Three adult Nanyang cattles were slaughtered from Biyin cattle farm Nanyang city, Henan province. Subcutaneous adipose tissue was obtained from calves for isolation of preadipocytes. The Animal Ethics Committees of Ningxia University approved the experimental design and the animal sample collection for the present study (permit number NXUC20211168). This study is reported in accordance with the ARRIVE guidelines.
Isolation and induced differentiation of bovine preadipocytes
Bovine preadipocytes were isolated using collagenase digestion, as described . Briefly, adipose tissue without blood vessels and connective tissues was minced into cubes with size ~1 mm3 and digested in 1 mg/mL collagenase type I (Sigma, C0130) in a water bath for 90 min at 37 ℃. Subsequently, preadipocytes were seeded and cultured on a 10 cm2 plate with a growth medium containing Dulbecco's modified eagle medium (DMEM), 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) at 37 ℃ with 5% CO2 for 24 h. Cells were then washed with PBS three times and fresh medium was replaced every two days.
When density reached 90%, cells were transferred to 6-well cell culture plates using trypsin. Subsequently, adipocytes were differentiated by feeding induction medium (IM) containing DMEM, 10% FBS, 10 µg/mL insulin, 1 µM rosiglitazone, 1 µM dexamethasone (DEXA, Sigma) and 0.5 mM 1-methyl-3-isobutylxanthine (IBMX, Sigma). Three days later, the medium was replaced with maintenance medium which was changed every two days until maturation.
Sequence analysis of bovine SGK1
Primers were designed to amplify the CDS region of SGK1 (Genbank No. NM_001102033.1) (Supplementary Table 1) and interacting proteins were predicted using STRING.
Recombinant adenovirus packaging
Endonucleases Kpn I (Takara, Dalian, China) and Hind III (Takara) were added to forward and reverse primers, respectively. Subsequently, the CDS region was inserted into the shuttle vector pAd-tract-CMV and was transformed into competent cells BJ5183. The recombinant vector was transfected into taraka (HEK293a). Subsequently, cells and culture medium were collected named (OE-SGK1) with the and negative control named (OE-NC) eight days after transfection. pAd-track-CMV and taraka were preserved in our laboratory.
Dual luciferase report analysis
Three short hairpin RNAs (shRNA1, shRNA2 and shRNA3) were designed using online software (http://rnaidesigner.thermofisher.com.rnaiexpress/) (Table 1) and were synthesized. They were connected to the pENTR/U6 plasmid and were named pENTR/U6-shRNA1, pENTR/U6-shRNA2 and pENTR/U6-shRNA3. The recombinant plasmid was constructed with psicheck II and the CDS region of SGK1 (psicheck II-SGK1). Cell transfection was performed using Lipofectamine 3000 (Thermo, Waltham, MA, USA) according to the manufacturer’s instructions. pENTR/U6-shRNA or pENTR/U6-NC were dissolved using Opti-MEM (Sigma, St. Louis, Missouri, USA) and incubated with psicheckII-SGK1 to form a DNA-liposome mixture. Subsequently, the DNA-liposome mixture was added to the culture medium and was incubated 48 hrs at 37°C and 5% CO2. The luciferase activity in 24 wells containing HEK293T cells were measured.
Table 1 short hairpin RNA sequences information of SGK1
Names of sequence
ShRNA1-F 5'-GATCC GCCAATAACTCCTATGCATGCTCAAGAGGCATGCATAGGAGTTATTGGC TTTTTT C -3'
ShRNA1-R 5'-TCGAG AAAAAA GCCAATAACTCCTATGCATGCCTCTTGAGCATGCATAGGAGTTATTGGC G -3'
ShRNA2-F 5'-GATCC GGAATGTTCTCCTGAAGAACGTCAAGAGCGTTCTTCAGGAGAACATTCC TTTTTT C -3'
ShRNA2-R 5'-TCGAG AAAAAA GGAATGTTCTCCTGAAGAACGCTCTTGACGTTCTTCAGGAGAACATTCC G -3'
ShRNA3-F 5'-GATCC GCCGAAACACAGCTGAGATGTTCAAGAGACATCTCAGCTGTGTTTCGGC TTTTTT C -3'
ShRNA3-R 5'-TCGAG AAAAAA GCCGAAACACAGCTGAGATGTCTCTTGAACATCTCAGCTGTGTTTCGGC G -3'
Note: GATCC and TCGAG were the sequence of endonuclease BamHl and Xhol, italic sequence indicated loop sequence.
