Figure Supplementary 1;Figure Supplementary 2;Figure Supplementary 3;Figure Supplementary 4;Figure Supplementary 5 from Cell cycle-dependent binding between Cyclin B1 and Cdk1 revealed by time-resolved fluorescence correlation spectroscopy
Creators: name: Barbiero, Martina
name: Cirillo, Luca
name: Veerapathiran, Sapthaswaran
name: Coates, Catherine
name: Ruffilli, Camilla
name: Pines, Jonathon
Publisher: name: The Royal Society
Year: 2022
Subjects: Biochemistry
Cell Biology

description: Figure Supplementary 1: Characterization of RPE-1 CCNB1-mEmerald+/+ cells. A) Anti-Cyclin B1 and anti-αTubulin immunoblots of lysates of parental RPE-1 or RPE-1 CCNB1-mEmerald+/+ cells showing that in the RPE-1 CCNB1-mEmerald cells all Cyclin B1 molecules are tagged with mEmerald and are expressed at the same level as untagged Cyclin B1. Molecular mass indicated on the right. B) Representative fluorescence confocal image of RPE-1 CCNB1-mEmerald+/+ cells in interphase. Scale bar corresponds to 10 µm. C) Representative fluorescence confocal images over time of a RPE-1 CCNB1-mEmerald+/+ cells progressing through mitosis. Time is expressed as mm:ss. Scale bars correspond to 10 µm. D) Dot plots of the mitotic timing of parental RPE-1 or RPE-1 CCNB1-mEmerald+/+ cells, untreated (left), treated with 55 nM nocodazole (middle) or 100 nM paclitaxel (right). Each dot represents one cell. Statistical significance was determined using an unpaired t-test. E) Representative chromosome spreads of parental RPE-1 and RPE-1 CCNB1-mEmerald+/+ cells. Scale bar corresponds to 10 µm. F) Dot plots of the chromosome number of parental RPE-1 or RPE-1 CCNB1-mEmerald+/+ cells. Each dot represents one cell. Statistical significance was determined using unpaired t-test with Welch's correction. In all plots the large dots represent the median of independent experiments. N = 3 independent experiments.;Figure Supplementary 2: Viscosity of RPE-1 cells. A) Representative fluorescence confocal image of RPE-1 cells expressing eGFP. Scale bar corresponds to 20 µm. B) Graph representing the autocorrelation function of eGFP over time. C) Dot plots representing the viscosity in the nucleus and the cytoplasm of RPE-1 cells expressing eGFP. Horizontal bars indicate medians, each dot corresponds to a single FCS measurement n≥33 FCS measurements. D) Dot plots representing the viscosity in the nucleus and the cytoplasm of RPE-1 cells expressing mVenus. n≥33 FCS measurements. Horizontal bars indicate medians, each dot corresponds to a single FCS measurement. Statistical significance was determined using an unpaired t-test. N = 3 independent experiments. ;Figure Supplementary 3: A fraction of Cyclin B1 is not in complex with Cdk1 in RPE-1 cells during G2 phase. A) Anti-Cyclin B1 and anti-Cdk1 immunoblot of G2 phase cell lysates before (1st) and after (2nd and 3rd lanes) immunodepleting Cdk1, compared with control immunodepletion with IgG (4th and 5th lanes). (Note that we do not have a definitive explanation for the apparent depletion of Cyclin B1 on control beads in the 2nd depletion.) B) Quantification of Cyclin B1 and Cdk1 levels measured by immunoblotting, before and after immunodepletion of Cdk1. C) Anti-Cyclin B1 and anti-Cdk1 immunoblot of G2 phase cell lysates before and after immunodepletion of Cdk1 to at or below detectable levels, or after control immunodepletion. D) Anti-Cyclin B1, anti-Cdk1 and anti-Cdk2 immunoblots of G2 phase cell lysates before (1st lane) and after immunoprecipitation with anti-Cyclin B1 antibody (2nd lane), or immunoprecipitation with control anti-IgG antibody (3rd lane) and the unbound fractions from the respective immunodepletions (4th and 5th lanes). For all graphs, individual dots represent biological replicates, horizontal lines indicate medians. For all panels, N=2 independent experiments. ID= immunodepletion, IP = immunoprecipitation, FT = Flow Through. Molecular mass indicated for all gels on the right.;Figure Supplementary 4: FCCS controls. A) Top panel: representative fluorescence confocal images of RPE-1 cells expressing an mEmerald-mScarlet fusion protein. Scale bar corresponds to 20 µm. Bottom panel: graph of the autocorrelation function of mEmerald and mScarlet and the cross-correlation function between the two. Note the positive cross-correlation signal, as expected. B) Top panel: representative fluorescence confocal images of RPE-1 CCNB1-mEmerald+/+ cells expressing mScarlet. Scale bar corresponds to 20 µm. Bottom panel: graph of the autocorrelation function of Cyclin B1-mEmerald and mScarlet and the cross-correlation function between the two. Note the lack of cross correlation between the molecules. C) Anti-Cyclin B1 and anti-Cdk1 immunoblots of RPE-1 CCNB1-mEmerald+/+ cells lysates before (1st lane) and after immunoprecipitation with anti-IgG antibody (2nd lane), anti-RFP antibody (3rd lane), anti-GFP antibody (4th lane) and lysates following the respective immunodepletions (5th, 6th and 7th lanes). Note that Cyclin B1-mEmerald co-precipitates with Cdk1-mScarlet. Molecular mass indicated on the right. N=1 experiment. For all F(C)CS experiments, n≥35 F(C)CS measurements in ≥15 cells,N=2 independent experiments.;Figure Supplementary 5: Time resolved Cyclin B1 FCS. A) Graph representing the FCS autocorrelation functions of Cyclin B1-mEmerald at different the indicated time points after release from Palbociclib-arrest. Each dot represents the measurement from one cell. B) Graph showing time resolved Cyclin B1-mEmerald concentration (left axis) and the fraction of Cyclin B1 in complex with Cdk1 (right axis) estimated from FCS measurements in the nucleus. Each data point represents the mean of ≥4 FCS measurements taken across 3-4 cells. Error bars indicate standard deviation. C) anti-Cyclin B1 and anti-Cdk1 immuno-blot of synchronized RPE-1 CCNB1-mEmerald+/+ cells lysates after immunoprecipitation with anti-IgG antibody. D) Representative widefield images over time of a RPE-1 CCNB1-mEmerald+/+ cells. Time is expressed at hh:mm. Scale bars correspond to 10 µm. For all panels, N=2 independent experiments, time indicates hours after Palbociclib release.