Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Interleukin 13 Promotes Maturation and Proliferation in Metaplastic Gastroids

doi: 10.1016/j.jcmgh.2024.101366

Figure Lengend Snippet: Antibodies Used

Article Snippet: ATF3 , Rabbit , Novus, NBP1-85816 , 1:500.

Techniques:

Journal: Journal of Lipid Research

Article Title: The flavoheme reductase Ncb5or protects cells against endoplasmic reticulum stress-induced lipotoxicity

doi: 10.1194/jlr.m900146-jlr200

Figure Lengend Snippet: Fig. 1. mRNA markers of ER stress. Primary hepatocytes were in- cubated for 12 h with fatty acid depleted BSA, 0.5 mM palmitate or 0.5 mM oleate. A: Xbp-1 mRNA splicing and Chop mRNA expression was determined by RT-PCR. Unspliced (u) and spliced (s) Xbp-1 products are indicated. Tunicamycin treated wild-type cells (Tun) served as a positive control for ER stress. Glyceraldehyde-3-phos- phate dehydrogenase (GAPDH) transcript levels served as internal control for RNA loading. B, C and D: Normalized Chop, ATF3, and ATF6 mRNA levels were measured in samples by quantitative PCR and are shown relative to levels in wild-type cells treated only with BSA. Asterisks represent statistically signifi cant differences between KO and WT: ** P < 0.01. (n = 3 animals/genotype). Error bars in- dicate mean ± SD.

Article Snippet: Anti-GRP78/BiP (GL-19) antibody was obtained from Sigma (St Louis, MO); anti-SCD1 (S-15) and anti-cytochrome b5 (H-114), anti-XBP-1 (M-186), anti-CHOP (GADD153 B-3), and anti-ATF3 (H-90) antibodies from Santa Cruz Biotechnology (Santa Cruz, CA), and anti- -actin antibody from Ambion, Inc. (Austin, TX).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Control, Real-time Polymerase Chain Reaction

Journal: Journal of Lipid Research

Article Title: The flavoheme reductase Ncb5or protects cells against endoplasmic reticulum stress-induced lipotoxicity

doi: 10.1194/jlr.m900146-jlr200

Figure Lengend Snippet: Fig. 2. Protein markers of ER stress. WT (Ncb5or+/+) or KO (Ncb5or/) primary hepatocytes were treated for 12 h with palmitic acid (0.5 mM) or fatty acid free BSA only. In addition WT hepatocytes were treated for 12 h with tunicamycin 1 g/ml. A: BiP, the cleaved N-terminal fragment of ATF6, CHOP, and ATF3 were ana- lyzed by Western blots. B: Top: Western blot determinations of XBP-1. Bottom: Western blot determinations of SCD1 and cytochrome b5. To normalize for protein loading, the same blots were reused for antibody de- tection of -actin.

Article Snippet: Anti-GRP78/BiP (GL-19) antibody was obtained from Sigma (St Louis, MO); anti-SCD1 (S-15) and anti-cytochrome b5 (H-114), anti-XBP-1 (M-186), anti-CHOP (GADD153 B-3), and anti-ATF3 (H-90) antibodies from Santa Cruz Biotechnology (Santa Cruz, CA), and anti- -actin antibody from Ambion, Inc. (Austin, TX).

Techniques: Western Blot

Journal: Journal of Lipid Research

Article Title: The flavoheme reductase Ncb5or protects cells against endoplasmic reticulum stress-induced lipotoxicity

doi: 10.1194/jlr.m900146-jlr200

Figure Lengend Snippet: Fig. 3. Dose response to palmitic acid (Palm) in wild-type and Ncb5or/- cells. A: WT and KO primary hepatocytes were treated with 0, 0.1, 0.25, and 0.5 mM palmitic acid for 12 h. Total RNA was extracted from cells and subjected to RT-PCR for analysis of XBP-1 splicing, Chop and GAPDH mRNA expression. B: ATF3 and C. ATF6 mRNA expression were determined by quantitative PCR. Asterisks represent statistically signifi cant differences between KO and WT: ** P < 0.01. (n = 3 animals/genotype). Error bars indicate mean ± SD.

Article Snippet: Anti-GRP78/BiP (GL-19) antibody was obtained from Sigma (St Louis, MO); anti-SCD1 (S-15) and anti-cytochrome b5 (H-114), anti-XBP-1 (M-186), anti-CHOP (GADD153 B-3), and anti-ATF3 (H-90) antibodies from Santa Cruz Biotechnology (Santa Cruz, CA), and anti- -actin antibody from Ambion, Inc. (Austin, TX).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction

Journal: Journal of Lipid Research

Article Title: The flavoheme reductase Ncb5or protects cells against endoplasmic reticulum stress-induced lipotoxicity

doi: 10.1194/jlr.m900146-jlr200

Figure Lengend Snippet: Fig. 6. Sensitivity to tunicamycin. Isolated primary hepatocytes from wild-type mice and NCB5OR null mice were treated for 12 h with increasing concentrations of tunicamycin. After cell col- lection, total RNA was extracted from each sample. Chop, ATF3, and ATF6 mRNA were determined by quantitative PCR. No sig- nifi cant differences were noted between KO and WT cells at each dose of tunicamycin (n = 3 animals/genotype). Error bars indi- cate mean ± SD. that CHOP-mediated apoptosis contributes signifi cantly to -cell loss in B6 Ncb5or / mice.

Article Snippet: Anti-GRP78/BiP (GL-19) antibody was obtained from Sigma (St Louis, MO); anti-SCD1 (S-15) and anti-cytochrome b5 (H-114), anti-XBP-1 (M-186), anti-CHOP (GADD153 B-3), and anti-ATF3 (H-90) antibodies from Santa Cruz Biotechnology (Santa Cruz, CA), and anti- -actin antibody from Ambion, Inc. (Austin, TX).

Techniques: Isolation, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Usp9X Regulates Cell Death in Malignant Peripheral Nerve Sheath Tumors

doi: 10.1038/s41598-018-35806-5

Figure Lengend Snippet: Usp9X inhibition causes Noxa increase and ER stress in MPNST cell lines. Ultrastructural analysis shows features of paraptosis. ( a , b ) ST88-14 cells were transfected for 24 h with either non-targeting (NT)-siRNA or Usp9X-siRNA ( a ) or treated with WP1130 at the concentration of 1.25 and 2.5 µM ( b ). Whole cell extracts were collected prior to Western blot analysis for ATF3, Noxa and ß-actin. Numbers shows protein quantification analyzed through ImageJ. N = 3. ( c – e ) Ultrastructural appearance of untreated control cells using TEM. ( f – h ) After treatment with WP1130 at the concentration of 2.5 µM ( f , g , h ) T265-2c cells showed extensive cytosolic vacuolization (f, red arrows) and swelling of ER (g, red arrowheads) and mitochondria (h, red arrows).

Article Snippet: Primary antibodies were obtained from the following sources: Usp9X (Cell Signaling, Danvers, MA #5751), Mcl-1 (Cell Signaling, Danvers, MA #5453), Noxa (Calbiochem, San Diego, CA #OP180), ATF4 (Cell Signaling, Danvers, MA #11815), ATF3 (Novus Biologicals, Littleton, CO #NBP1-85816), β-actin (Sigma, St. Louis, MO #A5316).

Techniques: Inhibition, Transfection, Concentration Assay, Western Blot, Control

Journal: Journal of Cell Science

Article Title: Key role for Rac in the early transcriptional response to extracellular matrix stiffness and stiffness-dependent repression of ATF3

doi: 10.1242/jcs.260636

Figure Lengend Snippet: Rac is dominant over Rho in the initial transcriptome-wide response to ECM stiffness and preferentially represses ATF3. (A,B) Venn diagrams of differentially expressed genes in MEFs cultured with 10% FBS for 1 h on stiff versus soft hydrogels, stiff hydrogels with or without EHT1864, or stiff hydrogels with or without CT04. (C,D) The genes regulated by ECM and Rac in A,B were compared to GO gene lists for transcription factors (TFs), transcription co-regulators (co-reg) and histone modifiers. (E,F) Log 2 (fold change) values and adjusted P -values of the genes regulated by ECM stiffness and Rac and contained within the indicated GO terms above. (G) Serum-starved MEFs were plated on soft or stiff FN-coated hydrogels with 10% FBS for 1 h with DMSO (Ctrl), EHT1864 or CT04. Atf3 mRNA levels were quantified by RT-qPCR. The graph shows mean+s.e.m. ( n =4) with results normalized to the expression level on soft hydrogels. * P <0.05; *** P <0.001 (two-tailed unpaired t -tests).

