Mitotracker red cmxros

Immunofluorescence was used to evaluate the effects of BG and Rb1 on fundamental protein, UCP1, and mitochondrial biogenesis using both 3T3-L1 cells and PWATs. For immunocytochemical analyses, differentiated adipocytes were fixed with 4% formaldehyde and permeabilized with 0.2% Triton X-100 (Sigma, St. Louis, MO, USA), followed by blocking with 1% BSA in PBST for 30 min. After that, the cells were incubated with anti-UCP1 antibody (1:250, Abcam) overnight at 4 °C. The cells were washed with PBS and incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit secondary antibody (1:500, Promega, Madison, WI, USA) in 1% BSA for 1 h. Mitochondria were stained using MitoTracker Red CMXRos (500 nM; Cell Signaling Technology) for 30 min according to the manufacturer’s protocol. Then, the cells were fixed and rinsed three times with PBS. The nuclei of the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, MA, USA). Fluorescence images were captured by using an inverted phase-contrast microscope with fluorescence (KI-2000F, Korea Lab Tech, Gyeonggi, Korea) and analyzed with image-processing software (OptiView 3.7, Korea Lab Tech, Gyeonggi, Korea).

Dulbecco's modified Eagle's medium (DMEM) was purchased from Gibco [Catalog (Cat.) No. 41966052] (Fisher Scientific), SHIP2 inhibitor AS19 from Sigma (Cat. No. SML1022), H₂O₂ from Sigma, PTEN inhibitor VO‐Ohpic trihydrate from Selleckchem (Cat. No. S8174), 2′,7′‐DCFDA from Sigma (Cat. No D6883), 3‐methyladenine from Sigma (Cat. No. M9281), MitoTracker Red CMXRos from Cell Signaling (Cat. No. 9082), and 4′,6‐diamidino‐2‐phenylindole dihydrochloride (DAPI) from Molecular Probes (Life Technologies, Carlsbad, CA, USA).

Cells were fixed with 4% paraformaldehyde in PBS for 8 min at room temperature, washed three times with 1X PBS and then blocked with 10% serum and 0.3% Triton X-100 in PBS for 1 h at room temperature. Cells were again washed three times with 1X PBS and incubated a 1:200 dilution of rabbit polyclonal antibody against LC3B (Cat^# ab63817, Abcam) in PBS containing 0.1% Triton X-100 and 5% horse serum and overnight at 4 °C. After washing, the cells were incubated with fluorescein (FITC)-conjugated AffiniPure goat anti-mouse IgG (H + L) (Cat# SA00003-1, Proteintech) for 2 h at room temperature. Samples were mounted with Immu-mount (Thermo Scientific).
Cells were stained with 50 nM MitoTracker® Red CMXRos (Cat# 9082S, Cell Signaling Technology) for 30 min at 37 °C, washed with 1X PBS, and then used to detect the colocalization of LC3 and MitoTracker.

A MitoTracker^® Red CMXRos (Cell Signalling Technology Inc., Danvers, MA, USA) staining was performed, as described in [11 (link)]. In brief, 1.5 × 10⁶ cells were stained in 750 µL of equiosmotic medium that contained either 100 nM of the dye or the growth medium as a vehicle control. The cells were then incubated for 30 min at 37 °C in the dark, counterstained with DAPI, and strained through cell mesh directly into a measuring tube, as described above.

Cells were seeded onto coverslips in 12-well plates and cultured for 24 h before drug treatment. The medium was removed and the cells were washed three times with PBS. Each sample was stained with MitoTracker Red CMXRos (9082) from Cell Signaling Technology, Boston, MA, USA) for 20 min and washed three times with PBS. Images were obtained with a laser scanning confocal microscope equipped with an argon laser (excitation wavelength: 555 nm) and a 63× objective lens.

Primary LKB1^−/−NIC mammary tumor cells were isolated as previously described [50 (link)] and plated onto coverslips. Treatments were performed using AZD8055 (100 nM), 2-deoxyglycose (10 mM) and a combination of both. Following this, cells were incubated for 20 min at 37°C with Mitotracker Red CMX/Ros (Cell Signaling) at a final concentration 100 nM. Cells were then washed 2x with PBS and prepared for analysis by fluorescence microscopy and flow cytometry. Fluorescence microscopy was conducted using a Nikon Eclipse TE 2000-E, mounted with a Q-Imaging CCD camera. Fluorescent images were acquired using Simple PCI software as previously described [3 , 49 (link)]. Flow cytometry was conducted using a Becton Dickinson FACS Calibur. Data was acquired using CellQuest software and analyzed using ModFitLT as previously described [49 (link)].