Journal: International Journal of Molecular Sciences

Article Title: Novel Benzoxazoles Containing 4-Amino-Butanamide Moiety Inhibited LPS-Induced Inflammation by Modulating IL-6 or IL-1β mRNA Expression

doi: 10.3390/ijms23105331

Figure Lengend Snippet: Compounds 5f and 4d attenuate inflammation in vitro. Effects of compounds (10 µM) on the protein levels of STAT3, IκB, and NF-κB in AML-12 cells. ( a ) Expression levels of inflammation related-proteins in vitro. ( b – d ) Protein expression levels were normal-ized against the indicated protein. * p < 0.05 compared with the group treated with the PBS (vehicle). + p < 0.05 compared with the group treated with the LPS. Data are pre-sented as the mean ± SD.

Article Snippet: The human keratinocytes HaCaT or the alpha mouse liver 12 (AML-12) cells were obtained from Cell Lines Service GmbH (Eppelheim, Germany).

Techniques: In Vitro, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Activation of SIRT1 by Resveratrol Represses Transcription of the Gene for the Cytosolic Form of Phosphoenolpyruvate Carboxykinase (GTP) by Deacetylating Hepatic Nuclear Factor 4? *

doi: 10.1074/jbc.M109.047340

Figure Lengend Snippet: Transcriptional repression of the PEPCK-C gene promoter by SIRT1 activators. HepG2 cells were co-transfected with 200 ng of p2000-Luc plasmid DNA and 50 ng of PKAc plasmid DNA or 50 ng of empty vector DNA, using the protocol described under “Experimental Procedures.” Cells were then treated with the indicated concentrations of SIRT1 activators, namely IsoNAM (panel A), resveratrol (panel B), and fisetin and butein (panel C) for 24 h. The inset in panel A was redrawn from data in Table 5 of Reshef and Hanson (58), in which fed rats were given 5 mmol of nicotinamide (NAM)/kg of body weight for 5 h and the activity of hepatic PEPCK-C determined. A unit of enzyme activity represents the conversion of 1 μmol of substrate to product per min. The IC50 was the concentration of IsoNAM required to achieve 50% inhibition of transcription from the PEPCK-C gene promoter. Values were expressed as mean ± S.E. of triplicate determinations. *, p < 0.001 and **, p < 0.023 as compared with untreated controls. Northern blotting (panel D) and RT-PCR (panel E) were used to determine endogenous mRNA levels of PEPCK-C in FTO-2B and HepG2 cells treated with IsoNAM for various times, or in AML12 mouse hepatocytes treated with IsoNAM at the indicated concentrations. The 18S rRNA stained with ethidium bromide or glyceraldehyde-3-phosphate dehydrogenase mRNA was used as loading controls. Panel F, IsoNAM-induced inhibition of PEPCK-C was reversible. FTO-2B cells were cultured in Krebs-Ringer bicarbonate buffer, supplemented with 2% of bovine serum albumin and 5 mm glucose in the absence (untreated) or presence of 5 mm IsoNAM (treated). After 6 h of treatment, IsoNAM was withdrawn from culture medium. Cells were collected at the time points indicated. PEPCK-C mRNA was measured using RT-PCR; the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as a control.

Article Snippet: The medium used to culture AML12 cells was also supplemented with insulin-transferrin-selenium (Invitrogen) and 40 ng/ml dexamethasone.

