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