Journal: Cell Death & Disease

Article Title: Semaphorin 7A promotes endothelial to mesenchymal transition through ATF3 mediated TGF-β2/Smad signaling

doi: 10.1038/s41419-020-02818-x

Figure Lengend Snippet: a ATF3 mRNA level was analyzed by qPCR normalized to GAPDH. Fold changes are shown. Data are mean ± SEM, N = 3, *** p < 0.001 b ATF3 protein expression were analyzed by western blotting, normalized to tubulin. Data are mean ± SEM, N = 3, *** p < 0.001. c TGF-β2 mRNA level was analyzed by qPCR normalized to GAPDH. Fold changes are shown. Data are mean ± SEM, N = 3, * p < 0.05; ** p < 0.01. d The concentration of TGF-β2 in cell supernatant was detected by ELISA among Con335-HUVECs, Sema7A-HUVECs, and Sema7A-HUVECs + ATF3-siRNA. N = 10. Data are mean ± SEM, * p < 0.05; *** p < 0.001. e Chip-qPCR product in agarose gel electrophoresis. f Chip-qPCR TGF-β2 percentage of input in con335-HUVECs and Sema7A - HUVECs were analyzed by qPCR normalized to IgG. Data are mean ± SEM, N = 3, ** p < 0.01. g Schematic graph of the constructed reporter plasmid. TGF-β2 mut indicates the TGF-β2 mutation promoter region in ATF3 binding site. The mutated nucleotides in TGF-β2 fragments are in red letters. h Luciferase reporter assays were performed on HEK 293 T cells. Data are mean ± SEM, N = 3, ** p < 0.01 vs negative control. i , j ATF3-overexpression plasmid was transfected to HUVECs, and the mRNA levels of TGF - β2 and TGF-β1 were performed by qPCR normalized to GAPDH. Fold changes are shown. Data are mean ± SEM, N = 3, ** p < 0.01. k , l P-Smad3 protein in cells treated with siRNA was analyzed by Western blotting, normalized to total Smad3. Data are mean ± SEM, N = 3, * p < 0.05; ** p < 0.01. m – q CD31 and α-SMA RNA and proteins in cells treated with siRNA or control was analyzed by qPCR and western blotting. Data are mean ± SEM, N = 3, * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The primary antibodies used in this study include mouse anti-human α-SMA (Abcam, UK, Cat#32575), rabbit anti-human CD31(Abcam, UK, Cat#28364), rabbit anti-human FSP-1 (Abcam, UK, Cat#41532), mouse anti-GAPDH (Abcam, UK, Cat#181602), rabbit anti-mouse VWF (Abcam, UK, Cat#6994), mouse anti-mouse fibronectin (Abcam, UK, Cat#6328), mouse anti-human vimentin (Abcam, UK, Cat#8069), rabbit anti-human Sema7A (Thermo Fisher Scientific, Waltham, USA, Cat#PA5-28971), rabbit anti-human ATF3 (CST, Danvers, USA, Cat#33593), rabbit anti-human VE-cadherin (CST, Danvers,USA, Cat#2500), mouse anti-β tubulin (CST, Danvers, USA, Cat#2146s), rabbit anti-human p-Smad3 (BOSTER, China, Cat#BM4033), rabbit anti-human Smad3 (BOSTER, China, Cat#BM3919), ATF3 (Novus Biologicals, USA, Cat# 85816), and CD31 antibody (BD Biosciences, New Jersey, USA, Cat#555446).

Techniques: Expressing, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, ChIP-qPCR, Agarose Gel Electrophoresis, Construct, Plasmid Preparation, Mutagenesis, Binding Assay, Luciferase, Negative Control, Over Expression, Transfection, Control

Journal: Cell Death & Disease

Article Title: Semaphorin 7A promotes endothelial to mesenchymal transition through ATF3 mediated TGF-β2/Smad signaling

doi: 10.1038/s41419-020-02818-x

Figure Lengend Snippet: a Cells were treated with β1 integrin antibody (P5D2), and ATF3 mRNA level was analyzed by qPCR normalized to GAPDH. Fold changes are shown. Data are mean ± SEM, N = 3, * p < 0.05; ** p < 0.0.1. b ATF3 protein expression was analyzed by western blotting, normalized to tubulin. Data are mean ± SEM, N = 3, * p < 0.05; ** p < 0.01. c TGF-β2 mRNA level was analyzed by qPCR normalized to GAPDH. Fold changes are shown. Data are mean ± SEM, N = 3, * p < 0.05; ** p < 0.0.1. d The concentration of TGF-β2 in cell supernatant was detected by ELISA for Con335-HUVECs, Sema7A-HUVECs, and Sema7A-HUVECs + P5D2. Data are mean ± SEM, N = 10, * p < 0.05; *** p < 0.001. e , f Smad3 phosphorylation were analyzed by western blotting, normalized to total Smad3. Data are mean ± SEM, N = 3, * p < 0.05. g , h CD31 and α-SMA mRNA level were analyzed by qPCR normalized to GAPDH. Fold changes are shown. Data are mean ± SEM, N = 3, * p < 0.05; ** p < 0.01; *** p < 0.001. i – k CD31 and α-SMA proteins were analyzed by western blotting, normalized to tubulin. Data are mean ± SEM, N = 3, * p < 0.05; ** p < 0.01; *** p < 0.001. l ATF3 overexpression plasmid was transfected into P5D2 incubated Sema7A-HUVECs, and TGF-β2 mRNA expression in Sema7A-HUVECs + P5D2 and Sema7A-HUVECs + P5D2 + ATF3 were analyzed by qPCR normalized to GAPDH. Fold changes are shown. Data are mean ± SEM, N = 3, ** p < 0.01. m – o CD31 and α-SMA protein expressions were analyzed by western blotting, normalized to GAPDH. Data are mean ± SEM, N = 3, ** p < 0.01 (Sema7A-HUVECs + P5D2 vs Sema7A-HUVECs + P5D2 + ATF3). P5D2 β1 integrin antibody.

