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: 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: KCC2 immunoreactivity in brainstem oculomotor, trochlear, abducens and facial motoneurons, in control and after axotomy, in the rat. A, C, E, G Confocal images of double immunofluorescence against ChAT (green) and pKCC2 (red) in control oculomotor (A), trochlear (C), abducens (E) and facial (G) motoneurons. B, D, F, H Confocal images of triple immunofluorescence against ChAT (green), KCC2 (red) and ATF3 (white) in the same brainstem nuclei, but after axotomy. Axotomized oculomotor, trochlear and facial motoneurons showed a marked reduction in pKCC2 and ChAT. ATF3 is a general marker of axotomized motoneurons and labels the cell nucleus, as can be observed in axotomized oculomotor (B), trochlear (D) and facial (H) motoneurons (some examples are indicated by white arrows). However, in axotomized abducens motoneurons (F), immunostaining for pKCC2 and ChAT showed a similar appearance to control (E), and ATF3 labeling was absent. I Quantification of KCC2 immunofluorescence in the four nuclei (oculomotor, trochlear, abducens and facial) and in the control and axotomy situation. Asterisks indicate significant (p < 0.001) difference between control and axotomized motoneurons within the same nucleus. Hashtag indicates significant difference (p < 0.001) between axotomized abducens motoneurons and the axotomized motoneurons of the other three nuclei (Two-way ANOVA followed by Holm-Sidak method; n = 35, 36, 33 and 39 for control and n = 33, 35, 44 and 42 for axotomized motoneurons of the oculomotor, trochlear, abducens and facial nuclei, respectively). Data in histograms represent mean ± SEM. Depicted to the right are the Cumming plots of estimated differences after bootstrap resampling with average difference indicated by a dot and 95% CI limit of the distribution by the vertical bars. Oculomotor, trochlear and facial motoneurons all showed significant reduction of 72%, 89% and 87% of the control value, respectively (two-sided permutation t-test p < 0.001 for all comparisons). There were no significant differences when comparing axotomized and control motoneurons in the abducens nucleus. Scale bar = 30 μm in H for A–H

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: Control, Immunofluorescence, Marker, Immunostaining, Labeling

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: KCC2 changes induced by axotomy in cat spinal motoneurons. A–H High magnification single plane confocal images of spinal motoneurons immunostained against ChAT, pKCC2 and ATF3. A–D corresponds to a control motoneuron and E–H to a motoneuron axotomized 21 days previously. Individual immunoreactivities are presented in isolation and then merged (D, H), as indicated in the figure. Axotomized motoneurons expressed ATF3 in the nucleus (arrow in G) and decreased ChAT immunoreactivity (E). They also lacked surface pKCC2 immunoreactivity in the cell body and proximal dendrite surfaces (F). I Axotomized spinal motoneuron immunolabeled for ChAT and ATF3. J KCC2b immunofluorescence of the same axotomized motoneuron as in I illustrating lack of KCC2b labeling in its somatic membrane. K Merge image of I and J. L Comparison of pKCC2 immunofluorescence between control and axotomized spinal motoneurons. Axotomized spinal motoneurons showed a significantly (asterisk) lower level of pKCC2 than control spinal motoneurons (t-test, p ≤ 0.001; n = 96 and 101 control and axotomized motoneurons, respectively). M Swarm dot plots of raw data (individual motoneurons) and differences between control (blue) and axotomy (yellow) shown in Cumming estimation plots. The distribution of differences obtained from bootstrap resampling are shown with the mean difference depicted as a dot and the 95% confidence interval indicated by the ends of the vertical error bars. A 70% significant decrease was detected in axotomized spinal motoneurons (two-sided permutation t-test p < 0.001). N Bar chat illustrating the results of a two-way ANOVA test and Holm-Sidak method comparing the following two factors: experimental condition (control versus axotomy) and type of KCC2 immunoreactivity (pKCC versus KCC2b). Data were gathered from one cat stained in serial sections with pKCC2 and KCC2b (control, n = 29 and 26 motoneurons; axotomy, n = 33 and 27 motoneurons for pKCC2 and KCC2b, respectively). Two-way ANOVA detected significant differences in control vs. axotomized motoneurons (asterisk, p < 0.001), but no difference between pKCC2 and KCC2b (p = 0.099, n.s.), or the interaction between axotomy and type of immunoreactivity (p = 0.334). Post hoc Holm-Sidak pairwise comparisons revealed significant difference between control and injured motoneuron for pKCC2 and KCC2b (#, p < 0.001 for both). O Swarm dot plots of raw data (individual motoneurons) and differences between pKCC2 in control (blue), KCC2b in control (yellow) and pKCC2 after axotomy (green) and KCC2b after axotomy (red) all shown in Cumming estimation plots. The distribution of differences obtained from bootstrap resampling are shown with the mean difference depicted as a dot and the 95% confidence interval indicated by the ends of the vertical error bars. No significant differences were found between KCC2b and pKCC2 in control motoneurons. Axotomized motoneurons showed a 70% significant decrease compared to their respective antibody matched controls (two-sided permutation t-test p < 0.001 in both comparisons). Scale bars = 30 μm in H for A–H; 20 μm in K for I–K

