Trnzol

RNA was extracted from cultured cells with TRNzol (DP424, TIANGEN Biotech, Beijing, China) following the manufacturer's protocol. After the concentration of RNA was determined, cDNA was synthesized from 1 µg of RNA per sample using the FastKing gDNA Dispelling RT SuperMix (KR118, TIANGEN Biotech). Amplification was performed using Talent qPCR PreMix containing SYBR Green (FP209, TIANGEN Biotech). PCR was performed using the following conditions: an initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 60 s. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was used as the internal reference gene for normalization. Relative expression was calculated using the 2^{−𝛥𝛥CT} method [26 (link)]. The primer sequences are shown in Supplementary Table S1.

The sh-IRP1 and sc cells plated in 15 cm dishes were grown to 80% confluency, then the cells were washed twice with ice-cold PBS and lysed with 1 ml polysome lysis buffer (20 mM Tris-HCl, pH 7.4, 10 mM NaCl, 10 mM MgCl₂, 1% Triton X-100, 2% Tween 20, and 1% sodium deoxycholate) for 10 min on ice. After centrifugation at 20,000 g for 15 min at 4 °C, the supernatant was collected and loaded onto 10%–50% (wt/vol) sucrose density gradients (buffered in 20 mM Tris-HCl, pH 7.4, 10 mM NaCl, 10 mM MgCl₂) and centrifuged at 36,000 rpm (SW 40 Ti rotor, Beckman Coulter) for 3 h at 4 °C. Gradients were fractionated and the optical density at 260 nm was continuously recorded using a fractionator. RNA from each fraction and input was extracted by using TRNzol (TianGen, DP405) according to the manufacturer's instructions, and the corresponding cDNA was synthesized by using a cDNA synthesis kit (Takara, 6130). Semi-quantitative PCR reactions were carried out on a PCR machine (Bio-Rad).

After the indicated treatments or transfections, 6.5 μg/ml actinomycin D was added into the cell medium for the indicated time. The total RNA was extracted by using TRNzol (TianGen, DP405), and cDNA was synthesized by using a cDNA synthesis kit (Takara, 6130). Quantitative RT-PCR was performed on diluted cDNA with SYBR green Master Mix (NEWBIO INDUSTRY, 308102) on a real-time PCR machine (Bio-Rad). The primers for Bcl-xL and reference Actin used in this study are listed in Supplementary Table 2.

TRNzol (#DP424) was acquired from Tiangen Biotech Co., Ltd. (Beijing, China); PrimeScript RT reagent Kit with gDNA Eraser (#RR047B), SYBR Premix Ex Taq II (Tli RNaseH Plus), and ROX plus (#RR82LR) were acquired from Takara Bio (Tokyo, Japan); an methyl thiazolyl tetrazolium (MTT) assay kit, bicinchoninic acid (BCA) protein quantification kit, and a NanoDrop 2000 spectrophotometer were acquired from Thermo Fisher Scientific (Waltham, MA, USA); a RIPA Total Protein Extraction Kit was acquired from Sigma-Aldrich (St. Louis, MI, USA); a mitochondria extract kit, enzyme-linked immunosorbent assay (ELISA) kits, and mitochondrial membrane potential JC-1 kit were acquired from Solarbio Science & Technology (Beijing, China); an adenosine triphosphate (ATP) kit, superoxide dismutase (SOD) kit, and methane dicarboxylic aldehyde (MDA) kit were acquired from Nanjing Jiancheng Bioengineering Institute (Nanjing, China); a FACSCalibur II Flow Cytometer was acquired from BD Biosciences (Franklin Lakes, NJ, USA); an XDS-2B inverted fluorescence microscope was acquired from Chongqing Optical & Electrical Instrument Co., Ltd. (Chongqing, China); and an ABI 7500 real-time PCR instrument was acquired from Applied Biosystems (Thermo Fisher Scientific); the autophagy inhibitor 3-methyladenine (3MA) was purchased from Selleck Chemicals (Houston, TX, USA).