Subject tissue samples
Based on the fundamental propositions laid out in the Declaration of Helsinki, we were granted permission from the adult subjects or guardians on behalf of the minors participants involved in our study by written before sample collection. Adipose tissue specimens were obtained from 6 overweight subjects, 17 obese subjects (14 females, 9 males) and 16 normal weight subjects (13 females, 3 males) (aged from 11 to 79 years, with a median of 45 years) who had an elective surgery at Xinqiao Hospital of Third Military Medical University, Chongqing, China. Demographic data of the studied subjects is shown in Supplementary Table S1, S2. All subjects, who were neither smoker nor under lipid lowering medications, went through thorough clinical phenotyping at an accelerated rate (12 h, overnight). To calculate the BMI (weight/height squared) (kg/m2), a measurement was taken of the weight (kg) of the subjects who was wearing cloth that weighed as little as 0.1 kg. Besides, the subjects’ height (bare foot) was determined using a stadiometer (cm). On the other hand, the distance around their abdomen was ascertained utilizing a measuring tape. In order to figure out how prevalent overweight and obesity was, a calculation was performed with a combination of the body mass index (BMI) (normal weight 18.5-23.9 kg/m2; overweight 24-27.9 kg/m2; obesity≥28 kg/m2) and the explanation of abdominal circumference (abdominal obesity: male≥90 cm, female≥85 cm) provided by Working Group on Obesity in China (16). Those subjects with cancer, HIV, collagen diseases and syphilis were excluded from the study. We received clearance granted by the studied participants. The project was granted approval from the local ethics committee of Third Military Medical University. Before the isolation of total RNA and protein, sampling of latest adipose tissue was conducted intraoperatively and immediately for freezing in liquid nitrogen and storage at −80 °C.
RNA isolation and RT-PCR
Human WAT with TRIzol® Reagent (Invitrogen) was involved for the extraction of total RNA and one microgram of total RNA was applied to synthesize cDNA by using the Revert Aid First Strand cDNA Synthesis Kit (Fermentas, K1611) in line with the requirements specified by the manufacturer, which was followed by PCR ampliﬁcation with speciﬁc forward and reverse primers for 35 to 45 cycles. To enable internal control, GAPDH was utilized. The specific primers were used for the human genes are listed (Supplementary Table S4). The mouse primers of KCTD15 used in the study are as follows:
A separation of the PCR products was conducted in 3% agarose gels for a subsequent visualization under UV light. Then, a quantitative analysis was performed to determine the density of the band using Quantity One software (Bio-Rad). The relative expression of all the genes detected were normalized to the GAPDH or β-actin.
Cell growing under controlled conditions culture and induction of differentiated adipocyte
While sustaining 3T3-L1 pre-adipocytes, the standard protocol was applied to facilitate them to differentiate (17). Briefly, Previous to conﬂuence, Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% newborn calf serum (SH30401.01, Hyclone) and 1% antibiotics (Penicillin/Streptomycin/Amphotericin B) was involved to grow 3T3-L1 pre-adipocytes (we named the cell confluence day as Day-2, D-2). With 48 hours past cell conﬂuence (Day 0, D0), the cells were induced to differentiation by exposing to differentiation medium containing 1 μmol/L dexamethasone (D2915, Sigma),0.5 mmol/L isobutyl methylxanthine (I5879, Sigma), 2 μmol/L rosiglitazone (R2408,Sigma), 10 μg/mL insulin (I6634, Sigma), and 10% fetal bovine serum (FBS; A15-151, PAA) for three days. When it approached the end of the 3rd day, DMEM supplemented only with 10 μg/mL insulin and 10% FBS was used as a substitute of the original substance to grow the cells, and was subjected to replenishment for every two days. Upon the completion of the process to differentiate, a minimum of 90% of the cells accumulated lipid droplets at Day 10, for which they were selected as mature adipocytes.
The siRNA transfection of 3T3-L1 pre-adipocytes was performed as indicated (18). GenePharma was applied to the design and synthesis of mouse KCTD15 siRNA (Shanghai, China). The siRNA sequences are indicated here:
Mouse KCTD15 siRNA #1 5’-GACGUCUCAAAACUGCUGCUU,
Negative control siRNA, 5’-UUCUCCGAACGUGUCACGU.
3T3-L1 preadipocytes were grown to 30–50% confluency in 35 mm dishes and transfected with siRNA oligonucleotides by applying Lipofectamine™ RNAiMAX (Invitrogen, 13778-150) according to the instructions of the manufacturer. Forty-eight hours later, the knockdown efficiency was detected by RT-PCR to facilitate differentiation, the standard differentiation protocol was applied.
Oil Red O dyeing
Subsequent to obtaining the mature adipocytes by in vitro differentiation, the lipid droplets in the mature adipocyte cells were monitored by adopting the approach of modified Oil Red O staining (19). In brief, the cells were washed twice with PBS before ﬁxed with 10% formaldehyde for a duration of 1h at room temperature. Afterwards, The cells were washed twice with 60% isopropanol and then dyed by applying Oil Red O solution for 1 h. Images of the Oil Red O-stained cells were derived from using with Leica DMI3000 B inverted microscope.
Triglycerides can be quantified with the triglyceride assay kit E1003-2 (Applygen Technologies Inc, Beijing, China). Briefly, cells ended up being harvested by trypsinization, rinsed three times with PBS and then lysed in lysis buffer. The supernatant was assayed in line with the requirements laid out by the manufacturer.
Total protein lysate was extracted from 3T3-L1 cells at different time points during differentiation with RIPA buffer (20 mM Tris-HCl, pH7.4, 20% glycerol, 0.5 % NP40, 1 mM MgCl2, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA.). Protein concentrations were measured using the Bradford Assay, and 100 μg protein extracts were subjected to SDS-PAGE. Then proteins were transferred to a PVDF membrane, blocked with 5% non-fat milk and incubated with first antibodies (anti-KCTD15, Abcam ab106373, 1: 1000; anti-β-Tubulin, Sigma t-5293, 1: 10000) for 1 h at R.T. After incubation with secondary antibody against mouse (Dakocytomation, p0161, 1:10,000) or rabbit (Dakocytomation, p0448, 1: 10,000) for 1 h at R.T, the membrane was visualized by ECL Western Blotting Detection Kit (RNP2232, GE healthcare).
All statistics were interpreted as the mean ± SEM. The correlation coefficient, Student’s t-test (for single comparison) and one-way ANOVA (for group comparisons) were involved to analyze data where appropriate using SPSS 16.0 for Windows (SPSS, part of IBM Corporation; Somers, NY). Tukey’s post hoc test was applied under the circumstance of a necessary comparison among individual groups. P <0.05 was regarded as being of statistical importance.
Permission was granted for this research by the Human Research Ethics of Xinqiao Hospital of Third Military Medical University, under the number of ChiCTR-RNRC-11001441, and all parents signed the informed consent form.