Journal: The Journal of biological chemistry

Article Title: Activation transcription factor 1 involvement in the regulation of murine H-2Dd expression.

doi: 10.1074/jbc.272.25.15993

Figure Lengend Snippet: FIG. 4. Gel supershift assay with anti-ATF-1, anti-c-Jun, and anti-ATF-2 antibodies. Nuclear extracts (1 mg) from the RadLV- induced, thymoma-derived cell line were incubated with end-labeled, double-stranded oligonucleotide (1 3 105 cpm) containing H-2 BF1 target sequences. Lane 1 had no antiserum added. Specific antibodies against ATF-1 (lane 2, sc-270x; lane 3, sc-243x), c-Jun (lane 4, sc-45x), and ATF-2 (lane 5, sc-187x) were mixed with parallel binding reactions, incubated for 1 h on ice, and loaded onto gel. All antibodies were purchased from Santa Cruz Biotechnology. Supershifts of the H-2 BF1 complex with anti-ATF-1 antibodies were observed: an asterisk (*) in lane 2 and double asterisks in lane 3.

Article Snippet: Specific monoclonal antibodies against ATF-1 (products sc-270x and sc-243x), ATF-2 (product sc-187x), and c-Jun (product sc-45x) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Derivative Assay, Incubation, Labeling, 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 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

Journal: International journal of molecular sciences

Article Title: Role of Activating Transcription Factor 4 in Murine Choroidal Neovascularization Model.

doi: 10.3390/ijms22168890

Figure Lengend Snippet: Figure 1. Expression of ATF4 in Laser-induced choroidal neovascularization (CNV). (A) Western blot analysis of ATF4 and β-actin in RPE–choroid–sclera complex with laser-induced CNV lesion at 1, 3, 5 and 7 days after the laser irradiation and without CNV treatment (normal, Nor) group. Data are shown as means ± standard error of the measurements (SEMs, n = 5). * p < 0.05 vs. normal group (Student’s t-test). (B) Hematoxylin and eosin (H&E) staining of a normal mouse (Normal) and the CNV model mouse at 3 days after the laser irradiation (CNV model). The red dotted line shows the laser irradiation site. (C) Immunohistochemistry of ATF4 (red), IB4 (green), and Hoechst 33342 (Blue) at 3 days after the laser irradiation [CNV lesion (Day 3)] and Normal. For the negative control, the cross-section of CNV lesion at 3 days after the laser irradiation was stained with secondary antibody and Hoechst 33342. The scale bars are 50 µm (B,C). ONL, outer nuclear layer; INL, inner nuclear layer.

Article Snippet: After washing with PBS for 5 min, the sections were incubated with ATF4 rabbit monoclonal antibody (1:50, Cell Signaling Technology, Danvers, MA, USA) and fluorescein-labeled Griffonia simplicifolia lectin I (GSL I) IB4 (20 μg/mL: Vector Laboratories, Burlington, VT, USA) overnight at 4 ◦C.

Techniques: Expressing, Western Blot, Irradiation, Staining, Immunohistochemistry, Negative Control

Journal: International journal of molecular sciences

Article Title: Role of Activating Transcription Factor 4 in Murine Choroidal Neovascularization Model.

doi: 10.3390/ijms22168890

Figure Lengend Snippet: Figure 3. ISRIB attenuates mRNA of ATF4 and pro-angiogenic factor induced by severe ER stress. (A–D) Level of expression of the mRNA of ATF4 (A), VEGF (B), FGF2 (C), and HIF-1α (D) induced by 100 nM of thapsigargin (Tg) for 6 h were determined by qRT-PCR. Thapsigargin was added 1 h after the ISRIB exposure (0.1 or 1 µM). Data are shown as means ± SEMs (n = 5 or 6). * p <0.05, ** p < 0.01 vs. Vehicle (Dunnett’s test), # p< 0.05, ## p < 0.01 vs. Control (Welch’s t-test).