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: Control, Isolation, Immunolabeling, Immunofluorescence, Labeling, Membrane, Comparison, Staining

Journal: Clinical and Translational Medicine

Article Title: ATF3 is a neuron‐specific biomarker for spinal cord injury and ischaemic stroke

doi: 10.1002/ctm2.1650

Figure Lengend Snippet: RNA‐Seq of mouse spinal cord after spinal cord injury. (a) Volcano plots of mouse spinal cord RNA‐sequencing (RNA‐Seq) results showing that Atf3 , highlighted in purple, is one of the most significantly upregulated genes 4 h after spinal cord injury (SCI) (adjusted Benjamini‒Hochberg false discovery rate [BHFDR] p < .05). (b) Top 20 in Gene Ontology (GO) analysis of differentially expressed genes (DEG) from RNA‐Seq, showing multiple major pathways including mitogen‐activated protein kinase (MAPK) cascade, positive regulation of cell death, and regulation of extrinsic apoptotic signalling pathway, and negative regulation of phosphorus metabolic pathway.

Article Snippet: Commercial mouse or human ATF3 enzyme‐linked immune‐sorbent assay (ELISA) kits (Aviva Systems Biology, Cat# OKDD00746 and OKDD01469) were used to quantitate ATF3 protein levels in plasma (mouse samples) or serum (human samples) using standard sandwich ELISA technology.

Techniques: RNA Sequencing

Journal: Clinical and Translational Medicine

Article Title: ATF3 is a neuron‐specific biomarker for spinal cord injury and ischaemic stroke

doi: 10.1002/ctm2.1650

Figure Lengend Snippet: Activating transcription factor 3 (ATF3) induction in the neurons of injured hemi‐cord after spinal cord injury (SCI). (a) Quantitative reverse transcriptase polymerase chain reaction (qRT‐PCR) confirms the remarkably increased Atf3 gene expression in mouse spinal cord 4 h after SCI. The results are normalized to Atf3 expression in control animals. Data are presented as mean ± SEM and were analysed with unpaired two‐tailed t ‐test, *** p < .001, n = 3 in each group. (b) Representative immunohistochemical staining of ATF3 and NeuN in control and injured hemi‐cord 1 day after SCI. All ATF3 + cells are NeuN + 1 day after SCI. Scale bar = 40 µm. The images are the magnification of the squared areas in Figure .

Article Snippet: Commercial mouse or human ATF3 enzyme‐linked immune‐sorbent assay (ELISA) kits (Aviva Systems Biology, Cat# OKDD00746 and OKDD01469) were used to quantitate ATF3 protein levels in plasma (mouse samples) or serum (human samples) using standard sandwich ELISA technology.

