Journal: bioRxiv

Article Title: TurboID-based proximity labeling discovers ABCF2 as an adhesion receptor for the zoonotic pathogen Pasteurella multocida

doi: 10.1101/2024.12.03.626657

Figure Lengend Snippet: (A, B) P. multocida infection induced increased levels of ABCF2 in NPTr and A549 cells evaluated by qPCR (A) and western-blotting (B) assays. (C) Western-blotting results showing the expression of ABCF2 in NPTr cells induced by P. multocida wild type strain (WT), the plpE -deleted strain (ΔPlpE), and plpE -complementary strains (CPlpE). (D) Results from western-blotting assays quantified by the image J software. (E) Western-blotting results showing the expression of ABCF2 and P65 phosphorylation (P-P65) in NPTr cells at 0 (mock), 1, 2, 6, and 12 hours post P. multocida inoculation. (F) Results from western-blotting assays quantified by the image J software. (G) Western-blotting results showing the expression of ABCF2 and P65 phosphorylation (P-P65) in NPTr cells treated with (+) or without (-) the NF-κB inhibitor (BAY11-7082), followed by P. multocida inoculation. (H) Results from western-blotting assays quantified by the image J software. (I) qPCR detecting the transcriptional levels of atf2 , stat1 , and abcf2 in NPTr cells at 12 hours post P. multocida inoculation. (J) Western-blotting results showing the P38 phosphorylation (P-P38) in NPTr cells at 0, 4, 8, 12, 16, and 20 hours post P. multocida inoculation. (K) Western-blotting results showing the expression of ABCF2 and P38 phosphorylation (P-P38) in NPTr cells treated with (+) or without (-) p38 inhibitor (BIRB796), followed by P. multocida inoculation. (L) P38 phosphorylation (P-P38) from western-blotting assays quantified by the image J software. (M) ABCF2 expression from western-blotting assays quantified by the image J software. (N) Dual luciferase assays demonstrating the regulation of atf2 on the expression of abcf2 . (O) qPCR assays verifying ATF2 overexpression (ATF2-OE) in NPTr cells with or without P. multocida infection. (P) qPCR assays showing ATF2 overexpression (ATF2-OE) contribute to ABCF2 expression after P. multocida infection. (Q) Western-blotting revealing ATF2 overexpression (ATF2-OE) contribute to ABCF2 expression after P. multocida infection. (R) ABCF2 expression from western-blotting assays quantified by the image J software. (S) Western-blotting showing inhibition of p38 MAPK signaling using the inhibitor BIRB796 decreases P65 phosphorylation (P-P65) after P. multocida infection. (T) The level of P65 phosphorylation (P-P65) from western-blotting assays quantified by the image J software. In all column charts, data were presented as mean ± standard deviation (SD). The significance level was set at P > 0.05 (no significance [ns]), P < 0.05 (*), P < 0.01 (**), or P < 0.001 (***).

Article Snippet: Proteins were transferred to PVDF membranes (Bio-Rad, Hercules, USA), blocked in PBST (containing 5%BSA) for 3 h at room temperature, and probed with specific antibodies overnight at 4 °C to examine the expression of ABCF2 (ABCF2 polyclonal antibody [1:200, MyBioSource, San Diego, USA]), Flag-ATF2 (DYKDDDDK tag polyclonal antibody [1:5,000, Proteintech, San Diego, USA]), p53 (p53 monoclonal antibody [1:10000, Proteintech, San Diego, USA]), P-p53 (phospho-p53 (Ser15) monoclonal antibody [1:2000, Proteintech, San Diego, USA]), Bcl-2 (Bcl-2 polyclonal antibody [1:2000, Proteintech, San Diego, USA]), BAX (BAX monoclonal antibody [1:10000, Proteintech, San Diego, USA]), Caspase3 (Caspase 3/p17/p19 polyclonal antibody [Proteintech, San Diego, USA]), Caspase9 (Caspase 9/p35/p10 polyclonal antibody [Proteintech, San Diego, USA]), and/or GAPDH (GAPDH polyclonal antibody [1:5000, Proteintech, San Diego, USA]).

