4 chamber slides

Five thousand cells were seeded on the 4 chamber slides (Thermo Fisher Scientific) one day prior to the immunofluorescence staining. After cell permeabilization and blocking, cells were probed with NFATc3 primary antibody overnight, then with Alexa Fluor 594 dye-conjugated secondary antibody and DAPI (blue-green) for confocal laser scanning. Confocal laser scanning microscopy was performed using Fluoview FV10i Confocal Microscope (Olympus), and images were captured with 60x oil objective under different gain settings. The laser diode 559 nm was used to capture NFATc3 staining, and the diode 405 nm laser was used to capture DAPI nuclear stain. Image acquisition and further adjustment of brightness was performed using Olympus Fluoview Ver. 4.2a. Fluorescent images of cells were taken as single channel images then converted to overlay images and all images were saved in TIFF format.

Stably-transfected cells were grown in 4-chamber slides (Thermo Scientific Nunc) until 60–80% confluence. Living cell fluorescent images were captured using an AMG EVOS FL Cell Imaging System with a Plan Fluorite 40×/0.65 objective.
Immunoblots were used to confirm the redistribution of HKII and mutated HKII proteins into the cytoplasm from the mitochondria. The enriched-mitochondrial fractions and cytoplasmic fractions were collected and processed as described previously [9 (link)]. Equal concentrations of protein were separated by 10% SDS/PAGE and transferred to Immobilon-P membrane (Millipore Corp.) by electroblotting. HKII, mitochondrial Hsp70 (mtHsp70), and β-actin were identified by immunoblotting using the following antibodies purchased from Thermo Scientific; anti-HKII, anti-mtHsp70, anti-β-actin, anti-mouse-HRP, anti-rabbit-HRP. Antibodies targeting β-actin and mtHsp70 were used as loading controls for cytoplasmic and mitochondrial fractions, respectively. Protein was detected by chemi-luminescent signalling (Pierce ECL) and the immunoblots were scanned and analysed by densitometry using UN-SCAN-IT software (Silk Scientific, Inc.). The densitometry results were expressed as a ratio of the intensity of HKII to that of β-actin or Hsp70 from the same source.

Hydrogen peroxide-induced oxidative stress was assessed using dihydroethidium (DHE, Sigma-Aldrich) and CellROX green reagent (Life Technologies, Grand Island, NY). After exposure to 500 μM hydrogen peroxide for 6 h, 10 μM DHE or 5 μM CellROX green reagent was added to each cell on 4 chamber slides (Thermo Fisher Scientific) and incubated with DHE for 15minutes or with CellROX green reagent for 30 min at 37°C in DMEM. Cells were then fixed in 4% paraformaldehyde and mounted with VECTASHIELD HardSet Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA). Images were obtained using a confocal laser scanning microscopy (LSM 710; Carl Zeiss, Oberkochen, Germany). Fluorescence intensities were measured using ImageJ software (http://rsb.info.nih.gov/ij/). For superoxide quantification, cells were treated with DHE dye as above and 1.0 μg/mL Hoechst 33342 (Wako Pure Chemical Industries) in black walled clear bottom 96 well plates (Corning Life Sciences, Tewksbury, MA) for 15 min and were scanned in a fluorescent plate reader (PerkinElmer Ensight: Perkin-Elmer Inc., Wellesley, MA) at λ_ex = 535 nm, λ_em = 610 nm for DHE, and λ_ex = 350 nm, λ_em = 460 nm for Hoechst 33342. The DHE fluorescence intensity per cell was determined by dividing DHE fluorescence by the Hoechst 33342 fluorescence.

0.03 × 10⁶ A549 cells were seeded at 4-chamber slides (Thermo, Nunc) and cultured at 37 °C overnight. Cells were treated with HDAC inhibitor SAHA and Lipoic acid or LM for 6 h, followed by 1 mM Arsenite for 30 min. Cells were fixed by 4% PFA and permeabilized by 0.5% Triton X-100/PBS. 10% Goat serum in PBS was used for blocking. After manually selecting an area with cell confluency of 50–80%, 16 pictures were taken randomly around the central point (ZEISS software in-built function). Stress granule marker G3BP1 (Aviva Systems Biology, ARP37713_T100) was visualized and quantified by ImageJ. Statistical analysis was performed with GraphPad Prism.