Article Snippet: The primary antibodies used in this study include mouse anti-human α-SMA (Abcam, UK, Cat#32575), rabbit anti-human CD31(Abcam, UK, Cat#28364), rabbit anti-human FSP-1 (Abcam, UK, Cat#41532), mouse anti-GAPDH (Abcam, UK, Cat#181602), rabbit anti-mouse VWF (Abcam, UK, Cat#6994), mouse anti-mouse fibronectin (Abcam, UK, Cat#6328), mouse anti-human vimentin (Abcam, UK, Cat#8069), rabbit anti-human Sema7A (Thermo Fisher Scientific, Waltham, USA, Cat#PA5-28971), rabbit anti-human ATF3 (CST, Danvers, USA, Cat#33593), rabbit anti-human VE-cadherin (CST, Danvers,USA, Cat#2500), mouse anti-β tubulin (CST, Danvers, USA, Cat#2146s), rabbit anti-human p-Smad3 (BOSTER, China, Cat#BM4033), rabbit anti-human Smad3 (BOSTER, China, Cat#BM3919), ATF3 (Novus Biologicals, USA, Cat# 85816), and CD31 antibody (BD Biosciences, New Jersey, USA, Cat#555446).

Techniques: Expressing, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Over Expression, Plasmid Preparation, Transfection, Incubation

Journal: Cell Death & Disease

Article Title: Semaphorin 7A promotes endothelial to mesenchymal transition through ATF3 mediated TGF-β2/Smad signaling

doi: 10.1038/s41419-020-02818-x

Figure Lengend Snippet: Carotid artery exposed to d-flow from Sema7A +/+ and Sema7A − /− mice were immunofluorescently stained for various endothelial–mesenchymal marker combinations as indicated ( a ) CD31 (red)/α-SMA (green), ( b ) CD31 (red)/FSP-1 (green), ( c ) VWF (red)/α-SMA (green), and DAPI (blue) 1 week after PCL, scale bar = 50 µm. The first column is the sham group, the second column is the single channel stained with CD31 or VWF, the third column is the merge channel stained with CD31 + SMA, CD31 + FSP, or VWF + SMA, and the fourth column is the enlarged image of the box in the third column. Co-positive cells per × 60 field were counted and shown on the right (upside). Quantification of CD31 or VWF fluorescence intensity of intima is shown on the right (downside). Data are mean ± SEM, ** p < 0.01; *** p < 0.001. d Carotid artery exposed to d-flow from Sema7A +/+ and Sema7A − /− mice were immunofluorescently stained for ATF3 (green) and CD31 (red). The first column is the single channel stained with ATF3, the second column is the enlarged image of the box in the first column, the third column is the merge channel (CD31 + ATF3), and the fourth column is the enlarged image of the box in the third column. Quantification of ATF3 fluorescence intensity of intima is shown on the right. Data are mean ± SEM, ** p < 0.01; *** p < 0.001.

Article Snippet: The primary antibodies used in this study include mouse anti-human α-SMA (Abcam, UK, Cat#32575), rabbit anti-human CD31(Abcam, UK, Cat#28364), rabbit anti-human FSP-1 (Abcam, UK, Cat#41532), mouse anti-GAPDH (Abcam, UK, Cat#181602), rabbit anti-mouse VWF (Abcam, UK, Cat#6994), mouse anti-mouse fibronectin (Abcam, UK, Cat#6328), mouse anti-human vimentin (Abcam, UK, Cat#8069), rabbit anti-human Sema7A (Thermo Fisher Scientific, Waltham, USA, Cat#PA5-28971), rabbit anti-human ATF3 (CST, Danvers, USA, Cat#33593), rabbit anti-human VE-cadherin (CST, Danvers,USA, Cat#2500), mouse anti-β tubulin (CST, Danvers, USA, Cat#2146s), rabbit anti-human p-Smad3 (BOSTER, China, Cat#BM4033), rabbit anti-human Smad3 (BOSTER, China, Cat#BM3919), ATF3 (Novus Biologicals, USA, Cat# 85816), and CD31 antibody (BD Biosciences, New Jersey, USA, Cat#555446).

Techniques: Staining, Marker, Fluorescence

Journal: Cell Death & Disease

Article Title: Semaphorin 7A promotes endothelial to mesenchymal transition through ATF3 mediated TGF-β2/Smad signaling

doi: 10.1038/s41419-020-02818-x

Figure Lengend Snippet: When exposed to d-flow, endothelial Sema7A expression is upregulated, potentially by the cAMP/CREB (cAMP response element-binding protein) pathway. Endothelial Sema7A upregulates the transcription factor ATF3 through interaction with β1 integrin, leading to TGF-β2 transcription, and activation of TGF/Smad3 signaling pathway, and thus inducing EndMT.

Article Snippet: The primary antibodies used in this study include mouse anti-human α-SMA (Abcam, UK, Cat#32575), rabbit anti-human CD31(Abcam, UK, Cat#28364), rabbit anti-human FSP-1 (Abcam, UK, Cat#41532), mouse anti-GAPDH (Abcam, UK, Cat#181602), rabbit anti-mouse VWF (Abcam, UK, Cat#6994), mouse anti-mouse fibronectin (Abcam, UK, Cat#6328), mouse anti-human vimentin (Abcam, UK, Cat#8069), rabbit anti-human Sema7A (Thermo Fisher Scientific, Waltham, USA, Cat#PA5-28971), rabbit anti-human ATF3 (CST, Danvers, USA, Cat#33593), rabbit anti-human VE-cadherin (CST, Danvers,USA, Cat#2500), mouse anti-β tubulin (CST, Danvers, USA, Cat#2146s), rabbit anti-human p-Smad3 (BOSTER, China, Cat#BM4033), rabbit anti-human Smad3 (BOSTER, China, Cat#BM3919), ATF3 (Novus Biologicals, USA, Cat# 85816), and CD31 antibody (BD Biosciences, New Jersey, USA, Cat#555446).

