Anti p ampk

pMSCs treated under different conditions were collected and then lysed with RIPA buffer along with PMSF protease inhibitor. The primary antibodies used for immunodetection included anti-OCT4 (Cat.#: AF0226; Affinity Biosciences, Changzhou, China), anti-p53 (Cat.#: AF0879; Affinity Biosciences, Changzhou, China; Cat.#: 10442-1-AP; Proteintech, Wuhan, China), anti-p62 (Cat.#: ab109012; Abcam, Cambridge, MA, USA), anti-LC3-I/II (Cat.#: NB100-2220; Novusbio, CO, USA), anti-p-AMPK (Cat.#: AF3423; Affinity Biosciences, Changzhou, China), anti-AMPK (Cat.#: sc74461; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-β-actin (Cat.#: sc8432; Signalway Antibody, Baltimore, MD, USA). The specific protein bands were visualized with the Odyssey Infrared Imaging System (LI-COR, Lincoln, Dearborn, MI, USA). The band density was analyzed using Image-Pro Plus 5.1 software (MEDIA CYBERNETICS, Silver Spring, MD, USA) using β-actin as an internal control and then normalized to the vehicle control.

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).

Fibroblast growth factor 21 standard protein was provided by Wenzhou Medical University (Wenzhou, China); metronidazole was purchased from Shenggong (Shanghai) Biotechnology Co., Ltd.; chloroquine (HY-17589A) was purchased from MCE Biotechnology Co., Ltd.; and glutathione (GSH), malondiazole aldehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) detection kits were purchased from Nanjing Jiancheng Institute of Bioengineering (Nanjing, China). Bicinchoninic acid (BCA) protein assay kits were purchased from Thermo Fisher Scientific (Rockford, Illinois, United States), and the ECL Plus kit was purchased from PerkinElmer Life Sciences (Waltham, MA, United States). The TUNEL apoptosis detection kit (Alexa Fluor 640) was purchased from Yeasen Biotechnology Co., Ltd. (Shanghai, China), and qPCR reagents were purchased from Takara (Dalian, China).
The following primary antibodies were purchased: PCNA and LC3B from Abmart Inc. (Shanghai, China); Anti-p53, Anti-bcl2, Anti-bax, Anti-AMPK, Anti-p-AMPK, Anti-mTOR, Anti-p-mTOR, and Anti-SQSTM1/p62 from Affinity Biosciences (Cincinnati, OH, United States); and 4’,6-diamidino-2-phenylindole (DAPI), protein loading buffer, and β-actin from Beyotime Biotechnology (Jiangsu, China).