Chamber slide

Cells were cultured in chamber slides (SPL Life Sciences), fixed with 4% PFA, permeabilized with 0.25% Triton X100, blocked in PBS containing 2% bovine serum albumin (Bio-Rad) and applied with primary antibodies to TrkA (LSBio), Lamp1 (GeneTex). Alexa Fluor antibodies (Life Technologies) were used as secondary antibodies. Isotype control antibodies were used as controls. Cell nuclei were visualized with DAPI (Santa Cruz Biotechnology). Images were captured using a Nexcope NE950 fluorescent microscope (Ningbo Yongxin Optics Co., United States).

Immunofluorescence was executed to analyze the expression level of HIF1A. H9c2 cardiac cells cultured in chamber slides (SPL Life Sciences, Korea) were challenged with Dox for 24 h. They were then fixed in 4% paraformaldehyde for 1 h, permeabilized with 0.1% Triton X-100 for 5 min followed by blocking for 1 h to avoid nonspecific binding. Next, H9c2 cells were incubated with the HIF1α primary antibody for 1 h and Alexa Fluor 488 Red anti-rabbit IgG secondary antibody for 1 h. Lastly, cells were washed, and stained with DAPI, and then imaged to analyze the expression of HIF1A.

Eight-week-old mice were injected with saline or 4 mg/kg BLM, intratracheally. Mice were sacrificed 7 days after BLM injection, and saline perfused lungs were treated using a mouse lung dissociation kit (Miltenyi Biotec, Auburn, CA, USA), in the gentleMACS^TM Dissociator (Miltenyi Biotec). Red blood cells (RBCs) were lysed with RBC lysis solution (Miltenyi Biotec), and isolated cells from the mouse lung were double stained with anti-CCSP/anti-EpCAM for club cells or anti-CD74/anti-EpCAM for AT2 cells. Isotype control antibodies (IgG2a κ-FITC and IgG2a κ-APC; eBioscience, San Diego, CA) were used to establish gating parameters for positively stained cells. Double-positive populations were sorted using a BD FACSAria^TM III cell sorter (BD Biosciences, San Jose, CA, USA). Sorted double-positive cells were subjected to an RNeasy Micro Kit (Qiagen, Germany) to obtain mRNA for qRT-PCR assays or plated on chamber slides (SPL Life Sciences, Korea) for in situ proximity ligation assay (PLA) analysis.

In order to confirm the cell death, the cells morphology changes were analyzed by the Acridine Orange/Ethidium Bromide double staining method. A number of 10⁴ SW480 cells were seeded into each well of chamber slide (30118, SPL, Korea) and incubated for 24 h in a humidified, 5% CO₂ at 37 °C. The cells were treated with various concentration of metabolites, stained with 10 μl Acridine Orange 50 mg ml^-1 (A6014, Sigma, Germany) and Ethidium Bromide 50 mg ml^-1 (E7637, Sigma, Germany) for 5 min and were washed with PBS. Ultimately, the cell death was characterized under a fluorescence microscope with 400× magnification.