GPAT2 overexpression increased TAG storage in CHO-K1 cells.

<p>CHO-K1 cells were transiently transfected with pcDNA3.1 empty vector (control), pcDNA3.1-GPAT1 (GPAT1) or pcDNA3.1-GPAT2 (GPAT2) constructs tagged with a FLAG epitope (Lanes 1–3 and 4–6 correspond to two different transient transfections). The expression of GPAT1 and GPAT2 was confirmed by western blot. Total particulate protein (50 µg) from GPAT1, GPAT2 and control cells was probed with anti-FLAG (A) and anti-GPAT2 (B) antibodies. The molecular mass of the expressed protein was 90 kDa (GPAT1) and 80 kDa (GPAT2). The membranes were probed with anti-β-actin antibody as a loading control. C) Lipid droplets were visualized in control, GPAT1, and GPAT2-overexpressing CHO-K1 cells by Oil-Red O staining. D) The average size of cellular lipid droplets and the average number of lipid droplets in each cell were quantified by Image Pro plus v5.1 software. Data represent mean ± SD of three independent experiments. (*<i>p</i><0.05).</p

GPAT2 overexpression increased arachidonoyl-CoA esterification.

<p>CHO-K1 cells were transiently transfected with pcDNA3.1 empty vector (control), pcDNA3.1-GPAT1 (GPAT1) or pcDNA3.1-GPAT2 (GPAT2) constructs. A) NEM-sensitive GPAT activity was measured with [³H]glycerol-3-phosphate and the acyl-CoA esters substrates palmitoyl-CoA (16∶0-CoA), oleoyl-CoA (18∶1-CoA), linoleoyl-CoA (18∶2-CoA), arachidonoyl-CoA (20∶4-CoA), eicosapentaenoyl-CoA (20∶5-CoA) and docosahexanoyl-CoA (22∶6-CoA) in CHO-K1 cells. GPAT2 overexpression significantly increased GPAT activity only when arachidonoyl-CoA was used as a substrate. B) GPAT activity was measured in both control and GPAT2-overexpressing CHO-K1 and Vero cells with [³H]glycerol-3-phosphate and arachidonoyl-CoA in the absence (Total GPAT activity) and presence (NEM-resistant, NEM+) of 2 mM NEM. NEM-sensitive GPAT activity (NEM−) was calculated by difference of the other two activity values. C) NEM-resistant GPAT activity was measured in control and GPAT1-overexpressing CHO-K1 cells with the substrates palmitoyl-CoA (16∶0-CoA) and arachidonoyl-CoA (20∶4-CoA). D) The ratio between NEM-sensitive GPAT activity in control and GPAT2-overexpressing CHO-K1 cells with the same fatty acyl-CoA substrates as in A) and Vero cells with palmitoyl-CoA and arachidonoyl-CoA was calculated. Bars represent the mean ± SD of three independent experiments (**<i>p</i><0.01).</p

TAG mass and AA content were maximal in testes from 30-d-old rats.

<p>Total lipids were isolated from testes from 19, 30, 40 and 60-d old rats. A) Lipids were separated by TLC and charred with 5% sulfuric acid in methanol, and the TAG spot was quantified by image processing. B) Fatty acid composition of the TAG fraction was determined by gas liquid chromatography at different stages of sexual development.</p

GPAT2 overexpression stimulated AA incorporation in TAG.

<p>CHO-K1 cells were transiently transfected with pcDNA3.1 empty vector (control) or pcDNA3.1-GPAT2 (GPAT2) constructs. A) Twenty-four h after transfection, cells growing in 6-well plates were incubated with 0.25 µCi of [¹⁴C]AA/well in standard medium plus 0.5% BSA for 3 h. Lipids were extracted and separated as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042986#s2" target="_blank">Materials and Methods</a>”. Bars represent the mean ± SD of three independent experiments; PE, phosphatidylethanolamine, PI, phosphatidylinositol, PS, phosphatidylserine, PC, phosphatidylcholine, LPC, lysophosphatidylcholine, TAG, triacylglycerol. B) Twenty-four h after transfection, total lipids were extracted from control and GPAT2-transfected cells incubated with medium plus 10% FBS, and separated by TLC to isolate the TAG fraction and analyze the fatty acid composition. Values represent the mean ± SD of three independent experiments (*<i>p</i><0.05, **<i>p</i><0.01).</p

GPAT2 overexpression increased phosphatidic acid synthesis.

