8 inhibitors or modulators of kinase activities were tested targeting: Akt, JNK, CDK2, RTK, PKA, JAK, mTOR and AMPK (see Figure 1). Two experimental screens were conducted: the first used 3T3-derived pre-adipocyte cells, and the second used LN71 glioblastoma cells. Cells were plated on fibronectin-covered 96-well plates, in serum-free conditions. Inhibitors were used at 0.1, 1 and 5 times the recommended working concentrations. Images were taken using the automated 3×3 gridscan mode, at an acquisition rate of 1 scan every 25 mins, for 40 h. After, images were stitched in FIJI, and segmented and analysed using tools developed in house.

Video 1. JNK inhibitor effect in pre-adipocytes compared to negative control

Nanolive imaging is the only label-free way to visualize lipid droplets (LDs). The absence of LDs allows to detect when LD reserves have been metabolized. In this experiment, cells were cultured in serum-free conditions (essentially starved). No LDs were present in the negative control (green) after 30 h; non-perturbed cells metabolize their LD reserves. In contrast, JNK (orange) and CDK2 (not shown) inhibited cells still have LDs at the end of the experiment, showing these treatments modify their metabolism.

Video 2. mTOR inhibitor effect in pre-adipocytes compared to negative control

• CDK2 drives the progression of cells into the S- and M-phases of the cell cycle.
• Inhibiting CDK2 caused severe nuclear disruption. Nuclear membranes were notably thicker than in the control and nucleoli were clearly disorganized structurally.
• Cells did divide when CDK2 was inhibited but the process was abnormal.

Figure 3. Drug-induced pathologies

• The high resolution of Nanolive cell imaging makes it possible to detect changes in the form and distribution of mitochondria in a cell.
• Mitochondria typically cluster around the nucleus, but CDK2-inhibition re-distributed them throughout the cell (Figure 4, A), with increasing densities present in the lamellipodia (Figure 4, B).
• The rounded morphology that mitochondria display towards the end of the experiment (Figure 4, C) shows that CDK2-inhibition clearly perturbs mitochondrial morphology.