Techniques: Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Gene Expression, Expressing, Control, Two Tailed Test, Immunohistochemical staining, Staining

Journal: Clinical and Translational Medicine

Article Title: ATF3 is a neuron‐specific biomarker for spinal cord injury and ischaemic stroke

doi: 10.1002/ctm2.1650

Figure Lengend Snippet: Activating transcription factor 3 (ATF3) is induced in the neurons in peri‐infarct area after ischaemic stroke. (a) Representative immunohistochemical staining of NeuN and ATF3 in control and peri‐infarct ischaemia region 1 day after permanent distal middle cerebral artery occlusion (pMCAO) in mice. All ATF3 + cells are NeuN + . (b) Representative immunohistochemical staining of ATF3 and Fluoro‐Jade C (FJC, a known marker for degenerating neurons) in the peri‐infarct ischemia region 1 day after pMCAO in mice. All FJC + cells are ATF3 + (arrowheads), but some ATF3 + cells are FJC − (arrows). Scale bar = 50 µm.

Article Snippet: Commercial mouse or human ATF3 enzyme‐linked immune‐sorbent assay (ELISA) kits (Aviva Systems Biology, Cat# OKDD00746 and OKDD01469) were used to quantitate ATF3 protein levels in plasma (mouse samples) or serum (human samples) using standard sandwich ELISA technology.

Techniques: Immunohistochemical staining, Staining, Control, Marker

Journal: Clinical and Translational Medicine

Article Title: ATF3 is a neuron‐specific biomarker for spinal cord injury and ischaemic stroke

doi: 10.1002/ctm2.1650

Figure Lengend Snippet: Increased plasma activating transcription factor 3 (ATF3) protein levels after rodent spinal cord injury (SCI) or ischaemic stroke. Enzyme‐linked immune‐sorbent assay (ELISA) results showing ATF3 protein level was detectable in mouse plasma, and its level was increased significantly post‐SCI (a) or ischaemic stroke (b). Data are presented as mean ± SEM and are analysed with unpaired two‐tailed t ‐test, *** p < .001, n = 4−7 in each group.

Article Snippet: Commercial mouse or human ATF3 enzyme‐linked immune‐sorbent assay (ELISA) kits (Aviva Systems Biology, Cat# OKDD00746 and OKDD01469) were used to quantitate ATF3 protein levels in plasma (mouse samples) or serum (human samples) using standard sandwich ELISA technology.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Clinical and Translational Medicine

Article Title: ATF3 is a neuron‐specific biomarker for spinal cord injury and ischaemic stroke

doi: 10.1002/ctm2.1650

Figure Lengend Snippet: Serum activating transcription factor 3 (ATF3) is elevated in clinical spinal cord injury (SCI) and ischaemic stroke patients 24 h after injury. (a) Human serum ATF3 levels were measured using a commercially available enzyme‐linked immune‐sorbent assay (ELISA) kit in healthy control ( n = 7), trauma control patients without SCI or traumatic brain injury (TBI) 24 h after injury ( n = 7), and SCI patients 24 h after injury ( n = 30). Serum ATF3 levels in SCI patients were significantly higher than those in healthy control and trauma control patients, with no statistical difference between healthy control and trauma control groups. (b) The serum ATF3 levels in patients with different severity of SCI. American Spinal Injury Association Impairment Scale (AIS) D represents mild SCI, while AIS A indicates the most severe SCI. (c) Human serum ATF3 levels were measured by ELISA from non‐stroke patient controls ( n = 8) and patients within 24 h of ischaemic stroke ( n = 21). Serum ATF3 levels were significantly elevated in stroke patients. (d) The serum ATF3 levels in patients with different NIH Stroke Score/Scale (NIHSS) at 24 h. Data are presented as mean ± SEM and are analysed with one‐way analysis of variance (ANOVA) with Bonferroni's multiple comparison tests (a, b and d) or unpaired two‐tailed t ‐test (c), **** p < .0001, *** p < .001, ** p < .01, * p < .05 and ‘ns’ as not statistically significant.

Article Snippet: Commercial mouse or human ATF3 enzyme‐linked immune‐sorbent assay (ELISA) kits (Aviva Systems Biology, Cat# OKDD00746 and OKDD01469) were used to quantitate ATF3 protein levels in plasma (mouse samples) or serum (human samples) using standard sandwich ELISA technology.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Comparison, Two Tailed Test

Journal: Clinical and Translational Medicine

Article Title: ATF3 is a neuron‐specific biomarker for spinal cord injury and ischaemic stroke

doi: 10.1002/ctm2.1650

Figure Lengend Snippet: Atf3 knockout (KO) mice had worse neurological outcomes in spinal cord injury (SCI) or ischaemic stroke models. (a) Paw placement in a cylinder task showing that Atf3 KO mice had worse functional recovery after SCI compared to wild‐type (WT) mice. Sticker removal time from right paw (b) and quantification of left turns in corner test (c) showing that Atf3 KO mice had more severe sensorimotor dysfunction than WT mice 3 days after left permanent distal middle cerebral artery occlusion (pMCAO). Data are presented as mean ± SEM, n = 7−12 in each group and are analysed with two‐way analysis of variance (ANOVA) and Sidak's multiple comparisons tests, **** p < .0001, *** p < .001 and ‘ns’ as not statistically significant.

Article Snippet: Commercial mouse or human ATF3 enzyme‐linked immune‐sorbent assay (ELISA) kits (Aviva Systems Biology, Cat# OKDD00746 and OKDD01469) were used to quantitate ATF3 protein levels in plasma (mouse samples) or serum (human samples) using standard sandwich ELISA technology.

Techniques: Knock-Out, Functional Assay

Journal: Clinical and Translational Medicine

Article Title: ATF3 is a neuron‐specific biomarker for spinal cord injury and ischaemic stroke

doi: 10.1002/ctm2.1650

Figure Lengend Snippet: Atf3 knockout (KO) mice had worse tissue injury after spinal cord injury (SCI) or ischaemic stroke. (a) SCI lesion area measured by Eriochrome cyanine (EC) staining, with the schematic outline, and (b) the quantification of the injury area in spinal cord of wild‐type (WT) and Atf3 KO mice 2 weeks after SCI. Scale bar = 200 µm. The injury size, presented as the percentage of ipsilateral lesion area in total contralateral uninjured area, was larger in Atf3 KO mice than WT mice 2 weeks post‐SCI. n = 6 or 7 in each group. Representative images of cresyl violet‐stained serial brain sections 3 days after permanent distal middle cerebral artery occlusion (pMCAO) (c) and their quantification (d) showing that Atf3 KO mice had larger infarct volume than WT mice. Scale bar = 1 mm. n = 6 in each group. (e) Atf3 KO mice had increased numbers of FJC + degenerating neurons 3 days after stroke. n = 6 in each group. Data are presented as mean ± SEM and are analysed with unpaired two‐tailed t ‐test, *** p < .001.

Article Snippet: Commercial mouse or human ATF3 enzyme‐linked immune‐sorbent assay (ELISA) kits (Aviva Systems Biology, Cat# OKDD00746 and OKDD01469) were used to quantitate ATF3 protein levels in plasma (mouse samples) or serum (human samples) using standard sandwich ELISA technology.

Techniques: Knock-Out, Staining, Two Tailed Test

Journal: Journal of Personalized Medicine

Article Title: Transcription Factor ATF3 Participates in DeltaNp63-Mediated Proliferation of Corneal Epithelial Cells

doi: 10.3390/jpm13040700

Figure Lengend Snippet: ATF3 expression is upregulated in ΔNp63 -overexpressing CECs.

Article Snippet: The pCMV- ATF3 plasmid (SC108959) carrying an ATF3 (NM_001674) human untagged clone was purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Expressing

Journal: Journal of Personalized Medicine

Article Title: Transcription Factor ATF3 Participates in DeltaNp63-Mediated Proliferation of Corneal Epithelial Cells

doi: 10.3390/jpm13040700

Figure Lengend Snippet: ΔNp63 upregulates ATF3 expression in CECs. ( A ) Human CECs were infected with Ad- ΔNp63 or Ad- GFP (control) for 6 h. Cells were harvested 2 days after infection, and total RNA was extracted to analyze the mRNA expression level of the genes; ( B ) Human CECs were treated with si- ΔNp63 , Ad- ΔNp63 , or control plasmids (si-control + Ad- GFP ). The Cells were harvested 2 days after transfection, and the cell lysates were used to assess the protein levels of ΔNp63 and ATF3 via western blot analysis. ** p < 0.01 versus control.