Techniques: Transfection, Plasmid Preparation, Activity Assay, Concentration Assay, Inhibition, Northern Blot, Reverse Transcription Polymerase Chain Reaction, Staining, Cell Culture, Control

Journal: Cells

Article Title: Ursodeoxycholic Acid Regulates Hepatic Energy Homeostasis and White Adipose Tissue Macrophages Polarization in Leptin-Deficiency Obese Mice

doi: 10.3390/cells8030253

Figure Lengend Snippet: Ursodeoxycholic acid (UDCA) alleviates high free fatty acid (HFFA)-induced hepatocyte lipogenesis, reactive oxygen species (ROS) production, and mitochondrial dysfunction in AML12 cells. AML12 cells were treated with 1 mM HFFA with 10, 30, 100 μM UDCA. ( A ) Lipid accumulation display using Oil Red O stain (red). ROS levels were measured using DCFH-DA (green) stain. Images of AML12 cells stained with Mito Tracker for mitochondria (red). qRT-PCR analysis of ( B ) Complex I, II, III, IV, and V mRNA expression in AML12 cells. Relative mRNA expression was normalized to Gapdh and then normalized to the controls. ( C ) Immunofluorescence analysis of SREBP1c (green), CD36 (red), NF-κB (green), and FXR (green) expression, and DAPI (blue) for nuclear. Scale bar, 25 μm. qRT-PCR analysis of ( D ) Srebp-1c, Fas , and Scd-1 mRNA expression in AML12 cells. In all panels, results are expressed as the mean ± S.E.M. of five independent experiments, and statistical significance of differences between means was assessed using an unpaired Student’s t -test (* p ≤ 0.05; 0 mM HFFA vs. 1 mM HFFA. # p ≤ 0.05; 1 mM HFFA vs. 1 mM HFFA+ 100 μM UDCA). UDCA, ursodeoxycholic acid; HFFA, high free fatty acid; ROS, reactive oxygen species; SREBP-1c, sterol regulatory element-binding protein-1c; CD36, cluster of differentiation 36; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; FXR, farnesoid X receptor; Fas , fatty acid synthase; Scd-1, stearoyl-CoA desaturase-1; qRT-PCR, quantitative real-time polymerase chain reaction; Gapdh , glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Alpha mouse liver 12 (AML12) cell line (passages 7–10, obtained from Bioresource Collection and Research Centre, Taiwan) was grown at 37 °C in Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 (DMEM/F12), 10% fetal bovine serum (FBS), 2 mmol/L l -Glutamine and 100 μg/mL penicillin and streptomycin (both from Gibco, AntiSel, Greece) at 5% CO 2 .

Techniques: Staining, Quantitative RT-PCR, Expressing, Immunofluorescence, Binding Assay, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: G9a/GLP-Sensitivity of H3K9me2 Demarcates Two Types of Genomic Compartments

doi: 10.1101/2020.06.26.173849

Figure Lengend Snippet: A. Western blotting with antibodies against H3K9me2, H3K9me3 and H3K9ac in DMSO and UNC0638 treated AML12 cells, with three biological repeats respectively (independent DMSO/UNC0638 treatment). H3 serves as the loading control. B. Relative grayscale of western blotting from (A) calculated by AlphaView software. P-values, t -test. ***, P < 0.001. C. Representative IF images of H3K9me3 and H3K9ac in DMSO and UNC0638 treated AML12 cells. Scale bar, 10 μm. D. Genome-wide alignments of H3K9me3 (left) and H3K9ac (right) ChIP-seq in DMSO and UNC0638 treated AML12 cells. The center of H3K9me3 heatmap is the boundary of its domain, and the center of H3K9ac heatmap is the called peak center. E. Western blotting with antibodies against H3K27ac, H3K27me3, H3K4me1 and H3K4me3 in DMSO and UNC0638 treated AML12 cells, with three biological repeats respectively (independent DMSO/UNC0638 treatment). H3 serves as the loading control. F. Representative IF images of H3K27ac, H3K27me3, H3K4me1 and H3K4me3 in DMSO and UNC0638 treated AML12 cells. Scale bar, 10 μm. G. Heatmap showing the log 2 fold enrichment of H3K27ac、H3K9ac、H3K9me3 and LB1 among five different subcompartments. H. Contact enrichment among the five subcompartments. Mean Interaction between five different compartments. I. Box plots showing the relative H3K9me2 levels (DMSO-UNC0638) at subcompartments after UNC0638 treatment in AML12 cells.