Article Snippet: The membranes were saturated with 5% BSA in 1× TBS (20 mM Tris-HCl, pH 7.5, 150 mM NaCl) with 0.1% Tween-20 and probed with primary antibodies to ATF3 (1:200, sc-188, Santa Cruz Biotechnology or 1:500, NBP1-85816, Novus Biologicals), cyclin D1 (1:200, sc-20044, Santa Cruz Biotechnology or 1:500, 681902, BioLegend), Rac1/2/3 (1:100, 2465, Cell Signaling Technology), RhoA/B/C (1:200, MA1-011, Thermo Fisher Scientific) or GAPDH (loading control; 1:1000, MA5-15738, Thermo Fisher Scientific).

Techniques: Cell Culture, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Key role for Rac in the early transcriptional response to extracellular matrix stiffness and stiffness-dependent repression of ATF3

doi: 10.1242/jcs.260636

Figure Lengend Snippet: Dose-dependent effects of ECM stiffness on Rac–GTP, Atf3 mRNA, cyclin D1 mRNA and S phase entry. Serum-starved MEFs were incubated in DMEM containing 10% FBS on FN-coated hydrogels of increasing stiffness (∼2, 8, 15 and 25 kPa). (A) Rac–GTP levels determined at 1 h and graphed relative to Rac activity on the softest hydrogel. Results show mean±s.e.m. ( n =4). (B,C) Atf3 and cyclin D1 mRNA levels determined after 9 h and graphed relative to the mRNA levels on the softest hydrogel. Results show mean±s.e.m. ( n =3). (D) The percentage of EdU-positive nuclei determined at 24 h and graphed relative to EdU incorporation on the softest hydrogel. Results show mean±s.d. ( n =3). Statistical significance for each panel was determined by one-way ANOVA; asterisks show the results of Dunnett's post-tests relative to the softest hydrogel. * P <0.05; *** P <0.001; **** P <0.0001.

Article Snippet: The membranes were saturated with 5% BSA in 1× TBS (20 mM Tris-HCl, pH 7.5, 150 mM NaCl) with 0.1% Tween-20 and probed with primary antibodies to ATF3 (1:200, sc-188, Santa Cruz Biotechnology or 1:500, NBP1-85816, Novus Biologicals), cyclin D1 (1:200, sc-20044, Santa Cruz Biotechnology or 1:500, 681902, BioLegend), Rac1/2/3 (1:100, 2465, Cell Signaling Technology), RhoA/B/C (1:200, MA1-011, Thermo Fisher Scientific) or GAPDH (loading control; 1:1000, MA5-15738, Thermo Fisher Scientific).

Techniques: Incubation, Activity Assay

Journal: Journal of Cell Science

Article Title: Key role for Rac in the early transcriptional response to extracellular matrix stiffness and stiffness-dependent repression of ATF3

doi: 10.1242/jcs.260636

Figure Lengend Snippet: ATF3 repression linked to stiffness-dependent cyclin D1 expression. (A) Serum-starved MEFs on soft or stiff FN-coated hydrogels in DMEM containing 10% FBS were treated with vehicle (DMSO) or EHT1864 for 9 h. Atf3 and cyclin D1 mRNA levels were determined from the same lysates and normalized to mRNA expression levels in cells on soft hydrogels. Results show mean±s.e.m. ( n =3). (B) MEFs were infected with adenoviruses encoding GFP (control) or Rac V12 , serum-starved, and cultured and analyzed as in panel A. Results show mean±s.e.m. ( n =3). (C) MEFs infected with adenoviruses (Ad) encoding GFP (control) or ATF3 were serum-starved, incubated on FN-coated hydrogels with 10% FBS for 15 h, and analyzed by immunoblotting for cyclin D1 and ATF3 with GAPDH as the loading control. (D) Quantification of the immunoblot results in C. The graph shows mean+s.d. with results normalized to GAPDH abundance and plotted relative to the normalized cyclin D1 signal on the soft hydrogels ( n =3). (E,F) Serum-starved ROSA and ATF3 KO MEFs were incubated on stiff FN-coated hydrogels with DMSO (Ctrl) or EHT1864 for 9 h. Lysates were analyzed for the levels of ATF3 and cyclin D1 by immunoblotting. GAPDH was used as the loading control. Panel E shows results from ROSA clone R12 and ATF3 KO clone 1-20, and panel F shows quantification of the combined results from ROSA clones R11, R12 and R15 and ATF3 KO clones 1-20 and 1-29. Data were accrued from four independent experiments, and the graph shows mean+s.d. with results normalized to GAPDH abundance and plotted relative to the normalized cyclin D1 signal in the ROSA control. (G) S phase entry was analyzed by EdU incorporation in ROSA clones (R3, R11, R12 and R15) and ATF3 KO clones (1-20, 1-29, 1-44, 1-48 and 1-49) after serum starvation and incubation on stiff FN-coated hydrogels in DMEM containing 10% FBS for 24 h with DMSO (Ctrl) or EHT1864. Results show mean+s.d. n =7 for the ROSA clones and n =8 for the ATF3 KO clones. (H) Model showing that cyclin D1 is regulated by ECM stiffness and Rac through ATF3. * P <0.05; ** P <0.01; **** P <0.0001 (D, two-tailed unpaired t -tests; F and G, one-tailed unpaired t -tests).