Article Snippet: After washing with PBS for 5 min, the sections were incubated with ATF4 rabbit monoclonal antibody (1:50, Cell Signaling Technology, Danvers, MA, USA) and fluorescein-labeled Griffonia simplicifolia lectin I (GSL I) IB4 (20 μg/mL: Vector Laboratories, Burlington, VT, USA) overnight at 4 ◦C.

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: International journal of molecular sciences

Article Title: Role of Activating Transcription Factor 4 in Murine Choroidal Neovascularization Model.

doi: 10.3390/ijms22168890

Figure Lengend Snippet: Figure 4. ISRIB attenuates the expression of ATF4 and VEGF induced by rhVEGF. HRMECs were cultured at a density of 4 × 104 cells/mL and incubated for 24 h. (A) Phosphorylated level of eIF2α induced by 10 ng/mL rhVEGF was determined by Western blotting. Samples were collected at 0.5, 1, 3, and 6 h after rhVEGF exposure. Data are shown as means ± SEMs (n = 5). ** p < 0.01 vs. Control (Dunnett’s test). (B,C) Level of expression of the mRNAs of ATF4 (B) and VEGF (C) induced by 10 ng/mL rhVEGF were determined by qRT-PCR. Samples were collected at 1, 3, and 6 h after rhVEGF exposure. Data are shown as means ± SEMs (n = 6). ** p < 0.01, * p < 0.05 vs. Control (Student’s t-test for B, Dunnett’s test for C). (D) Level of Expression of ATF4 induced by 10 ng/mL rhVEGF and the inhibitory effects of ISRIB were determined by qRT-PCR. For this, 1 µM ISRIB was added 1 h before the exposure to rhVEGF. Samples were collected at 1 h after rhVEGF exposure. Data are shown as mean ± SEM (n = 5 or 6). ** p < 0.01 vs. Vehicle (Dunnett’s test), ## p < 0.01 vs. Control (Student’s t-test). (E) Endogenous VEGF secretion into medium from HRMECs induced by rhVEGF and ISRIB. The level of expression of VEGF was determined by Western-blotting. Data are shown as means ± SEMs (n = 6). ** p < 0.05 vs. Vehicle (Dunnett’s test), ## p < 0.01 vs. Control (Student’s t-test).

Article Snippet: After washing with PBS for 5 min, the sections were incubated with ATF4 rabbit monoclonal antibody (1:50, Cell Signaling Technology, Danvers, MA, USA) and fluorescein-labeled Griffonia simplicifolia lectin I (GSL I) IB4 (20 μg/mL: Vector Laboratories, Burlington, VT, USA) overnight at 4 ◦C.

Techniques: Expressing, Cell Culture, Incubation, Western Blot, Control, Quantitative RT-PCR

Journal: Experimental & molecular medicine

Article Title: Upregulation of the serine palmitoyltransferase subunit SPTLC2 by endoplasmic reticulum stress inhibits the hepatic insulin response.

doi: 10.1038/s12276-022-00766-4

Figure Lengend Snippet: Fig. 2 Regulation of the ER stress markers Sptlc1 and Sptlc2 by tunicamycin in wild-type (WT) mice. Tunicamycin (2.5 µg/g body wt) was injected intraperitoneally into C57BL/6 J WT mice. The liver was isolated for mRNA expression analysis at 0, 1, 2, 4, 6 and 24 h after injection. The expression levels of ER stress markers, including ATF4 (a), ATF6 (b), unspliced XBP1 (uXBP1) (c), spliced XBP1 (d), CHOP (e), GRP78 (f), Sptlc1 (g), and Sptlc2 (h), were measured by qRT–PCR. *p < 0.05 vs. 0 h. n = 3.

Article Snippet: Antibodies against phospho-insulin receptor β subunit (IRβ) (Tyr1162/1163), XBP1 and ATF4 (CREB-2) were purchased from Santa Cruz Biotechnologies (Dallas, TX, USA); IRβ was purchased from Upstate (Burlington, MA, USA); Sptlc1 was purchased from BD Biosciences (Hampton, MA); and Sptlc2 was purchased from Abcam (Cambridge, UK).

Techniques: Injection, Isolation, Expressing, Quantitative RT-PCR