1. A Tumor Suppressor Function for the Lipid Phosphatase INPP4B in Melanocytic Neoplasms 
Rolando Perez-Lorenzo, Kamraan Z. Gill, Che-Hung Shen, Feng X. Zhao, Bin Zheng, Hans-Joachim Schulze, David N. Silvers, Georg Brunner, Basil A. Horst 
Journal of Investigative Dermatology 
Volume 134, Issue 5, Pages (May 2014)
DOI: /jid
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Inositol polyphosphate 4-phosphatase type II (INPP4B) protein is expressed at lower levels in malignant melanocytic tumors as compared with nevi. (a) INPP4B protein expression in a representative melanocytic nevus (Nevus) and primary melanoma (Prim Mel). Note strong cytoplasmic staining within nevus cells in the dermis (upper panel), and near-complete absence of staining in a primary melanoma (lower panel). H&E, hematoxylin and eosin. Bar=300μm; original magnification × 100. (b) Comparison of INPP4B protein tissue microarray expression scores between nevi (n=59), primary melanomas (Prim Mel, n=67), and metastatic melanomas (Met Mel, n=65). Proportions of INPP4B high (expression score 2+, 3+) and INPP4B low (0+, 1+) expressing lesions differed significantly between nevi and primary melanomas, as well as between nevi and metastatic melanomas. (c) Distribution of final INPP4B staining scores for patient-derived melanocytic neoplasms.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Forced expression of inositol polyphosphate 4-phosphatase type II (INPP4B) is associated with reduced pAkt levels, as well as proliferative and migratory potential of melanocytic cells. (a) Relative endogenous INPP4B, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), and pAkt protein levels in immortalized human melanocytes (HMEL), and three groups of melanoma cell lines, BRAF mutant, NRAS mutant, BRAF/NRAS wild type (wt; top). Relative INPP4B messenger RNA (mRNA) expression levels assessed by semiquantitative reverse-transcriptase–PCR (bottom). (b) Epidermal growth factor (EGF)-stimulated Akt phosphorylation levels in BRAF/NRAS wt and mutant melanoma cells overexpressing INPP4B. Representative of three independent experiments. (c) Proliferative potential of control versus FLAG-INPP4B-expressing BRAF/NRAS wt melanoma cells and HMEL cells; error bars, SD. (d) Migratory potential as assessed in wound-closure assays. Note significant inhibition of migration upon INPP4B overexpression; error bars (bottom), SD. (e) Migratory potential of BRAF and NRAS mutant melanoma cells overexpressing INPP4B; error bars, SD.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Inositol polyphosphate 4-phosphatase type II (INPP4B) depletion leads to increased Akt phosphorylation, impacting the invasive, proliferative, and migratory capacity of melanoma cells in a phosphoinositide-3 kinase (PI3K)-dependent manner. (a) PAkt (Ser473, Thr308) levels in melanoma cells and immortalized human melanocytes (HMEL) upon INPP4B knockdown. (b) INPP4B knockdown increases the invasive capacity of melanoma cells through collagen. Representative of three independent experiments performed in triplicate; error bars, SD. (c) Migratory potential is increased upon INPP4B knockdown (upper rows). Addition of the PI3K inhibitor LY29004 reverses increased migratory effect (lower row). (d) Quantification of migratory capacity from c; error bars, SD. (e) Proliferative capacity upon INPP4B knockdown in BRAF/NRAS wild type (wt; C8161), BRAF mutant (A375), and NRAS mutant (MM485) melanoma cells; error bars, SD.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Inositol polyphosphate 4-phosphatase type II (INPP4B) overexpression inhibits tumorigenesis in a xenograft model. (a) Tumor growth kinetics after subcutaneous injection of nude mice with C8161 melanoma cells expressing FLAG-INPP4B (n=5) or vector alone (n=5). (b) Kaplan–Meyer survival curves. Death is defined as tumor volume reaching 1,000mm3; follow-up period, 1.5 months. (c) Immunohistochemistry in xenografts. Weak cytoplasmic INPP4B expression is seen in controls, and moderate-to-strong staining in FLAG-INPP4B-expressing tumors. Note decreased pAkt (Ser473) levels and increased levels of cleaved caspase-3 in INPP4B-overexpressing tumors. Bars=150μm; original magnification × 400 (left panel), × 200 (right panels). (d) Representative animals injected with C8161 melanoma cells overexpressing INPP4B (FLAG-INPP4B) or vector alone. Bar=5mm. (e) Quantification of cleaved caspase-3 protein expression levels (left) and Ki67 index (right) in xenografts; error bars, SD.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Knockdown of inositol polyphosphate 4-phosphatase type II (INPP4B) promotes tumor growth in vivo. (a) Tumor growth kinetics after subcutaneous injection of C8161 melanoma cells expressing sh-INPP4B (n=10) or vector control (n=10). (b) Representative images of INPP4B and pAkt (Ser473) immunohistochemistry on INPP4B-knockdown xenografts and control tumors. Original magnification × 400 (left panel), × 200 (right panel). Scale bars=150 μm.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions