Journal: The Journal of Biological Chemistry

Article Title: BATF2-mediated control of astrocyte proliferation

doi: 10.1016/j.jbc.2025.110710

Figure Lengend Snippet: Loss of BATF2 increases expression of proliferative markers in astrocytes. A and B, wildtype ( A ) and Batf2 −/− ( B ) astrocytes labeled for H3Ser10ph, actin, and nuclei counterstained with DAPI. The scale bars represent 50 μm. C and D, wildtype ( C ) and Batf2 −/− ( D ) astrocytes labeled for Ki67, actin, and nuclei counterstained with DAPI. The scale bars represent 50 μm. E and F, wildtype ( E ) and Batf2 −/− ( F ) astrocytes labeled for pMCM2, actin, and nuclei counterstained with DAPI. The scale bars represent 50 μm. G, quantification of H3Ser10ph-positive nuclei for wildtype and Batf2 −/− mice. Data were normalized to the total nuclei count, and data points are representative of individual mice. ∗∗ p < 0.01 compared with wildtype samples by two-tailed Student's t test. The bars represent mean ± SEM. H, quantification of colocalization between H3Ser10ph and DAPI. Data points are representative of individual mice. ∗∗ p < 0.01 compared with wildtype samples by two-tailed Student's t test. The bars represent mean ± SEM. I, quantification of Ki67-positive nuclei for wildtype and Batf2 −/− mice. Data points were normalized to the total nuclei count and are representative of individual mice. ∗∗∗∗ p < 0.0001 compared with wildtype samples by two-tailed Student's t test. The bars represent mean ± SEM. J, quantification of colocalization between Ki67 and DAPI. Data points are representative of individual mice. ∗∗∗ p < 0.001 compared with wildtype samples by two-tailed Student's t test. The bars represent mean ± SEM. K, quantification of pMCM2-positive nuclei for wildtype and Batf2 −/− mice. Data points were normalized to the total nuclei count and are representative of individual mice. ∗∗∗ p < 0.001 compared with wildtype samples by two-tailed Student's t test. The bars represent mean ± SEM. L, quantification of colocalization between pMCM2 and DAPI. Data points are representative of individual mice. ∗ p < 0.05 compared with wildtype samples by two-tailed Student's t test. The bars represent mean ± SEM. M, quantification of Ccnb1 transcript expression in wildtype and Batf2 −/− astrocytes. Data points were normalized to the wildtype average and are representative of individual mice. ∗∗ p < 0.01 compared with wildtype samples by two-tailed Student's t test. The bars represent mean ± SEM. N, representative Western blot of whole-cell cyclin B1 and vinculin protein levels of wildtype and Batf2 −/− astrocytes. O, quantification of whole-cell cyclin B1 protein levels in wildtype and Batf2 −/− astrocytes shown in N , normalized to vinculin expression. Data points are representative of individual mice. ∗∗ p < 0.01 compared with wildtype samples by two-tailed Student's t test. The bars represent mean ± SEM. P, cell viability assay of wildtype and Batf2 −/− astrocytes over 96 h. Data points are representative of the combined average of individual mouse absorbance values at 450 nm ( Batf2 +/+ N = 4, Batf2 −/− N = 4 per timepoint). ∗ p < 0.05 compared with wildtype samples by one-way ANOVA. The bars represent mean ± SEM. Q, live cell counts of wildtype and Batf2 −/− astrocytes over 96 h. Data points are representative of the combined average of individual mouse live cell counts ( Batf2 +/+ N = 4, Batf2 −/− N = 4 per timepoint). ∗ p < 0.05, ∗∗ p < 0.01 compared with wildtype samples by one-way ANOVA. The bars represent mean ± SEM. BATF2, basic leucine zipper ATF-like transcription factor 2; DAPI, 4′,6-diamidino-2-phenylindole; pMCM2, phospho-mini chromosome maintenance protein 2.

