(A) Schematic showing the mouse Hes1 primary transcript, with 29 smFISH probes targeting exonic sequence (red) and 38 smFISH probes targeting intronic sequence (blue). (B) smFISH for Hes1 mRNA …
mRNA counts by smFISH in C17.2 and NS cells.
Quantification of miR-9 sensor activity.
Validation of smFISH accuracy.
Quantification of Tau-GFP signal intensity.
Quantification of miR-9 copy number in C17.2 cells by qRT-PCR.
Intensity of Ds-Red-miR-9 sensor (40 cells analysed) or Ds-Red-control sensor (20 cells analysed) relative to intensity of constitutive eGFP in Tuj1 positive (differentiated) or Tuj1 negative …
(A) To test the reliability of smFISH for accurate single-cell RNA quantification, we analysed spot number detected by two interleaved smFISH probe sets (each comprising 28 x 20 nt probes), …
Following smFISH in Tau-GFP NS cells, GFP protein was detected using anti-GFP primary (Life Technologies A11122) and goat anti-rabbit Alexa Fluor 488 secondary antibody (Life Technologies A11008). …
(A) Diagram showing the process of microRNA reverse transcription (Applied Biosystems TaqMan microRNA Reverse Transcription kit (4366596)) and TaqMan qRT-PCR (TaqMan Fast Advanced master mix reagent …
(A) Graphs showing qPCR standard curves generated from known concentrations of synthetic mature miR-9 (3 biological experiments) and linear regression. (B) Graph showing the number of miR-9 …
Schematic and image of stably infected NS-E cells containing (A) UbC-VENUS:HES1 reporter construct and (B) 2.7 kb-Hes1pr-VENUS:HES1 reporter construct. VENUS:HES1 signal in yellow. Scale bars show …
FCS data and Western blots.
(A) Time-series of Venus intensity fluctuations recorded in the confocal volume in NS-E UbC-VENUS:HES1 cells. (B) Auto-correlation function of time-series (A) and data fitting with a two-component …
(A) Time series of LUC2:HES1 reporter protein expression in a single primary NS cell determined by bioluminescence imaging. (B) Deterministic simulation of Hes1/miR-9 network at a single-cell level, …
MATLAB code to simulate the Hes1/miR-9 network with a deterministic or stochastic model (with dSSA).
Includes time series for experimental data.
Multiple time series examples of single-cell experimental data.
Corresponds to single-cell LUC2:HES1 time-series data shown in Figure 3A. 16 colours look-up table was used from ImageJ where hot (red) colours show high intensity and cold (blue) colours show low …
Traces contained in Figure 3—source data 2.
(A, D, G) Examples of stochastic simulations using the dSSA at =50, 5 and 1, respectively. Blue, mRNA; green, protein; red, miR-9. (B, E, H) The distribution of times to reach the low HES1 protein …
MATLAB code for measuring time to differentiation and systematic shift in mean.
The time course of HES1 protein and miR-9 expression, compared with the production of miR-9 at a system size of (A) =5 or (B) =1 and simulated with the CLE. Green, protein; red, miR-9; blue, …
MATLAB code to simulate Hes1/miR-9 network using the CLE with tracked miR-9 production and noise-induced delayed differentiation.
(A, B, C) The distribution of times to reach the low HES1 state of 3000 simulations using initial conditions of miR-9 r(0) = 0, 40 and 80, respectively. The initial conditions of mRNA and protein …
MATLAB code to simulate timing to differentiation with different initial conditions of miR-9.
Includes experimental data for the spread of differentiation of C17.2 cells.
Data for the speed up of differentiation in C17.2 cells with added miR-9.
Data for the speed up of differentiation in NS cells measured with FACS.
(A) Percentage of Tuj1 positive neurons in differentiated C17.2 cells after transfection of 50 nM miR-9 mimic or control mimic and culture in serum-free differentiation conditions. Significance …
(A) Representative flow cytometry plots showing the percentage of GFP positive neurons during neuronal differentiation of Tau-GFP NS cells after transfection of 40 nM miR-9 mimic or control mimic. …
(A) The areas of HES1 protein oscillations in the deterministic system, as a function of translation repression Hill co-efficient () and translation repression threshold (). (B) The areas of …
MATLAB code to scan parameters for deterministic and stochastic oscillations.
MATLAB code to generate power spectra of examples.
(A, C) The power spectrum of HES1 protein oscillations calculated using the average of 1000 simulations with the dSSA (blue), for = 3.5, = 600 and = 1.5, = 600, respectively. (B, D) HES1 …
(A, B, C) smFISH with an exonic Hes1 probe showing asymmetry in Hes1 mRNA number (white) between sister NS cells, just prior to their complete separation at cytokinesis. Aurora-B kinase antibody …
smFISH mRNA counts of cells at cell division.
(A) Schematic to show the difference between symmetric and asymmetric division. (B) The distribution of time to reach the low HES1 state with 50/50 initial conditions using deterministic dynamics as …
MATLAB code for deterministic and stochastic dynamics with wither 50/50 or binomial division.
Parameters of the model and their values.
Parameter | Set 1 | Set 2 | Interpretation | Reference |
---|---|---|---|---|
29 min | 29 min | Time delay in HES1 protein production | (Lewis, 2003) | |
1 min−1 | 1 min−1 | Hes1 transcription rate in absence of Hes1 protein | ||
1 min−1 | 2 min−1 | Translation rate | ||
1 min−1 | 1 min−1 | miR-9 transcription rate in absence of Hes1 protein | ||
390 | 390 | Amount of protein required to reduce Hes1 transcription by half | Fitted in (Goodfellow et al., 2014) | |
5 | 5 | Quantifies the step-like nature of | (Monk, 2003) | |
100 | 80 | Amount of miR-9 required to reduce Hes1 degradation by half | ||
5 | 5 | Quantifies the step-like nature of | ||
min−1 | min−1 | Lower bound for Hes1 mRNA half-life | (Bonev et al., 2012) | |
min−1 | min−1 | Upper bound for Hes1 mRNA half-life | (Bonev et al., 2012) | |
300 | 100 | Amount of miR-9 required to reduce HES1 protein translation by half | ||
5 | 5 | Quantifies the step-like nature of | ||
5 | 5 | Quantifies the step-like nature of | ||
22 min | 22 min | Half-life of HES1 protein | (Hirata et al., 2002) | |
260 | 280 | Amount of protein required to reduce miR-9 production rate by half | ||
1000 min | 1000 min | Half-life of miR-9 |