In vivo assessment of the GATA3 inhibition:
Liposomes preparation
Liposomes were prepared using the ethanol-based proliposome technology by adapting a previously published protocol (27). Briefly, 50mg of phospholipid was mixed with 100µl of absolute ethanol and dissolved at 70˚C. Then 50mg of cholesterol was added to the previous mixture and dissolved at 70˚C water bath. One ml of 0.1mg/ml DNAzyme “hgd40” was added to the phospholipid-cholesterol mixture with continuous vigorous mixing for 4 minutes. The resulting blend was left at room temperature for 2 hours followed by sonication for 10 minutes. The liposomes were centrifuged at 12000 rpm for 15 minutes to get rid of titanium particles released by the probe of the sonicator.
Animal care, experimental design and treatment:
Adult male (12-16 weeks old) BALB/c mice were provided by the Laboratory Animal Research Center (LARC) at Qatar University (QU). Animals were housed in individually ventilated cages (IVC) under standard husbandry conditions (room temperature 18-22℃, relative humidity 40-65% and 12/12 hrs light/dark cycle), provided with normal chow diet and drinking water ad libitum. All animal procedures were performed according to approved institutional ethical rules and regulations and were approved by Qatar University-Institutional Animal Care and Use Committee (QU-IACUC 024/2020). A total of 28 animals were used in this study and divided into three groups. (A) Vehicle Control Group with 8 animals that were treated with 100µl of DNAzyme-free liposomes, (B) Positive Control Group with 8 animals that were treated with liposome-loaded with 1µM of pioglitazone (40 mg/Kg), and (C) GATA3 inhibitor treated Group with 12 animals treated with liposome-loaded DNAzyme (10; µg/ml,hgd40). Treatments were administered subcutaneously to the right flank region (site of injection), twice a week for two weeks. The mice were housed under standard animal husbandry conditions with 12 hours dark and light cycle, were provided standard rodent chow and water ad libitum. Animals were weighed at the beginning and the end of the study. All the animals were euthanized as per AVMA guidelines and the subcutaneous adipose tissues from right flank (site of injection), left flank (opposite site) and omental were collected from scarified mice, weighed, and blood was drawn via a cardiac puncture.
Assessment of oxidative stress:
Oxidative stress was assessed by measuring the activity of anti-oxidant enzymes, including catalase and superoxide dismutase (SOD), in serum samples prepared from collected blood using the Catalase Assay Kit (Merck Millipore) and the SOD kit (Merck Millipore) following the manufacturer’s instructions. Measurements and data analysis was performed using the Cytation 5 Cell Imaging Multi-Mode Reader (BioTek, Germany).
In vitro effect of the GATA3 inhibition:
Recruitment criteria of participants were previously described (17). Approvals of the Institutional Research Board (IRB) committees of Hamad Medical Corporation and Qatar University for the proposed project were sought before onset of research, (MRC-03-21-154, and QU-IRB 1548-EA/21). Five patients undergoing maxillofacial surgeries were recruited, and information about the donors’ gender and BMI were collected. SVFs were isolated from buccal fat pad (BFP) biopsies collected from the recruited subjects as described. Stromal vascular fraction (SVF) was re-suspended in stromal media containing DMEM-F12 with 10% FBS, 1% Antibiotic-Antimycotic solution and 1% L-Glutamine (200 mM) and plated at 4x104 /cm2. The cells were then maintained in a humidified incubator at 37°C with 5% CO2. The media was changed every 2–3 days until the cells achieve 80–90% confluence. When confluent, cells were either harvested or induced to differentiate by changing the medium into differentiation medium (DMEM-F12, 3% FBS, 1% Antibiotic-Antimycotic solution, 1% L-Glutamine (200 mM), 1 µM dexamethasone, 0.25 mM IBMX, 0.2 µM Insulin, Biotin (66 µM), Rosiglitazone (PAPARγ agonist) (5 µM)) ) for 3-7 days, followed by 9-10 days in maintenance medium containing the same components as the differentiation medium except IBMX and rosiglitazone as we described previously (28). To investigate the effect of GATA3 inhibition; cells were grown as above and treated with GATA-3 inhibitor as mentioned previously (29) . Briefly, 24 hours after seeding, the cells were transfected with hgd21 (human GATA3 mRNA specific DNAzyme) and Lipofectamine 3000 transfection reagent. The cells were incubated for 6-8hours, followed by changing media to induce adipogenic differentiation.
Assessment of cell viability and adipogenic capacity
Cells were fixed with 4 % Formaldehyde (Thermo Scientific, 28908) and stained with DAPI (Molecular probs by life technonolies, D1306) and Lipidtox (Invetrogen, H34476) as previously described. Total number of nuclei (DAPI-positive) and differentiated adipocytes (Lipidtox-positive) were automatically scored in twenty five fields/well by Cytation 5 Cell Imaging Multi-Mode Reader (BioTek, Germany). While adipogenic capacity was assessed by calculating the percentage of Lipidtox-positive cells to total number of nuclei.
Assessment of gene expression from both in vitro and in vivo experiments:
For the in vivo experiments, stromal Vascular Fractions (SVFs) were isolated from adipose tissue biopsies collected from right/left thighs and omental depots using established protocol (30-32). Briefly, the collected adipose tissue biopsies (0.5g) were homogenized using gentleMACS™ Dissociator (Miltenyi Biotec) then digested using collagenase solution (0.1% collagenase I/1% BSA in PBS) for one hour at 37°C. Samples were then centrifuged at 1500 rpm for 5 minutes to separate SVF. The resulting cell pellet was then washed with 1% BSA, followed by erythrocyte lysis buffer for 10 minutes. TRizol reagent (Invitrogen) was added to the pellet for the RNA extraction using the TRizol method according to manufacturer's instructions. For the in vitro experiments, RNA was extracted from preadipocyte cultures prior and post induction of differentiation using TRIzol method (Invitrogen) according to manufacturer's instructions. Three μg of the resulting RNA from in vitro and in vivo experiments was used for First-strand cDNA synthesis using Superscript III first strand synthesis super mix kit (Invitrogen) according to manufacturer's instructions. Real-time PCR was carried out for gene expression analysis using 10 ng of the produced cDNA with the listed primers (Table I) using 7500 Real Time PCR System from Applied Biosystem. The PCR conditions were as follows: 1 cycle of 95 °C for 10 minutes, 45 cycles of 95 °C for 15 seconds, 55 °C for 40 s and 72 °C for 30 cycles and finally 60 °C for 15 seconds. Real-time PCR was carried out in triplicate and the GAPDH was used as a housekeeping gene for normalization of the amplified signals of the target genes. The data analysis was performed using the ΔΔCt based calculations (33).
Assessment of insulin signaling
Insulin signaling was measured by assessing the phosphorylation levels of IRS-1, GSK3B, IGF1R, Akt, Mtor, p70s6k, IR, PTEN, GSK3, TSC2, and RPS6, using a commercial Bio-Plex Pro™ Cell Signaling Akt Panel (Bio-Rad) using Luminex 200 technology (Thermo Fisher Scientific, USA) following manufacturer's instructions.
Statistical analysis
Comparisons were performed using t-test, One-way ANOVA, Two-way ANOVA or Linear regression models as appropriate using GraphPad prism. Data are presented as mean ± SEM.