Cultivation and treatment of cells

Non-tumorigenic mammary cell line (MCF-10A) and breast cancer cell lines (MDA-MB-231, SUM190PT) were all supplied by Otwo Biotech (Shenzhen, China). MDA-MB-231 cells were incubated in Leibovitz's L-15 medium (Life Technologies, Karlsruhe, Germany), while other cells were all grown in Roswell Park Memorial Institute (RPMI)-1640 medium (Trace Biosciences, Melbourne, Vic, Australia). In addition, breast cancer MCF-7 cells that were purchased from Typical Culture Preservation Commission Cell Bank, Chinese Academy of Sciences (Shanghai, China) were incubated in minimum essential medium (MEM; Life Technologies, Karlsruhe, Germany). All cells were cultivated in corresponding mediums containing 10% fetal bovine serum (FBS; Trace Biosciences, Melbourne, Vic, Australia) at 37 ℃ with 5% CO2.

MCF-7 cells were treated by autophagy inhibitors 3-methyladenine (3-MA; 2.5 mM; Selleck, USA) for 2 h (Cheng et al. 2019) or chloroquine (CQ; 20 μM; Selleck, USA) for 2 h (Shi et al. 2015; Tang et al. 2021) or ASK1 inhibitor (GS-4997; 1 µmol/L; Selleck, USA) for 1 h (Han et al. 2019). Then, small interfering RNA (siRNAs) targeting PDK4 (si-PDK4#1/2) and the scrambled control siRNA (si-NC) that were constructed by Hippo Biotechnology (Huzhou, China) were transfected into cells using XfectTM RNA transfection reagent (Takara, Dalian, China).

Immunofluorescence (IF) staining

After the immobilization by 4% paraformaldehyde for 30 min and the permeation with 0.5% Triton X-100 for 10 min, MCF-7 cells were blocked with PBS containing 1% BSA. Subsequently, cells were incubated with LC3 antibody (cat. no. #14,600-1-AP; 1/250; Proteintech), LC3B antibody (cat. no. #AF5402; 1/100; Affinity Biosciences), p62 antibody (cat. no. #AF5384; 1/100; Affinity Biosciences) overnight at 4 °C. On the next day, the cells were incubated with secondary antibody conjugated with Alexa Fluor 488 (cat. no. ab150077; 1/200; Abcam) for 1 h at room temperature. The nuclei were stained by 1 mg/ml DAPI. The intensity was recorded under a fluorescence microscope (Leica, Wetzlar, Germany).

Estimation of total iron level

The total iron content in the cell supernatants was detected using Iron Assay Kit (cat. no. ab83366; Abcam) according to the manufacturer’s instructions. The absorbance of samples was detected at 593 nm under a microplate reader (SLT Lab Instruments GmbH, Salzburg, Austria).

Measurement of oxidative stress levels

MCF-7 cells were incubated with DCFH-DA probe (10 μmol/l; Elabscience, Shanghai, China) at 37˚C for 30 min in the dark according to the manufacturer’s instructions. Following the wash with PBS, the intensity was detected under a fluorescence microplate reader (BMG Labtech, Offenburg, Germany) with the excitation as 500 nm and the emission as 525 nm.

After the centrifugation at 2000 rpm/min, the activities of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in MCF-7 cells were detected using MDA assay kits (cat. no. J20465; GILED, Wuhan, China), 4-HNE assay kits (cat. no. J21715; GILED, Wuhan, China), SOD assay kits (cat. no. J21118; GILED, Wuhan, China) and GSH-Px assay kits (cat. no. J20841; GILED, Wuhan, China) according to the manufacturer’s instructions. Under a microplate reader, the absorbance was detected at 450 nm.

C11 BODIPY 581/591 assay

MCF-7 cells were incubated with 5 μmol/L C11-BODIPY581/591 probe (Amgicam, Wuhan, China) at 37 ℃ in the dark for 1 h according to the manufacturer’s instructions. Under a fluorescence microscope, the intensity was recorded following PBS washing.

Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was prepared from cells using Trizol reagent (Ambion, Austin, TX) according to the manufacturer’s instructions, and then reverse-transcripted into cDNA through ReverTra Ace qPCR RT Kit (TOYOBO Life Science, Shanghai, China). PCR reaction was implemented using THUNDERBIRD® SYBR® qPCR Mix (TOYOBO Life Science, Shanghai, China). PDK4 expression was detected using 2−ΔΔCq approach with GAPDH as a normalizer.

Western blot

The total proteins were extracted using RIPA buffer (Applygen, Beijing, China) and the protein concentration was detected using BCA method (Applygen, Beijing, China). Following the separation with gel electrophoresis, the proteins were transferred to the PVDF membranes. The membranes were blocked by 5% BSA and then cultivated with primary antibodies targeting PDK4 (CAT. NO. #DF7169; 1/1000), light chain 3B (LC3B; CAT. NO. #AF4650; 1/1000), p62 (CAT. NO. #AF5384; 1/1000), autophagy related 5 (ATG5; CAT. NO. #DF6010; 1/1000), autophagy related 7 (ATG7; CAT. NO. #DF6130; 1/1000), acyl-CoA synthetase long-chain family member 4 (ACSL4; CAT. NO. #DF12141; 1/1000), glutathione peroxidase 4 (GPX4; CAT. NO. #DF6701; 1/1000), ferritin heavy chain 1 (FTH1; CAT. NO. #DF6278; 1/1000), solute carrier family 7a member 11 (SLC7A11; CAT. NO. #DF12509; 1/1000), nuclear receptor coactivator 4 (NCOA4; CAT. NO. #DF4255; 1/1000), apoptosis signal-regulating kinase 1 (ASK1; CAT. NO. #AF6477; 1/1000), phosphorylated apoptosis signal-regulating kinase 1 (p-ASK1; CAT. NO. #AF3477; 1/1000), c-Jun N-terminal kinase (JNK; CAT. NO. #AF6318; 1/1000), phosphorylated c-Jun N-terminal kinase (p-JNK; CAT. NO. #AF3318; 1/1000) and β-actin (CAT. NO. #AF7018; 1/3000) from Affinity Biosciences, followed by the incubation with HRP-linked secondary antibody (CAT. NO. #S0001; 1/3000; Affinity Biosciences). By means of ECL Chemiluminescence solution (Applygen, Beijing, China), the binding signals were scanned.

Statistics

All data were analyzed using GraphPad Prism 8.01 software (GraphPad Software Inc., CA, USA) and then presented as mean ± standard deviation. Differences among multiple groups were compared using one-way ANOVA followed by Tukey’s post hoc test. P less than 0.05 indicated statistical significance.