Journal: Journal of Biological Chemistry

Article Title: Fibronectin Type I Repeat Is a Nonactivating Ligand for EphA1 and Inhibits ATF3-dependent Angiogenesis

doi: 10.1074/jbc.m702164200

Figure Lengend Snippet: FIGURE 1. EphA1 is up-regulated in an ATF3-dependent manner. A, West- ern blotting of EphA1, EphA2, and ephrin-A1 in NP31 endothelial cells before and after tetracycline-regulated expression of ATF3 and in control organs (rat lung, liver, and muscle). B, the nucleotide 254–775 region of rat EphA1 cDNA (GenBankTM accession number XM_231658) and 1505–1941 region of rat EphA2 cDNA (GenBankTM accession number XM_001072610) were amplified by PCR. The specificity of the PCR products was verified by Southern hybrid- ization with the corresponding regions as probes. C, promoter assays were performed before and after ATF3 induction in NP31 endothelial cells with luciferase constructs containing rat EphA1 promoter region 1027 to 82 (GenBankTM accession number NW_047690, see legend of supplemental Fig. 1) or the PAI-1 promoter (20). Results are expressed as percentage of relative lucif- erase activity, mean S.D., from six independent experiments. *, p 0.01.

Article Snippet: Molecular Reagents—Mouse monoclonal antibody (mAb) against human EphA1 and all the Fc fusion proteins were purchased from R & D Systems; mouse mAb against glutathione S-transferase (GST), rabbit polyclonal antibody against EphA2 (C-20), ephrin-A1 (V-18), and ATF3 (H-90) were from Santa Cruz Biotechnology; rat mAb against CD31 (MEC13.3) was from Pharmingen; rabbit polyclonal antibody against Fn was from LSL; mouse mAb against actin (mAb1501) was from Chemicon; pGEX-4T-1 and pGEX-5X-3, HRP-conjugated sheep anti-mouse Ig, HRP-conjugated goat anti-rabbit Ig, and HRP-conjugated protein A were from GE Healthcare; TRITCand fluorescein isothiocyanate-conjugated secondary antibodies were from Jackson ImmunoResearch; MatrigelTM Matrix was from BD Biosciences; human plasma fibronectin and cellular fibronectin were from Sigma; doxorubicin hydrochloride (DXR) was from Calbiochem.

Techniques: Expressing, Control, Amplification, Luciferase, Construct, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Fibronectin Type I Repeat Is a Nonactivating Ligand for EphA1 and Inhibits ATF3-dependent Angiogenesis

doi: 10.1074/jbc.m702164200

Figure Lengend Snippet: FIGURE 3. Doxorubicin induces EphA1 expression. A, whole cell lysates from NP31 endothelial cells that were incubated with DXR at 1 M for the indicated time (in hours) were subjected to anti-EphA1, anti-ATF3, and anti- actin Western blotting. B, quantification of NP31 endothelial tubulogenesis by DXR at 125 nM.

Article Snippet: Molecular Reagents—Mouse monoclonal antibody (mAb) against human EphA1 and all the Fc fusion proteins were purchased from R & D Systems; mouse mAb against glutathione S-transferase (GST), rabbit polyclonal antibody against EphA2 (C-20), ephrin-A1 (V-18), and ATF3 (H-90) were from Santa Cruz Biotechnology; rat mAb against CD31 (MEC13.3) was from Pharmingen; rabbit polyclonal antibody against Fn was from LSL; mouse mAb against actin (mAb1501) was from Chemicon; pGEX-4T-1 and pGEX-5X-3, HRP-conjugated sheep anti-mouse Ig, HRP-conjugated goat anti-rabbit Ig, and HRP-conjugated protein A were from GE Healthcare; TRITCand fluorescein isothiocyanate-conjugated secondary antibodies were from Jackson ImmunoResearch; MatrigelTM Matrix was from BD Biosciences; human plasma fibronectin and cellular fibronectin were from Sigma; doxorubicin hydrochloride (DXR) was from Calbiochem.

Techniques: Expressing, Incubation, Western Blot

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: Cell Death & Disease

Article Title: Lipid desaturation-associated endoplasmic reticulum stress regulates MYCN gene expression in hepatocellular carcinoma cells

