Dulbecco’s modified Eagle’s medium (DMEM), antibiotics (penicillin and streptomycin), fetal bovine serum (FBS), and 0.25% trypsin were purchased from GE Healthcare Life Sciences (Logan, Utah). Insulin, dexamethasone (DEX),3-isobutyl-1- methylxanthine (IBMX), protease inhibitor cocktail, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO). Diazinon (Cas: 33–41-5,) was purchased from Chem Service with a purity of 99.2% (West Chester, PA). The name of International Union of Pure and Applied Chemistry (IUPAC) is O,O-Diethyl O-[4-methyl-6-(propan-2-yl)pyrimidin-2-yl] phosphorothioate.

3T3-L1 mouse preadipocytes were kindly gifted from Dr. Clifton Bailey’s laboratory at the University of Georgia. Cells were maintained in DMEM composed of high glucose, 10% FBS and 100 U/mL penicillin and streptomycin in a 37°C, 5% CO₂ humidified environment. The cultured cells were maintained in a sub-confluent condition and change of media every 2–3 days.

3T3-L1 cells were cultured to 100% confluence (M1 medium: DMEM containing 10% FBS) in a 12-well plate, 35 mm dish, or 96 well-plate, for RNA, protein, or high-content analysis, respectively. This time point is denoted as day 0. After post-confluence, cells were then incubated in adipogenic induction medium (M2 medium: DMEM containing 1 μM DEX, 0.5 mM IBMX, 167 nM insulin and 10% FBS) for two days, and then cultured in adipogenic differentiation medium (M3 medium: DMEM containing 167 nM insulin and 10% FBS) for another two days, followed by DMEM with 10% FBS (M1) for another four days. The cells cultured with DEX in the M2 medium were used as a positive control, in order to ensure the cell’s ability to differentiate into the adipocyte. To examine the effects of diazinon on adipogenesis of 3T3-L1 cells, diazinon was added to M1, M2 without DEX, and M3 media as indicated doses for a total 8 days. Cells in the vehicle (DMSO 0.05%) in M1, M2, and M3 media were set as the untreated control (CTL). Figure 1 illustrates the stages and treatment protocol.

3T3-L1 cells were seeded in a 12-well plate and treated with diazinon of various concentrations or a vehicle control from day 0 to day 8. On day 8, cells were fixed in fresh 4% paraformaldehyde, and the intracellular lipid droplets were stained with a filtered solution of 60% Oil Red O in 100% 2-isopropanol. Stained cells were observed with an Olympus IX71 (TH4–100) imaging system with 20X phase contrast objectives. Oil Red O was extracted from cells with isopropanol, and optical density (OD) was then measured at a wavelength of 510 nm (Gen5, BioTek). The cells were also stained with Nile Red using AdipoRed Assay according to the manufacturer’s instructions (Lonza, MD), the fluorescence intensity was measured at 572 nm (Gen5, BioTek). In order to determine triglyceride levels more specifically, the triglyceride levels from the cell lysates were quantified using Infinity Triglycerides Reagent (Thermo Scientific, MA) and Triglyceride Standard (Pointe Scientific, Canton, MI). The 3T3-L1 cells were cultured in the 6-well plate, and treated with various doses of Diazinon, DEX as a positive control (pos), and vehicle (DMSO 0.05%) as the negative control (Ctl). After treatment for 10 days, the cells were washed with cold PBS twice, and the cell lysates were re-suspended and homogenized in 5% NP-40 solution. The cell lysates were then harvested, and heated for 5 mins in the 95ºC water bath, and vortexed for 30 seconds. This step was repeated twice, and then cool samples to room temperature. The Triglyceride levels, then were assayed with Infinity Triglyceride Reagent according to the manufacturer’s instructions. The protein content of the cell lysates was also measured by the Bradford protein assay following the manufacturer’s instruction (BioRad, Hercules, CA). The final triglyceride level was normalized with the protein content. The experiment was performed with three technical replicates, repeated three times.

3T3-L1 cells were seeded in a black frame 96-well plate (Corning, NY), and treated with diazinon as indicated concentrations from day 0 to day 8. Cells were then fixed with 4% paraformaldehyde and then washed with PBS twice. A staining solution containing Hoechst 33342 (1 μM) for nuclear staining, and LipidTOX for neutral lipid staining (Life Technologies, NY) was added to each well and incubated at room temperature for 30 min. Image acquisition and image-based quantification were performed using ArrayScan® VTI HCS Reader with HCS Studio™ 2.0 Cell Analysis Software (Thermo Scientific, MA). Images were acquired with high resolution (1024 × 1024), and 49 fields for each well were extracted. After feature extraction, the cell-based data were exported to JMP (SAS) or GraphPad Prism (San Diego, CA) for further statistical analyses. Lipid droplets were detected with the SpotDetector® algorithm of BioApplication. The method was based on a two-channel acquisition assay, which uses a 20 × objective (NA 0.5), a Hamamatsu ORCA-ER digital camera in combination with a 0.63 × coupler, and Carl Zeiss microscope optics for automatic image acquisition. Channel one applies BGRFR 386–23 for Hoechst 33342 (nuclei), and the objects were identified. This nuclear identification was used as a measure of cell number, and the nuclear area was determined by the staining Hoechst 33342. The spots (lipid droplets) were detected in channel two (BGRFR 549–15 filter). The average fluorescence intensity of spot, spot totally intensity per spot count, spot average area and average intensity of FABP4 were reported. The setting of the SpotDetector algorithm (version 4.1) was optimized for LipidTox analysis in channel two, and the thresholds were set to ensure that only lipid droplets of certain size and intensity were selected for analysis. Lipid counts, the intensity of the spot and intensity of FABP4 was normalized to the number of the nucleus. The experiment was performed with 8 technical replicates and repeated twice.

3T3-L1 cells were seeded in a 12-well plate and treated with diazinon of various concentrations or vehicle control media from day 0 to day 8. Total RNAs were isolated at the end of each stage as indicated in Figure 1. The quality and quantity of total RNA were measured on a Nanodrop (Thermo Scientific, MA). cDNA was reverse transcribed from 2 μg total RNA using iScript Reverse Transcription (BioRad, Hercules, CA). Using an aliquot of the synthesized cDNA, qRT-PCR was conducted with SsoAdvanced Universal SYBR Green Supermix (BioRad, CA). The amplification conditions were initially denatured at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 30s, annealing at 55–57°C for 30s and elongation at 72°C for 30s. Melt-curve analysis was performed to confirm that the signal was not of possible primer-dimers but of the expected amplification product. Oligonucleotide primers were designed using Primer 3.0 software and UCSC Genome Bioinformatics (https://genome.ucsc.edu/) or according to the published sequences [61]. The level of glyceraldehydes-3-phosphate dehydrogenase (GAPDH) transcript was used to normalize each sample. Results are expressed as relative fold change of treatment over control. Two independent experiments were performed, and each test condition was conducted in triplicates.

3T3-L1 cells were seeded in 35 mm dishes and treated with diazinon of various concentrations or control media from day 0 to day 8. At the end of each stage, cells were harvested and lysed with ice-cold cell lysis buffer (Cell Signaling, Boston, MA). The cell lysates were sonicated on ice, and the soluble materials were collected from the supernatants after centrifugation at 13,000 rpm for 15 min at 4°C. The protein concentration was determined according to the manufacturer’s instructions (BioRad, CA). 10 μg of total protein were resolved by 4–12% Bis-Tris polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, MA). The specific protein expression was detected with monoclonal rabbit antibodies against Peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer binding protein α (C/EBPα), fatty acid synthase (FASN), Acetyl CoA (ACC), adiponectin, perilipin, and mouse antibody against housekeeping proteins β-actin for overnight at 4 °C. The blots were washed five times with Tris-buffered saline containing 0.05% Tween 20 (TBS-T), and then incubated with a horseradish peroxidase-conjugated secondary anti-rabbit or anti-mouse IgG antibodies (Jackson Immuno Research, PA) for 1.5 h at room temperature. Immunoreactivity bands were visualized by enhanced chemiluminescence (BioRad, CA). The intensity of individual bands was quantified using Image J densitometry software (NIH, 1.49), and results are expressed as relative fold change of treatment over control after normalization with housekeeping protein.

The data are shown as the mean ± standard deviation from multiple experiments. Statistical significance was determined using one-way analysis of variances, and Tukey's multiple comparison test, with statistically significant at the cutoff level of p < 0.05 or p<0.01 (GraphPad, Prism5, CA).

In order to select a sub-toxic dose of diazinon for a subsequent adipogenesis study in 3T3-L1 cells, the cell number was determined using an Arrayscan VTI HCS reader with HCS Studio 2.0 Target Activation BioApplication module (Thermo Scientific, MA). The nuclei were stained with Hoechst 33342, and fluorescence intensities of the nuclei were examined and compared in 3T3-L1 treated with Diazinon at concentrations of 1, 10, 25, 50 and 100 μM. It was found that diazinon treatments decreased the cell number slightly at 25 and 50 μM, but significantly decreased at 100 μM as illustrated in Figure 2. Therefore, the concentrations below 100 μM of diazinon were selected in the following experiments to examine the effect of diazinon on the adipogenesis without obvious cytotoxicity. To clarify whether nuclear condensation that occurs alongside the differentiation, we also found that the nucleus size was reduced by approximately 10% during adipocyte differentiation after diazinon treatment, which likely is associated with alteration of the chromosome [62].

In order to visualize diazinon’s adipogenic characteristics, Oil Red O staining was used to observe lipid accumulation in 3T3-L1 cells. DEX treatment, a positive control, shows approximately 90% Oil Red O staining cells in each field, indicating a high degree of lipid droplet accumulation (Figure 3). Diazinon treatment significantly increases lipid droplet accumulation in a dose-dependent manner as compared with the untreated control. During the differentiation process for 8 days, the 3T3-L1 preadipocytes underwent morphological changes from spindle-like to round, and accumulated intracellular lipids in both a positive control and diazinon treated conditions (Figure 3). Quantitative measurement of intracellular lipid droplets shows diazinon treatments above 50 μM significantly increase lipid staining as compared to control (p < 0.01) (Figure 3F). The triglyceride was stained with another dye-based Nile Red (AdipoRed), the content of triglyceride was increased starting at 10 μM of diazinon (Figure 3G). In order to distinguish stored triglyceride inside of the cells from the accumulated fat-soluble diazinon, the triglyceride levels in cell lysates were further quantified based on lipase enzyme-based assay using Infinity Triglycerides Reagent (Thermo Scientific, MA). The cellular triglyceride levels were determined by comparison to a triglyceride standard curve. Diazinon treatment increased the cellular triglycerides in a dose-dependent manner, and significantly increased cellular triglycerides at 50 μM (Figure 4). This result further confirmed that diazinon promotes the cellular triglycerides, not fat-soluble compound diazinon itself in the cells.

To further quantification of adipocyte differentiation at a single-cell level, a single-cell-based HCA was used to characterize multiparametric features in 3T3-L1 cell treated with diazinon. Lipid accumulation has been used as the primary indicator for the differentiation of preadipocytes into mature adipocytes. We applied a neutral lipid staining, LipidTOX, to investigate the adipogenic effects of diazinon treatment in a high-throughput and high-content format. We evaluated the effects of diazinon on the lipid droplet accumulation in a wide range of concentrations (1, 10, 25 50 and 100 μM). A single-cell-based HCA for the nuclear, lipid droplets and lipid-associated fatty acid binding protein 4 (FABP4) were quantified. Figure 5A (LipidTOX) and 5B (FABP4) show representative fluorescence images of the lipid droplets or FABP4 staining, respectively, after the treatments. As shown in Figure 4A, the cells in control show evenly distributed nuclei with barely detectable green fluorescent lipid. In the treated conditions, we observed a dose-dependent increase of LipiTOX (Figure 5A) or FABP4 fluorescence (Figure 5B), suggesting diazinon-induced adipogenic effect in 3T3-L1 cells as compared to relative control. Similar morphological changes were observed with increasing lipid accumulation with increasing dose of diazinon, while nuclei count did not decrease. Quantification of lipid droplet accumulation, as measured by the Spot Detector Algorithm, increases significantly, whereas the average intensity of FABP4 shows some parallel increases at the end of differentiation, but less extent (Figure 6D). Lipid droplets are the lipid storage organelles, and the size of lipid droplets is associated with the capacity of fat storage. The parameters for lipid droplet size, including Spot Total Intensity Per Spot Count and Spot Average Area, were determined (Figure 6C). As shown in Figure 5, the droplet size in 3T3-L1 cells treated with 1 (B), 25 (C), and 50 μM (D) significantly increased as compared with the relative controls. Many other parameters derived from the lipid droplet images also increased in a similar manner, including Spot Total Intensity Per Spot Count and Average Intensity of Spot. Overall, these data demonstrated that Diazinon induced the lipid droplets and its associated protein FABP4 (Figure E-H) with multiple adipogenesis specific endpoints.

In order to further examine diazinon’s adipogenic efficacy during the differentiation process, adipogenic protein expression was examined in 3T3-L1 cells treated with diazinon (1 μM, 10 μM, 100 μM). The adipogenesis associated proteins, including Fatty acid synthase (FASN), PPARγ, C/EBPα, acetyl-CoA carboxylase (ACC), perilipin and adiponectin, were investigated at the end of each stage of differentiation process (Figure 1, including induction, differentiation, and maturation). As shown in Figure 7A, all the positive controls of each stage show adipogenic proteins, except Perilipin and adiponectin, and exceptionally high abundance of expression levels at the end of the stage (Day 8, maturation). Adipogenic proteins significantly increased with increasing dose and exposure time in 3T3-L1 cells treated with diazinon (Figure 7B-H). Specifically, the protein expression of PPARγ and C/EBPα, two major transcriptional factors for adipogenensis, were increased with diazinon at a low dose of 10 μM starting from the second stage (day 4). As indicated in Figure 7B, PPARγ expression was significantly induced as early as the first stage at 100 μM treatment, and at the lowest dose of 1 μM at the third stage. C/EBPα protein expression was significantly increased in all stages after treatment with 10 μM and 100 μM of diazinon. All examined proteins had a statistically significant increase in expression of the third stage with treatment 10 and 100 μM diazinon.

In order to examine the adipogenic signaling pathway, we measured the adipogentic regulatory genes at the end of each stage of adipogenesis: induction (day 2), differentiation (day 4) and maturation (day 8). As shown in Figure 8, all tested gene expression in the positive control were significantly increased with dramatic 10 to 1000-fold increases in a time-dependent manner (only show the third stage). Diazinon treatment significantly induced all tested gene expression at 10 μM in the third stage (Day 8) as compared with the control. Adiponectin, adipocyte differentiation marker, was significantly induced at the early stage (day 2), with dramatic induction at both the differentiation (30 fold) and maturation stage (1000 fold). The enzyme that regulates adipogenesis, fatty acid synthase (FASN), was also significantly induced at 10 μM of diazinon during the maturation (day 8).