Perilipin, Adipophilin and TIP47 Expression in Hepatocellular Carcinoma

Beate K. Straub

Supplementary Table 1: Immunohistology for PAT-proteins in different tumors. tissue tumor type n perilipin adipophilin TIP47 staining staining staining* [0-3] [0-3] [0-3] adrenal gland pheochromocytoma 1 0 0.5 0.5 adrenal cortical adenoma 2 0 3.0 1.0 brain glioblastoma 3 0 1.0 0.5 colon tubular adenoma 25 0 1.22 0.96 adenocarcinoma 87 0 1.18 1.11 duodenum adenoma 1 0 1.5 0.5 adenocarcinoma 1 0 1.0 0.5 esophagus squamous cell carcinoma 3 0 1.17 0.83 adenocarcinoma 1 0 0.5 0.5 extrahepatic cholangiocarcinoma (Klatskin) 2 0 1.0 1.5 bile ducts gall bladder clear cell adenocarcinoma 1 0 2.0 0.5 adenocarcinoma 1 0 1.0 0.5 kidney papillary renal cell carcinoma 16 0 1.3 0.9 clear cell renal cell carcinoma 69 0 2.06 1.26 chromophobic renal cell 0 0.84 1.09 17 carcinoma mixed type renal cell carcinoma 4 0 2 1.25 angiomyolipoma 19 2.55 1.08 1.0 larynx/ pharynx squamous cell carcinoma 3 0 1.3 1.0 liver cholangiocellular carcinoma 5 0 1.2 0.7 hepatocellular carcinoma** 198 0.44 1.32 0.61 dysplastic nodules** 9 0.5 1.5 0.5 hepatocellular adenoma** 9 0.78 1.89 0.88 focal nodular hyperplasia** 6 1.58 1.67 0.5 lung squamous cell carcinoma 68 0 1.43 1.13 bronchiolo-alveolar carcinoma 1 0 1.0 1.0 adenocarcinoma 63 0 1.20 0.94 small cell lung carcinoma 2 0 0.75 0.5 large cell lung carcinoma 4 0 1.5 0.75 Sarcomatoid/ pleomorphic lung 4 0 0.875 1.25 carcinoma lymph node B-cell non Hodgkin lymphoma 1 0 0.5 0.5 mammary ductal carcinoma in situ 1 0 1.5 1.0 gland invasive ductal carcinoma 140 0 1.21 0.92 invasive lobular carcinoma 28 0 1.29 0.95 tubular carcinoma 10 0 1.05 0.9 apocrine carcinoma 1 0 1.5 0.5 medullary carcinoma 3 0 1.67 1.0 mucinous carcinoma 2 0 1.5 0.75 ovary borderline tumor of the ovary 1 0 1.5 0.5 serous-papillary adenocarcinoma 3 0 1.0 0.5 steroid cell tumor 1 0 2.5 2.0 pancreas ductal adenocarcinoma 4 0 1 0.5 prostate gland adenocarcinoma 4 0 0.63 0.75 salivary glands pleormophic adenoma 1 0 1.0 0 cystadenolymphoma 1 0 1.0 0 adenocarcinoma 1 0 0.5 0.5 skin squamous cell carcinoma 62 0** 1.23 0.77 carcinoma in situ 18 0 1.22 0.64 actinic keratosis 30 0 1.12 0.85 basal cell carcinoma 6 0 1.83 1.0 malignant melanoma, nodular 2 0 0.5 0.5 type

