Male healthy Chinese Red Steppe calves at 2 months of age were obtained from the Jilin Academy of Agricultural Sciences and slaughtered for preadipocyte isolation. The animal experiment strictly complied with ethical requirements and operating rules. All animal procedures, such as welfare and ethical issues, were approved by the Committee for the Ethics of Animal Experiments by AWEC 2019A05, 16 May 2019.

Subcutaneous white adipose tissue of calve was isolated and washed with PBS (Sigma-Aldrich, St. Louis, MO, US) containing 6% penicillin/streptomycin (Sigma). The samples were sliced into 1 mm² piece with sterile scissors and were digested with collagenase Type II (Sigma) and 0.25% trypsin (Sigma) for 1 h. The solution containing blocks and cells was filtered with a 40 μm filter to remove the undigested tissue pieces and obtain the preadipocytes. The cells were placed in a 37°C incubator (Thermo Fisher Scientific, Waltham, MA, United States) with 5% CO₂ to culture in a complete culture medium consisting of 10% fetal bovine serum (Gemini Bio-Products, Woodland, CA, United States), 1% penicillin/streptomycin (Sigma), and Dulbecco’s modified Eagle medium-basic medium (Sigma), the fresh culture medium was replaced once 48 h. After culturing for 6 h, the preadipocyte proliferation and differentiation at the end of proliferation was induced into the mature adipocyte with the exogenous supply of inducer containing inducer I and II. Inducer I solution consisted of 10 mg/mL insulin (Sigma), 1.0 mM dexamethasone (Sigma), 0.5 mM IBMX (Sigma) and was added to the culture medium to treat the cells for 2 days. Inducer II solution consisted of 10 mg/mL insulin (Sigma) and was added to the culture medium to treat the cells for 2 days. The cells were cultured in a fresh culture medium until preadipocyte maturation.

The lipid droplets in the mature adipocytes can be stained by Oil Red O dye to red color, and it is a general way to verify adipocyte maturation. The adipocytes were washed with cold PBS three times, treated with 4% paraformaldehyde for 30 min in a 37°C incubator, and then washed again. Oil Red O solution containing Oil Red O dye and ddH₂O in a volume ratio of 3:2 was used to treat cells for 30 min in an incubator. The mature adipocytes were photographed and analyzed under a microscope after being washed with PBS. Finally, an isopropanol solution was used to absorb the lipid droplets, then the solution was mixed with the lipid to determine the lipid droplet content at an OD value of 490 nm.

The primers to amplify the coding-sequence region (CDs) region of the MED4 gene were designed according to the bovine sequences (NM_001034486.1) available on the NCBI website. Overexpression plasmid pEX4 vector was used to construct the recombination vector containing MED4 CDs sequence at XhoI and EcoRI restriction endonuclease, and pEX4 empty plasmid was the negative control. Based on the sequence of MED4 mRNA, three small interference RNA sequences and negative control were designed and synthesized by GenePharma Company, Shanghai (Table 1). The gel image mapping the CDs region of MED4 gene and sequencing comparison results are showed in the Suppl. Fig. S1.

The plasmid or interference sequences were transfected into the preadipocytes when the cells fused 70% culture dishes. Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) reagent gets the room temperature before use. One hundred picomoles of interference sequences, 5 μL lip2 000, and 200 μL Opti-MEM (Invitrogen, Carlsbad, CA, USA) were mixed well for 10 min. The mixture was added into the cells with 1.8 mL fresh medium for 6 h and replaced with a fresh complete culture medium. Transfection efficiency was determined by quantitative polymerase chain reaction (qPCR) test after 48 h. FuGENE reagent (Roche Applied Science, Indianapolis, IN, USA) was used to mix with 400 μL Opti-MEM, 3 ng plasmid; it was placed at room temperature for 20 min and added into the cells for 48 h, the transfection efficiency was determined by qPCR test.

TRIzol reagent (Thermo Fisher Scientific) was used to extract total cellular RNA following the procedure. RNA contamination and degradation were validated via 1% agarose gels, and concentration was detected via a Quawell Q5000 spectrophotometer (Quawell Technology, San Jose, CA, USA). The reverse transcription kit (Takara, Japan) reversed the total RNA into cDNA. The reaction volume includes 2 μL of 5 × PrimeScript RT Master Mix, 500 ng of total RNA, and RNase Free ddH₂O up to 10 μL. Reaction condition: 37°C for 15 min, 85°C for 5 s, and the PCR products were normalized with ddH₂O and stored in a 4°C fridge. Roche LightCycler® 480 (Roche Applied Science), a quantitative fluorescent PCR device, detected the relative expression of the mRNA. Beta-actin was an internal control. The reaction volume: 10 μL of 2 × Light Cycler 480 SYBR Green I Master, 8 μL of ddH₂O, 1 μL of cDNA, and 0.5 μL of sense and anti-sense primers. The reaction condition: 40 cycles of 95°C for 5 min, 95°C for 10 s, 60°C for 15 s, and 72°C for 20 s, followed by 95°C for 5 s, 65°C for 1 min, and 40°C for 10 s. The detailed information on primers was showed in Table 2.

