TPD52 Expression Increases Neutral Lipid Storage Within Cultured Cells Alvin Kamili1,, Nuruliza Roslan1,2,, Sarah Frost1,2, Laurence C

RESEARCH ARTICLE TPD52 expression increases neutral lipid storage within cultured cells Alvin Kamili1,*, Nuruliza Roslan1,2,*, Sarah Frost1,2, Laurence C. Cantrill2,3, Dongwei Wang3, Austin Della-Franca1,2, Robert K. Bright4, Guy E. Groblewski5, Beate K. Straub6, Andrew J. Hoy7, Yuyan Chen1,2,‡ and Jennifer A. Byrne1,2,‡

ABSTRACT et al., 2014; Shehata et al., 2008b). TPD52 is the founding member ∼ Tumor protein D52 (TPD52) is amplified and/or overexpressed in of the TPD52-like protein family, whose members share 50% cancers of diverse cellular origins. Altered cellular metabolism sequence identity. At the molecular level, TPD52-like proteins (including lipogenesis) is a hallmark of cancer development, and exhibit functional redundancy, in that heterologous partners protein–protein associations between TPD52 and known regulators identified through yeast two-hybrid screens using a single of lipid storage, and differential TPD52 expression in obese versus TPD52-like bait also interact with related TPD52-like proteins non-obese adipose tissue, suggest that TPD52 might regulate (Wilson et al., 2001; Proux-Gillardeaux et al., 2003; Shahheydari cellular lipid metabolism. We found increased lipid droplet numbers et al., 2014). However, stable expression of TPD52 or its paralogue in BALB/c 3T3 cell lines stably expressing TPD52, compared with TPD52L1 in BALB/c 3T3 cells produced shared but also isoform- control and TPD52L1-expressing cell lines. TPD52-expressing 3T3 specific cellular effects (Lewis et al., 2007; Shehata et al., 2008a). cells showed increased fatty acid storage in triglyceride (from both de Exogenous TPD52 but not TPD52L1 expression increase the novo synthesis and uptake) and formed greater numbers of lipid proliferation and anchorage-independent growth of 3T3 cells, droplets upon oleic acid supplementation than control cells. TPD52 whereas both proteins produce similar morphological changes colocalised with Golgi, but not endoplasmic reticulum (ER), markers (Shehata et al., 2008a). Similarly, TPD52 but not TPD52L1 and also showed partial colocalisation with lipid droplets coated with transcript levels are significantly higher in breast carcinoma ADRP (also known as PLIN2), with a proportion of TPD52 being samples, relative to normal breast tissue (Shehata et al., 2008a). detected in the lipid droplet fraction. Direct interactions between These results suggest that isoform-specific functions for TPD52 not ADRP and TPD52, but not TPD52L1, were demonstrated using the shared by TPD52L1 underpin the oncogenic effects of TPD52 yeast two-hybrid system, with ADRP–TPD52 interactions confirmed overexpression. using GST pulldown assays. Our findings uncover a new isoform- A hallmark of cancer cells is deregulated cellular metabolism specific role for TPD52 in promoting intracellular lipid storage, which (Luo et al., 2009), with a number of studies focusing upon might be relevant to TPD52 overexpression in cancer. lipogenesis (Budhu et al., 2013; Kumar-Sinha et al., 2003; Wang et al., 2013). Actively proliferating cells require lipids to build new KEY WORDS: Lipid storage, Lipid droplet, Golgi, Tumor protein D52, membranes, lipid cofactors and lipid-modified proteins (Brasaemle, PAT proteins 2007; Vander Heiden et al., 2009), yet the cytotoxicity of many lipid species requires their conversion into and storage as neutral lipids INTRODUCTION (e.g. triglycerides, TAG; cholesterol esters) within lipid droplets TPD52 is a candidate oncogene located at chromosome 8q21.13, (Listenberger et al., 2003). Lipid droplets are highly complex, which is frequently amplified or gained in human cancer (Byrne dynamic organelles that actively participate in lipid metabolism et al., 2012, 2014; Shehata et al., 2008b). TPD52 overexpression has and cellular signalling, controlling intracellular lipid trafficking and been reproducibly associated with poor outcomes in breast interacting with other organelles (Walther and Farese, 2012). Lipid carcinoma (Byrne et al., 2014) and aggressive phenotypes in most droplets consist of a neutral lipid core surrounded by a phospholipid cancers examined (Adler et al., 2006; Bismar et al., 2006; Byrne monolayer, and are coated by one or more members of the perilipin (PAT) family [perilipin, ADRP, TIP47 and S3-12 (also known as PLIN1–PLIN4, respectively), and OXPAT (also known as MLDP 1Molecular Oncology Laboratory, Children’s Cancer Research Unit, Kids Research Institute, The Children’s Hospital at Westmead, Westmead, New South Wales 2145, and PLIN5)] (Brasaemle, 2007) and a diverse array of other proteins Australia. 2Discipline of Paediatrics and Child Health, University of Sydney, The (Krahmer et al., 2009; Walther and Farese, 2012). It is commonly Children’s Hospital at Westmead, Westmead, New South Wales 2145, Australia. 3 proposed that lipid droplets form within the endoplasmic reticulum Kids Research Institute Microscope Facility, The Children’s Hospital at Westmead, Westmead, New South Wales 2145, Australia. 4Department of Immunology and (ER) and are transported from the ER to the Golgi, where more TAG Molecular Microbiology and TTUHSC Cancer Center, Texas Tech University Health is loaded and more proteins are attached (Fujimoto and Parton, Sciences Center, Lubbock, TX 79430, USA. 5Department of Nutritional Sciences, University of Wisconsin, Madison, WI 53706, USA. 6Department of General 2011; Walther and Farese, 2012; Wilfling et al., 2014). Pathology, Institute of Pathology, Heidelberg 69120, Germany. 7Discipline of Lipid droplets are constitutively present in fat-storing cells, Physiology, School of Medical Sciences and Bosch Institute and Boden Institute of including adipocytes and steroidogenic cells. Although present in Obesity, Nutrition, Exercise and Eating Disorders, University of Sydney, Sydney, New South Wales 2006, Australia. low numbers in most other cell types, increased numbers of lipid *These authors contributed equally to this work droplets can occur in cancer cells (Bozza and Viola, 2010). A

‡ lipogenic phenotype has been particularly associated with ERBB2- Authors for correspondence ([email protected]; [email protected]) positive breast cancers. Increased fatty acid synthase (FASN) expression has been noted in response to exogenous ERBB2

Received 21 December 2014; Accepted 10 July 2015 expression in breast cancer cells (Kumar-Sinha et al., 2003), and Journal of Cell Science