Article Snippet: The membranes were saturated with 5% BSA in 1× TBS (20 mM Tris-HCl, pH 7.5, 150 mM NaCl) with 0.1% Tween-20 and probed with primary antibodies to ATF3 (1:200, sc-188, Santa Cruz Biotechnology or 1:500, NBP1-85816, Novus Biologicals), cyclin D1 (1:200, sc-20044, Santa Cruz Biotechnology or 1:500, 681902, BioLegend), Rac1/2/3 (1:100, 2465, Cell Signaling Technology), RhoA/B/C (1:200, MA1-011, Thermo Fisher Scientific) or GAPDH (loading control; 1:1000, MA5-15738, Thermo Fisher Scientific).

Techniques: Expressing, Infection, Control, Cell Culture, Incubation, Western Blot, Clone Assay, Two Tailed Test, One-tailed Test

Journal: Brain structure & function

Article Title: Preservation of KCC2 expression in axotomized abducens motoneurons and its enhancement by VEGF

doi: 10.1007/s00429-023-02635-w

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: ATF3 Used for injured motoneuron identification , Recombinant protein corresponding to aa 1-103 in human ATF3 , Mouse/monoclonal , Novus Clone 1685 NBP2-34489 , AB_2786997 Recognizes the epitope: ASAIVPCLSPPGSL (Manufacturer’s information) Not present in uninjured motoneurons , 1:200.

Techniques: Comparison, Expressing, Recombinant

Journal: Cell reports

Article Title: Optic nerve regeneration screen identifies multiple genes restricting adult neural repair

doi: 10.1016/j.celrep.2021.108777

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following primary antibodies were incubated with tissue sections overnight at 4°C: anti-Iba1 (1:200, Abcam), anti-CD68 (1:250, Bio-Rad), anti-GFAP (1:500, Abcam), anti-DLK (1:100, Genetex), anti-Atf3 (1:200, Novus Biologicals), anti-GFP (1:500, Abcam), anti-Il22Ra1 (1:200, Bioss), anti-Rbpms (1:100; PhosphoSolutions), anti-βIII-tubulin antibody (1:500, Promega).

Techniques: Virus, Recombinant, SYBR Green Assay, DNA Extraction, Enzyme-linked Immunosorbent Assay, cDNA Synthesis, Gene Expression, shRNA, Expressing, Plasmid Preparation, Software

Journal: Molecular Medicine Reports

Article Title: Role of indoleamine 2,3-dioxygenase in ischemia-reperfusion injury of renal tubular epithelial cells

doi: 10.3892/mmr.2021.12111

Figure Lengend Snippet: Effect of anoxia in the presence or absence of the IDO inhibitor 1-MT on ATF4, CHOP, ATF3, p-p53 and p53 levels. Representative western blots for the levels of (A) ATF4, (B) CHOP, (C) ATF3, (D) p-p53 and (E) p53. Semi-quantification of (F) ATF4, (G) CHOP, (H) ATF3, (I) p-p53 and (J) p53 protein levels. (K) p-p53/total p53 ratio. *P<0.05 vs. control; # P<0.05 vs. control with 1-MT; ^ P<0.05 vs. anoxia; & P<0.05 vs. anoxia with 1-MT. 1-MT, 1-DL-methyltryptophan; IDO, indoleamine 2,3-dioxygenase 1; ATF4, activating transcription factor 4; CHOP; C/EBP homologous protein; ATF4, activating transcription factor 3; p-, phosphorylated; OD, optical density.