Cell TG content was determined using a Triglyceride kit (Prilax, Beijing, China) following the manufacturer’s procedure. Clean mature adipocytes without medium were digested with 0.25% trypsin (Sigma-Aldrich), and the mixture was centrifuged at 1500 rpm for 20 min. The sediments were digested for 10 min with lysis solution, heated at 70°C for 10 min, then centrifuged at 2000 rpm for 5 min. The OD of the enzyme-labeled supernatant was detected using an instrument at 550 nm wavelength and TG content was calculated.

Total protein from the cells was extracted using a Protein extraction kit (Solarbio, Shanghai, China). Adipocytes without medium were lysed for 25 min on ice, and the mixture was transferred into a tube and centrifuged at 12000 rpm at 4°C for 30 min to collect the protein. The BCA assay kit was used to determine the protein concentration. All proteins of samples were normalized with those in the lysate solution. The proteins were heated at 99°C for 8 min and mixed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) buffer (Beyotime, Jiangsu, China). The upper and lower gels were made with the PAGE Gel Fast Preparation kit (EpiZyme, Shanghai, China). Thirty micrograms of protein in each sample and 6 μL standard marker (Thermo Fisher Scientific) were loaded to the gel wells and separated by electrophoresis at 80 V for 20 min. Then 120 V for 40 min, the gel with samples was transferred into a 0.45 mm Immobilon poly-vinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA) at 200 mA for 1 h. PVDF membrane was washed with TBST (Tris-HCl buffered saline with tween 20; Beyotime, Jiangsu, China), blocked with 5% skimmed milk for 2 h, then washed again, and incubated with antibody (dilution ratio at 1:1 500; Bioss Beijing, China) and placed overnight on a shaker at 4°C. The clean membrane was incubated with a secondary antibody (anti-rabbit or anti-mouse IgG-HRP, 1:5000) at room temperature for 2 h and washed again. ECL hypersensitive photoluminescence solution (Pripril, Beijing, China) was used for a chromogenic reaction with the proteins. Then, protein bands were determined using the ChemiScope 6000 Touch imaging system (Clinx Science Instruments, Shanghai, China), and quantified using the ImageJ software. β-actin was an internal reference protein.

SPSS 17.0 software was used for statistical analyses. Each experiment was repeated three times. All data was described as means ± SD. The results compared two groups by t-test, and multiple group comparisons by one-way ANOVA. The statistical significance levels were set at p < 0.05. The raw data and images are presented in the Suppl. Table S1 and Fig. S1.

The preadipocytes were isolated and cultured in a normal incubator, and they started proliferating until full fusion in the plates. Then, the inducer solution was added to promote the preadipocyte differentiation into mature adipocytes. The whole process of differentiation took around 10 days. The mature adipocytes were shown in Fig. 1.

To investigate the changing pattern of the MED4 gene in preadipocyte differentiation. RNA in different periods (days 0, 2, 4, 6, 8) was extracted to verify the level of MED4 gene expression by qPCR. The inducer I solution was added to the preadipocytes on day 0 and inducer solution II was added on day 2. On days 4 and 6, fresh medium was added into the preadipocytes. Some small lipid droplets we noticed in the preadipocytes on day 8 and the majority of preadipocytes became mature adipocytes on day 10. These results showed that the expression of the MED4 gene was constant from day 0 to day 6 and significantly increased on day 8 (Fig. 2).

After 48 h of transfection, the efficiency was determined by qPCR test. The recombination plasmid exhibited significantly increased expression of the MED4 gene compared to the empty plasmid (p < 0.05) (Fig. 3A). Subsequent assessment showed that three interference sequences had different effects, of which siR354 significantly decreased the MED4 gene expression compared to the negative control (p < 0.05) (Fig. 3B). The results of western blotting had a similar trend as the qPCR results (Figs. 3C–3F).

To assess the effect of the MED4 gene on adipocyte differentiation, the expression of differentiation-related biomarker, was detected by qPCR test in different periods (days 0, 4, 8), and the TG content and lipid droplets were measured on day 10. With the increase in the MED4 gene, the PPARγ gene expression significantly increased (p < 0.05). Meanwhile, the MED4 gene also improved the accumulation of the TG content and lipid droplets (p < 0.05) (Fig. 4) and may improve adipocyte differentiation by affecting the PPARγ gene expression.

To investigate the effect of the MED4 gene on adipocyte differentiation. The differentiation-related biomarker expression in different periods (days 0, 4, 8) was detected by qPCR, and the TG content and lipid droplets were measured on day 10. Inhibition of the MED4 gene significantly reduced the PPARγ gene expression (p < 0.05) and decreased the OD value of TG content and lipid droplets (p < 0.05) (Fig. 5). Thus, the MED4 gene may inhibit adipocyte differentiation by affecting the expression of the PPARγ gene.