Journal: bioRxiv

Article Title: Matrix stiffening promotes perinuclear clustering of mitochondria

doi: 10.1101/2023.06.29.547150

Figure Lengend Snippet: a . FLIM images of MCF-7 cells cultured on soft (top panel) and stiff (bottom panel) ECM showing mitochondria stained with Mito Flipper-TR. White boxes represent ROIs drawn in the perinuclear (PN) and peripheral (PL) regions that were used to calculate fluorescence lifetimes. Magnified view of ROIs (scale bar: 2 μm) shows higher fluorescence lifetimes on soft ECM. Scale bar:10 μm. b . Scatter dot plot showing average fluorescence lifetimes calculated in PN and PL regions of cells cultured on soft and stiff ECM. n= 82 (soft PN), 68 (soft PL), 67 (stiff PN), 50 (stiff PL) ROIs from cells (83 cells on soft ECM, 76 cells on stiff ECM). c . Confocal images of MCF-7 cells transfected with mAppFLNA (in red) and cultured on soft ECM (top panel) and with dnFLNA (in red) and cultured on stiff ECM (bottom panel) showing mitochondria (in green) stained with Mito Flipper-TR. d . FLIM images of MCF-7 cells transfected with mAppFLNA and cultured on soft ECM (top panel); and with dnFLNA and cultured on stiff ECM (bottom panel) showing mitochondria stained with Mito Flipper-TR. White boxes represent ROIs drawn in the perinuclear (PN) and peripheral (PL) regions that were used to calculate fluorescence lifetimes. Magnified views of ROIs (scale bar: 5 μm) are shown for comparison. Scale bar:20 μm. e . Scatter dot plot showing average fluorescence lifetimes calculated in PN and PL regions of wild type (untransfected) and mAppFLNA transfected cells cultured on soft ECM (left graph); and wildtype (untransfected) and dnFLNA transfected cells cultured on stiff ECM (right graph). n=76 (wild type PN), 42 (wild type PL), 33 (mAppFLNA PN), 21 (mAppFLNA PL) ROIs from cells (42 wild type cells and 21 mAppFLNA transfected cells on 1.2 kPa); n=62 (wild type PN), 54 (wild type PL), 53 (dnFLNA PN), 32 (dnFLNA PL) ROIs from cells (47 wild type cells and 31 dnFLNA transfected cells on 90 kPa). Statistical significance was calculated using unpaired t-test with Welch’s correction (two-tailed) ( b , e ). Each data point represents the average lifetime in each ROI. Data are mean ± s.e.m ( b , e ) taken over two independent replicates. P-values are shown in the graphs. ROI: region of interest. Soft ECM: 1.2 kPa; Stiff ECM: 90 kPa

Article Snippet: For FLIM imaging with Mito Flipper-TR, cells were incubated with 1 μM of the Mito Flipper-TR (Spirochrome) for 15 min. Media conducive for live imaging conditions (fluorobrite DMEM with 10 % FBS, 1 mM Glutamine and 50 μM Hepes buffer) was added to dilute the probe concentration three times and imaging was done in a live cell chamber at 37°C.

Techniques: Cell Culture, Staining, Fluorescence, Transfection, Two Tailed Test