doi: 10.1038/s41419-020-2257-y

Figure Lengend Snippet: a ATF3 gene expression in JHH7 cells treated with 100 nM control siRNA (siCtl) or siSCD1 for 24 h. b ATF3 gene expression in FACS-isolated EpCAM + and EpCAM − JHH7 cells. c ATF3 gene expression in JHH7 cells grown as monolayers (2D) and spheres (3D). d Correlation between MYCN and ATF3 gene expression levels in HCC tissues of 372 patients obtained from the liver hepatocellular carcinoma TCGA PanCancer RNA-seq database. e Effect of ER stress inducer tunicamycin on ATF3 (left) and MYCN (right) gene expression in JHH7 cells. The cells were treated with tunicamycin at indicated concentrations for 4 h. f Expansion of the 1 H NMR spectrum of JHH7 cells treated with EtOH or 15 μM ACR for 4 h. The double bond region of unsaturated fatty acids from 5.20 to 5.45 ppm were highlighted. g Effect of ER stress inhibitor tauroursodeoxycholic acid (TUDCA) on ACR-mediated gene expression of ATF3 (left) and MYCN (right) in JHH7 cells. The cells were treated with EtOH or 15 μM ACR in the absence or presence of 1 mM TUDCA for 4 h. h Effect of siATF3 on ACR-mediated gene expression of ATF3 (left) and MYCN (right) in JHH7 cells. The cells were transfected with 100 nM siCtl or siATF3 for 24 h and then treated with EtOH or 15 μM ACR for 4 h. The data are presented as means ( n = 3 replicates) ± SD; * p < 0.05, Student’s t -test.

Article Snippet: A pool of 3 target-specific siRNAs targeting human SCD1 (sc-36464; siSCD1), human MYCN (sc-36003; siMYCN), human ATF3 (sc-44283; siATF3) and a control siRNA (sc-37007; siCtl) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Gene Expression, Control, Isolation, RNA Sequencing, Transfection

Journal: Nature

Article Title: Opposing roles for calcineurin and ATF3 in squamous skin cancer.

doi: 10.1038/nature08996

Figure Lengend Snippet: Figure 2 | Calcineurin/NFAT signalling negatively controls ATF3 expression. a, b, HKCs plus/minus CsA or VIVIT treatment (a) or CnB1 or Nfatc1 knockdown (b) were analysed in parallel with controls by immunoblotting. Similar results were obtained at mRNA level (Supplementary Fig. 8a–c). c, HKCs infected with retroviruses expressing constitutively active NFATC1 (1)19 or GFP control (2) plus/minus subsequent CsA treatment were analysed for ATF3 expression. Similar results wereobtained by real time RT–PCR (Supplementary Fig. 8f). d, HKCs plus/minus CsA/VIVIT treatment and cycloheximide (CHX) exposure were analysed at various times (hours) for Atf3 expression by real-time RT–PCR. Error bars represent mean 6 s.d. (n 5 3 replicates). e, Extracts of HKCs plus/ minus CsA treatment or Nfatc1 knockdown (left panel) or intact human epidermis (right panel) were processed for chromatin immunoprecipitation with anti-NFATC1 antibodies or non-immune IgGs, followed by real-time PCR of Atf3 promoter regions containing and lacking high-affinity NFATC1

Article Snippet: Biopsies of cutaneous SCCs from patients under CsA treatment and control patient population were collected for tissue array preparation, followed by immunohistochemical analysis of ATF3 protein expression using polyclonal rabbit anti-human ATF3 antibody (Atlas Antibodies) and 10 M citrate buffer at pH 6.0 as an antigen retrieval.

Techniques: Expressing, Knockdown, Western Blot, Infection, Control, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Nature

Article Title: Opposing roles for calcineurin and ATF3 in squamous skin cancer.

doi: 10.1038/nature08996

Figure Lengend Snippet: Figure 3 | ATF3 upregulation enhances keratinocyte tumour formation and suppresses cancer cell senescence. a, HKCs infected with ATF3-expressing (ATF3) and control retroviruses were analysed by real time RT–PCR for indicated genes. Error bars represent mean 6 s.d. (n 5 3 replicates). b, HKCs plus/minus infection with ATF3 and H-rasV12-expressing retroviruses were analysed for p53 expression by immunoblotting. c, HKCs plus/minus Atf3 knockdown and CsA/VIVIT treatment were analysed by real time RT–PCR for p53 and DcR2 (also known as Ccr6) expression. Additional results are shown in Supplementary Fig. 10a. d, HKCs plus/minus H-rasV12 expression, Atf3 knockdown and CsA treatment as indicated were analysed for ATF3 and p53 expression by immunoblotting. For densitometric quantification and

Article Snippet: Biopsies of cutaneous SCCs from patients under CsA treatment and control patient population were collected for tissue array preparation, followed by immunohistochemical analysis of ATF3 protein expression using polyclonal rabbit anti-human ATF3 antibody (Atlas Antibodies) and 10 M citrate buffer at pH 6.0 as an antigen retrieval.

Techniques: Infection, Expressing, Control, Quantitative RT-PCR, Western Blot, Knockdown

Journal: BMC Genomics

Article Title: Novel functional view of the crocidolite asbestos-treated A549 human lung epithelial transcriptome reveals an intricate network of pathways with opposing functions

doi: 10.1186/1471-2164-9-376

Figure Lengend Snippet: List of genes for which expression increased five-fold or greater or decreased two-fold or greater

Article Snippet: Antibodies for Egr-1 (sc-189) and ATF3 (sc-188) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and the antibody for β-actin was purchased from Sigma (St. Louis, MO).