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sebaceous carcinoma 1 2.5 2.5 2.5 sebaceous adenoma 3 1.0 2.67 1.17 small intestine neuroendocrine carcinoma 1 0 1.0 0.5 soft tissue alveolar rhabdomyosarcoma 1 0 1.0 0.5 fibromyxosarcoma 1 0 0 0.5 hibernoma 1 3 1.5 1.0 lipoma 2 3 0.5 0.5 dedifferentiated liposarcoma 1 2.5 2.0 n.d. pleomorphic liposarcoma 2 0.75 2.25 n.d. Myxoid/ round cell liposarcoma 1 2.5 2.5 n.d. stomach signet cell carcinoma 3 0 0.83 0.33 mucinous adenocarcinoma, 0 0.5 0.5 1 intestinal type Intestinal type adenocarcinoma 2 0 1.25 0.75 testis embryonal carcinoma 1 0 1.5 0.5 classical seminoma 2 0 1.0 0.75 leydig cell tumor 2 0 1.5 0.75 mature teratoma 1 0 0.5 0.5 thyroid gland follicular carcinoma 1 0 1.5 0.5 papillary carcinoma 3 0 1.17 0.63 tonsil squamous cell carcinoma 3 0 0.67 0.5 urinary bladder anaplastic carcinoma 1 0 0.5 0.5 (papillary) urothelial carcinoma 3 0 0.83 0.67 uterus endometrioid adenocarcinoma 3 0 0.67 1.0

Intensity of immunohistochemical stainings was grouped from 0 (no staining) to 3 (strong staining) [26]. *TIP47 staining was often diffusely cytoplasmic and not always seen in a typical lipid droplet pattern. ** Nuclear perilipin staining was observed in some SCC cases. n.d.: not done.

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Supplementary Table 2: Antibodies used in this study. antigen species/ type antibody/ clone company of antibody number actin mouse mAb clone C4 MP Biomedicals, Solon, OH adipophilin mouse mAb clone AP125 Progen Biotechnik, Heidelberg, Germany guinea pig pAb GP30 Progen Biotechnik guinea pig pAb GP31 Progen Biotechnik bcl-2 mouse mAb clone 124 DAKO, Carpinteria, CA, USA c-erbB-2 rabbit pAb - DAKO cytokeratin 8/18 guinea-pig pAb GP11 Progen Biotechnik cytokeratin (pan) mouse mAb clone KL1 DAKO Ki67 mouse mAb clone MIB1 DAKO p53 mouse mAb clone DO-7 DAKO perilipin mouse mAb clone Peri 117.12 Progen Biotechnik guinea-pig pAb GP29 Progen Biotechnik guinea-pig pAb GP33 Progen Biotechnik perilipin A rabbit pAb - Sigma-Aldrich, St. Louis, MO, USA perilipin / rabbit pAb ANTI-PLIN Atlas Antibodies, Stockholm, Sweden adipophilin TIP47 guinea-pig pAb GP30 Progen Biotechnik guinea-pig pAb GP31 Progen Biotechnik

Abbreviations: mAb: monoclonal antibody; pAb: polyclonal antibody.

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Figure Legends

Supplementary Figure 1. Immunohistochemical analysis of PAT-expression in different hepatocellular carcinoma. Examples of different immunohistochemical stainings of perilipin, adipophilin, and TIP47 are depicted in different human hepatocellular carcinoma (G2 to G3) with positivity for perilipin (a1), adipophilin (a2) and TIP47 (a3), negativity for perilipin (d1) and only sparse positive reactions for adipophilin (d2) and TIP47 (d3) and combinations of these (b1-3, c1-3). Magnification: 400x.

Supplementary Figure 2: Boxplot-analysis showing decreasing perilipin (a, orange) and increasing adipophilin (b, blue) staining intensities during hepatocarcinogenesis (see also table 1b).

Supplementary Figure 3: Immunoblot analysis showing cross-reactivity of a polyclonal anti- perilipin antibody with both perilipin and adipophilin. Following SDS-polyacrylamide gel electrophoreses and immunoblot, polyclonal antibody ANTI-PLIN R11723 (Atlas Antibodies; http://www.proteinatlas.org) reacts with both human recombinant adipophilin (recAP), and perilipin (recPeri), but not TIP47 (recTIP). For recombinant human perilipin, also degradation products are detected. Molecular mass markers are given on the right side.