Journal: ACS Omega

Article Title: New Synthetic Approach to C‑30 Ethers, Esters, and Amines of Betulin Using the Mitsunobu Reaction and Biological Evaluation of the Products

doi: 10.1021/acsomega.6c00716

Figure Lengend Snippet: Multiparametric flow cytometry analysis of the cellular response to compounds 30 and 37 in CCRF-CEM cells. (A) Cell cycle distribution. Representative PI histograms showing G1, S, and G2/M phase profiles after 24 h treatment with compounds 30 and 37 at 1 × IC 50 and 5 × IC 50 . PI fluorescence ( x -axis) was used to quantify total DNA content; only live, single-cell events were included. Untreated cells served as control. (B) Mitotic index (pH3Ser10). Flow cytometric detection of phospho-histone H-3 (Ser10)–positive cells as a measure of mitotic activity. Cells were stained with the anti-pH3Ser10 antibody following identical treatment conditions. Percentages represent the proportion of mitotic cells within the PI-gated population (region B). (C) DNA synthesis activity (BrdU incorporation). Cells were pulse-labeled with BrdU prior to harvesting. BrdU-positive cells (region B) reflect actively replicating populations. Dot plots show the relative BrdU incorporation under each treatment condition. (D) RNA synthesis activity (BrU incorporation). BrU-labeled nascent RNA was detected by flow cytometry using an anti-BrdU antibody cross-reactive with BrU. Region B denotes BrU-positive cells. Data illustrates treatment-dependent modulation of transcriptional activity. (E) Apoptotic cell death (sub-G1). Representative PI histograms showing the proportion of cells with sub-G1 DNA content following 24 h exposure to the compounds. The sub-G1 fraction quantifies apoptotic DNA fragmentation. All experiments were performed in biological triplicates with similar results. Flow cytometry data were processed and quantitatively evaluated using Kaluza software (Beckman Coulter). Collectively, these multiparametric analyses provide an integrated overview of how compounds 30 and 37 affect cell cycle dynamics, biosynthetic capacity, and cell death pathways in leukemia cells.

Article Snippet: After washing with PBS containing 0.5% Tween-20 and 1% BSA, BrdU incorporation was detected using a primary anti-BrdU antibody (EXBIO) followed by an FITC-conjugated secondary antimouse antibody (Sigma), with all incubations performed at room temperature in the dark.

Techniques: Flow Cytometry, Fluorescence, Single Cell, Control, Activity Assay, Staining, DNA Synthesis, BrdU Incorporation Assay, Labeling, Software