Biorupter pico

Cells for protein analysis were lysed with Pierce^® IP Lysis Buffer (Thermo Scientific). Western blots were performed using 25–50 μg of protein and primary antibodies against p53 (DO‐1; Santa Cruz Biotechnology) at 1:500 dilution and β‐actin (C4; Santa Cruz Biotechnology) at 1:3,000 dilution. HRP‐conjugated anti‐mouse secondary antibody (GE Healthcare) was used at 1:10,000 dilution. Immunoreactive proteins were visualized using SuperSignal^® West Pico Chemiluminescence Substrate (Thermo Scientific). MOLM13 cells were lysed with Biorupter Pico (Diagenode) using 30/30 s on/off for 10‐min program in Laemmli sample buffer (Bio‐Rad). Blots were probed with primary antibody against p53 (Sc‐126, DO‐1, Santa Cruz Biotechnology) at 1:1,000 dilution and β‐actin (Sc‐8432, C‐1; Santa Cruz Biotechnology) at 1:3,000 dilution.

For Hnf4g expression in WT and KO organoids: Organoid pellets were resuspended in 100 μl whole cell lysis buffer per well of a 6‐well plate (150 mM NaCl, 50 mM Tris pH 8, 10% glycerol, 1% NP‐40, 1 mM DTT, 1× CPI). Extracts were rotated for 1.5 h at 4°C followed by seven cycles of 30 s on and 30 s off sonication (Biorupter Pico, Diagenode). Afterward, the samples were spun at 16,200 × g for 5 min at room temperature and the supernatant was stored at −80°C. Samples were loaded on an 8% SDS–PAGE gel and afterward transferred to a nitrocellulose membrane. Membranes were incubated with primary antibody in 5% milk/TBST overnight at 4°C [anti‐beta actin 1:5,000 (Abcam, ab16039), and anti‐HNF4G 1:500 (Sigma, HPA005438)]. Afterward, membranes were incubated with secondary antibody [Polyclonal Swine Anti‐Rabbit Immunoglobulins/HRP 1:3,000 (Dako, P0399)] and imaged using chemiluminescent substrate (Thermo Fisher, 34580).
For mitochondrial ETC complexes: Organoids were washed once and Matrigel was mechanically removed in cold PBS. Total proteins were collected by direct lysis of organoids in Laemmli sample buffer. Proteins were run in SDS–PAGE and transferred to Polyscreen PVDF transfer membranes (PerkinElmer). Antibodies: Total OXPHOS Rodent WB Antibody Cocktail (ab110413, Abcam) and anti‐vinculin (V9131, Sigma).

T24 bladder cancer cells were cultured for 24 h in both 0.1% and 1% O₂. The protein–DNA interactions were cross-linked using ChIP cross-link gold (Diagenode, Denville, NJ, USA) and 1% formaldehyde before lysing the cells and shearing the chromatin into 200–300 bp fragments using a Biorupter Pico (Diagenode). Antibodies against HIF1α, HIF2α, and HIF1β, and Dynabeads Protein G were used for immunoprecipitation (Supplementary Table S1). The fragments were de-cross-linked and the DNA was eluted using the phenol–chloroform method. DNA with no immunoprecipitation was processed and sequenced in parallel as the input control. A qPCR was used to validate the ChIP experiment before the samples were sequenced and mapped by the CRUK Manchester Institute core facilities. Sequencing reads for all samples underwent quality control assessment and adapter removal with FASTQC [49 ] and Trim Galore [50 ] software, respectively. Trimmed fastq files were mapped against the hg19 reference assembly using bowtie2 with 1 allowed mismatch in seed alignment (-N set to 1). Resulting SAM files were converted into BAM format with samtools. Peaks were called with MACS2 software and subsequent annotation of identified peaks was performed with Homer (v4.10) where peak-to-gene annotations used the genes nearest to the transcriptional start site.

Cell lysates were sonicated using a Diagenode Biorupter Pico (Diagenode; Seraing, Belgium,). Lysates were subjected to 15 cycles of sonication (30 s on, 30 s off) at a temperature of 4 °C to produce chromatin fragments of 200–600 base pairs (bp). To determine the size of the chromatin fragments, decrosslinking solution (Table S2) was produced, and lysates were incubated at 65 °C for 1 h. Remaining chromatin sample volumes were then stored at −80 °C until required for immunoprecipitation. Chromatin fragments were then purified using the MinElute PCR Purification Kit (Qiagen; Manchester, UK) and following the protocol provided by the manufacturer. Chromatin fragments were then eluted in a 10 µL elution buffer (Table S3), and the chromatin fragment size was determined using a 1.5% agarose gel.