Journal: Communications biology

Article Title: The ATF2/miR-3913-5p/CREB5 axis is involved in the cell proliferation and metastasis of colorectal cancer.

doi: 10.1038/s42003-023-05405-w

Figure Lengend Snippet: Fig. 5 ATF2 transcriptionally regulates miR-3913-5p by binding to its promoter. a The CREB5 interacted with transcription factor proteins. b QPCR analyses of miR-3913-5p expression in CRC cells following treatment of ATF2, ATF7 or BATF3 interference. Student’s t test; *p > 0.05; ***p < 0.01; ****p < 0.001. c Cell lysates were immunoprecipitated by anti-CREB5 antibody or anti-ATF2 antibody. IgG was used as a control. Western blot analyses were performed using anti- CREB5 antibody or anti-ATF2 antibody. d The transcriptional factor ATF2-binding motif was predicted by informatics analysis. e Schematic illustration of the miR-3913-5p promoter with a potential ATF2 binding site. f Amplification of fragments containing ATF2-binding sites after ChIP assays using anti-ATF2 antibody is indicated in PCR gel. g Luciferase reporter assays were used to confirm the ATF2 binding to the promoter of miR- 3913-5p. Student’s t test; **p < 0.05; ***p < 0.01; ****p < 0.001.

Article Snippet: DNA fragments ranging from 400 to 500 bp were yielded via sonication and subjected to the immunoprecipitation process with anti-ATF2 antibody (Cell Signaling, #35031).

Techniques: Binding Assay, Expressing, Immunoprecipitation, Control, Western Blot, Luciferase

Journal: Communications biology

Article Title: The ATF2/miR-3913-5p/CREB5 axis is involved in the cell proliferation and metastasis of colorectal cancer.

doi: 10.1038/s42003-023-05405-w

Figure Lengend Snippet: Fig. 6 Inhibiting miR-3913-5p reverses the suppressions of CRC cell proliferation, migration and invasion induced by ATF2 interference. Colony formation assays (a) and EdU assays (b) revealed that inhibition of miR-3913-5p weakened the suppression of CRC cells growth by ATF2 interference. Student’s t test; ***p < 0.01; ****p < 0.001. Transwell assays showed that the suppression of CRC cells migration (c) and invasion (d) abilities induced by ATF2 interference was reversed by miR-3913-5p inhibitor. Student’s t test; ***p < 0.01; ****p < 0.001. Scale bars, 50 μm in (b–d).

Article Snippet: DNA fragments ranging from 400 to 500 bp were yielded via sonication and subjected to the immunoprecipitation process with anti-ATF2 antibody (Cell Signaling, #35031).

Techniques: Migration, Inhibition

Journal: Communications biology

Article Title: The ATF2/miR-3913-5p/CREB5 axis is involved in the cell proliferation and metastasis of colorectal cancer.

doi: 10.1038/s42003-023-05405-w

Figure Lengend Snippet: Fig. 7 miR-3913-5p is negatively correlated with ATF2 and CREB5 expression. Western blot assays (a) and qPCR assays (b) showed the CREB5, ATF2 and miR-3913-5p expression in 12 paired CRC tissues and their matched normal tissues. Student’s t test; **p < 0.05; ***p < 0.01; ****p < 0.001. c QPCR analyses of the CREB5 and ATF2 expression in 91 paired CRC tissues and their adjacent normal tissues. d The CREB5 and ATF2 expression levels in 91 pairs of CRC specimens and normal tissues. Student’s t test; **p < 0.05; ***p < 0.01. The expression correlations between CREB5 and ATF2 (e), between miR- 3913-5p and CREB5 (f), between miR-3913-5p and ATF2 (g) in CRC tissues. h ISH analyses of miR-3913-5p and IHC analyses of CREB5 and ATF2 in the CRC tissues and the corresponding normal tissues. i The illustration depicting the mechanism of ATF2/miR-3913-5p/CREB5 axis in CRC. Scale bars, 100 μm in (h).