Techniques: Expressing, Binding Assay, Virus, Activity Assay, Sequencing

Journal: BMC Genomics

Article Title: Novel functional view of the crocidolite asbestos-treated A549 human lung epithelial transcriptome reveals an intricate network of pathways with opposing functions

doi: 10.1186/1471-2164-9-376

Figure Lengend Snippet: Validation of the microarray data from crocidolite-treated human A549 cells

Article Snippet: Antibodies for Egr-1 (sc-189) and ATF3 (sc-188) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and the antibody for β-actin was purchased from Sigma (St. Louis, MO).

Techniques: Biomarker Discovery, Microarray

Journal: Journal of Neuroinflammation

Article Title: Tissue-resident M2 macrophages directly contact primary sensory neurons in the sensory ganglia after nerve injury

doi: 10.1186/s12974-021-02283-z

Figure Lengend Snippet: Antibodies used in the present study

Article Snippet: Anti-activating transcription factor 3 (ATF3) (a marker of damaged neuron) , HPA001562 , Rabbit , Polyclonal , 1:1000 , Atlas Antibodies, Stockholm, Sweden , AB_1078233.

Techniques: Concentration Assay, Marker, Binding Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: Macrophages amplify the spontaneous activity of damaged sensory neurons in a human co-culture model of neuropathic pain

doi: 10.1101/2025.06.05.656880

Figure Lengend Snippet: a iSNs 24hrs after axotomy and replating, illustrating expression of the canonical injury marker, ATF3, and evidence of rapid neurite regeneration. b Schematic of mechanical trauma model in which neurons have been transduced with AAV-hSyn-mCherry prior to axotomy and replating, and subsequent co-culture with ZsGreen iMacs. c Representative image of 7 div co-culture. Most iMacs aggregate around neuron soma clusters and appear to exhibit an ameboid profile ( i ), with iMacs not associated with these clusters exhibiting a larger, flattened morphology ( ii ).

Article Snippet: Primary antibodies used in this study: α-NF200 (1:1000, mouse, Sigma-Aldrich #266004), α-BRN3A (1:100, rabbit, Merck #AB5945), IBA1 (1:500, guinea pig, Synaptic systems #234 308), α-GFP (1:1000, chicken, Abcam #ab13970), α-ATF3 (1:500, rabbit, Novus Biologicals #NBP1-85816), α-FABP5 (1:200, rabbit, ProteinTech #12348-1-AP), and α-β-tubulin III (1:1000, mouse, Sigma-Aldrich #N5389).

Techniques: Expressing, Marker, Transduction, Co-Culture Assay

Journal: bioRxiv

Article Title: Macrophages amplify the spontaneous activity of damaged sensory neurons in a human co-culture model of neuropathic pain

doi: 10.1101/2025.06.05.656880

Figure Lengend Snippet: a UMAP plot of iMacs from all conditions showing four discrete clusters (n=1 differentiation and 1 iPSC line): total cells (3157), cultured alone (987 cells), with healthy iSNs for 3 (576 cells) and 7 days (791 cells), and for 7 days with injured iSNs (803 cells). b UMAP feature plots and corresponding violin plots of the genes used to describe each cluster. Each dot is a cell that was sequenced; green dots indicate those positive for a given gene. c Proportion of iMacs from different conditions assigned to each of the four clusters. Note that most iMacs from each condition were assigned to one cluster (or two in the case of co-culture with healthy iSNs). d Heatmap showing gene expression profile organised by iMac condition. The top fifteen differentially expressed genes per condition are shown. e (i) Violin plot and validatory immunoassay (ii) for Galectin-3, showing its upregulation in iMacs co-cultured with damaged iSNs. The immunoassay was performed on supernatant from div 7 co-cultures. Data represent mean ± SEM, with values for individual differentiations also shown (n=8, from 3 independent iPSC lines: black circles - SBAD2, grey circles - SBAD3 and white circles - ZsGreen ). Two-tailed unpaired t -test. *** P <0.05. f (i) Violin plot showing upregulation of the injury gene ATF3 in iMacs co-cultured with injured iSNs. Quantification of mean ATF3 signal intensity (ii) and representative images of iMacs co-cultured with healthy (iii) or injured iSNs (iv) for 7 days illustrating cells with nuclear ATF3 (white arrows). g (i) Violin plot for FABP5. Quantification of mean FABP5 signal intensity (ii) and representative images of iMacs co-cultured with healthy (iii) or injured iSNs (iv) for 7 days illustrating FABP5 expression. For quantification in f and g , paired values represent independent differentiations in which co-culture, ICC processing and image analysis were performed in parallel (n=6, from 3 independent iPSC lines: black circles - SBAD2, grey circles - SBAD3 and white circles - ZsGreen ). Two-tailed paired t -test. * P <0.05. Scale bars represent 25 µm.

