Mito stress test assay

Oxygen consumption and extracellular acidification rates were measured using an XFe96 Extracellular Flux Analyzer (Seahorse Bioscience) using the manufacturer’s established protocols. In brief, cells were plated overnight in Seahorse 96-well plates at 1 × 10⁴ per well in 80 μL of complete media. Cells were washed three times in non-buffered DMEM containing 25 mM glucose and 2 mM glutamine and incubated in this medium in a CO₂-free incubator at 37 °C for 2 h to allow for temperature and pH equilibration before loading into the XFe96 apparatus. In addition to basal measurements, the Mito Stress Test assay (Seahorse Bioscience, 103015-100) was performed according to manufacturer’s instructions. XFe assays consisted of sequential mix (3 min), pause (3 min), and measurement (5 min) cycles, allowing for determination of OCR/ECAR every 10 min. At the end of the assay media was removed from the plate which was then frozen at −80 °C for 24 h. CyQuant dye (Invitrogen, C7026) was then used according to the manufacturer’s instructions to generate DNA content values for data normalization. Data were analyzed using Wave software (Seahorse Bioscience).

Mitochondrial respiration oxygen consumption rates (OCR), which allow conclusions about different parameters of mitochondrial respiration, were recorded by using an extracellular flow analyser Agilent Seahorse XF24 (Seahorse Bioscience, North Billerica, MA, USA) [81 (link)] and the Mito Stress Test Assay (Seahorse Bioscience, North Billerica, MA, USA), which was performed according to the protocol described by Butler et al. [82 (link)]. Additionally, using the same instrument together with the company’s Extracellular Acidification Rate (ECAR) Assay, we studied the extracellular acidification rates of the respective cell cultures as indicators of energy-producing pathways of glycolysis [83 (link)]. The analyses of the OCR and ECAR of the untreated and light-exposed tumour cell cultures used were carried out under identical conditions, as previously described by us [35 (link)].

Antibodies against AnxA6, as well as secondary anti-mouse, anti-goat, and anti-rabbit horseradish peroxidase-conjugated antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The antibodies against COXIV, Cytc. C, Pyruvate dehydrogenase (PDH), and Superoxide dismustase (SOD1) were purchased from Cell Signaling Technology (Beverly, MA, USA). Oil Red-O, Rotenone, 2-Deoxy-D-Glucose, Etomoxir, and antibody against β-actin (ACTB) were purchased from Sigma Aldrich (St. Louis, MO, USA). A set of EGFR-TKIs including lapatinib-ditosylate, erlotinib, gefitinib, and canertinib were purchased from BioVision (Milpitas, CA, USA). The Mito Stress Test Assay, Palmitate FAO Assay, and the culture media were purchased from Agilent Technologies (Santa Clara, CA, USA). Except where otherwise indicated, all the other reagents were purchased from Sigma Aldrich and/or Cell Signaling Technology.