3223 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3223-3238 doi:10.1242/jcs.167692 genes encoding other regulators of lipid metabolism might be co- TPD52-expressing 3T3 cells form more lipid droplets amplified with ERBB2 at chromosome 17q (Kourtidis et al., 2010). following oleic acid supplementation TPD52 expression has been reproducibly associated with ERBB2 We next examined how 3T3 cell lines would respond to exogenous expression in human breast cancer cell lines and tissues, and in fatty acid supplementation. Following 400 µM oleic acid treatment mammary tissues from Erbb2 transgenic mice (Byrne et al., 2014; for 24 h, lipid droplet numbers per cell were significantly increased Kourtidis et al., 2010; Roslan et al., 2014). By contrast, knockdown in all 3T3 cell lines examined, relative to parallel cultures harvested of the Caenorhabditis elegans TPD52 orthologue F13E6.1 prior to oleic acid supplementation (Fig. 2A,B). However, significantly reduces lipid storage as assessed by a genome-wide significantly more lipid droplets per cell were measured in screening study (Ashrafi et al., 2003), and expression microarray TPD52-expressing cells, compared with parent and vector control analyses have identified increases in TPD52 levels in mouse and cells (Fig. 2A,B). Quantification of lipid droplet numbers showed human adipose tissue from obese versus lean subjects (Clement a significant increase (∼2-fold) in the mean lipid droplet number et al., 2004; Keller et al., 2008; Nadler et al., 2000). TPD52 was also per cell in all three TPD52-expressing cell lines post oleic acid identified as a perilipin-binding partner in a yeast two-hybrid screen treatment, compared with oleic-acid-treated vector control cells (Yamaguchi et al., 2006), and TPD52 co-immunoprecipitated with (Fig. 2B). All three TPD52-expressing 3T3 cells showed very TIP47 and other proteins (Zhang et al., 2007). These studies suggest similar mean lipid droplet numbers per cell after oleic acid the possible involvement of TPD52 in regulating lipid metabolism. treatment, despite different basal numbers of lipid droplets per Here, we show for the first time that TPD52 but not TPD52L1 cell (Fig. 2B). Individually, there were ∼18- and ∼14-fold increases expression increases lipid droplet numbers in cultured cells, and that in mean lipid droplet numbers per cell in parent and vector control TPD52 expression also promotes fatty acid storage in TAG. We also cells, respectively, after oleic acid treatment, but only ∼4-fold demonstrate that TPD52 colocalised with both Golgi markers and increases in D52-1-12 and D52-2-1 cells, and a 2-fold increase in ADRP-coated lipid droplets, with further evidence supporting a D52-2-7 cells (Fig. 2C). Increased lipid droplet numbers after 24 h direct interaction between TPD52 and ADRP. oleic acid treatment were associated with significantly increased lipid droplet areas in each cell line (Fig. 2D), with similar relative RESULTS fold changes in lipid droplet areas measured post oleic acid Stable TPD52 expression increased cellular lipid droplet treatment in all 3T3 cell lines analysed (Fig. 2E). However, numbers in 3T3 fibroblast and MDA-MB-231 breast cancer significantly larger lipid droplets were measured in two of the three cells TPD52-expressing cell lines (D52-1-12 and D52-2-7) post oleic We have previously reported the effects of TPD52 and TPD52L1 acid treatment, compared with oleic-acid-treated vector control cells expression in 3T3 fibroblast cells (Shehata et al., 2008a), whose (Fig. 2D). sequences alignments are shown in Fig. 1A. In the present study, we assessed lipid droplets in BALB/c 3T3 parental cells and those stably TPD52 expression alters fatty acid metabolism in 3T3 transfected with PG307 vector only (vector-2 and/or vector-3), fibroblast cells PG307-TPD52 (cell lines denoted D52-1-12, D52-2-1 and To explore the mechanisms by which TPD52 increases cellular lipid D52-2-7), or PG307-TPD52L1 (cell lines denoted D52L1-4 and storage, we compared fatty acid metabolism in 3T3 cell lines. D52-L1-6), using BODIPY 493/503 staining. Compared with parent Consistent with significant increases in lipid droplet numbers, the and vector (vector-3) cells, TPD52-expressing cell lines (D52-2-1, levels of cellular TAG, the main component of lipid droplets, were D52-2-7) showed strikingly increased cytosolic lipid droplet also significantly elevated in TPD52-expressing cells, most notably numbers (Fig. 1B; data not shown). TPD52L1-expressing 3T3 cell in D52-2-7 cells, where TAG levels were approximately four times lines showed similar lipid droplet staining to parental and vector-3 higher than those of vector controls (Fig. 3A). Cells were treated with control cell lines (Fig. 1B). Quantification of lipid droplets [3H]acetic acid, to trace fatty acid synthesis, and with [1-14C]oleate, confirmed a significant increase in mean lipid droplet numbers per which can be taken up by cells. The rates of de novo fatty acid cell in two TPD52-expressing 3T3 cell lines, but not in two incorporation into TAG (de novo lipogenesis) were significantly TPD52L1-expressing 3T3 cell lines, compared to vector control cells increased in D52-1-12 (by ∼2-fold) and D52-2-7 (by ∼1.5-fold) (Fig. 1C). This corresponded to an ∼10- and 4-fold increase in mean cells compared with parent and vector cells (Fig. 3B). Similarly, the lipid droplet numbers per cell in D52-2-7 cells and D52-2-1 cells, incorporation rates of medium-supplied [1-14C]oleate into TAG respectively, relative to vector-3 control cells (Fig. 1C). There was were also significantly increased in D52-1-12 (∼2-fold) and D52-2-7 also a smaller but significant increase in the mean lipid droplet area (∼1.5-fold) cells (Fig. 3C). In contrast, rates of [1-14C]oleate uptake (μm2) in D52-2-7 cells compared with vector-3 control cells differed significantly between vector control and parent 3T3 cells, (Fig. 1D). Levels of TPD52 or TPD52L1 in respective cell lines but not between vector control and D52-2-7 cells (Fig. 3D), despite were validated using western blot analyses (Fig. 1E). Electron the latter consistently demonstrating the highest number of lipid microscopy analyses of vector-3 and D52-2-7 cells further confirmed droplets per cell (Fig. 1B,C; Fig. 2A,B). lipid droplet accumulation in D52-2-7 cells (supplementary material To examine the molecular mechanisms contributing to increased Fig. S1A,B). Numerous whorled and laminated structures were also fatty acid incorporation into TAG observed in TPD52-expressing noted in D52-2-7 cells (supplementary material Fig. S1B), which cells, we examined the levels of key enzymes in 3T3 cell lines resembled phospholipid inclusions (O’Farrell et al., 2001). cultured without oleic acid supplementation, by using western blot To determine whether these phenotypes were confined to 3T3 analyses. There were no significant differences in the levels of cells, we examined a second stable TPD52-expressing cell line, FASN (Fig. 3E), acetyl-CoA carboxylase (Acc) (data not shown), derived from the breast cancer cell line MDA-MB-231 with low stearoyl-CoA desaturase 1 (Scd1) (Fig. 3E) or diacylglycerol endogenous TPD52 expression levels (Roslan et al., 2014). Again, acyltransferase 2 (DGAT2) (data not shown), according to TPD52 increased lipid droplets were detected in TPD52-transfected MDA- expression status. Similarly, there were no obvious differences in MB-231 cells (TPD52-H1D2) compared to vector control cells ADRP levels, the major PAT protein that regulates lipid droplets in

(supplementary material Fig. S1C). non-adipocytes (Brasaemle et al., 1997) (Fig. 3E), and no perilipin Journal of Cell Science

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Fig. 1. Increased lipid storage in TPD52- but not TPD52L1-expressing 3T3 cells. (A) Alignment of TPD52 (Uniprot identifier P55327-2) and TPD52L1 (Uniprot identifier Q16890-1) sequences, shown using the one-letter code, produced by the EMBOSS Needle algorithm. Numbers to the left and right of sequences refer to amino acid positions. Vertical lines and colons indicate identical and conserved residues, respectively, whereas hyphens represent gaps inserted to produce the alignment. PEST domains (red text) (Byrne et al., 1996), coiled-coil domains (underlined text) (Byrne et al., 1998), the TPD52 Ser136 phosphorylation site (bold text) (Thomas et al., 2010), and the TPD52L1 consensus 14-3-3-binding site (bold text) (Boutros et al., 2003) are indicated by arrows. A schematic diagram of the exon and domain composition of TPD52 isoforms has also been reported by Byrne et al. (2014). (B) Immunofluorescence analyses of control (3T3-parent, vector-3), TPD52-expressing (D52-2-1, D52-2-7) and TPD52L1-expressing (D52L1-4, D52L1-6) 3T3 cells, stained with BODIPY 493/503 (green) for lipid droplets and DAPI (blue) for nuclei. Images are representative of those obtained in three independent experiments. (C) Quantification of lipid droplet numbers per cell using Image-Pro Plus Version 5.1 software. Images of at least ten panels per cell line in each of three independent experiments were quantified, and data represent means±s.e.m. (D) Quantification of lipid droplet area (μm2) as described in C. P-values in C and D were calculated with a Student’s t-test. (E) Western blot analyses of 3T3 cells with antisera to the proteins shown (left). Gapdh served as a loading control. MW, molecular mass (right). Results are representative of three independent experiments. Journal of Cell Science

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Fig. 2. TPD52-expressing cells store more lipids upon oleic acid supplementation. (A) Indirect immunofluorescence analyses of control (3T3-parent, vector- 3), and TPD52-expressing (D52-1-12, D52-2-1, D52-2-7) 3T3 cells with (right panels, 24 h) or without (left panels, No) 400 μM oleic acid in fatty-acid-free BSA (OA). An enlarged image of a D52-2-7 cell post-supplementation (white boxed region) is shown. Lipid droplets were stained with BODIPY 493/503 (green) and nuclei were stained with DAPI (blue). Images shown are representative of those obtained in three independent experiments. (B) Quantification of lipid droplet numbers per cell using Image-Pro Plus 5.1 software as described in Fig. 1C. Data are presented as means±s.e.m. from three independent experiments. Lipid − droplet numbers were significantly increased in all cell lines post-oleic acid supplementation compared with no treatment (3T3-parent, P=5×10 5; vector-3, − − − P=2×10 5; D52-1-12, P=1×10 5; D52-2-1, P=3×10 6; D52-2-7, P=0.002). Comparisons between vector-3 and TPD52-expressing cells post-oleic acid treatment are also shown. (C) Relative fold changes in mean±s.e.m. lipid droplet numbers per cell (oleic acid-treated or untreated) for each cell type. (D) Quantification of lipid droplet area (μm2) as described in Fig. 1C. Lipid droplet areas were significantly increased in all cell lines post-oleic acid supplementation compared with − − − no treatment (3T3-parent, P=0.05; Vector-3, P=1×10 4; D52-1-12, P=2×10 6; D52-2-1, P=0.004; D52-2-7, P=4×10 4). Comparisons between vector-3 and TPD52-expressing cells post-oleic acid treatment are also shown. NS, not statistically significant, P>0.05. (E) Relative fold changes in mean lipid droplet area.

All P-values were calculated with a Student’s t-test. Journal of Cell Science

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Fig. 3. Altered fatty acid metabolism in TPD52-expressing 3T3 cells. (A) Triglyceride levels measured in parent, vector and TPD52-expressing 3T3 cells. Incorporation rates of (B) newly-synthesised free fatty acid (from [3H]acetate) into TAG, as a measure of de novo lipogenesis and (C) [1-14C]oleate into TAG, and (D) [1-14C]oleate uptake rates were measured in 3T3-parent, vector control, and TPD52-expressing 3T3 cells following 4 h incubation with [3H]acetic acid (1 µC/ml), [1-14C]oleic acid (0.5 µC/ml) and 0.5 mM unlabelled oleate. For A–D, three independent experiments were performed and differences between vector controls and TPD52-expressing cells were examined by Student’s t-test. NS, not statistically significant, P>0.05. The [1-14C]oleate uptake rate (D) was also significantly increased in parent versus vector control cell lines (P=0.002, Student’s t-test). (E) Western blot analyses of 3T3 cells cultured without oleic acid supplementation, with antisera to proteins shown (left). Gapdh served as a loading control. MW, molecular mass (right). Results are representative of at least three independent experiments. (F) MTT assays of parent, vector controls and TPD52-expressing 3T3 cells treated with 250 μM palmitic acid for 4 days. Results are presented as percentages of MTT absorbance at indicated time points relative to that at day 0. Data are means±s.e.m. from three independent experiments. Significant differences (Student’s t-test) were detected between D52-1-12 and vector controls (Day 1, P=0.03; Day 2, P=0.005; Day 3, P=0.01; Day 4, P=0.004),

and D52-2-7 and vector controls (Day 1, P=0.01; Day 2, P=0.003; Day 3, P=0.001; Day 4, P=0.004). Journal of Cell Science

3227 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3223-3238 doi:10.1242/jcs.167692 was detected as a marker of adipocyte differentiation (data not oleic acid treatment (Fig. 5B), and comparable results were obtained shown). in D52-1-12 and D52-2-1 cells (data not shown). As for Gm130, Excess free saturated fatty acids such as palmitic acid are toxic to quantification of the extent of colocalisation between ARL1 and cells, whereas unsaturated fatty acids can rescue palmitate-induced TPD52 (Fig. 5B) using Pearson’s correlation and Manders’ overlap apoptosis by channelling palmitate into triglyceride pools and away coefficients showed that this significantly increased from median from pathways leading to apoptosis (Listenberger et al., 2003). We values of 0.31 and 0.59 (no oleic acid, n=21) to 0.41 and 0.66 (24 h investigated whether TPD52 expression would provide protective oleic acid, n=21) (Pearson’s correlation coefficients, P=0.0022; effects to cells upon palmitic acid treatment. MTT assays performed Manders’ overlap coefficients, P=0.0003; Mann–Whitney U-test, over 4 days post 250 μM palmitic acid treatment showed data not shown). As the fixation and permeabilisation method significantly increased cell survival in D52-1-12 and D52-2-7 required for ARL1 detection did not allow for BODIPY 493/503 co- cells, compared with that of parent and vector cells (Fig. 3F). staining, we used ADRP as a surrogate for lipid droplet staining (Imamura et al., 2002), having also confirmed the co-incidence of Colocalisation of TPD52 with a Golgi but not an ER marker in ADRP and BODIPY 493/503 staining in TPD52-expressing 3T3 TPD52-expressing cells cells (data not shown). Western blot analyses showed no differences Studies have indicated that lipid droplet formation is initiated in in ARL1 or ARFRP1 levels between TPD52-expressing and control the ER (Martin and Parton, 2006). Indirect immunofluorescence cells (supplementary material Fig. S3C). analyses of TPD52 show granular cytoplasmic staining with a perinuclear accentuation in MCF-7 breast cancer (Balleine et al., Limited colocalisation of TPD52 and ADRP in TPD52- 2000) and TPD52-expressing 3T3 cells (Chen et al., 2013). As expressing 3T3 cells TPD52 further accumulated at the perinuclear region of TPD52- ADRP is the predominant PAT protein that coats small lipid droplets expressing 3T3 cells upon oleic acid treatment (supplementary in non-adipocytes (Bickel et al., 2009). We therefore compared the material Fig. S2B), we examined whether TPD52 could be subcellular distribution of ADRP and TPD52 in 3T3 cell lines. associated with the ER by staining for protein disulfide isomerase ADRP showed clustered ring structures in D52-2-7 cells, which (Pdi) (Ohsaki et al., 2008). Although Pdi staining was detected were largely excluded from the Golgi and TPD52 perinuclear region throughout the cytoplasm in both D52-2-7 and vector control cells, (Fig. 5; Fig. 6A,B, cell 1). However, a proportion of TPD52 was with or without 24 h of 400 µM oleic acid treatment (supplementary detected on ADRP-stained ring structures resembling lipid droplets material Fig. S2), Pdi did not obviously colocalise with TPD52 in in both untreated (Fig. 5B; Fig. 6A) and oleic acid-supplemented D52-2-7 cells (supplementary material Fig. S2B). D52-2-7 cells, with the latter showing larger grape-like ADRP- The Golgi complex also plays an important role in lipid droplet stained clusters (Fig. 5B; Fig. 6B, cell 2), than those detected in growth (Hesse et al., 2013; Kalantari et al., 2010), so we compared vector control cells (supplementary material Fig. S3A,B; data not TPD52 staining with that of the Golgi matrix protein Gm130 shown). Quantification of the extent of colocalisation between (Golgin 95, also known as GOLGA2) (Marra et al., 2001). Although ADRP and TPD52 (Fig. 6) using Pearson’s correlation and there were no obvious differences in Gm130 staining between Manders’ overlap coefficients showed that this significantly D52-2-7 (Fig. 4A) and vector control cells (supplementary material increased from median values of 0.15 and 0.17 (no oleic acid, Fig. S3A,B), perinuclear TPD52 staining colocalised with Gm130 n=25) to 0.29 and 0.33 (6 h oleic acid, n=19) (Pearson’s correlation in D52-2-7 cells (Fig. 4A). Following 6 h of 400 µM oleic acid coefficients, P<0.0001; overlap coefficients, P<0.0001; Mann– treatment, this colocalisation became more prominent in some cells Whitney U-test; data not shown). (Fig. 4B, cells 1 and 2). Quantification of the extent of colocalisation between Gm130 and TPD52 (Fig. 4) using Pearson’s correlation Altered lipid droplet distribution in TPD52-expressing versus and Manders’ overlap coefficients showed that this significantly vector control 3T3 cells increased from median values of 0.31 and 0.42 (no oleic acid, n=28) We also noted differences in the distribution of ADRP-stained lipid to 0.38 and 0.48 (6 h oleic acid, n=23) (Pearson’s correlation droplets between TPD52-expressing and vector control cells. coefficients, P<0.0001; Manders’ overlap coefficients, P<0.0001; ADRP-stained lipid droplets formed clusters towards the Mann–Whitney U-test, data not shown). Colocalisation of TPD52 and periphery of D52-2-7 cells, which became more prominent upon Gm130 was further confirmed in D52-1-12 and D52-2-1 cells with oleic acid treatment (Fig. 6; Fig. 7A), whereas lipid droplets and without oleic acid treatment (supplementary material Fig. S4). accumulated closer to the nuclei of vector-2 cells (Fig. 7A; supplementary material Fig. S3A,B). To further address this, we Colocalisation of TPD52 with ARL1 on Golgi in TPD52- quantified the distance between the centre of lipid droplets to the expressing cells centre of the respective cell nucleus, with more than 1900 lipid The GTPase ADP-ribosylation factor-like 1 (ARL1) coordinates droplets quantified for each setting. In vector control cells, 96.6% recruitment of Golgin proteins from the cytosol to Golgi membranes ADRP-stained bodies were distributed less than 100 pixels from the in a GTP-dependent manner (Gillingham and Munro, 2003), which centre of each nucleus (Fig. 7B). In contrast, 83.7% objects in D52- also requires ADP-ribosylation factor-related protein 1 (ARFRP1) 2-7 cells were distributed less than 100 pixels from the centre of (Zahn et al., 2006). It has been proposed that the ARFRP1–ARL1– each nucleus, with oleic acid treatment having no significant effect Golgin–Rab cascade participates in the control of lipid droplet on either distribution (Fig. 7B). Statistical analyses confirmed formation (Hesse et al., 2013; Hommel et al., 2010). We investigated significant differences in lipid droplet distributions between D52-2- whether ARL1 colocalised with Gm130 as well as TPD52. As 7 and vector cells with and without oleic acid treatment (Fig. 7C). expected (Lu et al., 2004), ARL1 showed incomplete peri-nuclear colocalisation with Gm130 in D52-2-7 cells with or without oleic Direct interactions between TPD52, but not TPD52L1, and acid treatment (Fig. 5A), and in vector control cells (supplementary ADRP or TIP47 material Fig. S3). ARL1 also colocalised with TPD52 in untreated Based on the colocalisation of TPD52 and ADRP, and possible roles

