Expanded View Figures

Transcription

1 Molecular Systems iology Immediate late genes decode ERK signal duration Florian Uhlitz et al Expanded View Figures Figure EV. cquired samples from HEK9ΔRF:ER cells. Microarrays - Untreated I +U Microarray time course data was used for model fitting and definition. RN-Seq time course data of metabolically labelled (SU) cells were used for determination of transcription rates and steady-state half-lives in untreated cells. C qpcr time course data were used for validation of the identified signal duration decoding principle. Data information: Numbers indicate independent biological measurements per condition and time point. +CYHX ctd +ctd Untreated II EGF FGF RN-Seq SU C qpcr Untreated U6 EV Molecular Systems iology : 9 7 ª 7 The uthors

2 Florian Uhlitz et al Immediate late genes decode ERK signal duration Molecular Systems iology wrss delayed χ =. α=.5 df = wrss immediate Figure EV. Model-based temporal gene identification. Sum of weighted squared residuals (wrss) for simple (immediate) and complete (delayed) model. The complete model was rejected for genes with v <. and Dt < min to only accept significantly better fitted genes for the complete model and to reflect time intervals in sampling. Mean fitting error [%] Mean prediction error [%] U6 EGF FGF Figure EV. Goodness of gene expression predictions. Mean fitting errors across temporal s, calculated as the mean of absolute residuals. Mean prediction errors across temporal s for tested signalling scenarios, calculated as the mean of absolute residuals. Data information: oxplots show median and inter-quartile range. IQR is extended with whiskers to the largest and smallest value respectively, but no further than.5 IQR from hinges. ª 7 The uthors Molecular Systems iology : 9 7 EV

3 Molecular Systems iology Immediate late genes decode ERK signal duration Florian Uhlitz et al HEKRF ctd derived mrn half life ON [min] rho =.7 rho.6 rho =.7 rho =.6 HEKRF ctd derived mrn half life OFF [min] Gene density.5..5 HEKRF SU derived mrn half life OFF [min] rho =.57 rho =. rho =.55 rho =. HEKRF ctd derived mrn half life OFF [min] Gene density.5..5 C Friedel (9) Yang () HEKRF median mrn half life [min] rho =.6 rho =. rho =.6 rho =. rho =.66 rho =.5 rho =.7 rho =.96 Gene density.6... Literature mrn half life [min] Temporal no Figure EV. mrn half-life comparisons for HEK9ΔRF:ER cells. Treatment comparison: ctd-derived half-lives in -pretreated versus untreated HEK9ΔRF:ER cells. Method comparison: ctd-derived half-lives compared to SU-derived half-lives in untreated HEK9ΔRF:ER cells. C Literature comparison: HEK9ΔRF:ER median mrn half-lives compared to published mrn half-lives in two different studies. Data information: Spearman correlation is indicated for all genes and for each gene. EV Molecular Systems iology : 9 7 ª 7 The uthors

4 Florian Uhlitz et al Immediate late genes decode ERK signal duration Molecular Systems iology transcription rate log [TPM/h] h h h h h rho =. rho =.67 rho =.6 rho =.55 rho =.5 Model derived mrn half life [min] nascent mrn total mrn log TPM 5 h h h h h h h h h h Figure EV5. nti-correlation of transcription rate and mrn half-life and comparison of nascent and total mrn levels in -stimulated HEK9ΔRF:ER. Comparison of absolute log transcription rate [TPM/h = transcripts per million per hour] after different periods of treatment and model-derived mrn half-life in HEK9ΔRF:ER. nti-correlation indicates that short mrn half-lives in s are compensated with high transcription rates. Comparison of nascent and total mrn levels after different periods of treatment in HEK9ΔRF:ER. s have higher nascent mrn levels after stimulation than s and s but end up at similar total mrn levels after prolonged activation. Data information: oxplots in () show median and inter-quartile range. IQR is extended with whiskers to the largest and smallest value respectively, but no further than.5 IQR from hinges. Figure EV6. Signal duration effects on mrn and protein level in -treated HEK9ΔRF:ER cells, and conservation of mrn response dynamics in rat PC and human MCF7 cells. mrn log fold changes in qpcr time course data for five different ERK signal durations in HEK9ΔRF:ER cells (cf. Fig EVC for treatment scheme). Representative Western blot for protein fold changes shown in Fig 6E. CLU and FOSL were measured on the same membrane; hence, lower GPDH control corresponds to both blots. C Comparison of HEK9ΔRF:ER and PC cells. Left panel: Spearman correlation of maximum mrn log fold changes in -treated HEK9ΔRF:ER cells and corresponding homologues in NGF-treated PC cells. Middle panel: Spearman correlation of mrn response times in -treated HEK9ΔRF:ER cells and peak expression time points of corresponding homologues in NGF-treated rat PC cells. Right panel: Spearman correlation of median mrn half-lives in HEK9ΔRF:ER cells and peak expression time points of corresponding homologues in NGF-treated rat PC cells. D Comparison of HEK9ΔRF:ER and MCF7 cells. ª 7 The uthors Molecular Systems iology : 9 7 EV

5 Molecular Systems iology Immediate late genes decode ERK signal duration Florian Uhlitz et al mrn log fc DUSP EGR EGR FOS FOS JUN RC CLU DUSP6 FOSL NR PTGS TFPI TNFRSF ZCCHC ZFP6 PPPR5 EGF Temporal Signal duration.5 h h 5h h h h 5h h EGR GPDH FOS GPDH CLU FOSL GPDH C HEKRF max log fc 6 rho =. HEKRF response time [h] rho =.7 HEKRF median mrn half life [h] rho =.6 SRG D 6 PC NGF max log fc PC NGF PC NGF HEKRF max log fc 6 rho =. HEKRF response time [h] rho =.6 HEKRF median mrn half life [h] rho =.6 SRG 6 MCF7 HRG max log fc MCF7 HRG MCF7 HRG Figure EV6. EV5 Molecular Systems iology : 9 7 ª 7 The uthors

6 Florian Uhlitz et al Immediate late genes decode ERK signal duration Molecular Systems iology EtOH +U6 n = 5; t = h n = 5; t = h n = 5; t = h FSC 7 6. % 9.6 %.7 % Cleaved Casp (FL ) Figure EV7. Gene Ontology enrichment and apoptosis in sustained versus transiently induced HEK9ΔRFER cells. Gene Ontology iological Process term enrichment for s, s and s. Score corresponds to significant enrichment in respective, where Score = log (P-value). Significant enrichments are highlighted with coloured border. ackground colour intensities correspond to denoted fractions and are normalised columnwise. FCS data to detect cleaved Casp-positive cells among untreated or treated HEK9ΔRF:ER cells as a marker for apoptosis. EtOH: no ERK signalling. : sustained ERK signalling. +U6: -h pulse ERK signalling. ª 7 The uthors Molecular Systems iology : 9 7 EV6