Article Snippet: Primary antibodies were specific the following proteins: IDO (1:200; cat. no. sc-25809), GCN2K (1:100; cat. no. sc-374609) (both from Santa Cruz Biotechnology, Inc.), phosphorylated at Thr899 GCN2K (p-GCN2K; 1:1,000; cat. no. ab75836; Abcam), eukaryotic translation initiation factor-2α (eIF2α; 1:100; cat. no. sc-133132; Santa Cruz Biotechnology, Inc.), p at Ser51 eIF2α (p-eIF2α; 1:1,000; cat. no. 9721; Cell Signaling Technology, Inc.), activating transcription factor 4 (ATF4; 1:500; cat. no. CSB-PA002272KA01HU), ATF3 (1:500; cat. no. CSB-PA020022) (both Cusabio Technology LLC), C/EBP homologous protein (CHOP; 1:1,000; cat. no. 5554), p53 (1:1,000; cat. no. 2524), p at Ser15 p53 (p-p53; 1:1,000; cat. no. 9284), Bax (1:1,000; cat. no. 5023) (all Cell Signaling Technology, Inc.), death receptor 5 (DR5; 1:500; cat. no. CSB-PA018500; Cusabio Technology LLC), activated cleaved caspase-3 (CC3; 1:1,000; cat. no. ab13847; Abcam), AhR (1:200; cat. no. sc-133088), cytochrome P450 family 1 subfamily A polypeptide 1 (CYP1A1; 1:500; cat. no. sc-25304) (both Santa Cruz Biotechnology, Inc.) and β-actin (1:2,500; cat. no. 4967; Cell Signaling Technology, Inc.).

Techniques: Western Blot, Control

Journal: Molecular Medicine Reports

Article Title: Role of indoleamine 2,3-dioxygenase in ischemia-reperfusion injury of renal tubular epithelial cells

doi: 10.3892/mmr.2021.12111

Figure Lengend Snippet: IDO-mediated anoxia-induced apoptosis and reoxygenation-induced ferroptosis molecular pathways. IDO-mediated anoxia-induced apoptotic molecular pathway is depicted on the left. IDO-mediated reoxygenation-induced ferroptotic molecular pathway is depicted on the right. AIMP3/p18, aminoacyl-tRNA synthetase-interacting multifunctional protein-3/p18; AhR, aryl-hydrocarbon receptor; ATF3, activating transcription factor 3; ATF4, activating transcription factor 4; ATM/ATR, ataxia-telangiectasia mutated/ataxia-telangiectasia and Rad3 related protein complex; CHOP, C/EBP homologous protein; CC3, cleaved caspase-3; CYP1A1, cytochrome P450 family 1 subfamily A polypeptide 1; DR5, death receptor 5; IDO, indoleamine 2,3-dioxygenase 1; Kyn, kynurenine; p-, phosphorylated; eIF2a, eukaryotic translation initiation factor-2α; GCN2K, general control nonderepressible-2 kinase; MRS, methionyl-tRNA synthetase; p53, p53; ROS, reactive oxygen species; Trp, tryptophan.

Article Snippet: Primary antibodies were specific the following proteins: IDO (1:200; cat. no. sc-25809), GCN2K (1:100; cat. no. sc-374609) (both from Santa Cruz Biotechnology, Inc.), phosphorylated at Thr899 GCN2K (p-GCN2K; 1:1,000; cat. no. ab75836; Abcam), eukaryotic translation initiation factor-2α (eIF2α; 1:100; cat. no. sc-133132; Santa Cruz Biotechnology, Inc.), p at Ser51 eIF2α (p-eIF2α; 1:1,000; cat. no. 9721; Cell Signaling Technology, Inc.), activating transcription factor 4 (ATF4; 1:500; cat. no. CSB-PA002272KA01HU), ATF3 (1:500; cat. no. CSB-PA020022) (both Cusabio Technology LLC), C/EBP homologous protein (CHOP; 1:1,000; cat. no. 5554), p53 (1:1,000; cat. no. 2524), p at Ser15 p53 (p-p53; 1:1,000; cat. no. 9284), Bax (1:1,000; cat. no. 5023) (all Cell Signaling Technology, Inc.), death receptor 5 (DR5; 1:500; cat. no. CSB-PA018500; Cusabio Technology LLC), activated cleaved caspase-3 (CC3; 1:1,000; cat. no. ab13847; Abcam), AhR (1:200; cat. no. sc-133088), cytochrome P450 family 1 subfamily A polypeptide 1 (CYP1A1; 1:500; cat. no. sc-25304) (both Santa Cruz Biotechnology, Inc.) and β-actin (1:2,500; cat. no. 4967; Cell Signaling Technology, Inc.).

Techniques: Control