Article Snippet: DNA fragments ranging from 400 to 500 bp were yielded via sonication and subjected to the immunoprecipitation process with anti-ATF2 antibody (Cell Signaling, #35031).

Techniques: Expressing, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Soluble IL-6 Receptor and IL-27 Subunit p28 Protein Complex Mediate the Antiviral Response through the Type III IFN Pathway.

doi: 10.4049/jimmunol.1600627

Figure Lengend Snippet: FIGURE 7. c-Fos and ATF1 are essential transcription fac- tors for activation of IFN-l1 gene expression. (A) Huh7 cells were cotransfected with IFN-l1 (0.2 mg) luciferase reporter constructs and pRL-TK (0.2 mg) together with the indicated transcription factor expression plasmids (0.6 mg). Twelve hours after transfection, cells were infected with Sendai virus (MOI = 1) for 12 h before reporter assays were performed. pRL-TK was used as an internal control. *p , 0.05. (B and C) Huh7 cells were cotransfected with IFN-l1 (0.2 mg) luciferase reporter constructs and pRL-TK (0.2 mg) together with the indicated expression plasmids (0.6 mg) (B) or the indicated shRNA (0.6 mg) (C). Twelve hours after transfection, cells were infected with Sendai virus (MOI = 1) for 12 h before reporter assays were performed. pRL-TK was used as an internal control. *p , 0.05, **p , 0.01. (D) A549 cells were infected with IAV (MOI = 1) for the indicated times. Cell lysates were examined by Western blot with c-Fos–specific Ab. (E) A549 cells were infected with IAV (MOI = 1) for the indicated times. Cell lysates were ex- amined by Western blot with ATF1 phosphospecific and total Abs. (F) Schematic diagram depicts the IFN-l1 promoter AP-1 regulatory elements and IFN-l1 site-specific mutants (left) and the results from the luciferase activity assays (right). Huh7 cells were cotransfected with pRL-TK and the indicated plasmids for 24 h prior to luciferase assays. pRL-TK was used as an internal control. **p , 0.01. (G) A549 cells were left untreated or in- fected with IAV (MOI = 1) for 12 h. ChIP assays for binding of c-Fos and ATF1 were performed (for ChIP primer location [see Fig. 6D], F3/R3, F4/R4, F5/R5). Numbers below the blots are the quantified OD; control blots were set as 10. All experiments were repeated at least three times with similar results. Graphs represent means 6 SD, n = 3. *p , 0.05.

Article Snippet: Abs against IL-6R, p28, TRAF6,MAVS, 29-59-oligoadenylate synthetase 1 (OAS1), PKR, IFNAR1, IFN-lRl, p65, p50, IRF3, IRF7, ATF1, and c-Fos (Santa Cruz Biotechnology); myxovirus resistance A (MxA) and VP1 (ProteinTech Group); p-ATF1, p38, and p-p38 (Cell Signaling Technology); hemagglutinin (HA) and Flag (Medical and Biological Laboratories, MBL); GAPDH, b-actin, and b-tubulin (Invitrogen) were purchased from the indicated manufacturers.

Techniques: Activation Assay, Gene Expression, Luciferase, Construct, Expressing, Transfection, Infection, Virus, Control, shRNA, Western Blot, Activity Assay, Binding Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Soluble IL-6 Receptor and IL-27 Subunit p28 Protein Complex Mediate the Antiviral Response through the Type III IFN Pathway.

doi: 10.4049/jimmunol.1600627

Figure Lengend Snippet: FIGURE 8. sIL-6R/p28 FP regulates IFN-l1 gene expression through p38 MAPK. (A) A549 cells were transfected with vector or sIL-6R/p28 FP expression plasmids for 24 h and were then infected with IAV (MOI = 1) for another 12 h. ChIP assays for binding of c-Fos and ATF1 were performed. *p , 0.05. (B) A549 cells were treated with SB 203580 (10 mM) or DMSO for 1 h and were then infected with IAV (MOI = 1) for another 12 h. ChIP assays for binding of c-Fos and ATF1 were per- formed. *p , 0.05. (C) A549 cells were transfected with vector or sIL-6R/p28 FP expression plasmids for 24 h before treatment with SB 203580 (10 mM) or DMSO for 1 h. Then cells were infected with IAV (MOI = 1) for another 12 h. Levels of IFN-l1 were measured by qRT-PCR. *p , 0.05. (D) A549 cells were transfected with vector or sIL-6R/p28 FP expression plasmids for 24 h and then infected with IAV (MOI = 1) for the indicated times. Cell lysates were examined by Western blot with p38 phosphospecific and total Abs. (E) A549 cells were transfected with vector or sIL-6R/p28 FP expression plasmids for 24 h and then infected with IAV (MOI = 1) for another 12 h (left). A549 cells were left untreated or treated with SB 203580 (10 mM) or DMSO for 1 h before infection with IAV (MOI = 1) for another 12 h (right). Cell lysates were examined by Western blot with c-Fos–specific Ab. (F) A549 cells were transfected with vector or sIL-6R/p28 FP expression plasmids for 24 h and were then infected with IAV (MOI = 1) for 1 h (left). Cells were left untreated or were treated with SB 203580 (10 mM) or DMSO for 1 h and then were infected with IAV (MOI = 1) for another 1 h (right). Cell lysates were examined by Western blot with ATF1 phosphospecific and total Abs. Numbers below the blots are the quantified OD; control blots were set as 10. All exper- iments were repeated at least three times with similar results.

Article Snippet: Abs against IL-6R, p28, TRAF6,MAVS, 29-59-oligoadenylate synthetase 1 (OAS1), PKR, IFNAR1, IFN-lRl, p65, p50, IRF3, IRF7, ATF1, and c-Fos (Santa Cruz Biotechnology); myxovirus resistance A (MxA) and VP1 (ProteinTech Group); p-ATF1, p38, and p-p38 (Cell Signaling Technology); hemagglutinin (HA) and Flag (Medical and Biological Laboratories, MBL); GAPDH, b-actin, and b-tubulin (Invitrogen) were purchased from the indicated manufacturers.

Techniques: Gene Expression, Transfection, Plasmid Preparation, Expressing, Infection, Binding Assay, Quantitative RT-PCR, Western Blot, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Soluble IL-6 Receptor and IL-27 Subunit p28 Protein Complex Mediate the Antiviral Response through the Type III IFN Pathway.

doi: 10.4049/jimmunol.1600627

Figure Lengend Snippet: FIGURE 9. Schematic of the proposed model for sIL-6R/p28 FP-mediated type I and type III IFN expression. Solid arrows represent signaling pathways identified in this study or in a previous study. Broken arrows indicate potential signaling pathways. Viruses (IAV, EV71, and HBV) induce sIL-6R and p28 expression. The sIL-6R/p28 FP associates with the MAVS/TRAF6 complex, leading to activation of type I IFN. In contrast, the sIL-6R/p28 FP and MAVS/TRAF6 complex induces IFN-l1 expression through regulation of the binding of IRF3, p50, and p65 to the IFN-l1 promoter. At the same time, sIL-6R/p28 FP induces the p38 MAPK signaling pathway, leading to the activation of c-Fos and ATF1, which bind to the IFN-l1 promoter and activate IFN-l1 expression.

Article Snippet: Abs against IL-6R, p28, TRAF6,MAVS, 29-59-oligoadenylate synthetase 1 (OAS1), PKR, IFNAR1, IFN-lRl, p65, p50, IRF3, IRF7, ATF1, and c-Fos (Santa Cruz Biotechnology); myxovirus resistance A (MxA) and VP1 (ProteinTech Group); p-ATF1, p38, and p-p38 (Cell Signaling Technology); hemagglutinin (HA) and Flag (Medical and Biological Laboratories, MBL); GAPDH, b-actin, and b-tubulin (Invitrogen) were purchased from the indicated manufacturers.