Article Snippet: The pCMV- ATF3 plasmid (SC108959) carrying an ATF3 (NM_001674) human untagged clone was purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Expressing, Infection, Control, Transfection, Western Blot

Journal: Journal of Personalized Medicine

Article Title: Transcription Factor ATF3 Participates in DeltaNp63-Mediated Proliferation of Corneal Epithelial Cells

doi: 10.3390/jpm13040700

Figure Lengend Snippet: ΔNp63 increases ATF3 promoter activity in CECs. ( A ) The p63-binding sites in the human ATF3 promoter region (−1249 to +12) were deduced using p63 motif analysis software (p63scan algorithm). Position weight matrices (matrix sites) were matched to identify p63 motifs (−1044 to −1025); ( B ) Human (HuLmP1) or rabbit (RbLmP1) CECs were co-transfected with β-gal plasmids (a transfection control) and either pGL3-Basic, wt-Hu ATF3 , del-Hu ATF3 , wt-Hu ATF3 +Ad- ΔNp63 or del-Hu ATF3 +Ad- ΔNp63 for 16 h. After 48 h of transfection, the cells were harvested and their luciferase activity and β-gal activity were measured. ** p < 0.01.

Article Snippet: The pCMV- ATF3 plasmid (SC108959) carrying an ATF3 (NM_001674) human untagged clone was purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Activity Assay, Binding Assay, Software, Transfection, Control, Luciferase

Journal: Journal of Personalized Medicine

Article Title: Transcription Factor ATF3 Participates in DeltaNp63-Mediated Proliferation of Corneal Epithelial Cells

doi: 10.3390/jpm13040700

Figure Lengend Snippet: ΔNp63 induces cell proliferation through an ATF3-dependent pathway. ( A ) Rabbit CECs were transfected with pCMV- ATF3 or control plasmids for 16 h. After transfection, cells were harvested and counted on days 1, 2, 3, and 4; ( B ) Cells were harvested on day 4 and analyzed for proliferation via BrdU assay and Ki-67 staining. Scale bar: 100 μm; ( C ) Ad- ΔNp63 -infected rabbit CECs were transfected with si- ATF3 or si-control for 16 h. Cells were harvested and counted on days 1, 2, 3, and 4 after transfection. ** p < 0.01.

Article Snippet: The pCMV- ATF3 plasmid (SC108959) carrying an ATF3 (NM_001674) human untagged clone was purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Transfection, Control, BrdU Staining, Staining, Infection

Journal: Journal of Personalized Medicine

Article Title: Transcription Factor ATF3 Participates in DeltaNp63-Mediated Proliferation of Corneal Epithelial Cells

doi: 10.3390/jpm13040700

Figure Lengend Snippet: ATF3 regulates the expression of cell-cycle–related genes in CECs. ( A ) Human CECs were transfected with pCMV- ATF3 or control plasmids for 48 h. After transfection, the cells were harvested, and the expression of cell-cycle–related genes was analyzed by qRT-PCR; ( B ) Human CECs were transfected with pCMV- ATF3 or control plasmids for 16 h. Cells were harvested on day 4 after transfection and assessed for cyclin D1 and p27 KipP1 protein expression via western blot analysis. ** p < 0.01.

Article Snippet: The pCMV- ATF3 plasmid (SC108959) carrying an ATF3 (NM_001674) human untagged clone was purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Expressing, Transfection, Control, Quantitative RT-PCR, Western Blot

Journal: Journal of Personalized Medicine

Article Title: Transcription Factor ATF3 Participates in DeltaNp63-Mediated Proliferation of Corneal Epithelial Cells

doi: 10.3390/jpm13040700

Figure Lengend Snippet: ATF3 did not alter the expression of keratinocyte differentiation-related proteins in human CECs. Human CECs were transfected with pCMV- ATF3 , si- ATF3 , or control plasmids for 16 h. Cells were harvested on day 4 after transfection and assessed for the expression of keratinocyte-differentiation–related proteins via western blot analysis.

Article Snippet: The pCMV- ATF3 plasmid (SC108959) carrying an ATF3 (NM_001674) human untagged clone was purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Expressing, Transfection, Control, Western Blot

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: