Anti mtor

Protein expression was assessed by immunoblot analysis of cell lysates (20–60 μg) in RIPA buffer in the presence of the following antibodies: including anti-p-AMPK (Affinity, AF3423), anti-AMPK (Affinity, AF6423), anti-p-ACC1 (Cell Signaling Technology, 11818s), anti-ACC1(Cell Signaling Technology, 4190s), anti-ACOX1 (Santa Cruz Biotechnology, sc-517306), anti-p-mTOR (Affinity, AF3308), anti-mTOR (Affinity, AF6308), anti-SGLT2 (Abcam, ab37296), anti-LC3B (Cell Signaling Technology, 12741S), anti-Beclin1 (Cell Signaling Technology, 4122s), anti-p62/SQSTM1 (Proteintech, 18420-1-AP), and anti-GAPDH (Abcam, ab8245).
Unless otherwise specified, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, United States). Dulbecco's modified Eagle’s medium (DMEM), fetal bovine serum (FBS, Gibco) were obtained from Gibco; palmitate (PA) was obtained from Sigma-Aldrich (P9767); compound C (Comp C) was purchased from AbMole (M2238), chloroquine (CQ, S4157) and dapagliflozin (S1548) were bought from Selleck; and Oil Red O and triglyceride detection kit (G1262; BC0625) were obtained from Solarbio.

As described previously, we extracted proteins from cells with RIPA lysis buffer, separated equal amounts of protein using SDS-PAGE, and then transferred the protein to a 0.45-µm PVDF membrane (Millipore, USA). Next, the membrane was blocked with 5% skim milk for 1 h at room temperature before it was washed 3 times with 1× TBST for 10 min each time. The membranes were then incubated with the following antibodies: rabbit anti-GAPDH (#5174), anti-Notch2 (#5732), anti-Bax (#0120), anti-Bcl-2 (#6139), anti-γH2AX (#8482), anti-Cyclin D1 (#0931), anti-AKT (#6261), anti-phospho-AKT (#0016), anti-mTOR (#6308), and anti-phospho-mTOR (#3308), all of which were purchased from Affinity Biosciences (OH, USA). A chemiluminescence reagent was used to visualize the protein bands, and the grayscale values of the bands were measured with Image Lab 6.0 software (Bio-Rad) [19 (link)].

First, the spleen paraffin sections were deparaffinized in xylene and graded ethanol. To block endogenous peroxidase activity, the sections were incubated with 10% hydrogen peroxide. For antigen retrieval, the sections were heated in 2% EDTA solution for 15 min, and allowed to cool for 2 h at room temperature. After washing with PBS, the slides were blocked with goat serum (Zhongshanjinqiao Biotechnology Co., Ltd., China) for 15 min, and then incubated with the diluted antibodies at 4℃ overnight. On the next day, the sections were incubated with corresponding secondary antibodies at room temperature for 15 min. The sections were then labeled with horseradish peroxidase, and the signals were detected using the DAB Peroxidase Substrate Kit (Solarbio). After staining with hematoxylin (Beyotime) for 30 s, the slides were dehydrated in graded ethanol and xylene. Finally, the sections were sealed with coverslips by resinene (Solarbio). The following primary antibodies were used for the assay: anti-GS (Affinity), anti-mTOR (Affinity), anti-Cyclin D1 (Affinity), anti-CDK4 (Affinity), anti-CDK6 (Affinity).

For Western blot analysis, the following antibodies were used: anti-LC3B (2775; Cell Signaling Technology), anti-SQSTM1/P62 (5114; Cell Signaling Technology), anti-ATG7 (DF6130; Affinity Biosciences), anti-ATG5 (CY5766; Abways), anti-fibronectin (ab32419; Abcam), anti-integrin α5 (CY5979; Abways), anti-integrin β1 (CY5469; Abways), anti-phospho-Beclin-1 (Ser15) (84966; Cell Signaling Technology), anti-BECN1 (ab207612; Abcam), anti-TLR2 (AF7002; Affinity Biosciences), anti-TLR4 (AF7017; Affinity Biosciences), anti-CST (AB0055; Abways), anti-Rab7 (9367; Cell Signaling Technology), anti-phospho-MTOR (Ser2448) (AF3308; Affinity Biosciences), anti-MTOR (AF6308; Affinity Biosciences), anti-phospho-ULK1 (Ser757) (6888; Cell Signaling Technology), anti-ULK1 (8054; Cell Signaling Technology), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5174; Cell Signaling Technology). Horseradish peroxidase-labeled goat anti-rabbit (ASS1009; Abgent) secondary antibodies were used. The transfection reagents were Lipofectamine 2000 (11688-019; Invitrogen) and HiPerFect (Y5-301705; Qiagen). The immunoprecipitation (IP) reagent was included in the Pierce classic magnetic IP/co-IP kit (88804; Thermo Scientific).

Since EVs carry microRNAs to modulate post-transcriptional gene expression in recipient cells, we applied microRNA sequencing to explore the mechanisms underlying the biological function of EVs. Small RNAs were extracted from CM-EV and OM-EV (n = 3 for each) using an Exosome RNA Purification Kit (EZBioscience, USA) following the manufacturer’s instructions. MicroRNA libraries were constructed and subjected to deep sequencing via the Illumina Hi-Seq 2500 platform at RiboBio Co. Ltd. (Guangzhou, China). Differentially expressed miRNAs with a 1.3-fold change in expression (P < 0.05) were analyzed. Kyoto Encyclopedia of Genes (KEGG) pathway analysis. Additionally, gene ontology (GO) analysis of target mRNA genes of differentially expressed miRNAs was performed to explore signaling pathways potentially involved in OM-EV function. To confirm whether the AMPK/mTOR pathway is involved in the regulation of OM-EV-mediated odontogenic differentiation, Western blotting was conducted to assess the expression of p-AMPK and p-mTOR in DPSCs after incubation with OM-EV for 7 days. The antibodies used were as follows: anti-AMPK (1:1000, Affinity, USA), anti-p-AMPK (1:800, Affinity, USA), anti-mTOR (1:1000, Affinity, USA), and anti-p-mTOR (1:800, Affinity, USA).