Journal: bioRxiv

Article Title: Glycogen synthase kinase 3 alpha/beta deletion induces precocious growth plate remodeling and cell loss in mice

doi: 10.1101/2020.04.04.025700

Figure Lengend Snippet: Presence of Sox9 positive cells in Gsk3a/Gsk3b KO and control mice at P36, P43 and P58 was assessed by immunohistochemistry on frontal knee joint sections (A). Bone marrow cavity-like regions are highlighted in black dotted line. Localized expression and loss of GSK3alpha (B&C) and GSK3beta (D&E) in Gsk3a/Gsk3b KO mice was evaluated by IHC at P36 (B&D) and P43 (C&E). Images are representative of 4-7 mice per group for Sox9, and 3-4 mice per group were used for GSK3alpha and GSK3beta at both time points.

Article Snippet: Primary antibodies used for immunolabelling are as follows: goat polyclonal Sox9 (AF-3075, R&D Systems), rabbit monoclonal GSK3alpha (ab40870, Abcam), rabbit polyclonal GSK3beta (12456S, Cell Signaling Technologies), mouse monoclonal type II collagen (Col2; 10R-C135b, Fitzgerald), rabbit polyclonal osteocalcin (ab93876, Abcam), rabbit polyclonal beta-catenin (bs-1165R, Bioss Antibodies) and rabbit polyclonal GLI1 (PA5-72942, Invitrogen, Thermo Fisher).

Techniques: Control, Immunohistochemistry, 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: Figure 4. Loss of BATF2 exacerbates EAE and enhances IRF1 and caspase-1 expression in vivo (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 mm. (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 mm. (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 mm. (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 mm. (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.

Article Snippet: Demyelinated lesions were identified and characterized using immunostaining for MBP (Abcam) and IBA1 (Wako) as previously described.9,24 Subsequent sections were exposed to antibodies specific for human BATF2 (Santa Cruz), IRF1 (Proteintech), Sox9 (R&D Systems), ALDH1L1 (Cell Signaling), and/or IBA1 (SPICA Dye 594-conjugated; Wako) for 4–5 days at 4 C. Sections were then washed with PBS-Triton-X-100, and secondary antibodies conjugated to Alexa Fluor 405, 488, and 647 (Thermofisher) were applied for 1h at room temperature as appropriate.

Techniques: Expressing, In Vivo, 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: Figure 5. BATF2 is expressed by astrocytes in MS lesions and colocalizes with IRF1 (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 mm. (B) Chronic active lesion labeled for BATF2 and IBA1. Dotted line indicates the border of the LR and LC. Scale bars, 50 mm. (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, 50mm. (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 mm. 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 mm. (Fii) Three-dimensional (3D) render of the high-magnification image from (Fi) labeled for BATF2, IRF1, and SOX9. Scale bar, 10 mm. (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 Table 1 (n = 6). Each data point is repre- sentative of an individual patient. LR, lesion rim; LC, lesion core.

Article Snippet: Demyelinated lesions were identified and characterized using immunostaining for MBP (Abcam) and IBA1 (Wako) as previously described.9,24 Subsequent sections were exposed to antibodies specific for human BATF2 (Santa Cruz), IRF1 (Proteintech), Sox9 (R&D Systems), ALDH1L1 (Cell Signaling), and/or IBA1 (SPICA Dye 594-conjugated; Wako) for 4–5 days at 4 C. Sections were then washed with PBS-Triton-X-100, and secondary antibodies conjugated to Alexa Fluor 405, 488, and 647 (Thermofisher) were applied for 1h at room temperature as appropriate.

Techniques: Labeling, Two Tailed Test