Antibodies and reagents: The following antibodies were used: Atg7 (Cell Signaling, #2631), p62 (Cell Signaling, #5114), LC3 (Cell Signaling, #4108), CREB (Cell Signaling, #9197), p-CREB (Cell Signaling, #9198), mTOR (Cell Signaling, #2972), p-mTOR (Cell Signaling, #2971), IRS-1 (Cell Signaling, #2382), p-IRS-1 (Cell Signaling, #2381), Akt (Cell Signaling, #4685), p-Akt (Cell Signaling, #4060), p-PERK (Cell Signaling, #3179), PERK (Cell Signaling, #5683), cleaved-ATF-6 (Santa Cruz Biotechnology, #sc-166659), GAPDH (Santa Cruz Biotechnology, #sc-47724) and peroxidase goat anti-rabbit IgG (Santa Cruz Biotechnology, #sc-2768). Insulin were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA). Mouse recombinant CaMKIV were obtained from Sino Biological (Sino Biological Inc. Wayne, PA, USA). Western Lightning Plus-ECL Enhanced Chemiluminescence Substrate Kit (PerkinElmer Inc., Richmond, CA, USA) was used to detect protein expression. Individual protein bands were quantified by ImageJ software. All other chemicals were obtained from standard resource and were of the highest grade available.
Cell culture and treatment
3T3-L1 were purchased from American Tissue Culture Collection (ATCC, Manassas, Virginia, USA) and maintained in DMEM (Gibco, Grand Island, New York, USA) with 10% fetal bovine serum (Gibco) at 37℃ in a humidified atmosphere with 5% CO2. For adipocytes differentiation, 100% confluent 3T3-L1 were induced with MDI induction media (0.5 mM 1-methyl-3-isobutylmethylxanthine, 200 nM dexamethasone, 160 nM insulin, and DMEM with 10% FBS) (day 0). Two days later media was changed to 10% FBS/ DMEM with 160 nM insulin. Cells were then fed with this maintenance medium every 2 days. Full differentiation is usually achieved on the 12th day. To induce ER stress, cells were treated with different concentration (0–5 µg/ml) of Tun for 4 hours. For the effect of CaMKIV, cells were treated with 100 ng/ml CaMKIV for 24 hours. For blocking mTOR signaling, cells were treated with 100 nM mTOR siRNA for 24 hours. For blocking CREB signaling, cells were treated with 100 nM CREB siRNA for 24 hours. For insulin signaling, cells were stimulated with 10 nM insulin for 10 minutes. Before each experiment, the medium was replaced by fresh medium.
Electron microscopy analysis
Cells were fixed in 4% paraformaldehyde/2% glutaraldehyde/0.1 M sodium cacodylate pH7.3, post-fixed in 1% osmium tetraoxide and embedded in epoxy resin (Epon). Ultrathin sections (80 nm) were stained with aqueous uranyl acetate and lead citrate and examined with a JEOL 2000FX transmission electron microscope (JEOL). For quantification of autophagolysosome-like vacuoles, the numbers of autophagolysosomal-like vacuoles were counted in each field and normalized by the surface area.
Small interfering RNAs (siRNAs) and transfection: Small interfering RNA (siRNA) for target genes (Atg7: sc-41448; CREB: sc-35111; mTOR: sc-35410, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or scrambled siRNA (CREB, sc-37007, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were transfected using Lipofectamine® RNAiMAX Transfection Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The transfected cells were cultured in medium containing 10% FBS for 48 hours after transfection. The knockdown efficiency was assessed by western blot.
Western blotting and immunoprecipitation
The cell lysates were extracted by RIPA Lysis and Extraction Buffer (Invitrogen; ThermoFisher Scientific, Inc., MA, USA) which contained 10% protease inhibitor (Thermo Scientific, USA), incubated on ice for 30 min, and then centrifuged at 14000 x g for 15 min at 4℃. The protein concentration determined using the BCA kit (Thermo Scientific, USA). The supernatants were mixed with equal volume of 4x SDS-PAGE sample loading buffer and then denatured at 95℃ for 10 min. The proteins were separated by SDS-PAGE gel, transferred to a polyvinylidene difluoride membranes, incubated with specific primary antibodies at 4℃ overnights, and detected with horseradish peroxidase (HRP)-conjugated secondary antibodies by using a VersaDoc Image System (BioRad, Hercules, CA, USA). For immunoprecipitation, the lysate was treated using the Dynabeads™ Protein G Immunoprecipitation Kit (Invitrogen; ThermoFisher Scientific, Inc., MA, USA) according to the protocol. The final precipitated proteins were analyzed via western blotting with the corresponding antibodies.
Data were analyzed by the Prism software, version 8.0 (GraphPad Software Inc., San. Diego, CA, US). Characteristics of subjects between 2 groups was performed using an unpaired, two-tailed Student t test for normally distributed variables. Multiple comparisons of quantitative variables among groups were made using one-way ANOVA testing. Data were presented as mean ± SD. N represents the number of animals used. A P value of༜0.05 or P value of༜0.01 was considered as significantly difference.