Article Snippet: Slides were blocked with 10% goat serum (Sigma) and 0.3% Triton X-100 for 15 min at room temperature and then incubated with primary antibodies for BATF2 (Santa Cruz; SC293274), cyclin D1 (Santa Cruz; SC717), phospho-histone H3 (Ser10) (Cell Signaling; 9701S), Ki67 (Abcam; AB15580), pMCM2 (Ser139) (D1Z8X) (Cell Signaling; 12958S), and CKS1B (Invitrogen; 36-6800) overnight at 4 °C.

Techniques: Expressing, Labeling, Two Tailed Test, Western Blot, Viability Assay

Journal: The Journal of Biological Chemistry

Article Title: BATF2-mediated control of astrocyte proliferation

doi: 10.1016/j.jbc.2025.110710

Figure Lengend Snippet: BATF2 binds to DNA motifs associated with cell cycle genes in astrocytes. A and B, IPA of top-regulated molecular functions ( A ) and diseases ( B ) by BATF2 in human astrocytes. C, adapted IPA S phase pathway. D, adapted IPA cell cycle regulation by BTG protein pathway. E–G, ChIP sequencing peak region counts of BATF2-binding events at the CKS1B ( E ) , CDK2 ( F ) , and CCND1 ( G ) gene loci and CpG islands in human astrocytes. BATF2, basic leucine zipper ATF-like transcription factor 2; ChIP, chromatin immunoprecipitation; IPA, ingenuity pathway analyses.

Article Snippet: Slides were blocked with 10% goat serum (Sigma) and 0.3% Triton X-100 for 15 min at room temperature and then incubated with primary antibodies for BATF2 (Santa Cruz; SC293274), cyclin D1 (Santa Cruz; SC717), phospho-histone H3 (Ser10) (Cell Signaling; 9701S), Ki67 (Abcam; AB15580), pMCM2 (Ser139) (D1Z8X) (Cell Signaling; 12958S), and CKS1B (Invitrogen; 36-6800) overnight at 4 °C.

Techniques: ChIP-sequencing, Binding Assay, Chromatin Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: BATF2-mediated control of astrocyte proliferation

doi: 10.1016/j.jbc.2025.110710

Figure Lengend Snippet: BATF2 regulates cell cycle machinery expression in astrocytes. A–C, quantification of Cks1b ( A ), Cdk2 ( B ), and Ccnd1 ( C ) transcript expression in wildtype and Batf2 −/− astrocytes. Data points were normalized to the wildtype average and are representative of individual mice. ∗ p < 0.05, ∗∗ p < 0.01 compared with wildtype samples by two-tailed Student's t test. The bars represent mean ± SEM. D, representative Western blot of whole-cell cyclin D1 and vinculin protein levels in wildtype and Batf2 −/− astrocytes. E, quantification of whole-cell cyclin D1 protein levels in wildtype and Batf2 −/− astrocytes shown in D , normalized to vinculin expression. Data points are representative of individual mice. The bars represent mean ± SEM. F and G, wildtype ( F ) and Batf2 −/− ( G ) astrocytes labeled for cyclin D1, actin, and nuclei counterstained with DAPI. The scale bars represent 50 μm. H and I, quantification of cyclin D1-positive area ( H ) and intensity ( I ) for wildtype and Batf2 −/− mice. Data points were normalized to actin expression and are representative of individual mice. ∗ p < 0.05 compared with wildtype samples by two-tailed Student's t test. The bars represent mean ± SEM. BATF2, basic leucine zipper ATF-like transcription factor 2; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Slides were blocked with 10% goat serum (Sigma) and 0.3% Triton X-100 for 15 min at room temperature and then incubated with primary antibodies for BATF2 (Santa Cruz; SC293274), cyclin D1 (Santa Cruz; SC717), phospho-histone H3 (Ser10) (Cell Signaling; 9701S), Ki67 (Abcam; AB15580), pMCM2 (Ser139) (D1Z8X) (Cell Signaling; 12958S), and CKS1B (Invitrogen; 36-6800) overnight at 4 °C.

