Fig 2: Profiling of cytosolic proteins from the prefrontal cortex of control and alcoholic subjects.Control (C) or alcoholic (A) prefrontal cortex cytosolic proteins were resolved by 1D PAGE and proteins stained with colloidal Coomassie. Alcoholic subjects displayed a prominent reduction in the level of protein staining at protein bands of ∼270 kDa and ∼50 kDa (upper panel). Protein levels were visualized by Western blotting (lower panel). Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PIMT, protein-L-isoaspartate O-methyltransferase.

Fig 3: PIMT inhibits ICAM-1-mediated signaling events in ECs.(A) Human umbilical vein endothelial cells (HUVECs) transduced with indicated adenovirus (moi = 10, 48 hr) were stimulated with either vehicle or TNF-a (10 ng/ml) for 12 hr. Left, expression of ICAM-1 on the surface of HUVECs was labeled by fluorescent antibody and determined by flow cytometry. Right, the statistical summary of fluorescence intensity. ***p<0.001, two-way ANOVA coupled with Tukey’s post hoc test, and n=4. (B) HUVECs were transduced with Ad-LacZ or Ad-PIMT (moi = 10) for 48 hr and then stimulated with TNF-a (10 ng/ml) for 12 hr and labeled with biotin. Precipitation was performed using streptavidin agarose beads, followed by western blot to detect the cell surface ICAM-1. (C) Western blot of ICAM-1 in different cellular fractions. HUVECs were fractionated after indicated virus transduction (moi, 10) and TNF-a (10 ng/ml, 12 hr) incubation. GAPDH, CD31, and acetyl-a-tubulin were used as cytosol, membrane, and cytoskeleton markers, respectively. (D) HUVECs were transduced with Ad-LacZ or Ad-PIMT (moi = 5, 48 hr) and stimulated with TNF-a (10 ng/ml) for 12 hr to induce both ICAM-1 bands, then subjected to the treatment with 10 µM cycloheximide (CHX) for indicated intervals. The half-life of ICAM-1 was determined by western blot (n=3), ***p<0.001, unpaired two-sided t test. (E) HUVECs transduced with adenovirus expressing LacZ or PIMT were stimulated with TNF-a (10 ng/ml, 12 hr) and then incubated with calcein-AM-labeled THP-1 for additional 1 hr. After washing, attached THP-1 cells were visualized and counted on an inverted fluorescent microscopy (n=6), **p<0.01, and two-way ANOVA coupled with Tukey’s post hoc test. Bars, 50 µm. (F) HUVECs pre-incubated with or without tunicamycin (TM, 2.5 µM) for 1 hr were exposed to indicated treatments and then subjected for ICAM-1 ligation using anti-ICAM-1 antibody followed by secondary antibody incubation. IgG were used as a negative cross-linking control. Phosphorylated and total ERK1/2 were determined by western blot and quantified by densitometric analysis (n=3), *p<0.05, **p<0.01, and two-way ANOVA coupled with Tukey’s post hoc test. (G) Schematic diagram of suppressing LPS-stimulated endothelial activation by PIMT. Figure 6—source data 1.Effect of PIMT on the membrane expression of ICAM-1. Figure 6—source data 2.Effect of PIMT on the levels of ICAM-1 in different cellular fractions. Figure 6—source data 3.Effect of PIMT on ICAM-1 protein stability. Figure 6—source data 4.Effect of PIMT on ERK1/2 activation after ICAM-1 ligation.