D52-2-7 cells, with more prominent colocalisation detected after for TPD52 in lipid droplet formation or trafficking, we investigated Journal of Cell Science

3228 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3223-3238 doi:10.1242/jcs.167692

Fig. 4. Colocalisation of TPD52 and the Golgi marker Gm130 in untreated and oleic-acid- treated D52-2-7 cells. Cells were untreated (A) or supplemented with 400 μM oleic acid in fatty-acid-free BSA (OA) for 6 h (B), fixed and subjected to indirect immunofluorescence analyses. Cells were colabelled with TPD52 (red), and Gm130 (green). Enlarged images of the regions indicated by white boxes are shown below, with colocalisation shown in yellow in the merge image. Images shown are representative of those obtained in at least three independent experiments.

whether TPD52 (Fig. 1A) directly interacts with ADRP. We first ADRP bait with TPD52L1 prey (Fig. 1A) resulted in no detectable employed the yeast two-hybrid system which has previously been yeast growth after 9 days (Fig. 8A). used to identify TPD52-binding partners (Boutros et al., 2003; To further confirm interactions between TPD52 and ADRP, we Byrne et al., 1998; Proux-Gillardeaux et al., 2003; Shahheydari performed GST pulldown assays. Given that the molecular mass of et al., 2014). Co-transfection of amino acids 1–415 ADRP or full- ADRP and GST-tagged TPD52 are both ∼50 kDa, GST proteins length TIP47 bait and TPD52 prey constructs produced detectable were cross-linked to GSH beads. ADRP was recovered by GST- Hf7c yeast growth on triple drop-out medium after 5 days and tagged mouse and human TPD52, but not by the GST tag alone,

3 days, respectively (Fig. 8A). However, co-transfection of TIP47 or from D52-1-12 and D52-2-7 3T3 cell lysates (Fig. 8B). Interactions Journal of Cell Science

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Fig. 5. Colocalisation of Gm130 or TPD52 with ARL1 and ADRP in untreated and oleic-acid-treated D52-2-7 cells. Cells were untreated or supplemented with 400 μM in fatty-acid-free BSA (OA) for 24 h, fixed, and subjected to indirect immunofluorescence analyses. Cells were colabelled with either (A) Gm130 (green) or (B) TPD52 (green), ARL1 (red), and ADRP (pseudo-coloured, red), with colocalisation shown in yellow in the merge image. Images shown are representative of those obtained in three independent experiments. between GST-tagged TPD52 and Rab5 were used as positive previously reported (Brasaemle and Wolins, 2006), lipid droplets controls (Shahheydari et al., 2014) (Fig. 8B). floated to the top of the gradient and were enriched in ADRP To examine whether TPD52 also localises in lipid droplet fractions, (Fig. 8C). A small proportion of both TPD52 and ARL1 were we isolated lipid droplet fractions from D52-2-7 cells treated with detected in this fraction, as was Rab5 (Brasaemle et al., 2004; Cho

400 μM oleic acid for 24 h using sucrose density centrifugation. As et al., 2007; Liu et al., 2007; Sato et al., 2006) (Fig. 8C). However, we Journal of Cell Science

3230 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 3223-3238 doi:10.1242/jcs.167692

Fig. 6. Colocalisation of ADRP and TPD52 in untreated and oleic-acid-treated D52-2-7 cells. Cells were prepared as described in Fig. 4. Cells were colabelled with TPD52 (green) and ADRP (pseudo-coloured, red). Enlarged images of the regions indicated by white boxes are shown below, with colocalisation shown in yellow. Images shown are representative of those obtained in three independent experiments. did not detect Snap23 or Vamp4 within this lipid droplet fraction 23, syntaxin-5 and syntaxin-6 and Vamp4 according to TPD52 (Fig. 8C), even after longer exposures (data not shown). This contrasts expression status in 3T3 cells (Fig. 8D). with the findings of Boström et al. (2007), who reported that Snap23 and Vamp4, as well as NSF, α-Snap and Syntaxin-5, were associated DISCUSSION with lipid droplets in ADRP-transfected NIH-3T3 cells. We Despite increasing recognition of TPD52 overexpression in various confirmed no obvious changes in the protein levels of Nsf1, Snap cancer types (Tennstedt et al., 2014; Byrne et al., 2014), little is Journal of Cell Science

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Fig. 7. Altered distributions of lipid droplets in untreated and oleic-acid-treated D52-2-7 cells versus vector control cells. (A) Representative images of Vector-2 and D52-2-7 cells with or without 400 μM oleic acid (OA) treatment. ADRP (pseudo-coloured, green), DAPI (blue). (B) Quantification of distances (pixels) between the centre of each lipid droplet object to the centre of the respective cell nucleus. At least 20 cells per cell type per treatment were assessed using images randomly chosen from three experiments, and frequencies (y-axis) of distances (x-axis) were plotted on histograms. (C) Statistical analyses confirmed significant differences in lipid droplet distributions between D52-2-7 and vector cells with or without oleic acid treatment (Mann–Whitney U-test). Boxes extend from the 25th percentile to the 75th percentile, with horizontal lines indicating median values. The whiskers show the range. Graphs were generated using GraphPad Prism 4. known about the molecular or cellular functions of TPD52. This results of high-throughput studies showing, for example, that study has provided experimental evidence that TPD52 regulates F13E6.1 (TPD52) knockdown in C. elegans significantly reduces cellular lipogenesis and lipid storage, which supports previous lipid storage (Ashrafi et al., 2003), and that there are increases in Journal of Cell Science

