Extended Data Fig. 3: Mitochondrial dynamics and respiratory activity are induced by ECM mechanics in pancreatic cancer cells and support invasive behavior.
In a-i, cells were cultured atop of 0.7-38kPa fibronectin-coated hydrogels and glass coverslips. (a) Glucose-derived labelled carbon incorporation in glucose and TCA cycle intermediates of KPC cells as indicated and treated with U-13C6-glucose for 3 hours. (b) GSH, GSSG levels and GSH/GSSG ratio of KPC cells cultured as indicated. (c) NADPH levels of cells from (b). (d) Glycolysis and TCA cycle metabolite levels of PANC-1 cells cultured as indicated. (e) GSH, GSSG levels and GSH/GSSG ratio of PANC-1 cells from (d). (f) Left; Mitochondrial mass (Mitotracker, MTG) of KPC cells as indicated. Values (gMFI) are mean ± SD relative to control (glass) from 3 independent experiments. Right; Representative histogram from left panel. (g) Left; Mitochondrial membrane potential (TMRE) of KPC cells cultured as indicated. CCCP; negative control. Values (gMFI) are mean ± SD relative to control (glass) from 3 independent experiments. Right; Representative histogram from left panel. (h) Left; Cellular ROS (CellROX) of KPC cells cultured as indicated. H2O2; positive, NAC + H2O2; negative control. Values (gMFI) are mean ± SD relative to control (glass) from 3 independent experiments. Right; Representative histogram from left panel. (i) Top; Maximum intensity projections of z-stack acquisitions of PANC-1 cells cultured as indicated showing labelled mitochondria. Scale bars, 10μm. Bottom; Magnification of areas indicated by a dashed box. Pictures representative of 2 independent experiments. Scale bars, 5μm. (j) Maximum intensity projections of z-stack acquisitions of KPC cells cultured on fibronectin-coated dishes (left) or invading 3D ECM (middle, right) showing labelled mitochondria. Pictures representative of 3 independent experiments. Scale bars, 7μm (left, right) and 8μm (middle). Values in a-e represent mean ± SD from 3 biological replicates performed on the same day. In b, d, e: statistical significance assessed by two-tailed unpaired t-test with Welch’s correction. In f, g, h: statistical significance assessed by two-tailed one-sample t-test on LN transformed values.