Techniques: Infection, Western Blot, Expressing, Software, Luciferase, Over Expression, Inhibition, Standard Deviation

Journal: Cell reports

Article Title: BATF2 is a regulator of interferon-γ signaling in astrocytes during neuroinflammation

doi: 10.1016/j.celrep.2025.115393

Figure Lengend Snippet: (A) PCA of RNA sequencing demonstrating distinct gene expression patterns of human spinal cord astrocytes treated with 10 ng/mL IFNγ, TNF-α, IL-1β, IL-17, GM-CSF, and media for 24 h. (B) Heatmap of top upregulated genes in human spinal cord astrocytes treated with 10 ng/mL of IFNγ for 24 h compared to TNF-α and IL-1β treatments. Data represent log2 fold change compared to media from 3 independent samples. (C) Top upregulated genes in human spinal cord astrocytes treated with 10 ng/mL IFNγ for 24 h compared to TNF-α and IL-1β treatments. Data represent the log2 fold change compared to media from 3 independent samples. (D) BATF2 transcript levels of human spinal cord astrocytes stimulated with 10 ng/mL IFNγ for 0–48 h. ** p < 0.01 and *** p < 0.001 compared to media-treated samples by one-way ANOVA. Data points represent mean ± SEM ( n = 3). (E) BATF2 transcript levels of human spinal cord astrocytes treated with 10 ng/mL of various inflammatory stimuli for 24 h. **** p < 0.0001 compared to media-treated samples by two-way ANOVA. Bars represent mean ± SEM ( n = 4). (F) Representative western blot of whole-cell BATF2 protein levels in human spinal cord astrocytes treated with 10 ng/mL of IFNγ for 24 h. (G) Quantification of whole-cell BATF2 protein levels in primary human spinal cord astrocytes shown in (F), normalized to β-actin expression. * p < 0.05 compared to media-treated samples by one-way ANOVA. Bars represent mean ± SEM ( n = 3).

Article Snippet: BATF2 Rabbit PolyAb , Proteintech , AB_2878285.

Techniques: RNA Sequencing, Gene Expression, Western Blot, Expressing

Journal: Cell reports

Article Title: BATF2 is a regulator of interferon-γ signaling in astrocytes during neuroinflammation

doi: 10.1016/j.celrep.2025.115393

Figure Lengend Snippet: (A) Merged peak region heatmap of BATF2 binding in human spinal cord astrocytes treated with 10 ng/mL IFNγ or media for 24 h. Scale bar indicates BATF2 binding events per merged region. (B) Peak tag numbers of BATF2 binding events in 1,289 merged peak regions of human spinal cord astrocytes treated with 10 ng/mL IFNγ or media for 24 h. (C) Peak location of BATF2 binding events in human spinal cord astrocytes treated with 10 ng/mL IFNγ or media for 24 h. hg38 binding events were used as an internal control. Data are the average peak locations between two independent samples per treatment. (D and E) Top BATF2 binding motifs of media- and IFNγ-treated human spinal cord astrocytes. Data shown in (A)–(E) are representative of two independent samples per treatment.

Article Snippet: BATF2 Rabbit PolyAb , Proteintech , AB_2878285.

Techniques: Binding Assay, Control

Journal: Cell reports

Article Title: BATF2 is a regulator of interferon-γ signaling in astrocytes during neuroinflammation

doi: 10.1016/j.celrep.2025.115393

Figure Lengend Snippet: (A) Ingenuity pathway analysis of top regulated pathways by BATF2 in human spinal cord astrocytes stimulated with 10 ng/mL IFNγ for 24 h. (B) Graphical summary of predicated targets regulated by BATF2 in human spinal cord astrocytes stimulated with 10 ng/mL IFNγ for 24 h. (C) Adapted ingenuity pathway analysis: IFNγ signaling pathway from (A). (D) Adapted ingenuity pathway analysis: multiple sclerosis signaling pathway from (A). (E) Peak region counts of BATF2 binding events upstream of the IRF1 gene locus and at CpG islands in human spinal cord astrocytes stimulated with 10 ng/mL IFNγ for 24 h (green) or media (gray). (F–K) Quantification of Irf1 and target gene transcript expression in Batf2 +/+ and Batf2 −/− primary murine spinal cord astrocytes stimulated with media or 10 ng/mL IFNγ for 48 h. Data are representative of 2 independent experiments. Individual data points were normalized to the media average and are representative of individual mice. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 compared to media-treated samples by two-way ANOVA. Bars represent mean ± SEM ( n =7). Data shown in (A)–(E) are representative of two independent samples per treatment.