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Fig. 8. Direct interactions between TPD52 and ADRP or TIP47. (A) Analyses of direct interactions between TPD52 or TPD52L1 and ADRP or TIP47, along with known positive and negative controls using the yeast two-hybrid system. (+), growth on solid SD/−His−Leu−Trp medium at 30°C after 5–6 days; (++), after 3–4 days; (+++), or after 1–2 days; (−), no visible growth after at least 7 days; ND, not determined. Results were obtained from three independent experiments where all interactions were tested in triplicate. (B) GST pulldown assays using GST-tagged full-length mouse Tpd52 or human TPD52 or GST tag, and D52-1-12 (left) or D52-2-7 (right) 3T3 cell lysates. Upper and middle panels show results of western blot analyses using anti-ADRP antibody and anti-Rab5 antisera, respectively. The lower panel shows Ponceau S staining to reveal mouse and human GST–Tpd52 and GST–TPD52, respectively, or the GST tag before cross- linking (arrows). At least three independent experiments were performed. (C) Western blot analyses [proteins detected are indicated on the left, molecular masses (MW) are indicated on the right] using lipid droplet (LD) fractions from D52-2-7 cells treated with 400 μM oleic acid in fatty-acid-free BSA for 24 h. Proteins detected in the lipid droplet fraction were compared with those detected in total protein from identically treated D52-2-7 cells. Gapdh served as a negative control. Results are representative of three independent experiments. (D) Detection of SNARE protein levels according to TPD52 expression status in 3T3 cell lines. Total proteins were extracted from 3T3 cells and subjected to western blot analyses. Antisera to the proteins examined are shown on the left, and molecular mass (MW) is shown on the right. α-Tubulin served as a loading control. Results are representative of three independent experiments. Journal of Cell Science

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Tpd52 or TPD52 levels in mouse and human adipose tissue from et al., 1998) but less frequently included in TPD52 transcripts obese versus lean subjects (Clement et al., 2004; Keller et al., 2008; (Boutros et al., 2003; Nourse et al., 1998; Wang et al., 2004). A 14- Nadler et al., 2000). Our findings are also consistent with TPD52 3-3-binding site was present in the TPD52L1 isoform expressed in being androgen-inducible in prostate cancer (Rubin et al., 2004; 3T3 cells and employed in interaction testing, but was absent from Wang et al., 2004), with androgen representing a potent regulator of the TPD52 isoform employed in these and other functional analyses lipogenesis and lipid metabolism in this disease (Massie et al., 2011; (Boutros et al., 2003; Shehata et al., 2008a). It is possible that Swinnen and Verhoeven, 1998). the presence of a 14-3-3-binding site in TPD52-like proteins acts Our results suggest that TPD52 enhances lipid storage through as a molecular switch, allowing interactions with 14-3-3 proteins more than one mechanism. Increased numbers of lipid droplets were and preventing interactions with PAT proteins. In support of this consistently noted in TPD52-expressing 3T3 fibroblastic cell lines hypothesis, another TPD52-like protein TPD52L2 has been and MDA-MB-231 breast cancer cells with exogenously increased reproducibly identified within lipid droplet fractions (Hodges and TPD52 levels, relative to vector controls. Following oleic acid Wu, 2010) from oleic-acid-treated A431 cells stably expressing supplementation, all three TPD52-expressing 3T3 cell lines also stomatin (Umlauf et al., 2004), Hep39 cells (Sato et al., 2006), U937 showed significantly increased lipid droplet numbers compared cells (Wan et al., 2007), differentiated 3T3-L1 cells (Cho et al., with both untreated cells and oleic acid-treated vector control cells 2007) and 3T3-L1 cells cultured to stimulate lipolysis (Brasaemle without corresponding differences in fatty acid uptake rates, which et al., 2004). Increased TPD52L2 levels have also been reported in could underpin these phenotypes. This suggests that TPD52 response to furan fatty acid treatment of Caco-2 cells (Lengler et al., contributes to lipid droplet formation within the ER, which is 2012), and in the first reported mouse knockout model for any supported by previous reports of TPD52 being an ER-associated Tpd52-like gene, smaller body length and absent or minimal hepatic protein (Hoja et al., 2000). However, TPD52 showed no detectable lipidosis were reported in Tpd52l2−/− females (Adissu et al., 2014). colocalisation with Pdi in TPD52-expressing 3T3 cells, but instead Like TPD52, TPD52L2 isoforms are predicted to lack 14-3-3- showed perinuclear colocalisation with the Golgi markers Gm130 binding sites in most tissues (Boutros et al., 2003). and ARL1, in the presence and absence of oleic acid supplementation. In addition to increased lipid droplet numbers that were measured TPD52, Gm130 and ARL1 detection became more prominent in all three TPD52-expressing 3T3 cell lines, two out of three of within this perinuclear compartment following oleic acid treatment, these cell lines (D52-1-12 and D52-2-7, the latter harbouring the as did the degree of colocalisation between TPD52 and Gm130 or highest TPD52 levels and lipid droplet numbers) also showed ARL1 in some cells. This suggests that rather than influencing significantly increased fatty acid incorporation into TAG from both lipid droplet formation within the ER, TPD52 might contribute to de novo fatty acid synthesis, and fatty acid uptake. Significantly lipid loading within the Golgi, leading to growth of lipid droplets larger lipid droplets were also noted in D52-1-12 and D52-2-7 cells within this compartment and facilitating their detection by light when they were supplemented with oleic acid, and in D52-2-7 cells microscopy. Expansion of the Golgi compartment and increased in the absence of oleic acid treatment. This indicates that in addition colocalisation between TPD52 and Gm130 and ARL1 in oleic-acid- to mechanisms regulating lipid droplet numbers, TPD52 can either supplemented 3T3 cell lines indicates that these cells increase their directly or indirectly alter other aspects of lipid metabolism within reliance upon Golgi function in response to increased exogenous cells, although TPD52 expression might not regulate fatty acid lipid loading. In support of a Golgi-located function for TPD52 in uptake rates. The functional significance of this phenotype was regulating lipid droplets, TPD52 has been reported to interact with demonstrated by D52-1-12 and D52-2-7 cells also showing members of two protein families involved in the ARFRP1–ARL1– improved cell survival in response to palmitic acid supplementation. Golgin–Rab cascade (Hesse et al., 2013), namely Rab and Golgin Lipids play diverse roles in building cellular structures, by forming proteins (Messenger et al., 2014; Shahheydari et al., 2014). membrane microdomains for functional scaffolding of protein In addition to localising to the Golgi complex in TPD52- complexes, serving as fat storage depots and acting as signalling expressing 3T3 cell lines, TPD52 showed limited, more peripheral, molecules (Baumann et al., 2013; Currie et al., 2013; Nomura and colocalisation with the lipid droplet protein ADRP. A small Cravatt, 2013; Zadra et al., 2013). All of these processes are important proportion of TPD52 was detected within the lipid droplet in cancer and, as such, altered lipid metabolism is now an established fraction of D52-2-7 cells, a finding that is supported by the hallmark of cancer (Baumann et al., 2013; Currie et al., 2013; Nomura Drosophila orthologue CG5174 having been previously detected and Cravatt, 2013; Zadra et al., 2013). Both breast and prostate cancers within the lipid droplet proteome (Cermelli et al., 2006). TPD52 show elevated levels of intracellular lipid (Fisher et al., 1977; Swinnen also bound both ADRP and TIP47 in a yeast two-hybrid system, and and Verhoeven, 1998), and are characterised by frequent TPD52 TPD52–ADRP interactions were validated using GST pulldowns. overexpression (Roslan et al., 2014; Rubin et al., 2004; Shehata et al., TPD52 was previously identified as a binding partner of perilipin 2008a; Wang et al., 2004), as well as TPD52 amplification in residues 1–240 in a yeast two-hybrid screen (Yamaguchi et al., clinically important disease subsets (Cornen et al., 2014; Guedj et al., 2006) and as a TIP47 co-immunoprecipitating protein (Zhang et al., 2012; Liu et al., 2013). As androgen upregulates lipogenesis in 2007). This indicates that TPD52 might directly co-operate with prostate cancer cells (Swinnen and Verhoeven, 1998; Massie et al., PAT proteins, to promote either lipid droplet trafficking and/or lipid 2011), our results suggest that androgen-induced TPD52 upregulation storage at the lipid droplet surface. might contribute to this important process. ERBB2-amplified breast Our results identified increased lipid droplet numbers in TPD52- cancers also show increased lipogenesis because ERBB2 both directly but not TPD52L1-expressing 3T3 cell lines, and although we and indirectly regulates FASN function (Kumar-Sinha et al., 2003; detected interactions between TPD52 and ADRP or TIP47, similar Menendez and Lupu, 2007), and the co-amplification of other interactions were not detected for TPD52L1. At present, it is not chromosome 17q genes encoding regulators of lipid metabolism known why TPD52 but not TPD52L1 can bind PAT proteins. (Kourtidis et al., 2010). As TPD52 is co-expressed with ERBB2 in TPD52-like genes commonly include alternatively-spliced exons human breast cancer cell lines and tissues (Byrne et al., 2014; encoding a 14-3-3-binding site (Boutros et al., 2003); the relevant Kourtidis et al., 2010; Roslan et al., 2014), and is a known survival exon is more commonly included in TPD52L1 transcripts (Nourse factor in ERBB2-amplified breast cancer cell lines (Kourtidis et al., Journal of Cell Science

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2010; Roslan et al., 2014), elevated TPD52 expression might similarly (v/v, 1:1) for 15 min at −20°C, and then incubated with affinity-purified advantage ERBB2-amplified breast cancer cells by increasing both rabbit polyclonal TPD52 antisera and GM130 antibody as described above, lipogenesis and lipid storage capacity. The isoform-specificity of these or with antibodies against TPD52 (mouse monoclonal, 1:10; Tiacci et al., functions might partly explain why TPD52 but not TPD52L1 is 2005), PDI (C81H6) rabbit monoclonal antibody (1:100, Cell Signaling targeted by gene amplification in cancer. Technology, Danvers, MA), ARL1 (EPR10595) rabbit monoclonal antibody (1:100, Abcam, VIC, Australia), and guinea pig polyclonal In summary, this study has identified a new isoform-specific role antibody against ADRP (1:200, Progen Biotechnik, Heidelberg, Germany). for TPD52 as a promoter of both lipid storage and lipogenesis that This method was not compatible with BODIPY 493/503 staining, but might contribute to an improved understanding of lipid droplet allowed ADRP detection at lipid droplets. Alexa-Fluor-488-conjugated anti- function under both physiological and pathological conditions. mouse-IgG (Life Technologies), Cy3-conjugated anti-rabbit-IgG (Jackson Given previous reports of increased TPD52 or Tpd52 transcript ImmunoResearch Laboratories Inc), and Alexa-Fluor-633-conjugated anti- levels in adipose tissue from obese versus non-obese subjects guinea-pig-IgG (Life Technologies) were utilised as secondary antibodies. (Clement et al., 2004; Keller et al., 2008; Nadler et al., 2000), All samples were viewed with a Leica LCS SP5 II confocal microscope inhibition of TPD52 function might have future relevance in the (NSW, Australia) using a 63× objective lens. management of obesity. As TPD52 amplification and Quantitative analysis of colocalisation between TPD52 and Gm130, overexpression is also frequent in lipogenic cancers such as breast ARL1 or ADRP with or without oleic acid supplementation was performed using Image-Pro Analyzer 7.0 (MediaCybernetics, Rockville, MD). and prostate cancer (Roslan et al., 2014; Rubin et al., 2004; Shehata Scatterplots displaying intensity ranges of red and green pixels were et al., 2008a; Wang et al., 2004), our results might also partially generated from 19–28 representative single focal plane images for each case explain TPD52 targeting in these diseases, and could similarly be without pre-processing (TPD52, Gm130 and ARL1), or after background applied to target lipogenic cancers in future. subtraction to obtain lipid droplet structures (ADRP), with same parameters applied to all ADRP images. Pearson’s correlation coefficients and ’ MATERIALS AND METHODS Manders overlap coefficients were calculated for each scatterplot Cell lines and cell culture (Zinchuk and Zinchuk, 2008). Human breast carcinoma cells (MDA-MB-231) were cultured in GIBCO® RPMI 1640 (Life Technologies, VIC, Australia) medium supplemented Electron microscopy with 10% fetal bovine serum (FBS; Life Technologies), 6 mM L-glutamine Vector-3 or D52-2-7 cells were fixed in 2% buffered glutaraldehyde (ProSciTech) for 2 h, washed in 0.1 M MOPS buffer, scraped from the (Life Technologies) in a humidified atmosphere containing 5% CO2 at 37°C. Cell line identities were confirmed through short tandem repeat culture flask, encapsulated in 10% BSA (Sigma-Aldrich), then cross-linked profiling by CellBank Australia (Westmead, NSW, Australia). Vector-, with glutaraldehyde to form a pellet. Small blocks were post-fixed in 2% TPD52- and TPD52L1-transfected BALB/c 3T3 cell lines are as previously buffered osmium tetroxide for 3 h followed by dehydration in a graded reported (Shehata et al., 2008a). ethanol series. After infiltration in epoxy resin with acetone, cells were embedded in epoxy resin (TAAB TLV) and polymerised for 10 h at 60°C. Semi-thin (500 nm) sections were cut on glass knives using a Leica Ultracut Derivation of stably TPD52-expressing MDA-MB-231 cell lines UC6 ultramicrotome (Leica Microsystems, Vienna) and stained with – The PG307-TPD52 plasmid was constructed by subcloning a SalI BamHI Methylene Blue for light microscopic evaluation. Selected areas were TPD52 fragment (GenBank NM_005079.3, encoding the TPD52 isoform trimmed and ultrathin (70 nm) sections were cut with a diamond knife ′ shown in Fig. 1A), representing the 91-bp 5 UTR, 555-bp coding sequence (Diatome, Switzerland), and collected onto 300 mesh thin bar copper ′ and 2599-bp 3 UTR, into the PG307 expression vector (Boutros et al., grids (ProSciTech). Grids were stained with 2% uranyl acetate in 50% ethanol, 2003). After verification of constructs using Sanger sequencing, the PG307- followed by Reynold’s lead citrate, and examined using a Philips CM120 TPD52 construct or PG307 vector alone were stably transfected into MDA- BioTWIN transmission electron microscope (FEI, The Netherlands) at MB-231 cells, as described previously (Boutros et al., 2003). 100 kV. Images were collected using an SIS Morada digital camera.

Indirect immunofluorescence analyses Cellular lipid droplet quantification Cells were plated onto glass coverslips in six-well plates, and cultured All image data intended for quantitative comparison were acquired using the overnight to reach 70–80% confluence the next day. For lipid droplet same sub-saturating settings on a single focal plane. Numbers of lipid quantification, untreated cells or cells supplemented with 400 µM oleic acid droplets per cell and diameters of each lipid droplet were quantified using (Sigma-Aldrich, NSW, Australia) complexed to 10% fatty-acid-free BSA Image-Pro Plus Version 5.1 software (MediaCybernetics, Rockville, MD). (Sigma-Aldrich) (Listenberger and Brown, 2007) for 24 h were fixed Lipid droplet areas were calculated from measured diameters. Based on in 3% formaldehyde in PBS for 30 min at room temperature, then stained expected lipid droplet sizes in non-adipocytes ranging from 0.2–1 μm with 1 µg/ml BODIPY 493/503 (Life Technologies) for 30 min at (Suzuki et al., 2011), objects with diameters smaller than 0.3 μm were room temperature. DNA was counterstained using 4′,6-diamidino-2- filtered to reduce noise from potential non-specific staining. The intensity phenylindole, dihydrochloride (DAPI; Sigma-Aldrich). range was set according to the BODIPY 493/503 fluorescence intensity For co-staining of lipid droplets and proteins, untreated cells or cells detected in one TPD52-expressing cell line, D52-2-7, and the same supplemented with 400 µM oleic acid in fatty-acid-free BSA for 6 h were fixed parameters were then used to quantify lipid droplets in all 3T3 cell lines. with 3% formaldehyde and 0.025% glutaraldehyde in PBS for 10 min at room Images of at least 10 panels per cell line were quantified in each of three temperature, treated with 50 mM ammonium chloride in PBS for 10 min, independent experiments. followed by permeabilisation using 0.1% Triton X-100 in PBS (Ohsaki et al., The dispersal of cellular lipid droplets was investigated using ADRP and 2005). Cells were stained with affinity-purified rabbit polyclonal TPD52 DAPI images. Measurements were carried out using Metamorph software antisera (1:100) (Balleine et al., 2000) and GM130 mouse monoclonal Version 7.7 (Molecular Devices, Sunnyvale, CA). After thresholding and antibody (1:1000, BD Transduction Laboratories, San Jose, CA), followed by background subtraction, the locations of the centre of each cell nucleus incubation in 3% BSA in PBS containing 1 µg/ml BODIPY 493/503 solution, and lipid droplet were used to calculate the distance between the centre of each Cy3-conjugated anti-rabbit-IgG (Jackson ImmunoResearch Laboratories Inc, lipid droplet and that of the respective cell nucleus, based on Pythagoras’ West Grove, PA), and Alexa-Fluor-633-conjugated anti-mouse-IgG (Life theorem. Technologies) secondary antibodies. For other indirect immunofluorescence analyses, cells treated with or Quantification of cellular TAG content without 400 µM oleic acid were fixed in 3% formaldehyde in PBS for Cellular total lipid was extracted using the Folch method (Folch et al., 1957)

15 min at room temperature, permeabilised with cold acetone and methanol and TAG concentrations were measured using a GPO-PAP kit (Roche Journal of Cell Science

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Diagnostics) according to the manufacturer’s instructions, and normalised EcoRI and SalI sites of pAS2.1. Sanger sequencing confirmed that pAS2.1- according to cellular protein levels. TIP47 construct encodes full-length TIP47 (434 amino acids), whereas pAS2.1-ADRP encodes ADRP residues 1–415 (of 437 residues) due to a Western blot analyses C>T mutation at nt 1456 (NM_001122.3) that produces a stop codon. Cells were lysed in 3% SDS lysis buffer as described previously (Boutros et al., 2003). Between 20 and 30 μg total protein extracts were resolved Yeast two-hybrid system by SDS-PAGE on 12.5% mini polyacrylamide gels or NuPAGE® Novex For direct interaction testing, Saccharomyces cerevisiae strain Hf7c cells were 4–12% Bis-Tris mini gels (Life Technologies, VIC, Australia). transformed with paired bait (pAS2.1) and prey (pACT2) constructs, and Affinity-purified rabbit polyclonal TPD52 (1:100) and TPD52L1 (1:100) cultured at 30°C on solid synthetic drop-out (SD) medium lacking Leu and Trp antisera were raised in-house (Balleine et al., 2000; Boutros et al., 2003). (SD/−Leu−Trp) (Byrne et al., 1998). Bait–prey interactions were scored Fatty acid synthase (FASN) mouse monoclonal antibody (A-5, 1:200) was according to the time (in days) until visible colony growth on solid SD medium purchased from Santa Cruz Biotechnology (Dallas, TX). SCD1 (M38, lacking His, Leu and Trp (SD/–His–Leu–Trp) (Boutros et al., 2003; 1:1000) rabbit polyclonal and Syntaxin 6 (C34B2, 1:1000) rabbit Shahheydari et al., 2014). Bait or prey constructs paired with the opposing monoclonal antibodies were purchased from Cell Signaling Technology. empty vector represented negative controls, whereas the interaction between ADRP (1:2000) guinea pig polyclonal antisera were obtained from Progen TPD52 bait and TPD52L1 prey was used as a positive control. Biotechnik. ARL1 (EPR10595, 1:2000) and ARFRP1 (EPR3899, 1:1000) rabbit monoclonal antibodies, mouse monoclonal anti-NSF (NSF-1, 1:2000) Pulldown assays and rabbit polyclonal anti-SNAP23 (1:500) antibodies were purchased from Pulldown assays employed glutathione S-transferase (GST) and His6-tagged Abcam. Syntaxin 5 (1:1000) and VAMP4 (1:1000) rabbit polyclonal recombinant mouse Tpd52 proteins expressed from pGEX3X (Byrne et al., antibodies were purchased from Synaptic Systems (Goettingen, Germany). 1998; Sathasivam et al., 2001), and GST-tagged recombinant human RAB5C rabbit polyclonal antibody (1:500) was purchased from Sigma- TPD52 expressed from pGEX6P-1 (Chen et al., 2013; Shahheydari et al., Aldrich, and has been previously shown by our laboratory to cross-react with 2014). Purification of mouse or human TPD52 fusion proteins and α other RAB5 isoforms (Shahheydari et al., 2014). Mouse monoclonal - respective GST controls was performed as previously described (Chen tubulin antibody (DM1A) (1:5000; Sigma-Aldrich) and mouse monoclonal et al., 2013; Shahheydari et al., 2014). Purified proteins were assessed by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (6C5, SDS-PAGE. Vector and TPD52-expressing 3T3 cells were lysed in lysis 1:10,000; Life Technologies; VIC, Australia) were used as loading controls. buffer (150 mM NaCl, 1 mM EDTA, 10 mM Tris-HCl pH 7.5, 0.1% deoxycholate, 1% Triton X-100) (Lopez-Verges et al., 2006). A total of Assessment of lipid metabolism 150 μg of recombinant TPD52 and respective GST proteins were cross- Cells were incubated for 4 h in GIBCO® DMEM (Life Technologies) linked to GSH–agarose (Glutathione Sepharose 4B, GE Healthcare) as containing 2% fatty-acid-free BSA, [3H]acetic acid (1 µC/ml; Perkin Elmer, described previously (Shahheydari et al., 2014), and incubated with 3T3 cell VIC, AU), [1-14C]oleic acid (0.5 µC/ml; Perkin Elmer, VIC, Australia) and lysates. Matrices were washed extensively before bound proteins were 0.5 mM unlabelled oleate (Sigma-Aldrich) to determine exogenous fatty acid eluted into SDS sample buffer and subjected to western blot analyses. oxidation and esterification into TAG, and TAG synthesis from de novo 14 lipogenesis. Fatty acid oxidation was determined by measuring CO2 in the Isolation of lipid droplets by cellular fractionation culture medium and acid soluble metabolite (ASM) production. To assess fatty Cellular lipid droplets were isolated essentially as previously described acid incorporation from de novo lipogenesis or exogenous oleate into TAG, (Brasaemle and Wolins, 2006). Briefly, D52-2-7 cells were treated with cellular lipids were extracted (Folch et al., 1957). After centrifugation of 400 µM oleic acid in fatty-acid-free BSA for 24 h and then lysed in chloroform-methanol extracts (v/v, 2:1) for 10 min at 1000 g, the lower phase hypotonic lysis medium [HLM; 40 mM Tris–HCl pH 7.4, 2 mM EDTA, was collected and evaporated under a stream of nitrogen. Lipids were separated 20 mM sodium fluoride, and EDTA-free protease inhibitor cocktail tablets using thin-layer chromatography by loading the dissolved pellet and an internal (Roche Applied Science, NSW, Australia)). After incubation on ice for TAG standard on silica plates (Merck Millipore, VIC, Australia), and then 10 min, cell lysates were briefly sonicated and subjected to centrifugation at immersing plates in heptane, isopropyl ether and acetic acid (60:40:3). Plates 1000 g for 10 min. The resulting supernatant with the floating fat layer was were subsequently sprayed with chlorofluorescein dye (0.02% w/v in ethanol) collected as a non-nuclear fraction into an ultracentrifuge tube and gently and lipids were visualised under long-wave UV light. TAG bands were scraped mixed with ice-cold HLM containing 60% sucrose (final 20% sucrose), then and radioactivity was counted. The rate of TAG synthesis from de novo overlaid sequentially with 15% sucrose in HLM, 5% sucrose in HLM and 3 lipogenesis was calculated as H counts (dpm/min) in the TAG fraction per mg HLM only to form discontinuous gradients. Gradients were centrifuged at of protein of cell lysates, and the rate of TAG synthesis from exogenously 28,000 g in a Beckman SW41Ti rotor for 45 min at 4°C, and the lipid 14 supplied oleate was calculated as C counts (pmol/min) in the TAG fraction droplet fraction was collected from the top layer. Proteins from the lipid per mg protein of cell lysates. The fatty acid uptake rate was calculated as the droplet fraction were concentrated by precipitation with 20% trichloroacetic 14 14 14 sum of C counts (pmol/min) funnelled into oxidation ( CO2+ASM) and C acid (TCA, Sigma-Aldrich) at −20°C overnight, and washed three times counts (pmol/min) in the TAG fraction, per mg of protein of cell lysates. with 100% acetone. After air drying, the pellet was dissolved in lysis buffer [150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl pH 7.5, 1% Triton X-100, Palmitic acid treatment and cell proliferation assays 50 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM Parental, vector control or TPD52-expressing 3T3 cell lines were plated in phenylmethylsulfonyl fluoride (PMSF), and EDTA-free protease inhibitor triplicate in 96-well plates (3×103 cells/well). After 24 h, cells were treated cocktail tablets]. Protein concentrations were measured using a Pierce™ or not with 250 μM palmitic acid (Sigma-Aldrich) in fatty-acid-free BSA. BCA Protein Assay Kit (Thermo Scientific, VIC, Australia), and 15 μg MTT assays were performed at indicated time points as described previously protein was subjected to western blot analyses. (Roslan et al., 2014). Statistical analysis Plasmid constructs The SPSS for Windows package (version 21; IBM) was used for most graph Plasmids encoding TPD52 bait protein, and TPD52, TPD52L1, RAB5C generation and statistical analyses. Values from lipid quantitation analyses and GOLGA5 prey proteins have been previously described (Byrne et al., were expressed as means±s.e.m. of three independent experiments, and 1998; Shahheydari et al., 2014). Bait constructs encoding human TIP47 comparisons between groups were made using a two-tailed, unequal variance (Genbank NM_005817.4) and ADRP (Genbank NM_001122.3) were Student’s t-test. The Mann–Whitney U-test (GraphPad Prism 4, GraphPad generated by subcloning respective EcoRI–XhoI fragments from pVP16- Software, La Jolla, CA) was utilised to compare the dispersal of cellular lipid TIP47 and pVP16-ADRP, which were kind gifts from Paul D. Bieniasz (The droplets, and Pearson’s correlation and Manders’overlap coefficients were

