Fig 2: Inhibition of mTOR by Torin1 improves cell injury and fibrogenesis in cyclosporine A‐treated HK‐2 cells. A, Quantitative cell cycle distribution analysis of CsA‐treated HK‐2 cells with or without addition of Torin1. B, Representative images of cell cycle analysis by flow cytometry. C, Flow cytometric analysis of apoptosis in HK‐2 cells after exposure to CsA with or without Torin1. D, Representative fluorescence images of p62 (red) and TGF‐β1 (green) staining in CsA‐treated HK‐2 cells with or without addition of Torin1. DAPI was used to stain nuclei. Scale bar: 10 µm. E, Quantitative analyses of p62 puncta in each cell. F, Quantitative analyses of TGF‐β1 fluorescence intensity in each cell. G‐I, Western blot analysis of FN and TGF‐β1 expression in HK‐2 cells after exposure to CsA with or without Torin1. Each bar represents the mean ± SEM. *P < .05, **P < .01 and ***P < .001

Fig 3: Phosphorylation of Akt was positively related to inhibition of autophagy by M. bovis. (A) Effects of CI-1033 on viability of bMECs in DMEM medium containing 10% FBS based on CCK-8 assays. (B) Cells were pre-treated with CI-1033 (1 μm) for 1 h and then control with DMSO, and cells infected with M. bovis (MOI = 30) were further cultured for 6 h. Thereafter, cell samples were analyzed by Western blotting with anti-Akt, anti-p-Akt, anti-LC3, anti-SQSTM1, and anti-GAPDH (loading control) antibodies. (C) Relative quantification of p-Akt protein levels compared to Akt protein, LC3-II protein levels compared to LC3-I protein, and SQSTM1 protein levels compared to GAPDH protein was determined by densitometry in M. bovis-infected bMECs in the absence or presence of CI-1033. (D) Transcriptional levels of PI3K-Akt–mTOR signaling pathway and autophagy-related genes including Akt, LC3-II and SQSTM1, were detected by real-time PCR in bMECs. The data represent the mean ± SD of 3 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 4: Evaluation of the effects of ACSL4 on O-GlcNAc-mediated HCC growth. Huh-7 and SK-HEP-1 cells were transfected with OE-ACSL4 or si-ACSL4, and then the cells were harvested for the western blotting assay to detect the expression of the following proteins. (A) OGT and O-GlcNAc in Huh-7 cells. (B) UAP1, GLUT1, GLUT2, GUCY1A3, CANT1 and SGLT1 in Huh-7 cells. (C, D) OGT, O-GlcNAc, UAP1, GLUT1, GLUT2, GUCY1A3, CANT1 and SGLT1 in SK-HEP-1 cells. (E) IP assay used to detect the interaction between O-GlcNAc and ACSL4 with an antibody against O-GlcNAc or ACSL4. IgG served as a negative control. Then, the si-ACSL4-transfected or untransfected Huh-7 and SK-HEP-1 cells were treated with PUGNAc, GlcNAc or nothing, and the following assays were carried out. (F, G) The levels of ACSL4, O-GlcNAc, mTOR and p-mTOR were determined by using a western blotting assay. (H, I) The protein stability was determined by western blotting after incubation with CHX (100 μg/ml) for 0, 1, 2, 4, 8 or 24 hours. (J, K) Cell proliferation was detected by CCK-8 assay. (L) Cell apoptosis was assessed by flow cytometry assay. (A–D) *P<0.05, si-ACSL4/OE-ACSL4 group compared with control group; (E–L)*P<0.05, PUGNAc/GlcNAc group compared with control group; #P<0.05, PUGNAc + si-ACSL4/GlcNAc + si-ACSL4 group compared with PUGNAc/GlcNAc group).

Fig 5: EPO Challenge in UT7epo-E cells. SILAC labeled UT7epo-E cells (forward labeled [light= -EPO, heavy= +EPO] and reverse labeled [light= +EPO, heavy= -EPO]; K + 8 and R + 10) were challenged with +/- 5U/mL EPO for 15 min. Western blots were conducted to validate target’s response for both light and heavy SILAC labeled cells as well as reverse labeled cells to confirm Affi-BAMS assays’ results (Lt = Light, Hv = Heavy, XX designates empty lane). The MALDI MS spectrum for select targets in the multiplexed Affi-BAMS assay are shown for the forward labeled pair, as well as the western blots for mTOR (Panel A), STAT3 (Panel B), and ERK1/2 (Panel C). The mass shift due to the incorporation of one heavy arginine is noted (+10) as well as the appropriate mass shift for phosphorylation (+80) that are found on the ERK1 and ERK2 peptides (see Table 2 for additional details). The red circles indicate the masses for the singly (labeled “1”) and doubly (labeled “2”) phosphorylated peptides for ERK2. The blue circles indicate the masses for the singly (labeled “1”) and doubly (labeled “2”) phosphorylated peptides for ERK1. Raw files were imported into mMass and normalized between replicates on the most intense peak to generate ratios between the light and heavy SILAC pairs.