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1.
FIG. 6.

FIG. 6. From: Human Papillomavirus Type 16 E6/E7 Upregulation of Nucleophosmin Is Important for Proliferation and Inhibition of Differentiation .

ARF is relocalized from the nucleolus to nucleoplasm in E6/E7-expressing cells with the knockdown of NPM. (a) Western blot showing an increase in ARF levels in E6/E7-expressing cells. (b) pBabe- and E6/E7-expressing HFKs were treated with either scrambled control or NPM siRNA. Cells were stained for ARF (green), nucleolar marker C23 (red), and DAPI (blue). ARF was found in the nucleoplasm and cytoplasm in control cells but in the nucleolus in E6/E7-expressing cells. However, ARF was relocalized in E6/E7-expressing cells from the nucleolus to nucleoplasm-cytoplasm when NPM was depleted using siRNAs E6 and E7 (E6/E7 siNPM), but no change was observed in the control cells (pBabe siNPM).

Rachel McCloskey, et al. J Virol. 2010 May;84(10):5131-5139.
2.
FIG. 7.

FIG. 7. From: Human Papillomavirus Type 16 E6/E7 Upregulation of Nucleophosmin Is Important for Proliferation and Inhibition of Differentiation .

ARF is relocalized in HFKs after inhibition of AKT or after depletion of both p53 and pRb. (a) ARF is relocalized from the nucleolus to nucleoplasm-cytoplasm in E6/E7-expressing cells treated with the AKT inhibitor AIV. (b) Western blot showing HFKs stably expressing shRNA for both p53 and Rb (shp53/Rb); a reduction in the levels of both proteins and a corresponding increase in NPM protein levels was seen compared to scrambled (shScr/shScr) control cells. (c) ARF localized to nucleoli in control HFKs with depleted levels of both Rb and p53, and the localization was the same as that seen in E6/E7-expressing cells.

Rachel McCloskey, et al. J Virol. 2010 May;84(10):5131-5139.
3.
FIG. 3.

FIG. 3. From: Human Papillomavirus Type 16 E6/E7 Upregulation of Nucleophosmin Is Important for Proliferation and Inhibition of Differentiation .

Half-life of NPM in control and E6/E7-expressing keratinocytes. (a) The half-life of NPM is increased in E6/E7-expressing cells compared to pBabe control cells but is reduced to pBabe levels in E6/E7-expressing cells when AKT is depleted by siRNA. Data in the graph represent averages of the results of 3 experiments from different time points; densitometry data represent percentages of the value for h 0 for the control (pBabe). (b) NPM localization is unchanged by AKT depletion in pBabe- and E6/E7-expressing cells. Scrambled control and AKT-depleted cells were stained for NPM (green), nucleolar marker C23 (red), and DAPI (blue). Images show no change in NPM localization with AKT knockdown in pBabe- and E6/E7-expressing cells.

Rachel McCloskey, et al. J Virol. 2010 May;84(10):5131-5139.
4.
FIG. 2.

FIG. 2. From: Human Papillomavirus Type 16 E6/E7 Upregulation of Nucleophosmin Is Important for Proliferation and Inhibition of Differentiation .

Increases in nucleophosmin levels in E6/E7-expressing cells occur at the protein level and may be due to increased AKT activity. (a) Real-time PCR was carried out on 3 different sets of pBabe- and E6/E7-expressing HFKs. Data shown in the graph represent averages of the results of the 3 experiments and show that NPM mRNA levels are not increased in E6/E7-expressing cells compared to pBabe control cells (P = 0.13). (b) Depletion of AKT by siRNA (siAKT) decreases NPM protein levels in pBabe- and E6/E7-expressing cells. (c and d) Reductions in AKT activity, but not total protein activity, by the use of an AKT inhibitor (AIV) and a PI3-kinase inhibitor (PI103) reduce NPM protein levels.

Rachel McCloskey, et al. J Virol. 2010 May;84(10):5131-5139.
5.
FIG. 5.

FIG. 5. From: Human Papillomavirus Type 16 E6/E7 Upregulation of Nucleophosmin Is Important for Proliferation and Inhibition of Differentiation .

Nucleophosmin knockdown induces K1 expression and increases p53 and pRb levels in E6/E7-expressing cells and organotypic rafts. HFKs were induced to differentiate either in media containing 1.5 mM calcium chloride or in organotypic rafts. (a) Western blot showing expression of NPM, K1 (an early marker of differentiation), and actin in E6/E7-expressing keratinocytes during induced differentiation at 0, 12, 24, and 36 h after calcium treatment. K1 levels were increased in cells with reduced levels of NPM (E6/E7shNPM) compared to the results seen with control cells (E6/E7shScr). (b) E6/E7-expressing organotypic rafts, sectioned and stained with H&E (upper panels) and K1 (lower panels), showing the induction of K1 with knockdown of NPM. (c) Western blots showing the expression of Rb and p53 in pBabe control and E6/E7-expressing cells with either a scrambled siRNA (siScr) or an siRNA directed to NPM (siNPM).

Rachel McCloskey, et al. J Virol. 2010 May;84(10):5131-5139.
6.
FIG. 1.

FIG. 1. From: Human Papillomavirus Type 16 E6/E7 Upregulation of Nucleophosmin Is Important for Proliferation and Inhibition of Differentiation .

Nucleophosmin levels are increased in E7- and E6/E7-expressing cells. HFKs were induced to differentiate by suspension in methylcellulose or organotypic rafts. A 2-D gel (a) and a magnified portion (b) showing an increase in NPM (circled in red and indicated with red arrows, respectively) in cells expressing E7 compared to pBabe control cells. (c and d) Western blots showing the expression of NPM in pBabe control cells and E7-expressing cells during keratinocyte differentiation (c) or in cycling cells (d). (e) pBabe- and E7- and E6/E7-expressing organotypic rafts stained for NPM and DAPI, confirming the findings of the 2-D gel analysis. (Merged images show overlays of DAPI and NPM staining images). Scale bar, 100 μm. (f) NPM staining of normal and CIN3 cervical sections. NPM is upregulated in the CIN3 lesions compared to the paired normal tissue. Scale bar, 100 μm.

Rachel McCloskey, et al. J Virol. 2010 May;84(10):5131-5139.
7.
FIG. 4.

FIG. 4. From: Human Papillomavirus Type 16 E6/E7 Upregulation of Nucleophosmin Is Important for Proliferation and Inhibition of Differentiation .

Knockdown of NPM decreases proliferation in E6/E7-expressing cells. NPM levels in cells were reduced by the presence of either short-hairpin molecules (shRNA) or small interfering siRNA. Organotypic rafts and cycling cells were stained with Ki67 and BrDU, positively staining cells were counted, and percentages compared to DAPI-positive cells were quantified. (a and b) Cycling cells showing a 40% reduction of BrDU incorporation in siNPM-expressing cells compared to scrambled control cells (siScr). Data represent results from 15 different fields of view over the course of 3 different experiments. (c and d) Organotypic rafts of cells stably expressing shRNA with respect to NPM (shNPM) showed a 50% reduction of Ki67 expression in stable shE6/E7-expressing cells compared to scrambled control cells (shScr). Ki67-positive cells were counted in 15 different fields of view. (e) Proliferation was also decreased by over 40% in pBabe-expressing cells with the knockdown of NPM siNPM compared to scrambled control cells (siScr). Data in graphs represent averages of the results of 3 experiments.

Rachel McCloskey, et al. J Virol. 2010 May;84(10):5131-5139.

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