<p>A) The reaction products of GPAT reaction measured in control (C), GPAT1 (G1) and GPAT2 (G2)-overexpressing CHO-K1 cells with [¹⁴C]glycerol-3-phosphate and palmitoyl-CoA (16∶0-CoA) or arachidonoyl-CoA (20∶4-CoA) were visualized by a Storm radioactivity scanner. DAG, diacylglycerol, PA, phosphatidic acid, LPA, lysophosphatidic acid. B) AGPAT activity was measured with oleoyl-CoA or arachidonoyl-CoA and [¹⁴C]oleoyl-lysophosphatidic acid. GPAT2 overexpression significantly increased both GPAT and AGPAT activities only when arachidonoyl-CoA was used as a substrate (*<i>p</i><0.05, **<i>p</i><0.01).</p

<i>Gpat2</i> was expressed in mouse primary spermatocytes.

<p>A) Adult mouse testis sections were hybridized in situ with a <i>Gpat2</i> specific antisense probe (left panels) and the corresponding sense probe (right panels). Magnification: 100× (first row) and 600× (second row). A strong signal was detected in primary spermatocytes (black arrow). B) GPAT2 protein was detected in adult rat testis by immunofluorescence in the presence (left panel) or absence (right panel) of a specific GPAT2 antibody (green signal). The GPAT2 signal was detected in spermatocytes as well as in spermatides. Nuclei were stained with propidium iodide (red signal). Magnification: 400×. The highest GPAT2 expression was detected in the spermatocytes. Bar: 50 µm.</p

Expression of GPAT2 protein was greatest in 30 d and 40 d rat testis.

<p>GPAT2 protein was detected in slides of testes from 19, 30, 40 and 60-d old rats by immunohistochemistry using an anti-GPAT2 antibody (brown signal). Nuclei were counterstained with haematoxylin (blue stain). Magnification: 400×. Bar = 80 µm. The results are representative of three independent experiments.</p

Phenotypic consequences of GPAT2 knock down in MDA-MB-231 cells.

<p>A) Total RNA was extracted from the MDA-MB-231 parent cell line, shRNA-Scr and shRNA-GPAT2 cells, subjected to cDNA synthesis and amplified by quantitative RT-PCR using primers for human GPAT2 gene, normalizing its expression level to that of TBP and β-actin housekeeping genes **p<0.01. B) shRNA-Scr and shRNA-GPAT2 cells were seeded at 10,000 cells/well on MW12 plates and incubated for 24, 48, and 72 h before estimating the cell proliferation rate by MTT proliferation assay *p<0.05. C) 5,000 cells from shRNA-Scr and shRNA-GPAT2 cells were seeded on 35-mm DMEM-agar plates and the number of colonies was quantified by fluorescent microscope after 14 d incubation under normal culture conditions ***p<0.001. D) shRNA-Scr and shRNA-GPAT2 cells were grown to confluence on 10 mm plates and the cell monolayer was wounded six times. The wound width was measured at 0, 2, 6 and 8 h under 100× magnification and the percentage of wound closure was calculated *p<0.05. E) shRNA-Scr and shRNA-GPAT2 cells were treated with apoptosis inducer staurosporine for 30 min or 2 h and the percentage of apoptotic cells was determined by counting the number of apoptotic and non-apoptotic cells using TUNEL assay and haematoxylin staining **p<0.01; ***p<0.001.</p

Phenotipic consequences of human and murine GPAT2 overexpression.

<p>A) Total RNA from pCMV6 and pCMV6-GPAT2 cells was extracted, subjected to cDNA synthesis and amplified by quantitative RT-PCR using primers for human GPAT2 gene, normalizing its expression level to that of TBP and β-actin housekeeping genes *** p<0.001. B) pCMV6 and pCMV6-GPAT2 cells were seeded at 10,000 cells/well on MW12 plates and incubated for 24, and 48 h before estimating the cell proliferation rate by MTT proliferation assay ***p<0.001. C) pCMV6 and pCMV6-GPAT2 cells were seeded in coverslips and 24 h later apoptosis was induced by 1 µM staurosporine treatment for 2 and 5 h. The percentage of apoptotic cells was determined by counting the number of apoptotic and non-apoptotic cells using TUNEL assay and haematoxylin staining **p<0.01; ***p<0.001. D) pcDNA3.1 (empty vector) and the cDNA coding for mouse Gpat2 cloned in pcDNA3.1 (pcDNA3.1-Gpat2) were transiently transfected in HeLa, Vero and HEK293 cells. Cell density was estimated 48 h post-transfection by crystal violet assay. ***p<0.001.</p

Effect of DAC treatment on mRNA GPAT2 expression in human cell lines.

<p>A) Relative mRNA expression of GPAT2 in human cell lines was assayed by quantitative RT-PCR. B) MCF7, HeLa, HEK-293 and MDA-MB-231 cells were treated with the methyltransferase inhibitor 5-aza-2′-deoxycitidyne 2 µM for 96 h (DAC) or with DMSO (control), and the mRNA relative expression of GPAT2 gene was assessed by quantitative RT-PCR. **p<0.01.</p