Article Snippet: BATF2 Rabbit PolyAb , Proteintech , AB_2878285.

Techniques: Binding Assay, Expressing

Journal: Cell reports

Article Title: BATF2 is a regulator of interferon-γ signaling in astrocytes during neuroinflammation

doi: 10.1016/j.celrep.2025.115393

Figure Lengend Snippet: (A and B) EAE was induced in Batf2 −/− and control mice. EAE (A) clinical course and (B) weights were blindly monitored. Data are a combination of 3 independent experiments and were analyzed using the Mann–Whitney U test for nonparametric data. ** p < 0.01 from day 2 and **** p < 0.0001. Data points represent mean ± SEM ( n = 8 per genotype). 25 days post-immunization, mice were sacrificed, and the CNS tissue was cryopreserved for immunofluorescent analysis. Ventral white matter tracts of the lumbar spinal cord were imaged using confocal microscopy. (C and D) Spinal cord tissue from (C) Batf2 +/+ and (D) Batf2 −/− mice labeled for MBP and nuclei counterstained with DAPI. Scale bars, 20 μm. (E) Quantification of MBP area normalized to the average of Batf2 +/+ control mice. ** p < 0.01 compared to Batf2 +/+ samples by two-tailed Student’s t test. Bars represent mean ± SEM. (F and G) Spinal cord tissue from (F) Batf2 +/+ and (G) Batf2 −/− mice labeled for CD45 and nuclei counterstained with DAPI. Scale bars, 20 μm. (H and I) Quantification of CD45 area and lesion area normalized to the average of Batf2 +/+ control mice. ** p < 0.01 compared to Batf2 +/+ samples by two-tailed Student’s t test. Bars represent mean ± SEM. (J and K) Spinal cord tissue from (J) Batf2 +/+ and (K) Batf2 −/− mice labeled for IRF1, SOX9, and nuclei counterstained with DAPI. Scale bars, 20 μm. (L–N) Quantification of IRF1 area normalized to the average of Batf2 +/+ control mice and IRF1 area in SOX9 + and SOX9 − cells normalized to lesion area and the average of Batf2 +/+ mice. * p < 0.05 and ** p < 0.01 compared to Batf2 +/+ samples by two-tailed Student’s t test. Bars represent mean ± SEM. (O) Colocalization of SOX9 with IRF1 for Batf2 +/+ and Batf2 −/− mice. ** p < 0.01 compared to Batf2 +/+ samples by two-tailed Student’s t test. Bars represent mean ± SEM. (P and Q) Spinal cord tissue from (N) Batf2 +/+ and (O) Batf2 −/− mice labeled for caspase-1, GFAP, and nuclei counterstained with DAPI. Scale bars, 20 μm. (R–T) Quantification of caspase-1 area normalized to the average of Batf2 +/+ control mice and caspase-1 area in GFAP + and GFAP − cells normalized to lesion area and the average of Batf2 +/+ mice. * p < 0.05 and *** p < 0.001 compared to Batf2 +/+ samples by two-tailed Student’s t test. Bars represent mean ± SEM. (U) Colocalization of GFAP with caspase-1 for Batf2 +/+ and Batf2 −/− mice. *** p < 0.001 compared to Batf2 +/+ samples by two-tailed Student’s t test. Bars represent mean ± SEM. Data in (C)–(U) are representative of 3 independent experiments and include mice that survived until endpoint at day 25 ( n = 8 for Batf2 +/+ , n = 7 for Batf2 −/− ). Each data point is representative of an individual mouse.

Article Snippet: BATF2 Rabbit PolyAb , Proteintech , AB_2878285.

