Fig. 1: Adipocyte progenitor proliferation can be driven by CDK4/6 or CDK2 activation.

a OP9 cells expressing mCitrine-PPARG and a nuclear marker (H2B-mTurquoise2) were differentiated over 4 days with the standard adipogenic DMI cocktail (Dexamethasone—1 μM, IBMX—250 μM, and Insulin—1.75 nM). Scale bar —50 μm. b Representative single-cell time courses of mCitrine-PPARG from a typical experiment. In the standard DMI-induced differentiation protocol, the DMI cocktail is added to cell culture media for 48 h (horizontal black bar), and the media is replaced after 48 h with fresh media containing just insulin for another 48 h (white horizontal bar). Fifty single-cell time courses are shown as examples. The differentiation commitment threshold is indicated by the dashed line. Cells go on to either differentiate by increasing PPARG levels above the threshold (green time courses) or remain undifferentiated with low PPARG levels (gray time courses). Distribution of PPARG levels at 96 h is shown on the right. Data shown is representative of >2000 cells tracked in the experiment and three biological replicates. c Schematic of APC/C cell cycle reporter construct and dynamics of the reporter’s nuclear fluorescence intensity within a cell during different phases of the cell cycle. d Dual reporter cells expressing mCitrine-PPARG and mCherry-APC/C-reporter were differentiated using the standard DMI protocol. Representative time courses from four single cells. The APC/C-reporter signal is low during the G1 phase, increases during the S/G2 phases, and drops off at mitosis. In differentiating cells, commitment to differentiation (yellow circle) occurs during the G1-phase, whereas undifferentiated cells continue to divide intermittently during the 96 h experimental window (last panel from the right). Data shown are representative of >2000 cells tracked in the experiment and three biological replicates. e CDK4/6 and CDK2 kinase-translocation reporter (KTR) constructs. NLS nuclear localization signal sequence, NES nuclear export signal sequence, S consensus serine phosphorylation site for CDKs. f Representative cells expressing both the CDK4/6 and CDK2 reporters. Increasing CDK activities during the cell cycle leads to the phosphorylation and translocation of the two reporters from the nucleus to the cytosol. Scale bar—10 μm. g Single-cell time courses from two proliferating OP9 progenitor cells expressing CDK4/6 and CDK2 reporters. CDK4/6 and CDK2 activities were measured as the ratio of cytosolic to nuclear signal intensity. ‘M’ marks mitosis events. ‘G1/S’ marks the transition from G1 to S-phase, defined as when CDK2 and CDK4/6 reporter activities reach a value of 0.8, as indicated by the dashed line. h Single-cell time courses of CDK4/6 (top) and CDK2 (bottom) activity from 30 OP9 progenitor cells that undergo mitosis (seen by the drop in CDK activities) within 6 h of starting the imaging. CDK activity bifurcates into high (orange and blue) and low states (gray) after mitosis. Representative of two biological replicates. i–l DMSO (control), CDK4/6 inhibitor (CDK4i, Palbociclib, 1 μM), CDK2 inhibitor (CDK2i, Tagtociclib, 1 μM), or both CDK4/6 and CDK2 inhibitors (Dual CDKi, 1 μM of each) were added to the culture media of proliferating OP9 progenitor cells 24 h after the start of imaging (dashed vertical line). Plots show single-cell time courses from 30 cells that had undergone at least one mitosis event (CDK2 activity >1) since the start of imaging and were in G1-phase when drugs were added. Datapoints represent independent wells from the same experiment, with over 100 cells analyzed per well. m Percent of cells for data represented in i–l that entered S phase, as defined by the CDK2 signal increasing above a value of 0.8 within 16 h of drug addition (see the “Methods” section). One-way ANOVA with Dunnett’s multiple comparisons test, P = 9.16 × 10⁻⁸ (Control vs. CDK4/6i), P = 0.0271 (Control vs. CDK2i), P = 4.75 × 10⁻¹² (Control vs. Dual CDKi).