Fig 4: PIMT suppresses LPS-induced endothelial NF-?B transactivation and proinflammatory effect.(A) EA.hy926 cells were transfected with NF-?B-luciferase reporter together with either empty or Myc-tagged PIMT plasmids. 36 hr after transfection, cells were treated with either vehicle or LPS (1 µg/ml) for additional 12 hr and then harvested for the luciferase assay. Results are presented relative to Renilla activity (n=4). Endogenous and ectopic expression of PIMT were determined by western blot. Vec, pCS2-6×Myc empty vector; WT, wild-type PIMT; Del, catalytic defective PIMT mutant. (B) EA.hy926 cells were co-transfected with NF-?B luciferase reporter together with increased amounts of Myc-tagged PIMT plasmid. 36 hr after transfection, cells were stimulated with either vehicle or LPS (1 µg/ml) for 12 hr and then harvested for luciferase assay (n=4). Expression of PIMT was determined by western blot. (C) EA.hy926 cells were transduced with lentivirus expressing control shRNA (sh-Ctrl) or PIMT shRNAs (sh1-PIMT or sh2-PIMT) for 36 hr and then transfected with NF-?B-luciferase reporter plasmid for additional 36 hr. Luciferase activity was determined after LPS (1 µg/ml) treatment for 12 hr (n=4). PIMT levels were determined by western blot. (D) Human umbilical vein endothelial cells (HUVECs) were transduced with lentivirus expressing sh-Ctrl or PIMT shRNAs (sh1-PIMT or sh2-PIMT) for 60 hr. The mRNA expression of ICAM-1, VCAM1, IL-1ß, IL-6, and Ccl2 was determined by quantitative real-time RT-PCR (qRT-PCR) after LPS (200 ng/ml) treatment for 12 hr (n=3). (E) HUVECs were transduced with Ad-LacZ or Ad-PIMT (moi = 10) for 48 hr and then stimulated with LPS (1 µg/ml) for indicated time points. Expression of PIMT and phosphorylation of the indicated proteins in toll-like receptor (TLR)-mediated NF-?B pathway was detected by western blot (above) and quantitated by densitometric analysis (n=3). (F), HUVECs were transduced with lentivirus expressing sh-Ctrl or PIMT shRNAs (sh1-PIMT or sh2-PIMT) for 72 hr and stimulated with LPS (200 ng/ml) for indicated time points. Expression and phosphorylation of indicated proteins were determined by western blot and quantitated by densitometric analysis (n=3). *p<0.05, **p<0.01, and ***p<0.001 (sh-Ctrl vs. sh1-PIMT). #p<0.05 and ###p<0.001 (sh-Ctrl vs. sh2-PIMT). Two-way ANOVA coupled with Tukey’s post hoc test. (G) HUVECs were transduced with Ad-LacZ or Ad-PIMT (moi = 10) for 48 hr and then treated with LPS (1 µg/ml) for 30 min. Levels of NF-?B p65 in the cytoplasmic and nuclear fractions were determined by western blot. Lamin A/C and a-Tubulin were used as nuclear and cytoplasmic markers. The representative images (left) and quantification (right, n=3) were shown. (H) HUVECs were transduced with Ad-LacZ or Ad-PIMT (moi = 10) for 48 hr, followed by LPS (1 µg/ml) stimulation for 30 min. Localization of p65 was determined by immunofluorescent staining. Nuclei were stained with DAPI. Scale bars, 20 µm. (I) HUVECs were transduced with Ad-LacZ or Ad-PIMT (moi = 10) for 48 hr and then stimulated with LPS (1 µg/ml) for 30 min. Nuclear fraction was extracted, and NF-?B DNA-binding activity was determined by electrophoretic mobility-shift assay (EMSA). Competitor was 50-fold unlabeled probe. Supershift was incubated with anti-p65 antibody. All data are representative of mean ± SD, *p<0.05, **p<0.01, ***p<0.001, and two-way ANOVA coupled with Tukey’s post hoc test. Figure 2—source data 1.Endogenous and ectopic expression of PIMT in EA.hy926 cells. Figure 2—source data 2.Expression of PIMT of PIMT in EA.hy926 cells transfected with Myc-PIMT plasmid. Figure 2—source data 3.Effect of PIMT shRNA on PIMT expression. Figure 2—source data 4.Effect of PIMT expression on NF-?B activation. Figure 2—source data 5.Effect of PIMT knockdown on NF-?B activation. Figure 2—source data 6.Effect of PIMT overexpression on NF-?B p65 cellular localization.

Fig 5: PIMT attenuates ICAM-1 expression and N-linked glycosylation in ECs.(A) Human umbilical vein endothelial cells (HUVECs) were transduced with increasing dosages of Ad-PIMT for 48 hr and then stimulated with TNF-a (10 ng/ml) for 12 hr. Expression of ICAM-1 and VCAM-1 was determined by western blot. Total protein levels of ICAM-1 and VCAM-1 and the ratio of high molecular weight (HMW) bands to lower molecular weight (LMW) bands were quantitated at different mois (normalized to GAPDH), n=3. **p<0.01, ***p<0.001, compared with the corresponding ICAM-1 or VCAM1 expression at moi of 0. # # #p<0.001, compared with the corresponding the ratio of HMW bands to LMW bands of ICAM-1 or VCAM-1 at moi of 0, two-way ANOVA coupled with Tukey’s post hoc test. (B) HUVECs transduced with indicated adenoviruses were exposed to TNF-a (10 ng/ml), IL-1ß (20 ng/ml), or LPS (1 µg/ml) for 12 hr, and expression of ICAM-1 and VCAM-1 was detected by western blot. (C) HUVECs and human lung microvascular endothelial cells (HLMVECs) were transfected with lentivirus expressing control shRNA (sh-Ctrl) or PIMT RNAs (sh1-PIMT or sh2-PIMT) for 72 hr, and expression of ICAM-1 was detected by western blot and quantitated by densitometric analysis. ***p<0.001, one-way ANOVA coupled with Tukey’s post hoc test, n=3. (D) HUVECs were transduced with Ad-PIMT (moi = 10) for 48 hr and then treated with TNF-a (10 ng/ml) for 12 hr. Immunoprecipitation was performed using anti-ICAM-1 antibody, followed by western blot to detect the interaction of ICAM-1 with PIMT. (E) HUVECs were transduced with Ad-PIMT (moi = 5) for 48 hr and then treated with TNF-a (10 ng/ml) for 12 hr. Immunoprecipitation was performed using anti-PIMT antibody, followed by western blot to detect the interaction of PIMT with ICAM-1. (F) Workflow in gel preparation of ICAM-1 from ECs for liquid chromatography and mass spectrometry (LC-MS/MS) analysis. Coomassie Blue R-250 staining showed LMW ICAM-1 immunoprecipitated from HUVECs transduced with Ad-PIMT. (G) Precursor ion peaks corresponding to two charged spectra, the m/z of the monoisotopic peak was exactly matched to the theoretical m/z (401.1649, z=3; above). Manually assigned MS/MS spectra of ICAM-1 peptide from PIMT overexpressed HUVECs (below). One representative spectrum is shown to exemplify the N-glycosite change. The asparagine (Asn) site was detected as aspartate (Asp). (H) Schematic demonstration of the N-glycosites of ICAM-1 (shown as lollipop patterns). Identified deamidated residuals were marked with red crosses. Figure 5—source data 1.Effect of PIMT on ICAM-1 and VCAM-1 expression. Figure 5—source data 2.Effect of PIMT on ICAM-1 and VCAM-1 expression in response to different inflammatory stimuli. Figure 5—source data 3.Effect of PIMT knockdown on ICAM-1 expression. Figure 5—source data 4.Interaction of PIMT with ICAM-1. Figure 5—source data 5.Interaction of PIMT with low molecular weight ICAM-1.