*2.4. Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR)*

Tissues (around 100 mg) were homogenized in liquid nitrogen with a glass homogenizer. Total RNA was extracted using QIAzol® Lysis Reagent (QIAGEN Science, Germantown, MD, USA) according to the manufacturer's instructions. RNA was converted into cDNA using the RevertAid™ first-strand cDNA synthesis kit (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. Next, we conducted a quantitative polymerase chain reaction using an ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA). The reaction solution (20 μL) contained 10 μL of SYBR Green master mix (New England Biolabs, Ipswich, MA, USA), 4 μL of cDNA, and 6 μL of primer set (200 nmol/L). The PCR reactions were conducted as follows: 2 min at 50 ◦C, 10 min at 95 ◦C, and 40 cycles at 95 ◦C for 15 s, followed by 1 min at 60 ◦C. The relative expression levels were determined as the Δcycle threshold (ΔCt) [19]. All primer sets used in the present study are shown in Supplemental Table S1.