Techniques: Control, MANN-WHITNEY, Confocal Microscopy, Labeling, Two Tailed Test

Journal: Cell reports

Article Title: BATF2 is a regulator of interferon-γ signaling in astrocytes during neuroinflammation

doi: 10.1016/j.celrep.2025.115393

Figure Lengend Snippet: (A) Human postmortem MS brain sections labeled for MBP and IBA1 to identify NAWM and chronic active lesions. Dotted line indicates the border of the LR and LC. Scale bars, 50 μm. (B) Chronic active lesion labeled for BATF2 and IBA1. Dotted line indicates the border of the LR and LC. Scale bars, 50 μm. (C) Quantification of BATF2 area. * p < 0.05 and ** p < 0.01 by two-tailed Student’s t test for each set of bars. Bars represent mean ± SEM. (D) Chronic active lesion labeled for IRF1 and IBA1. Dotted line indicates the border of the LR and LC. Scale bars, 50μm. (E) Quantification of IRF1 area. * p < 0.05 by two-tailed Student’s t test for each set of bars. Bars represent mean ± SEM. (F) Chronic active lesions labeled for BATF2 and SOX9 (top) and IRF1 and SOX9 (bottom). Dotted line indicates the border of the LR and LC. Scale bar, 50 μm. Green box indicates the area of interest within the LC. (Fi) High-magnification image of LC area of interest from (F) labeled for BATF2, IRF1, and SOX9. Scale bar, 10 μm. (Fii) Three-dimensional (3D) render of the high-magnification image from (Fi) labeled for BATF2, IRF1, and SOX9. Scale bar, 10 μm. (G) Quantification of colocalization between SOX9 and IBA1 with BATF2 (green) and IRF1 (blue). * p < 0.05 and ** p < 0.01 by one-way ANOVA for each set of bars. Bars represent mean ± SEM. (H) Quantification of colocalization of BATF2 and IRF1 in the LR and LC. Data were analyzed by a two-tailed Student’s t test. Bars represent mean ± SEM. Data in (C), (E), (G), and (H) are representative of 2 independent experiments and include all patients represented in ( n = 6). Each data point is representative of an individual patient. LR, lesion rim; LC, lesion core.

Article Snippet: BATF2 Rabbit PolyAb , Proteintech , AB_2878285.

Techniques: Labeling, Two Tailed Test

Journal: Cell reports

Article Title: BATF2 is a regulator of interferon-γ signaling in astrocytes during neuroinflammation

doi: 10.1016/j.celrep.2025.115393

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: BATF2 Rabbit PolyAb , Proteintech , AB_2878285.

Techniques: Recombinant, Control, Protease Inhibitor, Western Blot, Electron Microscopy, Amplification, Reverse Transcription, SYBR Green Assay, Bicinchoninic Acid Protein Assay, RNA Sequencing, Software, Real-time Polymerase Chain Reaction

Journal: Circulation research

Article Title: Cezanne regulates inflammatory responses to hypoxia in endothelial cells by targeting TRAF6 for deubiquitination

doi: 10.1161/CIRCRESAHA.111.300119

Figure Lengend Snippet: (A) HUVEC or HCMEC were exposed to hypoxia for 15 min-4 h or remained untreated (normoxia). Some cultures were then reoxygenated for 1 h. Cytosolic lysates were tested by Western blotting using anti-phosphorylated ATF2 antibodies or by using anti-α-tubulin antibodies to assess total protein levels. Data are representative of three independent experiments. (B) HUVEC were treated with siRNA sequences that target Cezanne (Cez 1, Dharmacon; Cez 2, SMARTpool), ATF2 (ATF2 1, Dharmacon; ATF2 2, SMARTpool) or SHP2, or with scrambled, non-targeting sequences as a control (Scr). They were then exposed to hypoxia (4 h) or remained untreated (normoxia). Levels of Cezanne or ATF2 were quantified by real-time PCR. Data were pooled from 3 independent experiments.

Article Snippet: Anti-Cezanne (Proteintech Europe Ltd), Anti-RelA (p65), anti-κBα, anti-TRAF6, anti-Lamin B (Santa Cruz Biotechnology), phosphorylated anti-ATF2 (Thr71), phosphorylated anti-RelA (Ser536) (Cell Signalling Technology), anti-ubiquitin (Invitrogen), anti- Lys63 polyubiquitin, anti-GAPDH (Merck-Millipore), anti-α-tubulin (Sigma-Aldrich) anti-kidney injury marker-1 (R&D Systems) and polyclonal goat anti-rabbit and anti-mouse conjugated horse radish peroxidise (HRP) (Dako) antibodies were obtained commercially.

Techniques: Western Blot, Control, Real-time Polymerase Chain Reaction