Article Snippet: Primary antibodies used in this study: α-NF200 (1:1000, mouse, Sigma-Aldrich #266004), α-BRN3A (1:100, rabbit, Merck #AB5945), IBA1 (1:500, guinea pig, Synaptic systems #234 308), α-GFP (1:1000, chicken, Abcam #ab13970), α-ATF3 (1:500, rabbit, Novus Biologicals #NBP1-85816), α-FABP5 (1:200, rabbit, ProteinTech #12348-1-AP), and α-β-tubulin III (1:1000, mouse, Sigma-Aldrich #N5389).

Techniques: Cell Culture, Co-Culture Assay, Gene Expression, Two Tailed Test, Expressing

Journal: bioRxiv

Article Title: Macrophages amplify the spontaneous activity of damaged sensory neurons in a human co-culture model of neuropathic pain

doi: 10.1101/2025.06.05.656880

Figure Lengend Snippet: a Representative image of monocyte-derived macrophages (MoMs) co-cultured with iSNs for 7 days under conditions identical to iMac co-culture. MoMs stain positive for the macrophage marker IBA1. b UMAP projection of MoMs pooled from 4 conditions; cultured alone (895 cells) and co-cultured with healthy iSNs for 3 (980 cells) and 7 (854 cells) days, or with injured iSNs for 7 days (1113 cells) (Note, n=1 differentiation and 1 donor). Four distinct clusters can be seen. c Dot plot showing the relative expression of markers used to define clusters. d Proportion of MoMs from different conditions corresponding to each of the four clusters. Most clusters consist of MoMs from one condition, similarly to iMacs. e We noted that unlike other clusters, the MRC1 Low LSP1 pos cluster had low expression of the female-biased gene XIST and enriched expression of the Y-linked gene RPS4Y1, leading to a reasonable suspicion that the female donor derived sample was partially (<10% cells) contaminated with male donor-derived MOMs concurrently in culture. We therefore excluded the MRC1 Low LSP1 pos cluster from subsequent analysis. f Venn diagram of genes upregulated in either iMacs or MoMs when co-cultured with healthy vs injured iSNs (FDR < 0.05 and log 2 FC > 0.5), showing an overlap of approximately 50%. This is far greater than expected by chance: hypergeometric p value = 3.289669e-238. g Violin plots of three injury-associated genes found to be upregulated in iMacs co-cultured with injured iSNs. ATF3 and LGALS3 are also upregulated in MoMs cultured with damaged iSNs, however no difference is observed in FABP5 expression.

Article Snippet: Primary antibodies used in this study: α-NF200 (1:1000, mouse, Sigma-Aldrich #266004), α-BRN3A (1:100, rabbit, Merck #AB5945), IBA1 (1:500, guinea pig, Synaptic systems #234 308), α-GFP (1:1000, chicken, Abcam #ab13970), α-ATF3 (1:500, rabbit, Novus Biologicals #NBP1-85816), α-FABP5 (1:200, rabbit, ProteinTech #12348-1-AP), and α-β-tubulin III (1:1000, mouse, Sigma-Aldrich #N5389).

Techniques: Derivative Assay, Cell Culture, Co-Culture Assay, Staining, Marker, Expressing

Journal: bioRxiv

Article Title: Macrophages amplify the spontaneous activity of damaged sensory neurons in a human co-culture model of neuropathic pain

doi: 10.1101/2025.06.05.656880

Figure Lengend Snippet: a Plots showing the expression level of marker genes in different cell clusters observed after sequencing mouse myeloid cells obtained from sciatic nerve either after sham surgery or 1-week or 15-weeks after nerve damage. Mφ = macrophages, p = phagocytic, i = injury-induced, Mo = monocytes, DC = dendritic cells and res = resident. b Violin plots of Lgals3, Atf3, Fabp5 and Ccl4 expression in the different injury conditions: samples taken one week after sham, crush or PSNL surgery (acute) vs. samples taken 15 weeks after crush or PSNL surgery (chronic).

Article Snippet: Primary antibodies used in this study: α-NF200 (1:1000, mouse, Sigma-Aldrich #266004), α-BRN3A (1:100, rabbit, Merck #AB5945), IBA1 (1:500, guinea pig, Synaptic systems #234 308), α-GFP (1:1000, chicken, Abcam #ab13970), α-ATF3 (1:500, rabbit, Novus Biologicals #NBP1-85816), α-FABP5 (1:200, rabbit, ProteinTech #12348-1-AP), and α-β-tubulin III (1:1000, mouse, Sigma-Aldrich #N5389).

Techniques: Expressing, Marker, Sequencing