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. 2021 Dec 22;12:782989. doi: 10.3389/fimmu.2021.782989

Figure 5.

Figure 5

Menthol promotes autophagy through activation of the AMPK/ULK1 or AMPK/Nrf-2 pathways. BMECs were pretreated with 200 μM menthol and CC (inhibitor of AMPK,10 μM) for 1 h and treated with 5 μg/ml LPS for 12 h. (A) Protein expression levels of p-ULK1, ULK1, p-AMPK, AMPK, T-Nrf-2, P62, HO-1, LC3B, N-Nrf-2 and Histone H3 were detected by western blotting (n≥3); T-Nrf-2 represents total intracellular Nrf-2, and N-Nrf-2 represents Nrf-2 in the nucleus. (B) Detection of Nrf-2 entry by immunofluorescence staining of the nucleus (blue) and Nrf-2 (green). (C-I) Bar graphs represent the quantitative results of the corresponding protein bands. The protein results were homogenized using β-actin as an internal reference protein. Histone H3 was used as the internal reference protein in the nucleus. (J) Results of qRT-PCR for mRNA levels of ULk1, P62 and LC3B (n≥3). β-actin was used as an internal reference to normalize the mRNA expression. The values are presented as the means ± SEM (*p < 0.05, **p < 0.01 and ∗∗∗p < 0.001).