n2b27 basal medium  (MedChemExpress)

Journal: bioRxiv

Article Title: HDAC3 prevents enhancer hyperactivation to enable developmental transitions

doi: 10.1101/2025.08.09.669452

Figure Lengend Snippet: a , Schematic of the complementation experiment design, involving transfection with an empty vector, HDAC3 WT , or the catalytically inactive mutant HDAC3 Y298F . b , Immunoblot analysis of the complementation experiment ( a ) with whole-cell extracts from HDAC3-mAID-FLAG degron ESCs treated with indole acetic acid (IAA) for 3 hours. Blots were probed with anti-FLAG (endogenous HDAC3), anti-HA (transfected HDAC3) and anti-VINCULIN antibodies c , Principal Component Analysis (PCA) plot of nascent transcriptomics data from the 3-hour HDAC3 depletion and complementation experiment. d , Violin plot illustrating the shrunk log 2 (fold change), in degron ESCs complemented with empty vector, HDAC3 WT , or HDAC3 Y298F upon treatment with or without IAA, for genes that were previously identified as upregulated genes upon HDAC3 loss . Statistical analysis was performed using Kruskal–Wallis rank sum test followed by Dunn’s test. P- values were corrected using the Benjamini–Hochberg method. e , Transcription levels of representative pluripotency-associated genes in ESCs degron line complemented by empty vector, HDAC3 WT , or HDAC3 Y298F and treated with IAA for 3-hours. f , Schematic of the dCas9-GCN4-based epigenome editing system. Constitutively expressed dCas9 fused to GCN4-binding sites and TagBFP was stably transfected with doxycycline-inducible vectors expressing sfGFP-scFV, HDAC3 WT -sfGFP-scFV, or HDAC3 Y298F -sfGFP-scFV. Guide RNAs were used to target a safe-harbor locus expressing mCherry-H2B under the Ef1a promoter. g–h , Bar plots showing the percentage of sfGFP+/TagBFP+/mCherry2+ positive cells and their median fluorescence intensity (A.U.) upon doxycycline treatment. Statistical analysis was performed using ANOVA followed by Šidák’s test for multiple comparisons. i , Schematic of the protocol for derivation of trophoblast stem cells (TSCs) from ESCs. j , Immunoblot analysis of whole-cell extracts from HDAC3-mAID-FLAG degron TSCs treated with indole acetic acid (IAA) for varying durations. Blots were probed with anti-FLAG and anti-VINCULIN antibodies. k , Schematic of the experimental design for the 3-hour HDAC3 depletion in TSCs cultured in N2B27 with FGF2, Activin A, XAV939 and Y27632 (FAXY). l , Volcano plot of genes in IAA-treated versus untreated TSCs with shrunk log 2 (fold change) and -log10(adjusted p- value) calculated using DESeq2. Differentially expressed genes with adjusted p-value<0.05 are highlighted in color. m , Violin plot showing nascent transcription levels in the untreated TSCs. Plotted are upregulated, downregulated and unchanged genes upon IAA treatment. Transcript levels are size-factor normalized fragments per kilobase per million reads (FPKMs). Statistical analysis was performed using the Kruskal–Wallis rank sum test followed by Dunn’s test. P- values were corrected using the Benjamini–Hochberg method..

Article Snippet: For mCherry2-H2B reporter line, cells were cultured on gelatin-coated plates in N2B27 basal culture medium (NDiff by Takara, y40002), supplemented with 1 μM PD0325901, 3 μM CHIR99021, 1,000 U mL −1 LIF (in-house production), 1% FBS (Millipore) and 1% penicillin-streptomycin (ThermoFisher).

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Binding Assay, Stable Transfection, Expressing, Fluorescence, Cell Culture