in Materials and Methods. A, Stat3 immunostaining of unexposed wild-type (WT), PKCq-overexpressing transgenic (TG224 and TG215), and PKCq knockout (KO) mice. B, UVR-exposed wild-type, PKCq-overexpressing transgenic (TG224 and TG215), and PKCq knockout mice. C, Stat3 immunostaining of mouse SCC, human SCC with or without blocking peptide, and normal human skin. Arrows, Stat3 nuclear localization. Bar, 50 Am. Magnification, 40. D, quantitation of Stat3-positive stained nuclei. Columns, mean from six different views; bars, SE.

Article Snippet: Subsequently, the slides were incubated overnight with a mixture of PKCq (goat polyclonal, 1:50 dilution) and Stat3 (rabbit polyclonal, 1:50 dilution) primary antibodies from Santa Cruz Biotechnology (Santa Cruz, CA) in a humidified chamber.

Techniques: Immunostaining, Transgenic Assay, Knock-Out, Blocking Assay, Quantitation Assay, Staining

Journal: Cancer Research

Article Title: Protein Kinase Cε, which Sensitizes Skin to Sun's UV Radiation–Induced Cutaneous Damage and Development of Squamous Cell Carcinomas, Associates with Stat3

doi: 10.1158/0008-5472.can-06-3350

Figure Lengend Snippet: Figure 3. UVR treatment leads to increased expression of Stat3-regulated genes. As described in the legend to Fig. 1, epidermal extract prepared after chronic UVR exposures was used to analyze cyclin D, c-myc, cdc25A, and COX-2 expression (A). The quantification of proteins (normalized to h-actin) was done by densitometric analysis using TotalLab Nonlinear Dynamics Image Analysis Software (Nonlinear USA; B). Y-axis, arbitrary numbers. Columns, mean of Western blots of epidermal extracts from four different mice; bars, SE. Shown is the Western blot of pooled epidermal extracts from four mice. Similar results were obtained in a repeat experiment.

Article Snippet: Subsequently, the slides were incubated overnight with a mixture of PKCq (goat polyclonal, 1:50 dilution) and Stat3 (rabbit polyclonal, 1:50 dilution) primary antibodies from Santa Cruz Biotechnology (Santa Cruz, CA) in a humidified chamber.

Techniques: Expressing, Software, Western Blot

Journal: Cancer Research

Article Title: Protein Kinase Cε, which Sensitizes Skin to Sun's UV Radiation–Induced Cutaneous Damage and Development of Squamous Cell Carcinomas, Associates with Stat3

doi: 10.1158/0008-5472.can-06-3350

Figure Lengend Snippet: Figure 4. PKCq associates with Stat3. Wild-type and PKCq-overexpressing transgenic mice were exposed to UVR [2 kJ/m2 four times (Monday, Wednesday, Friday, and Monday)]. Mice were sacrificed 1 h (A and B) or 3 h (C and D) post last UVR exposure. Epidermal protein extracts were prepared as described in the legend to Fig. 1. Twenty-five micrograms of protein were used for immunoprecipitation with the indicated antibodies. The immunoprecipitated (IP) samples were analyzed by immunoblot (IB) with the indicated antibodies. The indicated antibodies were pretreated overnight with the blocking peptide (1 Ag/mL) at 4jC before the immunoprecipitation of the epidermal extract (B). For colocalization of PKCq and Stat3 in UVR-exposed, PKCq-overexpressing mouse epidermis (D), PKCq (215) mice were exposed to UVR as described in the legend to Fig. 1. Mice were sacrificed at 3 h after the fourth UV exposure. Double immunofluorescence for PKCq and Stat3 was done as described in Materials and Methods. Immunofluorescence images of the representative localization of PKCq foci (green; top) and Stat3 foci (red; middle) following 3 h post chronic UVR exposure. Merging the images revealed that UVR-induced PKCq foci colocalized with Stat3 foci (white arrows; yellow foci; bottom). Bar, 25 Am (20 magnification).

Article Snippet: Subsequently, the slides were incubated overnight with a mixture of PKCq (goat polyclonal, 1:50 dilution) and Stat3 (rabbit polyclonal, 1:50 dilution) primary antibodies from Santa Cruz Biotechnology (Santa Cruz, CA) in a humidified chamber.

Techniques: Transgenic Assay, Immunoprecipitation, Western Blot, Blocking Assay, Immunofluorescence

Journal: Cancer Research

Article Title: Protein Kinase Cε, which Sensitizes Skin to Sun's UV Radiation–Induced Cutaneous Damage and Development of Squamous Cell Carcinomas, Associates with Stat3

doi: 10.1158/0008-5472.can-06-3350

Figure Lengend Snippet: Figure 5. PKCq is a Stat3Ser727 kinase. PKCq deletion inhibits phosphorylation of Stat3Ser727 (A). PKCq knockout (eKO), PKCq heterozygous (eHet), and wild-type mice were exposed to UVR four times (2 kJ/m2 per exposure on Monday, Wednesday, Friday, and Monday). Mice were sacrificed 3 h post last UVR exposure and the epidermal extracts were prepared as described in Materials and Methods. The protein extracts were analyzed for the expression levels of pStat3Ser727 and whole Stat3 by immunoblot analysis (A and B). A, lanes 1, 3, and 5, unexposed mice; lanes 2, 4, and 6, UVR-exposed mice. C and D, immunocomplex kinase assay. The dorsal skin of the PKCq transgenic mice was shaved and depilated 24 h before experimentation. The mice were euthanized, the dorsal skin was removed, and the epidermis was scrapped off on ice with a razor. PKC assay was done using a kit (Protein Kinase C enzyme Biotrak assay system) from Amersham Biosciences following the manufacturer’s protocol. The epidermis was placed in 0.5 mL of immunoprecipitation lysis buffer for immunoprecipitation with either PKCq or Stat3 antibodies. The immunoprecipitate was pelleted at 8,000 rpm in a microcentifuge, washed, and resuspended in 300 AL of assay buffer [50 mmol/L Tris (pH 7.4), 5 mmol/L EDTA (pH 8.0), 10 mmol/L EGTA (pH 7.9), 0.3% h-mercaptoethanol, 5 Ag/mL aprotinin, 5 Ag/mL leupeptin, and 50 Ag/mL phenylmethylsulfonyl fluoride]. Twenty-five microliters of the immunoprecipitate were assayed in kinase buffer containing 50 mmol/L Tris (pH 7.4), 8 mmol/L MgCl2, 0.136 mmol/L ATP with or without [g-32P]ATP, 3 mmol/L DTT, with or without 34 Ag/mL of l-a-phosphatidyl-L-serine, 3 Ag/mL TPA, and 1 mmol/L EGTA, and with or without 25 AL of Stat3. The PKC assays were run in the absence of added

Article Snippet: Subsequently, the slides were incubated overnight with a mixture of PKCq (goat polyclonal, 1:50 dilution) and Stat3 (rabbit polyclonal, 1:50 dilution) primary antibodies from Santa Cruz Biotechnology (Santa Cruz, CA) in a humidified chamber.

Techniques: Phospho-proteomics, Knock-Out, Expressing, Western Blot, Kinase Assay, Transgenic Assay, MMP-2 Human Biotrak Assay, Immunoprecipitation, Lysis

Journal: Cancer Research

Article Title: Protein Kinase Cε, which Sensitizes Skin to Sun's UV Radiation–Induced Cutaneous Damage and Development of Squamous Cell Carcinomas, Associates with Stat3

doi: 10.1158/0008-5472.can-06-3350

Figure Lengend Snippet: Figure 6. Working hypothesis illustrating how UVR-induced activation of PKCq is linked to growth factor expression, Stat3 activation, Stat3-regulated genes, and development of SCC. This hypothesis is based on our assumption that PKCq activation promotes the proliferation of UVR-induced initiated (preneoplastic) cells. This is accomplished by induction of growth factors (e.g., TNFa and EGF), which, via either an autocrine or a paracrine mechanism, activate cell survival signals. Here, Stat3 activation is the cell survival signal. PKCq may sensitize skin to UVR carcinogenesis by constitutive activation of Stat3. Increased transcriptional activity of Stat3 may be achieved by PKCq by Ser727 phosphorylation of Stat3 and by functioning as a transcriptional coactivator.

Article Snippet: Subsequently, the slides were incubated overnight with a mixture of PKCq (goat polyclonal, 1:50 dilution) and Stat3 (rabbit polyclonal, 1:50 dilution) primary antibodies from Santa Cruz Biotechnology (Santa Cruz, CA) in a humidified chamber.

Techniques: Activation Assay, Expressing, Activity Assay, Phospho-proteomics