P foxo3a

Protein lysate were extracted using RIPA lysis buffer (Thermo Fisher Scientific, China) contained protease and phosphatase inhibitors (1:100, Thermo Fisher Scientific, China). Subsequently, proteins were electrophoresed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes (PVDF, Millipore, Billerica, USA). After blocked with 5% bovine serum albumin for 1 hour, the membranes were incubated with following primary antibodies at 4℃ overnight: RPS9 (Proteintech, 1:1000); P- mTOR, mTOR , P-FAK, FAK, P-FoxO3a, FoxO3a, P-FoxO1, FoxO1, P-Stat3, Stat3, NF-κB, P-AMPK, AMPK, P-Smad2, Smad2, P-AKT, AKT, TGF-β, P-Erk and Erk (1:1000, Cell Signaling Technology, USA); β-actin (1:10000, Sigma-Aldrich, USA). Next day, after washed in PBST for three times, the membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit antibodies (1:5000, Sigma-Aldrich, USA) at room temperature for 1 hour. Finally, the bands were visualized and analyzed by SuperSignal West Femto Maximun Sensitivity Substrate (Thermo Fisher Scientific, USA).

Proteins were extracted from cells and 30 µg samples were fractionated by SDS-PAGE then transferred to nitrocellulose membranes. Membranes were blocked with 5% BSA in TBS-T (TBS with 0.05% Tween 20) and incubated with primary antibodies against p-Akt(473), Akt, p-Foxo3a, Foxo3a, raptor(1:1,000 dilution, Cell Signaling Technology, Danvers, MA, USA); Actin, RANKL, M-CSF, OPG,p16,p19,p27,p21,cdk2,p-NF-κB, NF-κB,p-STAT3, STAT3, α-SMA, and E-cadherin(1:1,000 dilution, Abcam).

Cultured mouse ovaries were lysed in WIP lysis solution (Cell Signaling Technologies, USA). Sample proteins were separated by electrophoresis by 10% SDS‐PAGE and transferred to PVDF (polyvinylidene fluoride) membranes (IPVH00010, Millipore, USA). Then, the membranes were blocked with 5% nonfat‐dry milk for 60 minutes and incubated at 4°C overnight with the following primary antibodies. The primary antibodies were as follows: p‐FOXO3a (9466, 97 kDa, 1:1000, Cell Signaling Technologies, USA), FOXO3a (12829, 82‐97 kDa, 1:1000, Cell Signaling Technologies, USA), p‐AKT (4060, 56 kDa, 1:1000, Cell Signaling Technologies, USA) and AKT (4691, 56 kDa, 1:1000, Cell Signaling Technologies, USA). The membranes were washed thoroughly with tris‐buffered saline with tween and incubated with the appropriate secondary antibody (1:5000, ZSGB‐BIO, China). The level of β‐actin (42 kDa, 1:1000, Sigma, USA) was used as an internal control. The membranes were visualized by the SuperSignal detection system (Prod 34080, Thermo, USA). To quantify the results of immunoblot, the image was quantified using Image J software.

Protein extracts were adjusted to a final concentration of 2.0 µg/µL in sample buffer (125 mM Tris–HCl, 4% SDS, 20% glycerol, 200 mM DTT, and 0.02% bromophenol blue at pH 6.8) and subjected to SDS-PAGE electrophoresis using 10% polyacrylamide gel. The proteins were transferred onto nitrocellulose membranes, blocked with 5% BSA, and incubated with primary antibodies against the following targets: PTEN (Cell Signaling Technology, 1:1000), AKT (Santa Cruz Biotechnology, 1:2000), p-AKT^T308 (BD Biosciences, 1:750), mTOR (Cell Signaling, 1:1000), p-mTOR (Cell Signaling, 1:1000), FoxO3a (Cell Signaling, 1:1000), p-FoxO3a (Cell Signaling, 1:1000), Beclin1 (Cell Signaling, 1:500), Bcl-xL (Cell Signaling, 1:333), Bax (Cell Signaling, 1:333), Atg7 (Cell Signaling, 1:500), and β-actin (Sigma-Aldrich, 1:10,000). Primary antibodies were diluted in 1% BSA, while secondary antibodies were diluted in 5% BSA (1:2000).

Cells were lysed in iced-cold Pierce M-PER buffer containing Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). After protein concentration was measured, protein expression was determined using an automated western blotting system according to the manufacturer's instructions (ProteinSimple, San Jose, CA, USA). Following capillary-based electrophoresis, primary antibodies against phosphorylated-Smad2 (p-Smad2, 1:50 dilution, #8828S; Cell Signaling Technology, Danvers, MA, USA), Smad2 (1:200 dilution, #5339S; Cell Signaling Technology), phosphorylated-Foxo3a (p-Foxo3a, 1:10 dilution, #5538S; Cell Signaling Technology), Foxo3a (1:50 dilution, #2497S; Cell Signaling Technology), phosphorylated-Akt (p-Akt, 1:10 dilution, #9271S; Cell Signaling Technology), Akt (1:100 dilution, #9272S; Cell Signaling Technology) and GAPDH (1:200 dilution, ab181602; Abcam) were infused into capillaries to probe target proteins and were visualized using labeled secondary antibodies. Protein expression was quantified using Compass Software (ProteinSimple).

After 24 h of treatment, mesangial cells were lysed with lysis buffer (1 M Tris (pH = 7.4), 1.5 M NaCl, 1% Triton-X, 10% Glycerol, 50 mM EDTA (pH = 8), 0.1 M Sodium vanadate, 0.1 M PMSF, 0.1% protease inhibitor cocktail (Calbiochem) Samples were boiled for 5 minutes, electrophoresed on 8% or 12% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with specific Antibodies (p-AKT, p-GSK3β, p-FoxO3a, PTEN, p-(rp)S6 and p-mTOR, (Cell Signaling) actin HRP (Sigma). Blots were developed using horseradish peroxidase-conjugated secondary Abs and the ECL detection system (Thermo scientific, Pierce research protein products (Rockford, IL, USA)).

Human prostate carcinoma cell lines, PC-3 and DU-145, were obtained from American Type Culture Collection and cultured (ATCC) and cultured according to the manufacturer instructions. No authentication was done by authors. Withaferin-A obtained from Nucleus Biopharma Inc. (Calbiochem, Billerica, MA, USA) was dissolved in dimethyl sulfoxide (DMSO), and the cells were treated with DMSO at a final concentration of 0.002%. The following antibodies obtained from Cell Signaling Technology (Danvers, MA, USA) were used for the immunoblotting: AKT, pAKT, FOXO3a, pFOXO3a, p27, 14-3-3, cleaved PARP, cleaved caspase-9, BCL2, and BAX. Antibodies for Par-4, pGSK-3β, 14-3-3, HA, GAPDH, β-actin, and tubulin, and secondary antibodies of anti-mouse, anti-goat, and anti-rabbit were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Annexin-FITC kit was purchased from BD Biosciences (San Diego, CA, USA). Propidium iodide was purchased from Sigma-Aldrich (St Louis, MO, USA). Alexa Fluor 488, Alexa Fluor 568, and Prolong gold antifade with DAPI mountant were purchased from Invitrogen (Grand Island, NY, USA). Mammalian expression plasmids for CT-FOXO3a, TM-FOXO3a, DBM-FOXO3a, AKT plasmids, and control vectors were obtained from Addgene (Cambridge, MA, USA). Wt-FOXO3a and Par-4 expression plasmids were obtained from Origene (Cambridge, MA, USA).