Techniques: Expressing, Binding Assay, Activation Assay

Journal: Experimental & Molecular Medicine

Article Title: A cell-penetrating peptide blocks Toll-like receptor-mediated downstream signaling and ameliorates autoimmune and inflammatory diseases in mice

doi: 10.1038/s12276-019-0244-0

Figure Lengend Snippet: a Cell viability was measured by the MTT assay upon the treatment of RAW 264.7 cells with various concentrations of TIP1 (12.5, 25, or 50 µM) for 24 h. Etoposide (ETO) served as a positive control because of its high cytotoxicity. b , c The cells were treated with TIP1, followed by treatment with LPS as indicated. The amounts of p-p65, Iκ-Bα, p-TBK1, TBK1, p-IRF3, IRF3, ATF3, p-ERK, ERK, p-JNK, JNK, p-p38, and p38 were measured by western blotting in the total protein extract. β-Actin served as a loading control. d The amount of p-p65 was measured by immunofluorescent staining and confocal microscopy. Hoechst 33258 was used for nucleus staining (scale bar represents 10 μm). e – g Cells were treated with TIP1 (12.5, 25, or 50 µM) in the presence or absence of LPS for 24 h. The secretion levels of TNF-α, IL-6, and IFN-β were measured by ELISAs. h The expression levels of iNOS and COX2 were measured by western blotting, and β-actin served as a loading control. i , j The production levels of intracellular and cytosolic NO were measured by DAF-FM staining and an NO secretion kit, respectively. k The production of intracellular ROS was measured by DCF-DA staining. l The secretion of TNF-α was measured by an ELISA after the treatment of RAW 264.7 cells with different concentrations of TIP1 not conjugated with CPP (12.5, 25, and 50 µM; TIP1 without CPP). m – o mBMDM cells were treated with TIP1 for 1 h in the presence or absence of LPS for 24 h. The secretion levels of ( m ) TNF-α and ( n ) IL-6 were measured by an ELISA, and the production of NO ( o ) was evaluated using an NO secretion kit. p mBMDM cells were treated with TIP1 for 1 h, followed by treatment with Poly(I:C) for 24 h. The secretion of IFN-β was measured by an ELISA. q hPBMCs were treated with TIP1 in the presence or absence of LPS. The amounts of p-ERK, ERK, p-JNK, JNK, p-p38, p38, p-p65, and Iκ-Bα were measured by western blotting. β-Actin was used as a loading control. ( r ) The secretion of TNF-α was measured by an ELISA during treatment with PAM 3 CSK 4 (affecting TLR1/2), FSL-1 (affecting TLR2/6), Poly(I:C) (affecting TLR3), R848 (affecting TLR7–TLR8), or CpG-ODN (affecting TLR9) at diverse concentrations. s The secretion of IFN-β was measured by an ELISA during Poly(I:C) (TLR3) treatment. t Cell viability was assessed by the MTT assay after the treatment of hBMNCs with various concentrations of TIP1 (12.5, 25, or 50 µM) for 24 h. u , v The secretion levels of IL-6 and TNF-α were measured by ELISAs after treatment of the hBMNCs with different concentrations of TIP1 for 1 h and LPS (affecting TLR4), PAM 3 CSK 4 (affecting TLR1/2), FSL-1 (affecting TLR2/6), Poly(I:C) (affecting TLR3), R848 (affecting TLR7 and/or TLR8), or CpG-ODN (affecting TLR9) for 24 h. The data shown represent at least three independent experiments ( n ≥ 3), and bars denote mean ± SEM (* P < 0.05, ** P < 0.01). N.D.: not detected. N.S.: not significant

Article Snippet: The membranes were immunoblotted with specific primary antibodies (1:500–1000) against p-p65, p65, p-JNK, JNK, p-IRF3, IRF3, p-TBK1, TBK1, p-ERK, ERK, p-p38, p38, Iκ-Bα, and human IL-1β (Cell Signaling Technology Inc., Danvers, MA, USA); ATF3, COX2, caspase 1, and β-actin (Santa Cruz Biotechnology Inc., Dallas, TX, USA); iNOS (BD Biosciences); IL-6 and mouse IL-1β (R&D Systems Inc.); TNF-α (Thermo Fisher Scientific, Inc.); or NLRP3 (Adipogen, San Diego, CA, USA) with gentle shaking at 4 °C overnight.

Techniques: MTT Assay, Positive Control, Western Blot, Control, Staining, Confocal Microscopy, Expressing, Enzyme-linked Immunosorbent 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: 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: Cell Death & Disease

Article Title: Semaphorin 7A promotes endothelial to mesenchymal transition through ATF3 mediated TGF-β2/Smad signaling

doi: 10.1038/s41419-020-02818-x

Figure Lengend Snippet: a The top 30 upregulated genes in Sema7A-HUVECs compared with Con335-HVUECs. b TGF-β2 mRNA level was analyzed by qPCR normalized to GAPDH. Fold changes are shown. Data are mean ± SEM, N = 3, *** p < 0.001. c The concentration of TGF-β2 in cell supernatant was detected by ELISA. N = 10. Unpaired two-tailed Student’s t tests was used to analysis the data. Data are mean ± SEM, ** p < 0.01. d GSEA based on gene ontology (GO) pathway database showed TGF-β signaling pathway was enrich in Sema7A-HUVECs. e , f Cells were treated with Oxymatrine (Oxy) (20 μmol/l) or T4442 (1 μg/ml) and the lysates were analyzed by western blotting for Smad3 phosphorylation, normalized to total Smad3. Data are mean ± SEM, N = 3, * p < 0.05; ** p < 0.01. g , h CD31 and α-SMA mRNA in cells treated with inhibitors were analyzed by qPCR normalized to GAPDH. Fold changes are shown. Data are mean ± SEM, N = 3, * p < 0.05; ** p < 0.01; *** p < 0.001. i – k CD31 and α-SMA proteins in cells treated with inhibitors were analysis by western blotting, normalized to tubulin. Data are mean ± SEM, N = 3, * p < 0.05; ** p < 0.01; *** p < 0.001. T4442: TGF-β2 blocking antibody; Oxymatrine (Oxy): TGF/Smad signaling pathway inhibitor.