Short hairpin RNA lentivirus packaging
The selected shRNA1 for single target lentivirus packaging and the negative control were named sh-SGK1 and sh-NC, respectively. Recombinant vector c-shRNA1 and helper vectors psPAX2 and pMD2G were co-transfected with Lipofectamine 3000 into HEK293T cells according to the manufacturer’s instructions. Fluorescence was detected with an inverted microscope after 24 h. The supernatant were collected at 48 h and 72 h to concentrate and purify the virus.
Preadipocytes were seeded onto 6-well plates overnight to reach 70% confluence. The OE-SGK1 and sh-SGK1 virus solutions were added to adipocyte culture plates (n = 3) with an optimal MOI (multiplicity of infection) value determined in a preliminary experiment, using OE-NC and sh-NC, respectively, as controls. After 48 h incubation at 37°C and 5% CO2, fluorescence was detected with an inverted microscope after 24 h and the medium was replaced by high glucose medium containing 10% FBS.
Total RNA extraction and RT-qPCR
Total RNA was extracted from adipocytes or tissues using Trizol reagent. cDNA was synthesized by using a PrimescriptTM RT reagent kit (TaKaRa) and SYBR premix Ex Taq II kit (TaKaRa) was used to perform the RT-qPCR reaction on a Bio-Red CFX 96 Touch instrument (Bio-Rad, Hercules, CA, USA). The 2−ΔΔCt method was used to analyze the data. Primers of adipogenic and cell cycle genes were designed by Primer Premier 5 according to the primer designing criteria (Supplementary Table 1). Relative expression levels were normalized with the internal control GAPDH .
Total protein extraction and immunoblotting
Total protein was extracted using whole cell Lysis Assay (KeyGEN BioTECH, Nanjing, China) and quantified using a BCA protein assay kit (KeyGEN BioTECH, Nanjing, China). Equal amounts of protein (10 µg per lane) were run on 5% and 12% or 8% SDS-PAGE and were transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore Corporation, Bedford, MA, USA). Membranes were blocked with 3% bovine serum albumin (Sigma, WXBD4881V) for 2 h at room temperature and incubated with the primary antibody overnight at 4°C. Primary antibodies were: anti-CDK2 (abbexa 1:500, cat. no. abx009457), anti-PPARγ (abbexa, 1:500, cat. no. abx104516), anti-C/EBPα (abbexa, 1:500, cat. no. abx009679), anti-Cyclin D2 (Abmart, 1:500, cat. no. TA5410), anti-FOXO1 (Abmart, WB: 1:500, cat. no. PA1431; pS256: 1:500, cat. no. PA5293), anti-FOXO3 (Abmart, WB: 1:500, cat. no. PA5311; pS322+S325/pS318+S321: 1:500, cat. no. PA5855) and anti-GAPDH (ZSGB-BIO, 1:500, cat. no. ZB-2301). Then membranes were incubated with anti-rabbit IgG (ZSGB-BIO, 1:5000, ZB-2301) secondary antibodies for 2 h at room temperature. Finally, blots were visualized by ECL reagent and captured using the Tanon-5200 imaging system (Shanghai, China).
Adipocytes infected by OE-SGK1 or OE-NC and induced differentiation for 6 days. Subsequently, cells were used to perform deep sequencing (n = 4) using Illumina xten completed by BioMarker Co (Qingdao, China).
RNA-seq data analysis
RNA-seq data was analyzed in R (R x64 4.1.2) using the R package (such as pheatmap and GOplot) and visual analysis was performed in Cytoscape (cytoscape_3.9.0).
For each group at least three independent experiments were performed and data was expressed as mean ± standard deviation (SD). GraphPad Prism v8.0.2 (GraphPad Software, Inc., La Jolla, CA, USA) was used to analyze the experimental data. Comparisons among multiple groups were performed with two-way analysis of variance (ANOVA). P<0.05 was considered to indicate a statistically significant difference, and P<0.01 was considered to indicate that the difference was extremely significant.