Lsm900 scanning microscope

For immunocytochemistry, 30,000 cells were seeded in a four-well chamber (Thermo Fisher Scientific) and treated with glucose as described above. After fixation with 4% paraformaldehyde for 15 min at room temperature, cells were blocked with blocking buffer (Cell Signaling Technology) for 60 min at room temperature. Then cells were incubated with p-MLC2 antibody (Cell Signaling Technology, 3671) diluted 1:50 overnight at 4°C. Secondary antibody conjugated with Alexa Fluor 488 (Molecular Probes, R37116) was used for visualization. For costaining with filamentous actin (F-actin), cells were stained with Acti-stain 555 phalloidin (Cytoskeleton, PHDH1) as described in the manufacturer’s instructions. Then DAPI (Invitrogen, R73606) was added for nucleus staining. Confocal microscopy images were acquired using LSM900 scanning microscope (Carl Zeiss) equipped with 63×, 40×, and 20× magnification objectives. To determine subcellular localization of pMLC2 and F-actin, confocal microscopy images were analyzed with line profiles using ZEN 3.4 software (Carl Zeiss). The colocalization of pMLC2 and F-actin signals was evaluated with Pearson’s correlation coefficient (r).

To analyze F-actin levels quantitively, we used two methods, confocal microscopy and Cellinsight High-Content Analysis, to acquire and analyze F-actin-stained cell images. For confocal microscopy, 30,000 or 10,000 cells were seeded in a four-well chamber or an optical 96-well plate, respectively. After overnight incubation, cells were treated with glucose as described above. F-actin was stained with Acti-stain 488/555 phalloidin (Cytoskeleton, PHDG1/PHDH1) for fixed cells or an SiR-actin probe for live cells as described in the manufacturer’s instructions (Cytoskeleton, CY-SC001). DAPI or Hoechst 33342 (Invitrogen) was added for nucleus staining. Representative confocal microscopy images were acquired using an LSM900 scanning microscope (Carl Zeiss). For quantification of F-actin levels, confocal microscopy images were randomly captured with a 20× objective and the total fluorescence intensity from only the area covered by cells from each image was quantified using ZEN 3.4 software (Carl Zeiss) and normalized to the number of nuclei. For unbiased quantification of F-actin levels as an independent validation, we used a High-Content Analysis (HCA) system (Cellinsight CX7, Thermo Fisher Scientific). Nine images were acquired with a 10× objective from each well. F-actin quantitation was computed using a dedicated proprietary algorithm (Cellomics Target Activation V4).