Article Snippet: The primary antibodies used in this study include mouse anti-human α-SMA (Abcam, UK, Cat#32575), rabbit anti-human CD31(Abcam, UK, Cat#28364), rabbit anti-human FSP-1 (Abcam, UK, Cat#41532), mouse anti-GAPDH (Abcam, UK, Cat#181602), rabbit anti-mouse VWF (Abcam, UK, Cat#6994), mouse anti-mouse fibronectin (Abcam, UK, Cat#6328), mouse anti-human vimentin (Abcam, UK, Cat#8069), rabbit anti-human Sema7A (Thermo Fisher Scientific, Waltham, USA, Cat#PA5-28971), rabbit anti-human ATF3 (CST, Danvers, USA, Cat#33593), rabbit anti-human VE-cadherin (CST, Danvers,USA, Cat#2500), mouse anti-β tubulin (CST, Danvers, USA, Cat#2146s), rabbit anti-human p-Smad3 (BOSTER, China, Cat#BM4033), rabbit anti-human Smad3 (BOSTER, China, Cat#BM3919), ATF3 (Novus Biologicals, USA, Cat# 85816), and CD31 antibody (BD Biosciences, New Jersey, USA, Cat#555446).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Western Blot, Phospho-proteomics, Blocking Assay

Journal: Cell Death & Disease

Article Title: Semaphorin 7A promotes endothelial to mesenchymal transition through ATF3 mediated TGF-β2/Smad signaling

doi: 10.1038/s41419-020-02818-x

Figure Lengend Snippet: a ATF3 mRNA level was analyzed by qPCR normalized to GAPDH. Fold changes are shown. Data are mean ± SEM, N = 3, *** p < 0.001 b ATF3 protein expression were analyzed by western blotting, normalized to tubulin. Data are mean ± SEM, N = 3, *** p < 0.001. c TGF-β2 mRNA level was analyzed by qPCR normalized to GAPDH. Fold changes are shown. Data are mean ± SEM, N = 3, * p < 0.05; ** p < 0.01. d The concentration of TGF-β2 in cell supernatant was detected by ELISA among Con335-HUVECs, Sema7A-HUVECs, and Sema7A-HUVECs + ATF3-siRNA. N = 10. Data are mean ± SEM, * p < 0.05; *** p < 0.001. e Chip-qPCR product in agarose gel electrophoresis. f Chip-qPCR TGF-β2 percentage of input in con335-HUVECs and Sema7A - HUVECs were analyzed by qPCR normalized to IgG. Data are mean ± SEM, N = 3, ** p < 0.01. g Schematic graph of the constructed reporter plasmid. TGF-β2 mut indicates the TGF-β2 mutation promoter region in ATF3 binding site. The mutated nucleotides in TGF-β2 fragments are in red letters. h Luciferase reporter assays were performed on HEK 293 T cells. Data are mean ± SEM, N = 3, ** p < 0.01 vs negative control. i , j ATF3-overexpression plasmid was transfected to HUVECs, and the mRNA levels of TGF - β2 and TGF-β1 were performed by qPCR normalized to GAPDH. Fold changes are shown. Data are mean ± SEM, N = 3, ** p < 0.01. k , l P-Smad3 protein in cells treated with siRNA was analyzed by Western blotting, normalized to total Smad3. Data are mean ± SEM, N = 3, * p < 0.05; ** p < 0.01. m – q CD31 and α-SMA RNA and proteins in cells treated with siRNA or control was analyzed by qPCR and western blotting. Data are mean ± SEM, N = 3, * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The primary antibodies used in this study include mouse anti-human α-SMA (Abcam, UK, Cat#32575), rabbit anti-human CD31(Abcam, UK, Cat#28364), rabbit anti-human FSP-1 (Abcam, UK, Cat#41532), mouse anti-GAPDH (Abcam, UK, Cat#181602), rabbit anti-mouse VWF (Abcam, UK, Cat#6994), mouse anti-mouse fibronectin (Abcam, UK, Cat#6328), mouse anti-human vimentin (Abcam, UK, Cat#8069), rabbit anti-human Sema7A (Thermo Fisher Scientific, Waltham, USA, Cat#PA5-28971), rabbit anti-human ATF3 (CST, Danvers, USA, Cat#33593), rabbit anti-human VE-cadherin (CST, Danvers,USA, Cat#2500), mouse anti-β tubulin (CST, Danvers, USA, Cat#2146s), rabbit anti-human p-Smad3 (BOSTER, China, Cat#BM4033), rabbit anti-human Smad3 (BOSTER, China, Cat#BM3919), ATF3 (Novus Biologicals, USA, Cat# 85816), and CD31 antibody (BD Biosciences, New Jersey, USA, Cat#555446).

Techniques: Expressing, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, ChIP-qPCR, Agarose Gel Electrophoresis, Construct, Plasmid Preparation, Mutagenesis, Binding Assay, Luciferase, Negative Control, Over Expression, Transfection, Control

Journal: Cell Death & Disease

Article Title: Semaphorin 7A promotes endothelial to mesenchymal transition through ATF3 mediated TGF-β2/Smad signaling

doi: 10.1038/s41419-020-02818-x

Figure Lengend Snippet: a Cells were treated with β1 integrin antibody (P5D2), and ATF3 mRNA level was analyzed by qPCR normalized to GAPDH. Fold changes are shown. Data are mean ± SEM, N = 3, * p < 0.05; ** p < 0.0.1. b ATF3 protein expression was analyzed by western blotting, normalized to tubulin. Data are mean ± SEM, N = 3, * p < 0.05; ** p < 0.01. c TGF-β2 mRNA level was analyzed by qPCR normalized to GAPDH. Fold changes are shown. Data are mean ± SEM, N = 3, * p < 0.05; ** p < 0.0.1. d The concentration of TGF-β2 in cell supernatant was detected by ELISA for Con335-HUVECs, Sema7A-HUVECs, and Sema7A-HUVECs + P5D2. Data are mean ± SEM, N = 10, * p < 0.05; *** p < 0.001. e , f Smad3 phosphorylation were analyzed by western blotting, normalized to total Smad3. Data are mean ± SEM, N = 3, * p < 0.05. g , h CD31 and α-SMA mRNA level were analyzed by qPCR normalized to GAPDH. Fold changes are shown. Data are mean ± SEM, N = 3, * p < 0.05; ** p < 0.01; *** p < 0.001. i – k CD31 and α-SMA proteins were analyzed by western blotting, normalized to tubulin. Data are mean ± SEM, N = 3, * p < 0.05; ** p < 0.01; *** p < 0.001. l ATF3 overexpression plasmid was transfected into P5D2 incubated Sema7A-HUVECs, and TGF-β2 mRNA expression in Sema7A-HUVECs + P5D2 and Sema7A-HUVECs + P5D2 + ATF3 were analyzed by qPCR normalized to GAPDH. Fold changes are shown. Data are mean ± SEM, N = 3, ** p < 0.01. m – o CD31 and α-SMA protein expressions were analyzed by western blotting, normalized to GAPDH. Data are mean ± SEM, N = 3, ** p < 0.01 (Sema7A-HUVECs + P5D2 vs Sema7A-HUVECs + P5D2 + ATF3). P5D2 β1 integrin antibody.

Article Snippet: The primary antibodies used in this study include mouse anti-human α-SMA (Abcam, UK, Cat#32575), rabbit anti-human CD31(Abcam, UK, Cat#28364), rabbit anti-human FSP-1 (Abcam, UK, Cat#41532), mouse anti-GAPDH (Abcam, UK, Cat#181602), rabbit anti-mouse VWF (Abcam, UK, Cat#6994), mouse anti-mouse fibronectin (Abcam, UK, Cat#6328), mouse anti-human vimentin (Abcam, UK, Cat#8069), rabbit anti-human Sema7A (Thermo Fisher Scientific, Waltham, USA, Cat#PA5-28971), rabbit anti-human ATF3 (CST, Danvers, USA, Cat#33593), rabbit anti-human VE-cadherin (CST, Danvers,USA, Cat#2500), mouse anti-β tubulin (CST, Danvers, USA, Cat#2146s), rabbit anti-human p-Smad3 (BOSTER, China, Cat#BM4033), rabbit anti-human Smad3 (BOSTER, China, Cat#BM3919), ATF3 (Novus Biologicals, USA, Cat# 85816), and CD31 antibody (BD Biosciences, New Jersey, USA, Cat#555446).

Techniques: Expressing, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Over Expression, Plasmid Preparation, Transfection, Incubation

Journal: Cell Death & Disease

Article Title: Semaphorin 7A promotes endothelial to mesenchymal transition through ATF3 mediated TGF-β2/Smad signaling

doi: 10.1038/s41419-020-02818-x

Figure Lengend Snippet: When exposed to d-flow, endothelial Sema7A expression is upregulated, potentially by the cAMP/CREB (cAMP response element-binding protein) pathway. Endothelial Sema7A upregulates the transcription factor ATF3 through interaction with β1 integrin, leading to TGF-β2 transcription, and activation of TGF/Smad3 signaling pathway, and thus inducing EndMT.

Article Snippet: The primary antibodies used in this study include mouse anti-human α-SMA (Abcam, UK, Cat#32575), rabbit anti-human CD31(Abcam, UK, Cat#28364), rabbit anti-human FSP-1 (Abcam, UK, Cat#41532), mouse anti-GAPDH (Abcam, UK, Cat#181602), rabbit anti-mouse VWF (Abcam, UK, Cat#6994), mouse anti-mouse fibronectin (Abcam, UK, Cat#6328), mouse anti-human vimentin (Abcam, UK, Cat#8069), rabbit anti-human Sema7A (Thermo Fisher Scientific, Waltham, USA, Cat#PA5-28971), rabbit anti-human ATF3 (CST, Danvers, USA, Cat#33593), rabbit anti-human VE-cadherin (CST, Danvers,USA, Cat#2500), mouse anti-β tubulin (CST, Danvers, USA, Cat#2146s), rabbit anti-human p-Smad3 (BOSTER, China, Cat#BM4033), rabbit anti-human Smad3 (BOSTER, China, Cat#BM3919), ATF3 (Novus Biologicals, USA, Cat# 85816), and CD31 antibody (BD Biosciences, New Jersey, USA, Cat#555446).

Techniques: Expressing, Binding Assay, Activation Assay

Journal:

Article Title: Tat-Induced FOXO3a Is a Key Mediator of Apoptosis in HIV-1-Infected Human CD4 + T Lymphocytes 1

doi:

Figure Lengend Snippet: Cell cycle profiles of apoptotic human CD4+ T cells infected with VSV-G-pseudotyped HIVΔ2GFP and HIVΔ3GFP and sorted for GFP positivity. A, Percentage of annexin V−GFP+ (left panel), annexin V+GFP+ (middle panel), and annexin V+GFP− (right panel) cells. B, Flow cytometric analysis of cell cycle profiles in the cell populations reported in A determined by staining with an Ab against Ki-67 and 7AAD. C, Production of p24 in human CD4+ T cells infected with HIVΔ2GFP or HIVΔ3GFP. Production of HIV-1 p24 was measured in supernatants from infected, sorted cultures on days 2, 3, and 4 postinfection using a p24 ELISA. The results are expressed as the means ± SE of three independent donors. *, p < 0.05 related to decrease or increase.

Article Snippet: Other Abs used in the studies were against HIV-1 p24 (KC57-FITC) (Beckman Coulter), human Ki67 (BD Biosciences), human Egr1 (early growth response protein-1), PTEN (phosphate and tensin homolog deleted on chromosome 10), p-Akt (Ser 473 ), FOXO3a, ATM (mutated in ataxia-telangiectasia) (Cell Singaling Technology), human activating transcription factor 3 (ATF3), cyclin E, p130 (Santa Cruz Biotechnology), and human cyclin A-PE (BD Pharmingen).

