Nuclease free water

RNA extraction, reverse transcription, and qPCR were performed according to the procedures described by Sun et al. [74 (link)]. Total RNA was extracted from the skin samples using the RNAiso Plus kit (Takara, Dalian, China), according to the manufacturer’s instructions, and treated with DNAse I (D7073, Beyotime, Shanghai, China). RNA quality and quantity were determined using 1% agarose gel electrophoresis and Nanodrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA), respectively. RNA was reverse-transcribed to generate cDNA using a PrimeScript™ RT Reagent Kit (Takara, Dalian, China). qPCR was performed on a Real-Time System (QuanStudio 5, Life Technologies, Carlsbad, CA, USA) using a reaction mixture containing 1 μL of cDNA, 0.2 μL of ROX, 5 μL of SYBR Premix Ex Taq, 0.4 μL of primers, and 3.4 μL of nuclease-free water (Vazyme, Nanjing, China). The relative gene expression was normalized to that of β-actin (internal control) and calculated using the 2^−ΔΔCT method, according to Livak and Schmittgen [75 (link)]. The specific primers used for qPCR are listed in Supplementary Table S4.

Muscle RNA extraction, reverse transcription, and qPCR were performed following the procedures established for our laboratory [35 (link)]. RNA was extracted using RNAiso Plus (Takara Bio, Kusatsu, Japan), treated with DNase I (Beyotime), and analyzed for purity and quantity via agarose gel electrophoresis and spectrophotometry (A_260/280 ratio), respectively. Reverse transcription was performed using the PrimeScript™ RT Reagent Kit (Takara Bio). The reaction system for qPCR contained 1 µL complementary DNA, 0.2 µL ROX, 5 µL SYBR Premix Ex Taq, 0.4 µL primers, and 3.4 µL nuclease-free water (Vazyme, Nanjing, China). The reaction was run in a QuanStudio 5Real-Time System (Life Technologies, Carlsbad, CA, USA) according to manufacturer protocol. The oligonucleotide primers used are presented in Table S4. The 2^−ΔΔCT method was used to calculate the changes in target gene expression relative to β-actin levels.

Skim milk powder (lactose > 51.8%, protein > 35.8%) was provided by Bright Dairy Co., Ltd. (Shanghai, China). Chemicals, enzymes, and bile were purchased from Sigma Aldrich (Shanghai, China). Pepsin (Sigma P6887) and pancreatin (Sigma, P7545 8 USP) were of porcine origin, whereas bile (Sigma B8631) was of bovine origin. Lactobacillus helveticus CCFM1263 was isolated from naturally fermented dairy products in China and was deposited in the Culture Collection of Food Microorganisms (CCFM) of Jiangnan University (Wuxi, China). The AIN-93G rodent diet was purchased from Jiangsu Xietong Biology Co., Ltd. (Nanjing, Jiangsu, China). Nuclease-free water, FastPure cell/tissue total RNA isolation kit V2, HiScript III RT SuperMix for qPCR (+gDNA wiper), and ChamQ Universal SYBR qPCR Master Mix were purchased from Vazyme Biotech Co., Ltd. (Nanjing, Jiangsu, China). The primers for TRPV5, TRPV6, PepT1, Calbindin-D9k, Na⁺/Ca²⁺ exchange mechanism (NCX), PMCA1b and β-actin were compounded by the Genewiz Biotechnology Co., Ltd. (Suzhou, Jiangsu, China).