Trizol reagent

Total RNA was extracted from kidney tissues (n = 6) using a Trizol reagent according to the manufacturer’s instructions (CW0580, CoWin Biotech Co., Inc., Beijing, China). Then, 1000 ng of total RNA was reverse-transcribed in a final 20 μL with HiScript Ⅱ First-Strand cDNA Synthesis Kit (R212-01, Vazyme Biotech Co., Ltd., Nanjing, China). The reverse transcription reaction was processed at 50 °C for 15 min and 85 °C for 2 min. qRT-PCR amplification was performed using AceQ qPCR SYBR Green Master Mix (Q111-02, Vazyme Biotech Co., Ltd., Nanjing, China) on StepOne Real-time PCR systems (ABI, Boston, MA, USA) in triplicate, and non-template controls were run under the same conditions for each assay. The PCR reaction was carried out with an initial denaturation step of 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. Relative mRNA expression levels were calculated using the 2^—ΔΔCt method and normalized to Gapdh expression levels. The primer sequences used are shown in Table 1.

Total RNA of each sample was extracted using the Trizol Reagent (Cowin Biosciences, Beijing, China) according to the manufacturer’s protocol from three biological replicates of each treatment (WT and Transgenic) resulting in 6 samples. The total RNA concentration of each sample was estimated using a Agilent 2100 Bioanalyzer (Agilent Technologies Inc.). The integrity of the RNA samples was assessed with 1.2% agarose gel electrophoresis. The six samples were sequenced using BGISEQ-500 Sequencing performed by Beijing Genomics Institute (BGI)-ShenZhen, China according to the manufacturer’s instructions. Detailed BGISEQ-500 sequencing experimental process and transcriptome data analysis method refer to Additional file 6.

UA232 was synthesized in our laboratory with a purity of > 98%. UA, 3-methyladenine (3-MA), and CA-074 methyl ester (CA-074-me) were purchased from MedChemExpress (Monmouth Junction, NJ, USA), and 4-phenylbutyric acid (4-PBA) was purchased from Energy Chemical (Shanghai, China). Crystal violet, 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), 4% paraformaldehyde fix solution, Lyso-Tracker Red dye, and Cell Counting Kit-8 (CCK-8) were purchased from Beyotime Biotechnology (Shanghai, China). The cell cycle detection kit and Annexin V-FITC/PI kit were purchased from KeyGen Biotech (Nanjing, China). Primary antibodies against cyclin D1, CDK4, Bax, Bcl-2, caspase-8, caspase-3, PARP1, CHOP, GRP78, ATF4, LC3, P62, and LAMP2 were purchased from Proteintech Group (Rosemont, IL, USA). Antibodies against PERK, p-PERK, eIF2a, and p-eIF2a were purchased from Bioss (Beijing, China). Antibodies against cathepsin B were purchased from Abcam (Cambridge, MA, USA). β-actin, horseradish peroxidase conjugated secondary antibodies (goat anti-mouse or goat anti-rabbit), and fluorescently labeled secondary antibodies (IgG Alexa Fluor® 488 or 594 goat-anti-rabbit) were obtained from ZSGB BIO (Beijing, China). Trizol reagent, cDNA synthesis kit, and UltraSYBR mixture were purchased from CoWin Biotech (Beijing, China).

Total RNA was extracted using TRIzol reagent (CoWin Biosciences, Beijng, China) and transcribed into cDNA with a Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, USA), according to the manufactures’ instructions. Then, real-time quantitative PCR (RT-qPCR) was performed using SYBR Green Master Mix (CoWin Biosciences, Beijng, China). The reaction conditions were: pre-denaturation at 95 °C for 10 min, then 40 cycles of denaturation at 95 °C for 15 s and annealing at 60 °C for 30 s. The level of GAPDH cDNA (F: 5′-CCTCGTCCCGTAGACAAAATG-3′, R: 5′-TGAGGTCAATGAAGGGGTCGT-3′) was used as internal reference to quantify the level of SIRT-1 (F: 5′-TTCAGAACCACCAAAGCGGA-3′, R: 5′-TCCCACAGGAGACAGAAACCC-3′) and PGC-1ɑ (F: 5′-CGAGAAGCGGGAGTCTGAAAG-3′, R: 5′-GAGCAGCGAAAGCGTCACA-3′) cDNA. The relative mRNA expression of SIRT-1 and PGC-1ɑ was calculated using the 2^-ΔΔCt formula. All results were normalized to the GAPDH expression level.

Total RNA was extracted using TRIzol reagent (CW0580S, CoWin Biosciences, China) according to the manufacturer’s instructions. The HiScript II Q RT SuperMix for qPCR (R223-01, Vazyme, China), miRNA cDNA synthesis kit (CW2141S, Cowin Biosciences, China), and miRNA qPCR assay kit (CW2142S, Cowin Biosciences, China) were used for qPCR. The primer sequences and their targets are detailed in Table 1. The 2^−ΔΔCT method was used to calculate the relative expression levels of genes.

Total RNA was extracted from the liver samples with TRIzol reagent (CoWin Biotech Co., Ltd., Taizhou, China). Nano drop, Qubit 2.0, and Agilent 2100 were used to analyze the purity, concentration, and integrity of the RNA samples, respectively. Single-strand cDNA was synthesized using the HiFiScript cDNA Synthesis Kit (CoWin Biotech Co., Ltd., Taizhou, China) following the instructions. The LightCycler^® 480 II RT-qPCR instrument (Roche, Switzerland) was used to detect gene transcripts. Primer 6.0 software was used to design primers (Table 1). A quantity of 20 µL of reaction system included 10 µL of SYBR Premix Ex Taq (2×), 2 µL of each primer, 2 µL of cDNA, and 6 µL of ddH₂O. Cycling was performed at 95 °C for 5 min, followed by 40 cycles at 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 30 s. The 2^−ΔΔCt method was used to analyze the data. Three replicates of each group were performed.