Techniques: Expressing, Two Tailed Test, Western Blot, Labeling

Journal: The Journal of Biological Chemistry

Article Title: BATF2-mediated control of astrocyte proliferation

doi: 10.1016/j.jbc.2025.110710

Figure Lengend Snippet: Overexpression of BATF2 in U87-MG cells limits the expression of target cell cycle genes. A–D, quantification of BATF2 ( A ), CKS1B ( B ), CDK2 ( C ), and CCND1 ( D ) gene expression in human astrocytes and U87-MG cells. Data points were normalized to the human astrocyte average and are representative of replicates from two independent experiments. ∗ p < 0.05, ∗∗ p < 0.01 compared with human astrocyte samples by two-tailed Student's t test. The bars represent mean ± SEM. E and F, human astrocytes ( E ) and U87-MG cells ( F ) labeled for BATF2, actin, and nuclei counterstained with DAPI. The scale bars represent 50 μm. G and H, quantification of BATF2-positive area ( G ) and intensity ( H ) for human astrocytes and U87-MG cells. Data points were normalized to actin expression and are representative of replicates from two independent experiments. ∗ p < 0.05 compared with human astrocyte samples by two-tailed Student's t test. The bars represent mean ± SEM. I, representative Western blot of whole-cell BATF2, cyclin D1, and vinculin protein levels in human astrocytes and U87-MG cells. J–K, quantification of whole-cell BATF2 ( J ) and cyclin D1 ( K ) protein levels of human astrocytes and U87-MG cells shown in I , normalized to vinculin expression. Data points are representative of replicates from two independent experiments. The bars represent mean ± SEM. BATF2, basic leucine zipper ATF-like transcription factor 2; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Slides were blocked with 10% goat serum (Sigma) and 0.3% Triton X-100 for 15 min at room temperature and then incubated with primary antibodies for BATF2 (Santa Cruz; SC293274), cyclin D1 (Santa Cruz; SC717), phospho-histone H3 (Ser10) (Cell Signaling; 9701S), Ki67 (Abcam; AB15580), pMCM2 (Ser139) (D1Z8X) (Cell Signaling; 12958S), and CKS1B (Invitrogen; 36-6800) overnight at 4 °C.

Techniques: Over Expression, Expressing, Gene Expression, Two Tailed Test, Labeling, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: BATF2-mediated control of astrocyte proliferation