Techniques: Expressing, Protein-Protein interactions, Activation Assay, Binding Assay

Journal: Biomolecules & Therapeutics

Article Title: ATF3 Mediates Anti-Cancer Activity of Trans -10, cis -12-Conjugated Linoleic Acid in Human Colon Cancer Cells

doi: 10.4062/biomolther.2014.107

Figure Lengend Snippet: t 10, c 12-CLA increases ATF3 expression and apoptosis in different human colorectal cancer cells. (A, C) HCT116, LoVo and HT-29 cells were incubated with media containing indicated concentrations of t 10, c 12-CLA for 24 h. Western analysis was performed for ATF3, cleaved PARP, caspase-3, and actin as described in Methods . Data represent one experiment. (B) Apoptosis of LoVo and HT-29 cells were analyzed with Cell Death Detection ELISA PLUS Kit (Roche Diagnostics). * p <0.05 versus vehicle-treated cells.

Article Snippet: HCT-116 cells were transfected with control or ATF3 small interference RNA (siRNA; Santa Cruz; Cat# sc-29757) at a concentration of 100 nM using TransIT-TKO transfection reagent (Mirus, Madison, WI, USA), as described previously ( Yamaguchi et al ., 2004 ).

Techniques: Expressing, Incubation, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Biomolecules & Therapeutics

Article Title: ATF3 Mediates Anti-Cancer Activity of Trans -10, cis -12-Conjugated Linoleic Acid in Human Colon Cancer Cells

doi: 10.4062/biomolther.2014.107

Figure Lengend Snippet: t 10, c 12-CLA increases transcriptional activity of ATF3 gene. (A) HCT-116 cells were treated with different doses of t 10, c 12-CLA for 24 h. Total RNA was isolated and RT-PCR was performed as described in Methods . Data represent one experiment. (B) The pATF3-1850/+34 construct (0.5 μg) was co-transfected with pRL-null vector (0.05 μg). The cells were treated with indicated doses of t 10, c 12-CLA for 24 h and then luciferase activity was measured as described in Methods . Values are expressed as mean ± SD of 3 replicates. * p <0.05 versus vehicle-treated cells. (C) HCT116 cells were transfected with indicated ATF3 deletion promoter constructs (0.5 μg) with pRL-null vector (0.05 μg). The cells were treated with vehicle or 50 μM of t 10, c 12-CLA for 24 h and luciferase activity was measured. Values are expressed as mean ± SD of 3 replicates. * p <0.05 versus vehicle-treated cells.

Article Snippet: HCT-116 cells were transfected with control or ATF3 small interference RNA (siRNA; Santa Cruz; Cat# sc-29757) at a concentration of 100 nM using TransIT-TKO transfection reagent (Mirus, Madison, WI, USA), as described previously ( Yamaguchi et al ., 2004 ).

Techniques: Activity Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Construct, Transfection, Plasmid Preparation, Luciferase

Journal: Biomolecules & Therapeutics

Article Title: ATF3 Mediates Anti-Cancer Activity of Trans -10, cis -12-Conjugated Linoleic Acid in Human Colon Cancer Cells

doi: 10.4062/biomolther.2014.107

Figure Lengend Snippet: t 10, c 12-CLA does not influence ATF3 stability (A) HCT-116 cells were treated with vehicle or t 10, c 12-CLA for 24 h, and subsequently co-treated with actinomycin D. At the indicated times, total RNAs were isolated and mRNA was examined by RT-PCR. (B) The band signals were quantified with Scion Image software. The relative level of ATF3 mRNA (relative to the level of GAPDH) was calculated and the results plotted as the % of the mRNA level present at time 0 of actinomycin D treatment. Data represent one experiment.

Article Snippet: HCT-116 cells were transfected with control or ATF3 small interference RNA (siRNA; Santa Cruz; Cat# sc-29757) at a concentration of 100 nM using TransIT-TKO transfection reagent (Mirus, Madison, WI, USA), as described previously ( Yamaguchi et al ., 2004 ).

Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Software

Journal: Biomolecules & Therapeutics

Article Title: ATF3 Mediates Anti-Cancer Activity of Trans -10, cis -12-Conjugated Linoleic Acid in Human Colon Cancer Cells

doi: 10.4062/biomolther.2014.107

Figure Lengend Snippet: Dependency of ATF3 in t 10, c 12-CLA-induced GSK3β and NAG-1 expression, and apoptosis (A) HCT116 cells were exposed to different doses of t 10, c 12-CLA for 24 h and Western blot was performed GSK3β and actin. (B) The HCT116 (upper) and HT-29 (lower) cells were treated with vehicle or t 10, c 12-CLA (50 μM) for 24 h and stimulated with IGF-1 (100 ng/mL). Western analysis was performed using antibodies for phospho-Akt (Ser 473 ), total Akt and actin. (C, D) HCT-116 cells were transfected with control or ATF3 siRNA (100 nM) for 24 h and treated with t 10, c 12-CLA (50 μM) for 24 h. Western analysis was performed for ATF3, NAG-1, GSK3β, PARP, and actin. Data represent one experiment.

Article Snippet: HCT-116 cells were transfected with control or ATF3 small interference RNA (siRNA; Santa Cruz; Cat# sc-29757) at a concentration of 100 nM using TransIT-TKO transfection reagent (Mirus, Madison, WI, USA), as described previously ( Yamaguchi et al ., 2004 ).

Techniques: Expressing, Western Blot, Transfection, Control

Journal: The Journal of Clinical Investigation

Article Title: Mechanisms and treatments of neuropathic itch in a mouse model of lymphoma

doi: 10.1172/JCI160807

Figure Lengend Snippet: ( A ) Double immunostaining for ATF3 and IB4 in cervical DRGs of naive and CTCL mice at day 60. Scale bar: 25 μm. ( B ) Double immunostaining of ATF3 and the neuronal markers NF200, P2X3, and CGRP in cervical DRGs from CTCL mice at day 60. Scale bar: 25 μm. ( C ) Percentage of ATF3 + , IB4 + , NF200 + , P2X3 + , and CGRP + neurons in the cervical DRG of CTCL mice at different time points. n = 3. Two-way ANOVA, F (12, 30) = 9.15, P < 0.0001. ( D ) Percentage of ATF3 colocalization with P2X3 + neurons in the cervical DRG of CTCL mice at different time points. n = 3. One-way ANOVA, F (3, 8) = 31.89, P < 0.0001. ( E ) Pie chart showing ATF3 colocalization with NF200 + , P2X3 + , and CGRP + neurons in the cervical DRG from a CTCL mouse on day 60. Data were collected from 3 animals. ( F ) Double immunostaining for CGRP and IB4 in the cervical spinal cord dorsal horn from CTCL mice at day 60. Arrowheads indicate the loss of IB4 + primary afferents in the IIi. Scale bar: 100 μm. Data are expressed as the mean ± SEM. One- or 2-way ANOVA with Bonferroni’s post hoc test, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 versus the naive group.

Article Snippet: The primary antibodies included anti-CGRP antibody (goat, 1:400, Bio-Rad, catalog 1720-9007), anti-ATF3 antibody (mouse, 1:400, Santa Cruz Biotechnology, catalog sc-81189), anti-NF200 antibody (rabbit, 1:5,000, Abcam, catalog ab8135), anti-P2X3 antibody (mouse, 1:500, Neuromics, catalog Gp10108), anti–β–tubulin III antibody (mouse, 1:500, R&D Systems, catalog MAB1195), anti-CD68 antibody (rat, 1:500, BioLegend, catalog 137011), anti–Gr-1 antibody (rat, 1:500, BioLegend, catalog 127623), anti-CD11c antibody (Armenian hamster, 1:500, BD Pharmingen, catalog 550283), cleaved caspase 3 antibody (rabbit, 1:400, Cell Signaling Technology, catalog 9661), and anti–IB4–Alexa Fluor 488 conjugate (Invitrogen, Thermo Fisher Scientific, 1:1,000, catalog 121411).

Techniques: Double Immunostaining