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

Journal: Oncotarget

Article Title: ATF3 functions as a novel tumor suppressor with prognostic significance in esophageal squamous cell carcinoma

doi:

Figure Lengend Snippet: (A) Expression of ATF3 in the progression from normal epithelium to carcinoma of esophagus. Scale bar, 50μm. (B) Expression of ATF3 protein in four randomly selected, paired ESCC samples and matched normal tissues was analyzed by Western blotting. Signal intensity for the expression of ATF3 was quantified by densitometric scanning and normalized by internal control (β-actin). (C) ATF3 levels in whole-cell extracts were determined in various ESCC cell lines and immortalized esophageal epithelial cell lines. EC171, EC9706, KYSE150, EC109 and KYSE510 were ESCC cell lines. NE1, NE2 and NEcA6 were immortalized esophageal epithelial cell lines. (D) Immunofluorescence analysis of ATF3 expression in KYSE150 cells, an ESCC cell lines with high-expression of ATF3 (400×). (E) Comparison for the invasive capability of cells lines with different ATF3 expression level.

Article Snippet: Rabbit anti-human ATF3 polyclonal antibody (Rockland, Pennsylvania, USA) and Mouse anti-human ATF3 polyclonal antibody (ABGENT, San Diego, USA) were used.

Techniques: Expressing, Western Blot, Immunofluorescence

Journal: Oncotarget

Article Title: ATF3 functions as a novel tumor suppressor with prognostic significance in esophageal squamous cell carcinoma

doi:

Figure Lengend Snippet: (A) Forced expression of ATF3 in EC109 and KYSE510 ESCC cell lines were addressed by Western blotting analysis. ATF3-1# and ATF3-2# were two different ATF3-transfected cell clones; Vector was cells transfected with vector control. (B) Colony formation assay was used to evaluate the growth of ATF3-expressing cells. (C) Invasiveness assay was used to determine the effect of ATF3 forced expression on cell invasion. Representative tumor cells invaded were photographed (400×), data represent mean ± SD of triplicates. (D) RNAi-mediated knockdown (siATF3) and re-expression of ATF3 (siATF3/ATF3) in KYSE150 cells were determined by Western blotting. Colony formation assay (E) and invasiveness assay (F) were employed to address the alterations of cell growth and invasion upon ATF3 knockdown and re-expression.

Article Snippet: Rabbit anti-human ATF3 polyclonal antibody (Rockland, Pennsylvania, USA) and Mouse anti-human ATF3 polyclonal antibody (ABGENT, San Diego, USA) were used.

Techniques: Expressing, Western Blot, Transfection, Clone Assay, Plasmid Preparation, Colony Assay

Journal: Oncotarget

Article Title: ATF3 functions as a novel tumor suppressor with prognostic significance in esophageal squamous cell carcinoma

doi:

Figure Lengend Snippet: (A) & (B) ATF3 forced expression EC109 cells and the control cells were implanted subcutaneously in nude mice. Tumor volume in different time points (A) and average weight of the tumors (B) were analyzed. (C) & (D) & (E) ATF3 forced expression EC109 cells and the control cells were inoculated via tail veins of the SCID mice. (C) The photomicrographs of H&E-stained lung tissues. Representative fields were shown and metastatic colonizations were marked with arrows. (D) and (E) Quantitative analysis of the number of surface lung metastasis colonization. *, P < 0.05.

Article Snippet: Rabbit anti-human ATF3 polyclonal antibody (Rockland, Pennsylvania, USA) and Mouse anti-human ATF3 polyclonal antibody (ABGENT, San Diego, USA) were used.

Techniques: Expressing, Staining

Journal: Oncotarget

Article Title: ATF3 functions as a novel tumor suppressor with prognostic significance in esophageal squamous cell carcinoma

doi:

Figure Lengend Snippet: (A) Western blotting analysis of MMP-2 expression in ATF3 forced expression or knockdown cells. β-actin served as a loading control. (B) Zymography of the conditioned medium for the activity of MMP-2. The coomassie staining of total protein in conditioned media were used to demonstrate that equal numbers of cells were present during the conditioning of the media. (C) Immunohistochemical staining of MMP-2 in the subcutaneous tumor tissues. Scale bar, 50μm. (D) Transcriptional level of MMP-2 was addressed by real time RT-PCR. (E) SiRNA targeted ATF3 (siATF3) and siRNA targeted MMP-2 (siMMP-2) were co-transfected into KYSE150 cells. (F) MMP-2 silencing inversed the increased cell invasion mediated by ATF3 knockdown.

Article Snippet: Rabbit anti-human ATF3 polyclonal antibody (Rockland, Pennsylvania, USA) and Mouse anti-human ATF3 polyclonal antibody (ABGENT, San Diego, USA) were used.

