Stepone real time pcr

Cells treated with or without KDM4D inhibitor were prepared for RNA extraction. Briefly, RNA was extracted by TRIpure total RNA extraction reagent (ELK Biotechnology, Wuhan, China). EntiLink™ 1st Strand cDNA Synthesis Kit (ELK Biotechnology, Wuhan, China) was used to synthesize cDNA according to the manufacturer’s instructions. Quantitative real-time PCR was performed using the StepOne™ Real-Time PCR (Life technologies, Wuhan, China) with specific primers (sequences were shown in Supplementary Table 1). The relative expression of RNAs was calculated using 2 − ΔΔCt method.

Immunoblotting analysis and qPCR were performed according to previous articles (29 (link)). In brief, Protein-extracts of snap-frozen eWAT and whole-cell lysates of SVF were prepared using standard procedures. Protein concentrations in the supernatants were measured using Bicinchoninic acid (BCA) assay (ASPEN, USA). Proteins were separated on SDS-polyacrylamide gels and transferred to PVDF membranes. After blocking with QuickBlock™ Western (P0252, Beyotime Biotechnology, China), the membranes were incubated with the primary antibodies overnight at 4°C, washed in PBST three times, and incubated with a secondary goat anti-rabbit polyclonal antibody (SA00001-2, Proteintech Group) at room temperature for 1h. Finally, the signals were tested by WesternBright™ Sirius ECL kit (K-12043-D20, Advansta, USA). Protein expression levels were normalized to β-actin.
Total mRNA was extracted from eWAT or SVF cells with GeneJET RNA Purification Kit (K0731, Thermo Fisher Scientific, USA). Reverse transcribed into cDNA using RevertAid First strand cDNA Synthesis kit (K1622, Thermo Fisher Scientific, USA). The StepOne Real-Time PCR (Life tech, Alameda, CA) was used for real-time qPCR analysis. The primers used are described in Table S1. β-actin was used as an internal control. The relative expression quantity 2^-ΔΔCt value was calculated to compare the differences among groups.

TRIzol (ELK Biotechnology, Wuhan, China) were used to extract total RNA from cells. Later on, complimentary DNA (cDNA) was synthesized from these extracted RNA using EntiLink™ 1st Strand cDNA Synthesis Kit (ELK Biotechnology). RT-qPCR was performed using EnTurbo™ SYBR Green PCR SuperMix Kit (ELK Biotechnology) and run on the StepOne™ Real-Time PCR (Life Technologies, Carlsbad, CA, USA).

For RNA extraction, muscle tissue was homogenized using a PRO200 homogenizer (PRO Scientific Inc, Oxford, USA). Regarding blood samples, the Blood RNA kit (PreAnalytiX GmbH, 8634 Hombrechtikon, Switzerland) was used starting from frozen Pax tubes. From here, the same protocol was used for samples of animals and patients.
RNA was extracted with TRIzol reagent (Invitrogen, Prat de Llobregat, Spain) and treated with Turbo DNA-free kit (Ambion, Madrid, Spain) to eliminate genomic DNA. Once purified, RNA was retrotranscripted using the Superscript First Strand kit (Invitrogen, Prat de Llobregat, Spain). Quantitative real-time PCR (qRT-PCR) was performed from 1:10 diluted cDNA in triplicates using StepOne Real-Time PCR (Life Technologies; Waltham, MA, USA). The TaqMan probes (Applied Biosystems, Madrid, Spain) used in this study are indicated in Table 2.
Gapdh and β-actin were used as housekeeping genes for normalization of animal data, and GAPDH, HPRT1 and TBP were used to normalize patient data. The relative gene expression was determined using the 2-ΔΔCT method [60 (link)].

Caki-1 cells were harvested, and total RNA was extracted. Then, cDNA was synthesized by EntiLink™ 1st Strand cDNA Synthesis Kit (ELK Biotechnology, Wuhan, China) according to the manufacturer's instruction. Quantitative real-time PCR was performed using the StepOne™ Real-Time PCR (Life technologies, Wuhan, China) with the following primer sequences: JMJD4 sense primer, 5′-AGATGGTGTTTGTGCCCAGT-3′; JMJD4 antisense primer, 5′-CATGTTGGCCAGGTTGAAGC-3′; β-actin sense primer, 5′-CATCATCCCTGCCTCTACTGG-3′; and β-actin antisense primer, 5′-GTGGGTGTCGCTGTTGAAGTC-3′.