Two photon microscope

IF images were acquired using a Leica SPE confocal microscope, a Leica SP8 confocal microscope or an in house-built Olympus two-photon microscope (Multiphoton Microcopy Core, Massachusetts General Hospital), depending on the experiment. IHC and ISH images were acquired with a Olympus DP72 microscope. Metabolites in the colorectal adenoma were imaged using a timsTOF fleX mass spectrometer (Bruker Daltonics, Billerica, MA). Graphpad Prism 6 and Microsoft Excel v16.15 were used for statistical analyses throughout this study. ImageJ 1.51 u was used to process IF images. STAR was used to map RNA-sequencing data. HTseq was used to assign reads to Gencode vM9. Pathway enrichment analysis was carried out using GSEA as detailed in the experimental procedures. MSI data were visualized using SCiLS Lab software (version 2021c premium, Bruker Daltonics, Billerica, MA). Flow cytometry data was analyzed by FlowJo vX.0.7.

Organoids were collected with cold PBS and transferred to a 15-ml tube. They were spun down at 300 g for 3’ and fixed with 4% PFA for 1 h at room temperature. After washing twice with PBS, organoids were permeabilized with PBS-1% Triton-X-100 for 20’ at 4 °C and incubated in blocking solution (PBS + 1% BSA + 3% goat serum + 0.2% Triton-X-100) for 1 h at 4 °C. Organoids were then incubated with primary antibodies in working solution (PBS + 0.1% BSA + 0.3% goat serum + 0.2% Triton-X-100) over night at 4 °C. The primary antibodies were removed, and the samples were washed three times for 5 min each with PBS before incubating them with secondary antibodies (1:500) in working solution for 1 h at room temperature. Organoids were stained with DAPI, resuspended with mounting medium and mounted on slides. Images were acquired using a Leica SPE confocal microscope, a Leica SP8 confocal microscope or an in house-built Olympus two-photon microscope (Multiphoton Microcopy Core, Massachusetts General Hospital), depending on the experiment. For live imaging of Pdk1-mCherry, organoids were plated in Matrigel drops in 6-cm plates and imaged by two-photon microscopy using a water immersion 25× objective. To measure ROS levels in organoids, we incubated them with CellRox Green (Life Technologies, C10444) for 1 h, fixed them with 2% PFA and imaged them using a Leica SPE confocal microscope.

BALB/c nu/nu mice (5 weeks old) were anaesthetized by an intraperitoneal injection of sodium pentobarbital (50 mg/kg). Two symmetrical 5‐mm‐diameter full‐thickness skin wounds were created on the back with a skin biopsy punch as our previously study described.15, 17, 18 We mixed 10⁶ epidermal cells and 2 × 10⁶ dermal cells (per wound) derived from neonatal K14‐H2B‐GFP mice in 20% PSS or equal volume of saline and encapsulated them in 10 μL Matrigel (BD, USA). After incubation for 30 minutes at 37°C, the cells‐Matrigel was implanted into an excisional wound. The wound was then covered with Tegaderm (3M, USA) transparent dressing, which was further covered with self‐adhering elastic bandage. After 3 weeks, the grafts were examined under dissecting microscope (Leica) and two‐photon microscope (Olympus) to logical analysis.