Techniques: Infection, Staining, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: LncRNA ZNFTR functions as an inhibitor in pancreatic cancer by modulating ATF3/ZNF24/VEGFA pathway

doi: 10.1038/s41419-021-04119-3

Figure Lengend Snippet: A The schematic diagram showed the position relationship between ZNFTR and ZNF24. The data were searched from the UCSC Genome Browser. B After transfected with siNC or siZNFTR #1/2, and pcDNA-Vector or pcDNA-ZNFTR in BxPC-3 and PANC-1 cells, the expression of ZNF24 both at mRNA and protein levels was detected by qRT-PCR and western blot, respectively. C After co-transfected with pcDNA-ZNFTR and siNC/siZNF24 or co-transfected with siZNFTR and pcDNA-vector/pcDNA-ZNF24 in PANC-1 cells, the expression of ZNF24 both at mRNA and protein levels was measured by qRT-PCR and western blot, respectively. D After co-transfected with siZNFTR and pcDNA-vector/pcDNA-ZNF24 or co-transfected with pcDNA-ZNFTR and siNC/siZNF24 in PANC-1 cells, the proliferation of transfected PANC-1 cells was analyzed via CCK-8 assay for 5 days. E , F To assess the invasive and migrative abilities of PANC-1 cells, co-transfected with pcDNA-ZNFTR and siNC/siZNF24 or co-transfected with siZNFTR and pcDNA-vector/pcDNA-ZNF24, Transwell assay ( E ) and Wound healing ( F ) were conducted, respectively. The histograms were showed as quantized relative invaded cells or wound size. Five fields were selected randomly and averaged. All data were revealed as means ± standard deviation (SD) for no less than three independent experiments. Significant P values showed as * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: We designed short interference RNAs for human ZNFTR (siZNFTR), ZNF24 (siZNF24), ATF3 (siATF3), HIF-1α (siHIF-1α), HDAC1 (siHDAC1), and paired siRNA negative control (siNC) were purchased from RiboBio (Guangzhou, China) and transfected with a final concentration of 50 nM.

Techniques: Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Transwell Assay, Standard Deviation

Journal: Cell Death & Disease

Article Title: LncRNA ZNFTR functions as an inhibitor in pancreatic cancer by modulating ATF3/ZNF24/VEGFA pathway

doi: 10.1038/s41419-021-04119-3

Figure Lengend Snippet: A , B After transfected with siNC or siZNFTR #1/2, and pcDNA-ZNFTR or pcDNA-Vector in PANC-1 cells, the expression of ATF3 both at mRNA and protein levels was detected by qRT-PCR and western blot, respectively. C ChIP assay analyzed the enrichment level of the ZNF24 promoter after co-transfected pcDNA-ZNFTR with or without pcDNA-ATF3 in PANC-1 cells. D The activity of ZNF24 promoter after co-transfected pcDNA-ZNFTR with or without pcDNA-ATF3 was assessed via luciferase reporter assay in PANC-1 cells. E ChIP assay analyzed the enrichment level of the ZNF24 promoter after co-transfected siZNFTR with or without siATF3 in PANC-1 cells. F The activity of the ZNF24 promoter after co-transfected siZNFTR with or without siATF3 was assessed via luciferase reporter assay in PANC-1 cells. G After co-transfected pcDNA-ZNFTR with or without pcDNA-ATF3 in PANC-1 cells, the expression of ZNF24 was detected via qRT-PCR or western blot. H After co-transfected siZNFTR with or without siATF3 in PANC-1 cells, the expression of ZNF24 was detected via qRT-PCR or western blot. WT: wild type, MUT-1: Site1 mutated, MUT-2: Site2 mutated, MUT-3: Both Site1 and Site2 mutated. All data were revealed as means ± standard deviation (SD) for no less than three independent experiments. Significant P values showed as * P < 0.05 and ** P < 0.01. n.s. means the difference was not significant.

Article Snippet: We designed short interference RNAs for human ZNFTR (siZNFTR), ZNF24 (siZNF24), ATF3 (siATF3), HIF-1α (siHIF-1α), HDAC1 (siHDAC1), and paired siRNA negative control (siNC) were purchased from RiboBio (Guangzhou, China) and transfected with a final concentration of 50 nM.

Techniques: Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, Activity Assay, Luciferase, Reporter Assay, Standard Deviation

Journal: Cell Death & Disease

Article Title: LncRNA ZNFTR functions as an inhibitor in pancreatic cancer by modulating ATF3/ZNF24/VEGFA pathway

doi: 10.1038/s41419-021-04119-3

Figure Lengend Snippet: A After transfected with pcDNA-Vector or pcDNA-ZNF24, and siNC or siZNF24 #1/2 in PANC-1 cells, the expression of VEGFA both at mRNA and protein levels was detected by qRT-PCR and western blot, respectively. B After transfected with pcDNA-Vector or pcDNA-ZNF24, and siNC or siZNF24 #1/2 in PANC-1 cells, the VEGFA secretion level in supernatants was detected by ELISA assay. C ChIP assay analyzed the enrichment level of VEGFA promoter after co-transfected siZNFTR with or without pcDNA-ZNF24 in PANC-1 cells. D After transfected with a vector containing wild type (WT) or mutant binding site (MUT) of VEGFA promoter, the activity of the VEGFA promoter after co-transfected siZNFTR with or without pcDNA-ZNF24 was assessed via luciferase reporter assay in PANC-1 cells. E Left: Co-transfected siZNFTR with or without pcDNA-ZNF24, the expression of VEGFA was analyzed by qRT-PCR and western blot, respectively. Right: Co-transfected pcDNA-ZNFTR with or without siZNF24, the expression of VEGFA was analyzed by qRT-PCR and western blot, respectively. F After co-transfected siZNFTR with or without pcDNA-ZNF24, and co-transfected pcDNA-ZNFTR with or without siZNF24, VEGFA secretion level in supernatants was detected by ELISA assay. G Representative images and quantifications of the tube formation ability of HUVEC treated with different condition medium collected from transfected cells (co-transfected siZNFTR with or without pcDNA-ZNF24, co-transfected pcDNA-ZNFTR with or without siZNF24) was performed by tube formation assay. All data were revealed as means ± standard deviation (SD) for no less than three independent experiments. Significant P values showed as * P < 0.05 and ** P < 0.01. n.s. means the difference was not significant.