Article Snippet: AML12 cells were treated with 8 μM of UNC0638 (Selleck, S8071, Houston, TX, USA) for five days, and the same amount of DMSO (Sigma, D2650) was used as the control.

Techniques: Western Blot, Control, Software, Genome Wide, ChIP-sequencing

Journal: bioRxiv

Article Title: G9a/GLP-Sensitivity of H3K9me2 Demarcates Two Types of Genomic Compartments

doi: 10.1101/2020.06.26.173849

Figure Lengend Snippet: A. Representative IF images of H3K9me2 in DMSO and UNC0638 treated AML12 cells. Scale bar, 10 μm. B. Representative ChIP-seq tracks of H3K9me2 upon UNC0638 treatment in AML12 cells; PC1 values and smoothed LB1 DamID signals of AML12 cells . Below: zoom-in view of the highlighted region. C. Aligned H3K9me2 profiles of mirrored border regions of A/B compartments (left) and LADs (right) in DMSO and UNC0638 treated AML12 cells. D. Box plots showing relative H3K9me2 levels (DMSO-UNC0638) at GSRs and non-GSRs in AML12 cells. The numbers at the bottom show the genomic bins of 1 kb. E. Percentages of GSRs in LADs/iLADs (upper) and A/B compartments (below) in AML12 cells.

Article Snippet: AML12 cells were treated with 8 μM of UNC0638 (Selleck, S8071, Houston, TX, USA) for five days, and the same amount of DMSO (Sigma, D2650) was used as the control.

Techniques: ChIP-sequencing

Journal: bioRxiv

Article Title: G9a/GLP-Sensitivity of H3K9me2 Demarcates Two Types of Genomic Compartments

doi: 10.1101/2020.06.26.173849

Figure Lengend Snippet: A. Representative tracks of H3K9me2 ChIP-seq and difference (DMSO-UNC0638) in DMSO and UNC0638 treated AML12 cells; PC1 values and smoothed LB1 DamID signals of AML12 cells . Below: zoom-in view of the highlighted region. B. Correlation between H3K9me2 levels and PC1 values in DMSO (left) and UNC0638 (right) treated AML12 cells. C. Correlation between H3K9me2 levels and LB1 DamID signals in DMSO (left) and UNC0638 (right) treated AML12 cells. D. Correlation between PC1 values and H3K9me3 (left) / H3K27me3 (right) levels in AML12 cells. E. Correlation between LB1 DamID signals and H3K9me3 (left) / H3K27me3 (right) levels in AML12 cells. The 40 kb bins were used for the correlation analyses shown from C to F.

Article Snippet: AML12 cells were treated with 8 μM of UNC0638 (Selleck, S8071, Houston, TX, USA) for five days, and the same amount of DMSO (Sigma, D2650) was used as the control.

Techniques: ChIP-sequencing

Journal: bioRxiv

Article Title: G9a/GLP-Sensitivity of H3K9me2 Demarcates Two Types of Genomic Compartments

doi: 10.1101/2020.06.26.173849

Figure Lengend Snippet: A. Representative electron micrographs of DMSO and UNC0638 treated AML12 cells. CP, cytoplasm; NP, nucleoplasm; NL, nucleolus. Black arrowheads indicate the nuclear periphery chromatin. Scale bar, 2 μm. B. Representative tracks of smoothed LB1 DamID signals and H3K9me2 ChIP-seq data of DMSO and UNC0638 treated AML12 cells. Black bars represent locations of LADs, and colored shadows indicate switched regions. C. Proportions of switched and stable LADs in AML12 cells upon UNC0638 treatment. D. Box plots showing relative expression changes of genes associated with loss or gain of LADs in AML12 cells upon UNC0638 treatment. P-values, Wilcoxon rank-sum test. E. Proportions of the four sections (GSRs/LADs, GSRs/iLADs, non-GSRs/LADs and non-GSRs/iLADs) in the genome of AML12 cells. F. Box pots of LB1 DamID signals of the four sections in DMSO and UNC0638 treated AML12 cells. The numbers at the bottom show the probes counts of LB1 DamID. P-values, t -test.

