Figure 5. Dynamic changes in condensin dependency during lymphocyte differentiation.

(A) Chronological representation of the BrdU pulse chase assay to measure the efficiency of cell division in primary cell types cultured ex vivo. Lymphocyte isolation and culture protocols are detailed in Materials and methods. Quantifying the % of BrdU⁺ cells (B) that complete mitosis and halve their DNA content (C) allows the efficiency of a single-cell division to be quantified under normal or acute condensin deficient conditions. The appearance of BrdU⁺G1 cells can be seen at 3 and 5 hr. (D) Representative DNA content profiles, gated on BrdU⁺ as shown in panel B, from cycling early (thymic/marrow) or activated mature (Splenic) T and B lymphocytes, measured following a 3.5-hr chase in the presence or absence of condensin I or II. (E) Quantification of division efficiency, based on the % of BrdU⁺ cells in G1 after 3.5 hr (n = 3 biological replicates from at least 2 independent experiments). Corresponding condensin depletion levels for each experiment are shown in Figure 5—figure supplement 1C (F). Quantification of the effect of NCAPH or NCAPH2 degradation on cell division across cell types in panel E. For each cell type, division efficiency (panel E) in the vehicle only control condition was set to 100%, and the same parameter in IAA-treated cells was expressed relative to this. Asterisks represent p values from paired t-tests ***p < 0.01, **p < 0.05, *p < 0.1.

Figure 5—figure supplement 1. Dynamic changes in condensin dependency during lymphocyte differentiation.

(A) The % of cells engaged in DNA replication (BrdU+) is not significantly different following acute depletion of NCAPH or NCAPH2. Each point shows the average % of cells incorporating BrdU following a 30-min pulse following 2 hr of culture in 500 μM IAA or vehicle, measured by flow cytometry. Bone marrow B cells required an extra hour of IAA treatment (3 h total) to achieve robust depletion. Experimental schematic is shown in Figure 5A. (B) Acute depletion of NCAPH or NCAPH2 does not induce the DNA damage marker ˠH2AX in interphase cells undergoing DNA replication. Each point represents the average fluorescence intensity from at least 1000 single CD8⁺ thymocytes single cells with DNA content between 2N and 4N (presumed to be in S phase). * indicates significant differences at p < 0.05 based on two-tailed unpaired t-tests. Positive control wildtype cells were treated with 500 μM hydroxyurea for 3 hr to induce replication fork collapse. (C) Mean depletion levels of NCAPH and NCAPH2 proteins in the BrdU pulse chase experiments shown in Figure 5. Clover was quantified by flow cytometry in S/G2/M cells at the start (0 hr) and end (3.5 hr) of the chase period, with the +IAA value expressed as a % of vehicle only control after correcting for background autofluorescence. Where mean Clover fluorescence was lower than autofluorescence in the +IAA condition, a mean value of 0% was assigned. (D) Reduced proliferation in peripheral B cells following acute degradation of NCAPH or NCAPH2, measured by Cell Trace flow cytometry assays. Contour plots show Cell Trace dye dilution via cell division following stimulation with LPS + IL4 for 48 hr in the continuous presence of 500 μM IAA. Condensin-AID:Clover signal is shown on the y-axis to visualise degradation.