Techniques: Western Blot, Expressing, Zymography, Activity Assay, Staining, Immunohistochemical staining, Quantitative RT-PCR, Transfection

Journal: Oncotarget

Article Title: ATF3 functions as a novel tumor suppressor with prognostic significance in esophageal squamous cell carcinoma

doi:

Figure Lengend Snippet: (A) Transfected EC109 cells and KYSE510 cells were treated with MG132 or NH 4 Cl, and then harvested for Western blotting analysis of MMP-2. (B) Expressions of MDM2, total P53 and nuclear P53 in ATF3 forced expression EC109 cells was addressed by Western blotting. β-actin and Nucleoporin p62 served as loading controls. (C) Increased expression of MDM2 was confirmed in the subcutaneous tumor tissues by immunohistochemical staining. Scale bar, 50μm. (D) ATF3 forced expression EC109 cells and the control cells were treated with MG132, MDM2 inhibitor or both MDM2 inhibitor and MG132 and then used for co-immunoprecipitation. The ubiquitinated and the total level of MMP-2 were addressed by Western blotting. (E) The reciprocal protein complexes involving ATF3, MDM2 and MMP-2 in the ATF3-expressing cells. An antibody against MMP-2 was subjected to co-IP experiment and expression of ATF3, MDM2 or MMP-2 was addressed. β-actin was served as a loading control.

Article Snippet: Rabbit anti-human ATF3 polyclonal antibody (Rockland, Pennsylvania, USA) and Mouse anti-human ATF3 polyclonal antibody (ABGENT, San Diego, USA) were used.

Techniques: Transfection, Western Blot, Expressing, Immunohistochemical staining, Staining, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Oncotarget

Article Title: ATF3 functions as a novel tumor suppressor with prognostic significance in esophageal squamous cell carcinoma

doi:

Figure Lengend Snippet: (A) Cytotoxicity of Cisplatin treatment on EC109 cells was determined by MTT assay. (B) Time course analysis for the expressions of ATF3, MDM2, MMP-2, P53 and p-ERK1/2 in EC109 cells treated with Cisplatin (4μg/ml). (C) Effect of Cisplatin on the formation of the ATF3/MDM2/MMP-2 complex. (D) ATF3 was silenced by RNAi method in the Cisplatin-treated cells. (E) MTT assay was employed to determine the effect of ATF3 silencing on Cisplatin-induced inhibition of cell growth. (F) Role of ATF3 in the Cisplatin-induced inhibition of cell invasion was addressed by invasiveness assay.

Article Snippet: Rabbit anti-human ATF3 polyclonal antibody (Rockland, Pennsylvania, USA) and Mouse anti-human ATF3 polyclonal antibody (ABGENT, San Diego, USA) were used.

Techniques: MTT Assay, Inhibition

Journal: Oncotarget

Article Title: ATF3 functions as a novel tumor suppressor with prognostic significance in esophageal squamous cell carcinoma

doi:

Figure Lengend Snippet: Proposed model illustrating opposing regulatory influences of ATF3 on MMP-2 degradation and cancer cell invasion and metastasis

Article Snippet: Rabbit anti-human ATF3 polyclonal antibody (Rockland, Pennsylvania, USA) and Mouse anti-human ATF3 polyclonal antibody (ABGENT, San Diego, USA) were used.

Techniques:

Journal: Scientific Reports

Article Title: Mechanism of Saikosaponin D in regulating ferroptosis in patient-derived lung adenocarcinoma organoids via upregulation of ATF3/CHOP/CHAC1 signaling

doi: 10.1038/s41598-025-27251-y

Figure Lengend Snippet: SSD induces ferroptosis via downregulation of GPX4 through ER stress. (A) Western blot analysis of GPX4 in PDO and A549 cells treated with 2 µM SSD and control groups, β-Actin was used as a loading control. (B) Statistical analysis of GPX4 expression levels from three independent experiments ( n = 3). (C) Molecular docking of SSD and ATF3 protein. (D) DARTS-Western blot analysis showed the resistance of ATF to pronase E digestion under the treatment of SSD (10 µM), n = 3. (E) RT-qPCR analysis of mRNA expression levels of PDO-related genes ATF3 , CHOP , and CHAC1 treatment with SSD ( n = 3). (F , G) Western blot analysis of protein expression (ATF3, CHOP, CHAC1) in A549 cells and PDOs after treatment with 2 µM SSD( n = 3). (H) Mechanism of SSD acting on lung adenocarcinoma. (Data are presented as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001). ER, endoplasmic reticulum; PDO, patient-derived organoids; SSD, saikosaponin D. SD, standard deviation.

Article Snippet: Then blocked with 5% BSA for 1 h. Reaction with specific antibodies against the following proteins: β-Actin (A00730, Genscript, China), ATF3 (DF3110, Affinity, USA), GPX4 (ET1706-45, HUABIO, China), CHOP (15204-AF6277, Affinity, USA), and CHAC1 (15207-1-AP, Proteintech, China).

Techniques: Western Blot, Control, Expressing, Quantitative RT-PCR, Derivative Assay, Standard Deviation