Article Snippet: AML12 cells were treated with 8 μM of UNC0638 (Selleck, S8071, Houston, TX, USA) for five days, and the same amount of DMSO (Sigma, D2650) was used as the control.

Techniques: ChIP-sequencing, Expressing

Journal: bioRxiv

Article Title: G9a/GLP-Sensitivity of H3K9me2 Demarcates Two Types of Genomic Compartments

doi: 10.1101/2020.06.26.173849

Figure Lengend Snippet: A. Mapping statistics of Hi-C deep sequencing data of DMSO and UNC0638 treated AML12 cells, with two biological repeats respectively (independent treatments, Hi-C assay and sequencing). B. Pearson correlation coefficients of PC1 values derived from compartment analysis between replicates. C. Representative contact matrices of DMSO and UNC0638 treated AML12 cells. D. Average contact enrichment between pairs of 250 kb bins ranked by PC1 values in DMSO (1 st column) and UNC0638 (2 nd column) treated AML12 cells, and the difference between them (3 rd and 4 th columns), with two biological repeats respectively.

Article Snippet: AML12 cells were treated with 8 μM of UNC0638 (Selleck, S8071, Houston, TX, USA) for five days, and the same amount of DMSO (Sigma, D2650) was used as the control.

Techniques: Hi-C, Sequencing, Derivative Assay

Journal: bioRxiv

Article Title: G9a/GLP-Sensitivity of H3K9me2 Demarcates Two Types of Genomic Compartments

doi: 10.1101/2020.06.26.173849

Figure Lengend Snippet: A. Representative Hi-C contact matrices (obs/exp) in DMSO (left) and UNC0638 (right) treated AML12 cells. B. Average contact enrichment between pairs of 250 kb bins ranked by PC1 values in DMSO (left) and UNC0638 (middle) treated AML12 cells, and the difference between them (right). C. Box plots showing compartmentalization strength across chromosomes in DMSO and UNC0638 treated AML12 cells. P-value, two-factor ANOVA. D. Violin plots showing TAD boundary strength changes within A (left), B (middle) and between A/B (right) in AML12 cells upon UNC0638 treatment. E. Box plots showing interaction scores of the four fractions (GSRs/LADs, GSRs/iLADs, non-GSRs/LADs and non-GSRs/iLADs) in DMSO and UNC0638 treated AML12 cells. P-values, two-factor ANOVA.

Article Snippet: AML12 cells were treated with 8 μM of UNC0638 (Selleck, S8071, Houston, TX, USA) for five days, and the same amount of DMSO (Sigma, D2650) was used as the control.

Techniques: Hi-C

Journal: bioRxiv

Article Title: G9a/GLP-Sensitivity of H3K9me2 Demarcates Two Types of Genomic Compartments

doi: 10.1101/2020.06.26.173849

Figure Lengend Snippet: A. Heatmap of differentially expressed genes between DMSO and UNC0638 treated AML12 cells, with two biological replicates (independent DMSO/UNC0638 treatment, RNA libraries preparation and sequencing) for each sample. B. Top ten Gene Ontology (GO) terms of up-regulated genes upon UNC0638 treatment in AML12 cells. C. Quantification of expression changed/unchanged genes in four fractions (GSRs/LADs, non-GSRs/LADs, GSRs/iLADs and non-GSRs/iLADs) in AML12 cells. The percentages of up-regulated genes in each fractions were shown. D. Clusters of gene-centric signal changes of H3K9me2 ChIP-seq signals, PC1 values, DamID signals and ATAC-seq signals upon UNC0638 treatment in AML12 cells. Relative signal changes were calculated at promoters with bin size of 5 kb. E. Box plots showing gene expression changes in four clusters upon UNC0638 treatment in AML12 cells. F. Proportions of the cluster 4 genes in four sections (GSRs/LADs, GSRs/iLADs, non-GSRs/LADs and non-GSRs/iLADs) in AML12 cells.