doi: 10.1016/j.jbc.2025.110710

Figure Lengend Snippet: Knockdown of BATF2 upregulates cell cycle genes in U87-MG cells. A–D, quantification of BATF2 ( A ), CKS1B ( B ), CDK2 ( C ), and CCND1 ( D ) gene expression in U87-MG cells treated with siControl or si BATF2 for 72 h. Data points were normalized to the siControl average and are representative of replicates from two independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001 compared with siControl samples by one-way ANOVA. The bars represent mean ± SEM. E–G, U87-MG cells treated with siControl ( E ) and si BATF2 ( F and G ) labeled for BATF2, actin, and nuclei counterstained with DAPI. The scale bars represent 50 μm. H and I, quantification of BATF2-positive area ( H ) and intensity ( I ) for U87-MG cells treated with siControl or si BATF2 . Data points were normalized to actin expression and are representative of technical replicates. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 compared with siControl samples by one-way ANOVA. The bars represent mean ± SEM. J–L, U87-MG cells treated with siControl ( J ) and si BATF2 ( K and L) labeled for cyclin D1, actin, and nuclei counterstained with DAPI. The scale bars represent 50 μm. M and N, quantification of cyclin D1-positive area ( M ) and intensity ( N ) for U87-MG cells treated with siControl or si BATF2 . Data points were normalized to actin expression and are representative of technical replicates. ∗ p < 0.05, ∗∗ p < 0.01 compared with siControl samples by one-way ANOVA. O–Q, U87-MG cells treated with siControl ( O ) and si BATF2 ( P and Q ) labeled for Ki67, actin, and nuclei counterstained with DAPI. The scale bars represent 50 μm. R and S, quantification of Ki67-positive nuclei for siControl or si BATF2 -treated U87-MG cells. Data points in R were normalized to the total nuclei count. Data shown in R and S are representative of technical replicates. ∗ p < 0.05 compared with siControl samples by one-way ANOVA. The bars represent mean ± SEM. T, quantification of CCNB1 transcript expression in siControl or si BATF2 -treated U87-MG cells. Data points were normalized to the siControl average and are representative of replicates from two independent experiments. ∗∗∗∗ p < 0.0001 compared with siControl samples by one-way ANOVA. The bars represent mean ± SEM. U, cell viability assay of siControl or si BATF2- treated U87-MG cells over 96 h. Data points are representative of the combined average of absorbance values at 450 nm from individual replicates from two independent experiments (siControl N = 4–6, si BATF2 (149104) N = 5–6, si BATF2 (36727) N = 4 to 6 per timepoint). ∗∗ p < 0.01 compared with siControl samples by one-way ANOVA. The bars represent mean ± SEM. V, live cell counts for siControl or si BATF2 -treated U87-MG cells over 96 h. Data points are representative of the combined average of live cell counts of individual replicates from two independent experiments. (siControl N = 4, si BATF2 (149104) N = 3–4, si BATF2 (36727) N = 4 per timepoint). ∗∗∗ p < 0.001 compared with siControl samples by one-way ANOVA. The bars represent mean ± SEM. BATF2, basic leucine zipper ATF-like transcription factor 2; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Slides were blocked with 10% goat serum (Sigma) and 0.3% Triton X-100 for 15 min at room temperature and then incubated with primary antibodies for BATF2 (Santa Cruz; SC293274), cyclin D1 (Santa Cruz; SC717), phospho-histone H3 (Ser10) (Cell Signaling; 9701S), Ki67 (Abcam; AB15580), pMCM2 (Ser139) (D1Z8X) (Cell Signaling; 12958S), and CKS1B (Invitrogen; 36-6800) overnight at 4 °C.

Techniques: Knockdown, Gene Expression, Labeling, Expressing, Viability Assay

Journal: The Journal of Biological Chemistry

Article Title: BATF2-mediated control of astrocyte proliferation

doi: 10.1016/j.jbc.2025.110710

Figure Lengend Snippet: BATF2 expression negatively correlates with cyclin D1 levels in GBM. A, overall survival outcomes relative to BATF2 expression in GBM patients. B, quantification of BATF2 expression between GBM and nontumor samples (GBM N = 163, nontumor N = 207). Data represented in A and B were obtained from TCGA and GTEx publicly available data sets and were analyzed using Gepia2. ∗ p < 0.05 compared with nontumor samples. The bars represent mean ± SD. C and D, quantification of BATF2 ( C ) and CCND1 ( D ) gene expression in human astrocytes and GBM patient samples. Data points were normalized to the human astrocyte average. Human astrocyte data points are representative of technical replicates. GBM data points are representative of individual patients. The bars represent mean ± SEM. E, regression analysis of CCND1 versus BATF2 gene expression in GBM patients. Data points were analyzed by simple linear regression and are representative of individual patients. F, representative Western blot of whole-cell BATF2, cyclin D1, and beta-actin protein levels in human astrocytes and GBM patient samples. G, regression analysis of cyclin D1 versus BATF2 protein expression in GBM patients. Data points were analyzed by simple linear regression and are representative of individual patients. BATF2, basic leucine zipper ATF-like transcription factor 2; GBM, glioblastoma multiforme; GTEx, Genotype-Tissue Expression; TCGA, The Cancer Genome Atlas.

Article Snippet: Slides were blocked with 10% goat serum (Sigma) and 0.3% Triton X-100 for 15 min at room temperature and then incubated with primary antibodies for BATF2 (Santa Cruz; SC293274), cyclin D1 (Santa Cruz; SC717), phospho-histone H3 (Ser10) (Cell Signaling; 9701S), Ki67 (Abcam; AB15580), pMCM2 (Ser139) (D1Z8X) (Cell Signaling; 12958S), and CKS1B (Invitrogen; 36-6800) overnight at 4 °C.

Techniques: Expressing, Gene Expression, Western Blot