Rockefeller University, New York, NY) (Eastman et al., 2009), into the calculated for quantitative analysis of immunofluorescence staining. Journal of Cell Science

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Acknowledgements Brasaemle, D. L., Dolios, G., Shapiro, L. and Wang, R. (2004). Proteomic analysis We thank Dr Emma Kettle and Dr Ross Boadle (Electron Microscopy Unit, Westmead of proteins associated with lipid droplets of basal and lipolytically stimulated 3T3- Hospital, Westmead, NSW, Australia) for electron microscopy assistance, and L1 adipocytes. J. Biol. Chem. 279, 46835-46842. Dr Enoch Tay (Westmead Millennium Institute, NSW, Australia) and Associate Budhu, A., Roessler, S., Zhao, X., Yu, Z., Forgues, M., Ji, J., Karoly, E., Qin, L.-X., Professor Geraldine O’Neill (Kids Research Institute, NSW, Australia) for expert Ye, Q.-H., Jia, H.-L. et al. (2013). Integrated metabolite and gene expression advice and discussions. Professor Paul D. Bieniasz (The Rockefeller University, profiles identify lipid biomarkers associated with progression of hepatocellular USA) kindly provided pVP16-TIP47 and pVP16-ADRP plasmids, and Dr Enrico carcinoma and patient outcomes. Gastroenterology 144, 1066-1075.e1. Tiacci (University of Perugia, IT) provided TPD52 monoclonal antibody for this study. Byrne, J. A., Mattei, M.-G. and Basset, P. (1996). Definition of the Tumor Protein The Leica SP5 II in the CLEM Suite at the Kids Research Institute was supported by D52 (TPD52) gene family through cloning of D52 homologues in human (hD53) the following grants: Cancer Institute New South Wales Research Equipment [grant and mouse (mD52). Genomics 35, 523-532. number 10/REG/1-23], NHMRC [2009-02759], the Ian Potter Foundation [grant Byrne, J. A., Nourse, C. R., Basset, P. and Gunning, P. (1998). Identification of homo- number 20100508], the Perpetual Foundation [grant number 730], Ramaciotti and heteromeric interactions between members of the breast carcinoma-associated Foundation [grant number 3037/2010], and the Sydney Medical School Research D52 protein family using the yeast two-hybrid system. Oncogene 16, 873-881. Byrne, J. A., Chen, Y., Martin La Rotta, N. and Peters, G. B. (2012). Challenges in Infrastructure Major Equipment Scheme. Ultrastructural studies were performed in identifying candidate amplification targets in human cancers: chromosome 8q21 the Electron Microscope Laboratory, Westmead, a joint facility of the Institute for as a case study. Genes Cancer 3, 87-101. Clinical Pathology and Medical Research and the Westmead Research Hub. Byrne, J. A., Frost, S., Chen, Y. and Bright, R. K. (2014). Tumor protein D52 (TPD52) and cancer-oncogene understudy or understudied oncogene? Tumour Competing interests Biol. 35, 7369-7382. The authors declare no competing or financial interests. Cermelli, S., Guo, Y., Gross, S. P. andWelte, M. A. (2006). The lipid-droplet proteome reveals that droplets are a protein-storage depot. Curr. Biol. 16, 1783-1795. Author contributions Chen, Y., Kamili, A., Hardy, J., Groblewski, G., Khanna, K. K. and Byrne, J. A.K., N.R. and Y.C. performed experiments, analysed data and wrote the (2013). Tumor protein D52 represents a negative regulator of ATM protein levels. manuscript. S.F., A.J.H. and D.W. performed experiments, and analysed data. Cell Cycle 12, 3083-3097. L.C.C., A.D.-F., R.K.B., G.E.G. and B.K.S. provided advice regarding experimental Cho, S. Y., Shin, E. S., Park, P. J., Shin, D. W., Chang, H. K., Kim, D., Lee, H. H., design and/or reagents. J.A.B. conceived of the study and wrote the manuscript. All Lee, J. H., Kim, S. H., Song, M. J. et al. (2007). Identification of mouse Prp19p as authors read and approved the final manuscript. a lipid droplet-associated protein and its possible involvement in the biogenesis of lipid droplets. J. Biol. Chem. 282, 2456-2465. Funding Clement, K., Viguerie, N., Poitou, C., Carette, C., Pelloux, V., Curat, C. A., This work was supported by a postgraduate scholarship from the Government of Sicard, A., Rome, S., Benis, A., Zucker, J.-D. et al. (2004). 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TPD52 Expression Increases Neutral Lipid Storage Within Cultured Cells Alvin Kamili1,*, Nuruliza Roslan1,2,*, Sarah Frost1,2, Laurence C

TPD52 Expression Increases Neutral Lipid Storage Within Cultured Cells Alvin Kamili1,, Nuruliza Roslan1,2,, Sarah Frost1,2, Laurence C