Article Snippet: AML12 cells were treated with 8 μM of UNC0638 (Selleck, S8071, Houston, TX, USA) for five days, and the same amount of DMSO (Sigma, D2650) was used as the control.

Techniques: Sequencing, Expressing, ChIP-sequencing, Gene Expression

Journal: American journal of physiology. Cell physiology

Article Title: Nicotinamide N-methyltransferase upregulation via the mTORC1-ATF4 pathway activation contributes to palmitate-induced lipotoxicity in hepatocytes.

doi: 10.1152/ajpcell.00195.2021

Figure Lengend Snippet: Figure 1. Palmitate upregulates NNMT expression in he- patocytes. A: NNMT catalyzes SAM-dependent methyla- tion/degradation of nicotinamide, which serves as a predominant endogenous precursor for NAD þ biosyn- thesis via the salvage pathway. B: AML12 cells were treated with the indicated concentrations of palmitate for 16 h. NNMT gene expression was quantified using real time-qPCR. Data are expressed as means ± SD, n = 4 separate experiments. Differences between the 2 groups were determined using Student’s t test (P < 0.05; P < 0.001 vs. control). C: AML12 cells were treated with the 0.4 mM palmitate for 16 h. Protein abun- dance of NNMT was detected by Western blotting. The signal of NNMT protein band was measured by densi- tometry and then divided by the signal of its correspond- ing actin abundance in the same sample. Data are expressed as means ± SD, n = 3 separate experiments. Student’s t test was used for statistical evaluation (P < 0.001 vs. control). D: AML12 cells were treated with the 0.4 mM palmitate for 16 h. Intracellular total NAD (tNAD) concentrations were measured. Data are expressed as means ± SD, n = 3 separate experiments. Student’s t test was used for statistical evaluation (P < 0.05 vs. control). NNMT, nicotinamide N-methyltransferase.

Article Snippet: Cells were grown at 80% confluence before the exposure of treatments in various experiments. siRNA Transfection Cultured AML12 hepatocytes were transfected with mouse ATF4 siRNA (Santa Cruz, sc-35113).

Techniques: Expressing, Gene Expression, Control, Western Blot

Journal: American journal of physiology. Cell physiology

Article Title: Nicotinamide N-methyltransferase upregulation via the mTORC1-ATF4 pathway activation contributes to palmitate-induced lipotoxicity in hepatocytes.

doi: 10.1152/ajpcell.00195.2021

Figure Lengend Snippet: Figure 3. mTORC1 activation upon palmitate exposure contributes to NNMT upregulation. A: AML12 cells were pretreated with or without Torin1 (0.25 mM) or rapamycin (Rapa at 50 nM) for 2 h before palmitate (0.4 mM) expo- sure for 16 h. Total protein was extracted. Protein abun- dance of phosphorylated (p-)S6, total-S6, and actin were detected by Western blotting. The signal of p-S6 protein band was measured by densitometry and then divided by the signal of its corresponding actin abundance in the same sample. Data are expressed as means ± SD, n = 3 separate experiments. Student’s t test was used for statistical evaluation (P < 0.001 vs. control). B and C: AML12 cells were pretreated with or without Torin1 (0.25 mM) (B) or rapamycin (Rapa at 50 nM) (C) for 2 h before palmitate (0.4 mM) exposure for 16 h. Total RNA was extracted. NNMT gene expressions were detected by real time-qPCR. Data are expressed as means ± SD, n = 3 separate experiments. Differences between the two groups were determined using Student’s t test (P < 0.01; P < 0.001 vs. control). NNMT, nicotina- mide N-methyltransferase.

Article Snippet: Cells were grown at 80% confluence before the exposure of treatments in various experiments. siRNA Transfection Cultured AML12 hepatocytes were transfected with mouse ATF4 siRNA (Santa Cruz, sc-35113).

