Ti a1r

Images were acquired with a Nikon TI A1R inverted confocal microscope with a CFI60 Plan Apochromat Lambda 10x objective lens. Images were acquired with the following scan parameters: a “frame” scan mode of 1024 × 1024 pixels with a 16 bit depth and a grating of 3 rotations. Three-dimensional projections were obtained through Z stack images with 0.4700 μm between each image.

BT25 cells and COS-7 cells were plated on 8-well chamber slides with a glass bottom (Ibidi) and then were transfected with Lipofectamine 3000 (Invitrogen) with constructs expressing GFP-NIK, mCherry-Drp1 (mch-Drp1), and mito-BFP. After 18 h, time-lapse images were acquired every 5 s for 3 min on a Nikon TI A1R inverted confocal microscope with 60× Plan-Apochromat lenses and Stage-top incubator system. Random fields of mitochondria were imaged (n > 19 cells each) and analyzed using NIS-Elements imaging software (Nikon, Japan). The diffuse cytosolic images were threshold-adjusted by subtracting background signal in the cytosol. For analysis of COS-7 cells under metabolic switching conditions, cells were seeded at a density of 50,000 cells into a collagen-coated (50 μg/ml), 8-chamber slide (Ibidi) with 1% FBS. Twenty-four hours later, cells were transfected with GFP-NIK, mCherry-DRP1, Mito-BFP (40 ng each) using lipofectamine (Lipofectamine 3000, Invitrogen); 24–48 h after transfection, cell media was either switched to media containing 25 mM galactose (without glucose) or media containing 25 mM glucose. Cells were live imaged as described above. Correlation analysis was done on RBG images using NIS Elements software. Mander’s correlation coefficient of the pixel overlap was measured in individual frames from enhanced video.

BMEs were fixed in 4% paraformaldehyde for 40 minutes at room temperature and stained with Human Mesenchymal Stem Cell Kit's anti-CD19 and anti-CD44 component (Merck Millipore, Germany) according to the manufacturer's instructions. The experiments were performed in case of two patients' four-four BMEs after 5 days' incubation in FCS, HAS, or serum-free media. Samples were imaged in PBS buffer in glass bottom dishes using a Nikon Ti A1R confocal scanning microscope with 4x and 10x objective (Plan Fluor, NA = 0.3).