Article Snippet: We designed short interference RNAs for human ZNFTR (siZNFTR), ZNF24 (siZNF24), ATF3 (siATF3), HIF-1α (siHIF-1α), HDAC1 (siHDAC1), and paired siRNA negative control (siNC) were purchased from RiboBio (Guangzhou, China) and transfected with a final concentration of 50 nM.

Techniques: Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Mutagenesis, Binding Assay, Activity Assay, Luciferase, Reporter Assay, Tube Formation Assay, Standard Deviation

Journal: Cell Death & Disease

Article Title: LncRNA ZNFTR functions as an inhibitor in pancreatic cancer by modulating ATF3/ZNF24/VEGFA pathway

doi: 10.1038/s41419-021-04119-3

Figure Lengend Snippet: A The schematic diagram showed the position relationship between ZNFTR and ZNF24. The data were searched from the UCSC Genome Browser. B After transfected with siNC or siZNFTR #1/2, and pcDNA-Vector or pcDNA-ZNFTR in BxPC-3 and PANC-1 cells, the expression of ZNF24 both at mRNA and protein levels was detected by qRT-PCR and western blot, respectively. C After co-transfected with pcDNA-ZNFTR and siNC/siZNF24 or co-transfected with siZNFTR and pcDNA-vector/pcDNA-ZNF24 in PANC-1 cells, the expression of ZNF24 both at mRNA and protein levels was measured by qRT-PCR and western blot, respectively. D After co-transfected with siZNFTR and pcDNA-vector/pcDNA-ZNF24 or co-transfected with pcDNA-ZNFTR and siNC/siZNF24 in PANC-1 cells, the proliferation of transfected PANC-1 cells was analyzed via CCK-8 assay for 5 days. E , F To assess the invasive and migrative abilities of PANC-1 cells, co-transfected with pcDNA-ZNFTR and siNC/siZNF24 or co-transfected with siZNFTR and pcDNA-vector/pcDNA-ZNF24, Transwell assay ( E ) and Wound healing ( F ) were conducted, respectively. The histograms were showed as quantized relative invaded cells or wound size. Five fields were selected randomly and averaged. All data were revealed as means ± standard deviation (SD) for no less than three independent experiments. Significant P values showed as * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: We designed short interference RNAs for human ZNFTR (siZNFTR), ZNF24 (siZNF24), ATF3 (siATF3), HIF-1α (siHIF-1α), HDAC1 (siHDAC1), and paired siRNA negative control (siNC) were purchased from RiboBio (Guangzhou, China) and transfected with a final concentration of 50 nM.

Techniques: Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Transwell Assay, Standard Deviation

Journal: Cell Death & Disease

Article Title: LncRNA ZNFTR functions as an inhibitor in pancreatic cancer by modulating ATF3/ZNF24/VEGFA pathway

doi: 10.1038/s41419-021-04119-3

Figure Lengend Snippet: A ATF3 showed potential mutual interaction with ZNFTR and the promoter of ZNF24 through intersecting from the catRAPID and JASPAR database. B RNA interaction profile showed the putative binding position between ZNFTR and ATF3. C RIP assay was performed to verify the interaction between ATF3 and ZNFTR with anti-ATF3 antibody in BxPC-3 and PANC-1 cells. The co-precipitated RNAs were detected by qRT-PCR. Anti-SNRNP70 rabbit polyclonal antibody co-precipitated with U1 snRNA was used as a positive control. The qRT-PCR products were separated by 2% agarose gel electrophoresis. D The RNA-pulldown assay was conducted to demonstrate the interaction between ATF3 and ZNFTR by biotinylated ZNFTR probe in BxPC-3/PANC-1 cells. Then the pulldown protein was detected by immunoblot assay with anti-ATF3 and anti-HuR antibody. RNA from the 3′ untranslated-region (UTR) of the androgen receptor (AR), involving UC-rich HuR binding areas, was used as a positive control. E Biotinylated ZNFTR full length, antisense, and part of it were conducted, and RNA-pulldown assay was performed to verify the region of ZNFTR interacted with ATF3. F The schematic diagram exhibited two predicted binding sites between ATF3 and promoter of ZNF24. G ChIP assay was conducted to verify the interaction between ATF3 and promoter of ZNF24. The qRT-PCR products were separated by 2% agarose gel electrophoresis. H The vectors containing the wild type (WT) or three mutants (MUT) of ATF3 binding sites were co-transfected with empty vector or pcDNA-ATF3 in BxPC-3 and PANC-1 cells to perform luciferase reporter assay. WT: wild type, MUT-1: Site1 mutated, MUT-2: Site2 mutated, MUT-3: Both Site1 and Site2 mutated. All data were revealed as means ± standard deviation (SD) for no less than three independent experiments. Significant P values showed as * P < 0.05 and ** P < 0.01. n.s means the difference was not significant.

Article Snippet: We designed short interference RNAs for human ZNFTR (siZNFTR), ZNF24 (siZNF24), ATF3 (siATF3), HIF-1α (siHIF-1α), HDAC1 (siHDAC1), and paired siRNA negative control (siNC) were purchased from RiboBio (Guangzhou, China) and transfected with a final concentration of 50 nM.