Techniques: Activation Assay, Western Blot, Control

Journal: American journal of physiology. Cell physiology

Article Title: Nicotinamide N-methyltransferase upregulation via the mTORC1-ATF4 pathway activation contributes to palmitate-induced lipotoxicity in hepatocytes.

doi: 10.1152/ajpcell.00195.2021

Figure Lengend Snippet: Figure 4. mTORC1 activation is an upstream event in palmitate induced ATF4 activation. A: AML12 cells were pretreated with or without Torin1 (0.25 mM) or rapamycin (Rapa at 50 nM) for 2 h before palmitate (0.4 mM) exposure for 16 h. Total protein was extracted. Protein abundance of ATF4 and actin were detected by Western blotting. The signal of ATF4 protein band was measured by densitometry and then divided by the sig- nal of its corresponding actin abundance in the same sample. Data are expressed as means ± SD, n = 3 separate experiments. Student’s t test was used for statistical evaluation (P < 0.001 vs. control). B: AML12 were pretreated with Torin1 (0.25 mM) for 2 h before a 16-h palmitate (0.4 mM) exposure. Total RNA was extracted. ATF4 mRNA levels were detected by real time-qPCR. Data are expressed as means ± SD, n = 4 sep- arated experiments. Differences between the two groups were deter- mined using Student’s t test (P < 0.001 vs. control). ATF, activating transcription factor.

Article Snippet: Cells were grown at 80% confluence before the exposure of treatments in various experiments. siRNA Transfection Cultured AML12 hepatocytes were transfected with mouse ATF4 siRNA (Santa Cruz, sc-35113).

Techniques: Activation Assay, Quantitative Proteomics, Western Blot, Control

Journal: American journal of physiology. Cell physiology

Article Title: Nicotinamide N-methyltransferase upregulation via the mTORC1-ATF4 pathway activation contributes to palmitate-induced lipotoxicity in hepatocytes.

doi: 10.1152/ajpcell.00195.2021

Figure Lengend Snippet: Figure 5. mTORC1 activation contributes to palmi- tate-induced ER stress and NNMT upregulation. A and B: AML12 cells were pretreated with Torin1 (0.25 mM) for 2 h before the palmitate (0.4 mM) exposure for 16 h. Total RNA was extracted. The gene expres- sions of Xbp1, Xbp1s, and Xbp1u were quantified by real time-qPCR and Xbp1s/Xbp1u ratio calculated. Data are expressed as means ± SD, n = 4 different experiments. Differences between the two groups were determined using Student’s t test (P < 0.001vs. control). C: AML12 cells were pretreated with Tornin1 for 2 h before tunicamycin (10 μm) treat- ment for 16 h. Protein abundance of p-S6 and actin was detected by Western blotting. The signal of p-S6 protein band was measured by densitometry and then divided by the signal of its corresponding actin abundance in the same sample. Data are expressed as means ± SD, n = 5 separate experiments. Student’s t test was used for statistical evaluation (P < 0.01; P < 0.0001 vs. control). D: Ten- week-old male C57BL/6N mice were injected with tunicamycin (2 mg/kg body wt ip) or isovolumic vehi- cle (150 mM dextrose) and 16 h later livers were har- vested. Protein abundance of ATF4, p-S6 and actin was detected by Western blotting. E: AML12 cells were pretreated with Torin1 (0.25 mM) for 2 h before tunicamycin (10 μm) treatment for 16 h. Protein abun- dance of ATF4 was detected by Western blotting. F: AML12 cells were pretreated with Torin1 for 2 h before tunicamycin (10 μm) treatment for 16 h. Total RNA was extracted and NNMT gene expression quantified by real time-qPCR. All data were expressed as means ± SD, n = 4 separated experiments. Differences between the two groups were determined using Student’s t test (P < 0.01; P < 0.001; P < 0.0001 vs. control). NNMT, nicotinamide N-methyl- transferase; XBP1, X-box binding protein 1.

Article Snippet: Cells were grown at 80% confluence before the exposure of treatments in various experiments. siRNA Transfection Cultured AML12 hepatocytes were transfected with mouse ATF4 siRNA (Santa Cruz, sc-35113).

Techniques: Activation Assay, Control, Quantitative Proteomics, Western Blot, Injection, Gene Expression, Binding Assay

Journal: American journal of physiology. Cell physiology

Article Title: Nicotinamide N-methyltransferase upregulation via the mTORC1-ATF4 pathway activation contributes to palmitate-induced lipotoxicity in hepatocytes.

doi: 10.1152/ajpcell.00195.2021

Figure Lengend Snippet: Figure 6. NNMT inhibition protects against palmitate-induced cell death. A: AML12 cells were pretreated with either JBSNF-000088 (20 mM) or II399 (20 mM) at the indicated concentrations for 4 h before palmitate (0.4 mM) exposure for 16 h. Cell viability was determined by LDH release mea- surement. Data are expressed as mean ± SD, n = 3 separated experi- ments. Bars with different character differ significantly (P < 0.05). B: AML12 cells were transfected with either scramble siRNA or NNMT siRNA for 24 h and treated with palmitate at 0.4 mM for 16 h. Cell death was determined by LDH release measurement. Data are expressed as means ± SD, n = 3 separated experiments. Differences between the two groups were determined using Student’s t test (P < 0.01; P < 0.001; P < 0.001 vs. control). NNMT, nicotinamide N-methyltransferase.

Article Snippet: Cells were grown at 80% confluence before the exposure of treatments in various experiments. siRNA Transfection Cultured AML12 hepatocytes were transfected with mouse ATF4 siRNA (Santa Cruz, sc-35113).

Techniques: Inhibition, Transfection, Control

Journal: American journal of physiology. Cell physiology

Article Title: Nicotinamide N-methyltransferase upregulation via the mTORC1-ATF4 pathway activation contributes to palmitate-induced lipotoxicity in hepatocytes.

doi: 10.1152/ajpcell.00195.2021

Figure Lengend Snippet: Figure 7. Protein kinase A (PKA) inhibition compro- mises the protective effect of NNMT inhibition against palmitate-induced cell death. A and B: AML12 cells were treated with either JBSNF-000088 (25 mM) or II399 (25 mM) for 6 h. Total proteins were isolated and PKA substrates detected by Western blotting. The signal of PKS substrates was measured by densitometry and then divided by the signal of its corresponding actin abundance in the same sample. Data are expressed as means ± SD, n = 3 separate experiments. Student’s t test was used for statistical evaluation (P < 0.05; P < 0.01 vs. untreated cells). C: AML12 cells were pretreated with either JBSNF-000088 (25 mM) or II399 (25 mM) at the pres- ence/absence of PKA inhibitor, either SQ22536 (200 mM) or H89 (10 mM) for 4 h before palmitate exposure for 16 h. Cell death was determined by LDH release. All data are expressed as means ± SD, n = 3 sepa- rated experiments. Differences between the two groups were determined using Student’s t test (P < 0.05; P < 0.01; P < 0.001 vs control). D: sche- matic illustration of the role and mechanism of NNMT upregulation in palmitate-induced hepatocyte lipotox- icity. The mTORC1-ATF4 pathway activation contrib- utes to palmitate-elicited NNMT upregulation and protein kinase A (PKA) activation contributes to NNMT inhibition-conferred protection against hepatolipotox- icity. NNMT, nicotinamide N-methyltransferase.

Article Snippet: Cells were grown at 80% confluence before the exposure of treatments in various experiments. siRNA Transfection Cultured AML12 hepatocytes were transfected with mouse ATF4 siRNA (Santa Cruz, sc-35113).

Techniques: Inhibition, Isolation, Western Blot, Control, Activation Assay