1. Activation of Protein Kinase C Triggers Irreversible Cell CycleWithdrawal In Human Keratinocytes 
Shalini S. Tibudan, Yihua Wang, Mitchell F. Denning 
Journal of Investigative Dermatology 
Volume 119, Issue 6, Pages (December 2002)
DOI: /j x
Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
PKC inhibition protects from suspension-induced growth arrest. (A) Keratinocytes were either cultured on plastic, or placed into suspension culture with the indicated kinase inhibitors: PP-1 at 10 μm, GF109203X at 5 μm, LY at 25 μm, and Wortmannin at 10 μm. After 1 d, cells were replated and allowed to grow for 7 d, and dishes stained with crystal violet. Note the dramatic protection from growth arrest by the PKC inhibitor GF109203X. (B) Keratinocytes were cultured as indicated for 2 d, replated, and allowed to grow for 3 d before being photographed. (C) Keratinocytes were cultured as indicated for 1 d, counted, and replated as several densities for colony-forming assays. After 5 d of growth, dishes were stained and colonies counted. Note that the loss of colony-forming efficiency in suspension culture is partially inhibited by GF190203X. Similar results were obtained in two additional experiments.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Induction of differentiation markers during suspension culture. (A) Keratinocytes were cultured in suspension for 1 or 2 d in the presence or absence of the PKC inhibitor GF109203X at 5 μm, and cornified envelopes assayed. The data presented is the average of two independent cultures, with error bars denoting range. (B) Keratinocytes were cultured as indicated and total protein harvested for western blotting with profilaggrin, involucrin, and actin. Note the induction of profilaggrin and involucrin after 2 d in suspension culture that is blocked by GF109203X. (C) Keratinocytes were suspended in methylcellulose medium containing 3H-thymidine in the presence or absence of 5 μm GF109203X. At the indicated times, aliquots were removed and thymidine incorporation measured. The data presented are the average of four measurements, with error bars denoting standard deviation. Similar results were observed in one additional experiment.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Kinetics of PKC activation and commitment to growth arrest. (A) Keratinocytes were removed from suspension culture after the indicated times and replated to assay for growth. After 3 d of growth, cells were counted. Note the steep decline in growth potential after 4–8 h in suspension culture. The data presented are the average of two independent cultures, with error bars denoting range. (B) Keratinocytes were harvested from suspension culture after the indicated times and assayed for PKC activity in the presence of calcium (Calcium-Dependent) or absence of calcium (Calcium-Independent). Note the increase in calcium-dependent PKC activity 2–3 h after suspension.
Journal of Investigative Dermatology  , DOI: ( /j x)
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5. Figure 4
Protection from suspension-induced growth arrest by PKCα inhibition, Ca2+chelation, and Bryostatin I pretreatment. (A) Keratinocytes were either cultured on plastic, or placed in suspension for 1 d in the presence of 5 μm rottlerin or 5 μm Gö6976. The cells were then replated and allowed to grow for 5 d before being stained with crystal violet. Note the protection from growth arrest by Gö6976, but not rottlerin. (B) Keratinocytes were either cultured on plastic, placed into suspension, or pretreated with 50 μm BAPTA for 30 min before suspension for 1 d. Cells were then replated and allowed to grow for 6 d before dishes were stained. Note the partial protection from growth arrest by BAPTA pretreatment. (C) Keratinocytes were either cultured on plastic, placed into suspension, or pretreated with 1 nm or 10 nm Bryostatin I overnight before suspension in methylcellulose for 1 d. Cells were then replated and allowed to grow for 2 d before the dishes were stained. Note the protection from growth arrest by 10 nm Bryostatin I.
Journal of Investigative Dermatology  , DOI: ( /j x)
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6. Figure 5
Direct activation of PKC by TPA induces irreversible growth arrest. (A) Keratinocytes were fed with DMEM/10 FBS, containing 0–100 nm TPA, cultured for 1 d, and then replated in Medium 154. After 2 d of growth, the cells were counted. One day in DMEM/10% FBS did not trigger irreversible growth arrest. Similar results were obtained in one additional experiment. (B) Keratinocytes were fed with DMEM/10% FBS in the presence or absence of 100 nm TPA. For GF190203X treatment, cells were pretreated with the PKC inhibitor 15 min before TPA addition. After 1 d of exposure, cells were replated, allowed to grow for 3 d, and the dishes were stained. Note the protection from TPA-induced growth arrest by GF109203X. (C) Keratinocytes were fed with DMEM/10% FBS in the presence or absence of 10 nm TPA. For GF109203X and Gö6976 treatments, cells were pretreated with inhibitors at 5 μm for 15 min before TPA. After 1 d exposure, cells were replated in large dishes, allowed to grow for 5 d, and the viable cells counted. Cells were treated and assayed in duplicate, and error bars denote the range. Similar results were observed in four additional experiments. (D) Keratinocytes were pretreated overnight with 0–100 nm Bryostatin I to downregulate PKC, and then fed with DMEM/10% FBS in the presence or absence of 100 nm TPA as indicated. After 1 d of exposure, cells were replated, allowed to grow for 2 d, and then counted. Note the protection from TPA-induced growth arrest by 10–100 nm Bryostatin I.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
Suspension-induced changes in cell cycle machinery are blocked by PKC inhibition. Keratinocytes were placed in suspension culture in the presence or absence of 5 μm GF109203X, and harvested at the indicated times for western blot analysis. The phosphoform-2 of p130 is indicated by the arrow, and its accumulation is prevented by GF109203X. Also note that the induction of p21 and p27, and downregulation of p107 were blocked by GF109203X.
Journal of Investigative Dermatology  , DOI: ( /j x)
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8. Figure 7
Localization of PKCα in normal human skin. The upper left panel shows PKCα immunohistochemical staining in normal human skin. The lower left panel is the same section stained without the PKCα antibody. The right panel shows PKCα localization in green and laminin V in red. Note the membrane localization of PKCα between keratinocytes.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions