Fig. 5: Validation of computationally inferred velocities by cell tracking and labeling experiments.

a,b, Posterior estimate plot of constant (a) and periodic (b) cell cycle speed in dHFs⁴⁰. c, Top: schematic of time-lapse microscopy to track consecutive cell divisions. Bottom: example images at multiple time points to illustrate tracking a single segmented dHF (pink) through two divisions. Following division of the mother cell (16:40 h), one daughter cell (indicated by white arrow) is tracked for 15 h until dividing itself (31:40 h). d, Histogram of cell cycle period for 282 dHFs tracked by live imaging. e, Violin plot of dHF cell cycle speed, stratified by categorical phase assignment (G1, 514 cells; S, 383 cells; G2M, 325 cells). Median velocities are indicated by black lines (G1, 0.35; S, 0.37; G2M, 0.48). f, Dual-axis plot of the correspondence between unspliced–spliced (U–S) expression delay (left) and velocity (right). Left: genes were grouped by phase into 20 equal bins to calculate unspliced–spliced delay. The solid red line indicates binned mean delay; red bands indicate one standard deviation. Right: scaled velocity estimate from b. Bottom: categorical phase assignment probability. g, Gene expression scatterplots for genes peaking in S and M phases. Vertical lines correspond to the peak phase of spliced (blue) and unspliced (red) counts. h, Posterior estimate plot of periodic cell cycle speed in RPE1 cells. i, Images tracking a single RPE1 cell from birth (3:20 h) to subsequent division (20:00 h). j, Histogram of cell cycle period for 337 RPE1 cells tracked by live imaging. k, Diagram of the cumulative EdU/p21 experiment. Cells were continuously exposed to EdU, fixed at different time points and subjected to EdU detection and p21 immunostaining. l, Left: images of p21 (green), 4,6-diamidino-2-phenylindole (DAPI) (cyan) and EdU (magenta) staining after cumulative EdU labeling for 2 h, 8 h and 36 h (representative from one of three experimental replicates). Scale bar, 100 μm. Right: images of individual cells with different staining combinations. Scale bar, 10 μm. m, Schematic of cumulative EdU labeling during cell cycle progression. n, Dot plot representing the average percentage of p21+ cells along the different time points. The black horizontal line indicates the mean (min, 0.24, max, 0.76, mean, 0.52), with an error bar for s.d.; each dot represents the percentage of p21+ cells for a single replicate (n = 29; a total of three replicates with ten, ten and nine time points). o, Top: line plot of the fraction of EdU+ cells at 13 time points (from 30 min to 73 h). Data show the mean of three replicates (except for 2 h, which is from two) and error bars indicate the s.d. Bottom: line plot of fraction of EdU-positive cells among quiescent cells (p21+) as a function of time. A, x value at the intersection between growth and plateau; B, y intercept of the linear fit; GF, y value of the plateau. p, Illustration of scEU-seq⁴³ experimental design, which generates 24 tables used by Dynamo⁸ to produce a gold standard cell cycle period estimate. RFP, red fluorescent protein; GFP, green fluorescent protein; RPEs, retinal pigmented epithelium. q, Left: schematic of the different experimental measurements, manifold and cell path inference approaches taken by VeloCycle, Dynamo (without metabolically labeled information) and Dynamo-Metabolic (with metabolically labeled information) models. Right: plot showing the estimated cell cycle period obtained by VeloCycle, Dynamo and Dynamo-Metabolic models. The violin plot displays the posterior distribution output by VeloCycle and the circles are individual evaluations of the LAP from different start/end cells; red stars indicate the means. The red dashed line indicates the median in d and j. The white dashed line indicates the mean of 500 posterior predictions and the black bar indicates the credibility interval (5th to 95th percentile) in a,b,f (right) and h. NS, not significant; **P < 0.01.