Foxo1 antibody

WT and TAZ-KO C2C12 cells were grown on coverslips in 24-well dishes. The cells were washed three times with PBS and then fixed in 4% PFA + 8% sucrose (3:1) at 37 °C for 20 m. After fixation, the cells were washed twice with PBS and then permeabilized in PBS containing 0.3% TritonX100. The cells were washed with PBS three times, then incubated in blocking solution (5% BSA diluted in PBS) at room temperature for 1 h before incubating with FOXO1 antibody (#2880, Cell Signaling; diluted 1:150 in 5% BSA/ sc374427, Santa Cruz; 1:100) at 4 °C for a minimum of 16 h. Cells were then washed three times with PBS before being incubated with Alexa Fluor 488 conjugated secondary antibody (A32723, Thermo Fisher; diluted 1:200 in 5% BSA) at room temperature for 1 h. After three final washes with PBS, the slides were mounted with ProLong™ Diamond Antifade Mountant containing DAPI (Invitrogen). All images were captured using a Leica TCS SP8 confocal microscope.

The frozen liver tissues were homogenized in radioimmunoprecipitation assay buffer containing protease inhibitors. The lysates were pre-cleared with protein G agarose at 4 °C for 2 h. The supernatants were incubated overnight with 2 μg of FoxO1 antibody (Cell signaling technology, Danvers, MA, USA) at 4 °C. Added protein G agarose to this, and then incubated for 2 h at 4 °C. The immune-complexes were washed three times with lysis buffer (20 mmol/L Tris, 150 mmol/L NaCl, 1% NP-40, 0.5% SDS, 0.1% SDS, and proteinase inhibitor cocktail) and subjected to western blot analysis. For western blot analysis, 1 μg/mL of FoxO1 (Santa Cruz, Dallas, TX, USA) or acetyl lysine (Abcam, Cambridge, UK) antibodies were used.

For immunofluorescence analysis, control and FoxO1 over-expressed C2C12 cells were grown on cover slides held in 12 wells dishes. Cell were washed 3 times with PBS and then fixed in 100% methanol at room temperature for 10 min or in 4% paraformaldehyde for 15 min. After fixation, cells were washed 3 times with PBS and then quenched in PBS containing 50 mM NH₄Cl to avoid the deleterious effect of the methanol on the antibodies. Cells were then incubated in blocking solution (2% goat serum and 2% BSA diluted in PBS) at room temperature for 30 min before incubated with FoxO1 antibody (#9462, Cell Signaling Technology; diluted 1∶200 in blocking solution) at 4°C for at least 16 hr. Then cells were washed 3 times with PBS before incubated with Alexa Fluor 488 conjugated secondary antibody (A11008, Molecular Probes; diluted 1∶200 in blocking solution) at room temperature for 2–3 hr and then washed 4 times with PBS. To visualize the nuclei, cells were incubated with DAPI (1: 5000 dilution in PBS) at room temperature for 10 min after the secondary antibody incubation and were washed with PBS thoroughly afterward. All the images were observed and photographed under the Carl Zeiss Axio Observer A1 fluorescence microscope with AxioVision software.

For HepG2 siRNA knockdown experiments, cells were seeded in 6 well plates for 24 h prior to siRNA transfection with RNAiMAX for 24 h. Cells were then reseeded in 24-well plates with coverslips for an additional 24 h. Cells were serum deprived for 3 h, fixed and immunostaining was conducted as previously described (Frendo-Cumbo et al., 2019 (link)). Briefly, cells were permeabilized and blocked in PBS containing 0.2% saponin (Calbiochem) and 10% normal goat serum (SS-PBS) for 30 min. Subsequently cells were incubated for 1 h with FOXO1 antibody (#2880; Cell Signaling) in SS-PBS, washed three times with PBS and incubated with secondary Alexa Fluor conjugated antibodies and phalloidin-565 (Invitrogen) for 1 h. Cells were washed three times with PBS and mounted in fluorescence mounting medium (Dako). Images were acquired using a Quorum spinning disk confocal scan head (Leica DMI 6000 B inverted fluorescence microscope, Hamamatsu ORCA Flash 4 sCMOS and color camera) equipped with a 63× objective. FOXO1 nuclear localization was quantified using ImageJ software. Briefly, the nuclear compartment was marked using DAPI and the cell border was determined using phalloidin. The cytosolic region was isolated by removing the DAPI-nuclear mask from the phalloidin-whole cell mask. FOXO1 fluorescence was then measured in the nuclear and cytosolic regions and a ratio was calculated.

Western blotting was performed by following the lab-established protocol (Dai et al., 2007 (link); Dai et al., 2009 (link)). Briefly, whole cell extracts were prepared by lysing the cell pellets with CelLytic™ M Cell Lysis Reagent (Sigma-Aldrich). Protein lysates were separated by 4%–15% Criterion™ TGX™ Precast sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad Laboratories, Inc.) and then transferred onto a polyvinylidene difluoride (PVDF) membrane. Membranes were blocked with 5% non-fat milk in PBST, incubated with a primary antibody, and subsequently a secondary horseradish peroxidase-conjugated antibody. After applying ECL plus western blot substrate (GE Healthcare, Cleveland, OH, United States), the blot images were captured with iBright CL1000 Imaging System (ThermoFisher Scientific). The Foxo1 antibody was purchased from Cell Signaling Technology, Inc. United States. β–actin antibody (Santa Cruz Biotechnology) was used as a reference protein.

Ishikawa cells were seeded overnight in the growth media in 6‐well plates and transfected the next day with 10 nmol/L non‐specific scramble siRNA control (#6568; Cell Signaling Technology) or FOXO1‐specific siRNA (#6256; Cell Signaling Technology) by use of Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's recommendations. At 48 h after transfection, cells were incubated in RPMI‐1640 medium supplemented with 10% FBS in the absence or presence of 2 mM metformin for 24 h. Cell number was counted before cells harvested with RIPA buffer for Western blot analysis of total protein. Silencing of FOXO1 was verified by Western blot analysis with FOXO1 antibody (1:1000; Cell Signaling Technology).