Fig 2: PHGDH inhibition promotes Beclin-1-dependent canonical autophagy mediated by p-AMPK in a p-mTOR-independent manner.a NT2/D1 cells, with scrambled control or PHGDH shRNA, were subjected to western blot (WB) analysis for (i) p-mTOR, mTOR, p-Akt and Akt, (ii) p-p70S6K, p70S6K, p-4EBP1, 4EBP1. b NT2/D1 cells, with scrambled control or PHGDH shRNA, were treated with rapamycin and subjected to WB analysis for (i) SQSTM1, LC3A-II, LC3B-II, and (ii) caspase-3. c NT2/D1 cells, with scrambled control or PHGDH shRNA, were subjected to WB analysis for p-AMPK, p-TSC2, TSC2, p-Beclin-1. d Schematic diagram depicting the activation of autophagy through AMPK-Beclin-1 activation due to energy depletion. e NT2/D1 cells, with scrambled control or siBeclin-1 siRNA, were treated with PHGDH inhibitor CBR-5884 and chloroquine (CQ), alone or in combination, and the levels of LC3A-II and LC3B-II were analyzed by WB

Fig 3: miR-744 interferes with AMPK signaling to inhibit cell growth. miR-744 knockdown activated the AMPK signaling pathway as determined by (A) AMPKα, and (B) AMPKβ1 phosphorylation. (C) miR-744 knockdown regulated TSC2 activity by phosphorylation at S1387. (D) miR-744 knockdown inhibited mTOR activation. Overexpression of miR-744 inhibited the AMPK signaling pathway as determined by (E) AMPK and (F) AMPKβ1 phosphorylation. (G) Upregulation of miR-744 regulated TSC2 inhibition by phosphorylation at S1387. (H) Overexpression of miR-744 caused mTOR activation. AMPK, adenosine monophosphate-activated protein kinase; miR, microRNA; TSC2, tuberin; mTOR, mammalian target of rapamycin; NC, negative control.

Fig 4: Macrophage AMPK signals through autophagy and is linked to autophagic protein expression. Signaling and flux through autophagy in response to AMPK expression and activation (A-769662 100 µM) and/or autophagy inhibition (chloroquine 50 µM) for 5 h. Total and phosphorylated proteins were determined from duplicate gels. Relative quantification for select targets is shown on the right. pmTOR/mTOR (280 kDa), pTSC2/TSC2 (200 kDa), pULK1/ULK1 (150 kDa), pRAPTOR/RAPTOR (150 kDa), p62 (62 kDa), AMPKα (60 kDa), LC3I/II (17/14 kDa), and β actin (37 kDa). Data are the mean ± SEM and is representative of at least three independent experiments from BMDM isolated from separate mice, where *, ** and *** represent p < 0.05, p < 0.01, and p < 0.001 compared to vehicle control for within genotype.

Fig 5: Relationship between phosphorylation of intracellular signal kinases and lysophosphatidylcholine (lysoPC) effects through long‐chain acyl‐coenzyme A synthases 5 (ACSL5). Levels of phosphorylated (p‐) phosphoinositide 3‐kinase (PI3K), Akt, mTOR, GSK‐3β, TSC2, Raf, MEK, ERK, RSK and MSK proteins were measured in SPC‐A1 cells 6 h after treatment with lysoPC. Regulatory effects of PI3K and ERK in reactive oxygen species (ROS) production quantified by DCF fluorescence intensity (B), ACSL5 gene expression (C) and cell proliferation rate (D) were assayed in SPC‐A1 cells pretreated with PI3K inhibitor LY294002 or ERK inhibitor GDC0994 for 2 h and followed by lysoPC treatment for 6 h (n = 6) or in ACSL5 NC or ACSL5 KD cells pretreated with inhibitors of PI3K and ERK for 2 h (n = 6). Protein levels of ACC and FASN in SPC‐A1 cells pretreated with vehicle or PI3K/ERK inhibitors were measured 6 h after lysoPC treatment (E). * and ** or # and ## stand for the p‐values less than .05 and .01, respectively, as compared with cells treated with vehicle or lysoPC