Techniques: Binding Assay, Quantitative RT-PCR, Positive Control, Agarose Gel Electrophoresis, Western Blot, Transfection, Plasmid Preparation, Luciferase, Reporter Assay, Standard Deviation

Journal: Cell Death & Disease

Article Title: LncRNA ZNFTR functions as an inhibitor in pancreatic cancer by modulating ATF3/ZNF24/VEGFA pathway

doi: 10.1038/s41419-021-04119-3

Figure Lengend Snippet: A , B After transfected with siNC or siZNFTR #1/2, and pcDNA-ZNFTR or pcDNA-Vector in PANC-1 cells, the expression of ATF3 both at mRNA and protein levels was detected by qRT-PCR and western blot, respectively. C ChIP assay analyzed the enrichment level of the ZNF24 promoter after co-transfected pcDNA-ZNFTR with or without pcDNA-ATF3 in PANC-1 cells. D The activity of ZNF24 promoter after co-transfected pcDNA-ZNFTR with or without pcDNA-ATF3 was assessed via luciferase reporter assay in PANC-1 cells. E ChIP assay analyzed the enrichment level of the ZNF24 promoter after co-transfected siZNFTR with or without siATF3 in PANC-1 cells. F The activity of the ZNF24 promoter after co-transfected siZNFTR with or without siATF3 was assessed via luciferase reporter assay in PANC-1 cells. G After co-transfected pcDNA-ZNFTR with or without pcDNA-ATF3 in PANC-1 cells, the expression of ZNF24 was detected via qRT-PCR or western blot. H After co-transfected siZNFTR with or without siATF3 in PANC-1 cells, the expression of ZNF24 was detected via qRT-PCR or western blot. WT: wild type, MUT-1: Site1 mutated, MUT-2: Site2 mutated, MUT-3: Both Site1 and Site2 mutated. All data were revealed as means ± standard deviation (SD) for no less than three independent experiments. Significant P values showed as * P < 0.05 and ** P < 0.01. n.s. means the difference was not significant.

Article Snippet: We designed short interference RNAs for human ZNFTR (siZNFTR), ZNF24 (siZNF24), ATF3 (siATF3), HIF-1α (siHIF-1α), HDAC1 (siHDAC1), and paired siRNA negative control (siNC) were purchased from RiboBio (Guangzhou, China) and transfected with a final concentration of 50 nM.

Techniques: Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, Activity Assay, Luciferase, Reporter Assay, Standard Deviation

Journal: Cell Death & Disease

Article Title: LncRNA ZNFTR functions as an inhibitor in pancreatic cancer by modulating ATF3/ZNF24/VEGFA pathway

doi: 10.1038/s41419-021-04119-3

Figure Lengend Snippet: A After transfected with pcDNA-Vector or pcDNA-ZNF24, and siNC or siZNF24 #1/2 in PANC-1 cells, the expression of VEGFA both at mRNA and protein levels was detected by qRT-PCR and western blot, respectively. B After transfected with pcDNA-Vector or pcDNA-ZNF24, and siNC or siZNF24 #1/2 in PANC-1 cells, the VEGFA secretion level in supernatants was detected by ELISA assay. C ChIP assay analyzed the enrichment level of VEGFA promoter after co-transfected siZNFTR with or without pcDNA-ZNF24 in PANC-1 cells. D After transfected with a vector containing wild type (WT) or mutant binding site (MUT) of VEGFA promoter, the activity of the VEGFA promoter after co-transfected siZNFTR with or without pcDNA-ZNF24 was assessed via luciferase reporter assay in PANC-1 cells. E Left: Co-transfected siZNFTR with or without pcDNA-ZNF24, the expression of VEGFA was analyzed by qRT-PCR and western blot, respectively. Right: Co-transfected pcDNA-ZNFTR with or without siZNF24, the expression of VEGFA was analyzed by qRT-PCR and western blot, respectively. F After co-transfected siZNFTR with or without pcDNA-ZNF24, and co-transfected pcDNA-ZNFTR with or without siZNF24, VEGFA secretion level in supernatants was detected by ELISA assay. G Representative images and quantifications of the tube formation ability of HUVEC treated with different condition medium collected from transfected cells (co-transfected siZNFTR with or without pcDNA-ZNF24, co-transfected pcDNA-ZNFTR with or without siZNF24) was performed by tube formation assay. All data were revealed as means ± standard deviation (SD) for no less than three independent experiments. Significant P values showed as * P < 0.05 and ** P < 0.01. n.s. means the difference was not significant.

Article Snippet: We designed short interference RNAs for human ZNFTR (siZNFTR), ZNF24 (siZNF24), ATF3 (siATF3), HIF-1α (siHIF-1α), HDAC1 (siHDAC1), and paired siRNA negative control (siNC) were purchased from RiboBio (Guangzhou, China) and transfected with a final concentration of 50 nM.

Techniques: Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Mutagenesis, Binding Assay, Activity Assay, Luciferase, Reporter Assay, Tube Formation Assay, Standard Deviation

Journal: Cell Death & Disease

Article Title: LncRNA ZNFTR functions as an inhibitor in pancreatic cancer by modulating ATF3/ZNF24/VEGFA pathway

doi: 10.1038/s41419-021-04119-3

Figure Lengend Snippet: A The relationship between ATF3 and ZNFTR (green) or ZNF24 (blue), ZNF24, and ZNFTR (green) or VEGFA (purple) in PC tissues were analyzed by Pearson’s correlation respectively. B Pearson’s correlation analysis and chi -square test identified a positive association between ZNFTR and ZNF24 in the PC samples. The chi -square test identified a negative association between ZNFTR and VEGFA in the PC samples. C The expression level of ZNF24 and VEGFA was analyzed by immunohistochemistry (IHC) in the “ZNFTR-HIGH” or “ZNFTR-LOW” group. Forty-eight patients were separated into “HIGH” and “LOW” two groups according to the expression level of ZNFTR at the median. D Overall survival analysis in 48 patients with PC was shown by the Kaplan–Meier curve. Forty-eight patients were separated into “HIGH” and “LOW” two groups according to the expression level of ZNF24 at the median. E Schematic diagram of the mechanism by which ZNFTR regulated PC in normal and hypoxia environments.

Article Snippet: We designed short interference RNAs for human ZNFTR (siZNFTR), ZNF24 (siZNF24), ATF3 (siATF3), HIF-1α (siHIF-1α), HDAC1 (siHDAC1), and paired siRNA negative control (siNC) were purchased from RiboBio (Guangzhou, China) and transfected with a final concentration of 50 nM.

Techniques: Expressing, Immunohistochemistry