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. 2021 Aug 28;23:107–123. doi: 10.1016/j.omto.2021.08.013

Figure 6.

Figure 6

OA selectively inactivates SOD1 to degrade HGPRT and 5′-NT via the ROS/AMPK/mTORC1/macroautophagy/lysosome pathway

(A) Western blot showing the time course of the effects of OA treatment (200 μM) on the abundance of phospho-AMPK (Thr172), AMPK, phospho-ACC (Ser79), ACC, phospho-ULK1 (Ser555), phospho-ULK1 (Ser757), ULK1, phospho-4EBP1 (Thr37/46), 4EBP1, and LC3-I/II in A549 cells. (B) The effects of two ULK1 inhibitors, ULK-101 (1 μM) and MRT68921 (1 μM), on levels of phospho-ATG14 (Ser29), ATG14, LC3-I/II, HGPRT, and 5′-NT in OA-treated A549 cells. (C) Western blot showing the influence of 3-MA (1 mM) on HGPRT, 5′-NT, and LC3-I/II expression in OA-treated A549 cells. (D) Western blot showing the time course of the effects of 200 μM OA treatment on the expression of phospho-AMPK (Thr172), AMPK, phospho-ACC (Ser79), ACC, phospho-ULK1 (Ser757), ULK1, phospho-4EBP1 (Thr37/46), 4EBP1, LC3-I/II, HGPRT, and 5′-NT in A549 cells with and without PRKAA1 KO. (E) Measurement of reactive oxygen species (ROS) generation induced by OA (200 μM) and ROS removal following N-acetyl-l-cysteine (NAC) (5 mM) treatment in A549 cells. (F) Time course of the reversion of phospho-AMPK (Thr172), phospho-ACC (Ser79), ACC, HGPRT, 5′-NT, and LC3-I/II in OA-treated A549 cells after NAC (5 mM). The values of phospho-AMPK (Thr172) were normalized as follows: the raw abundance of phospho-AMPK (Thr172) of each lane was first normalized by the corresponding actin, and the actin-normalized proteins of all lanes were then further normalized by the actin-normalized protein on the fourth lane (control group treated by OA for 8 h). Normalized phospho-ACC (Ser79) was acquired by the same computation approach. (G) Restoration of impaired cell proliferation and PCNA expression by NAC with OA treatment (200 μM). Viable cell proliferation was measured with an ATPlite kit. (H) Measurement of ROS generation in OA-treated A549 cells with and without SOD1 overexpression. (I) Under OA treatment, SOD1 overexpression enhanced HGPRT and 5′-NT stability and restored PCNA expression. Error bars represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, Student’s t test. Ns, no significance.