Nature Biotechnology: doi: /nbt Supplementary Figure 1. Technique validation.

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1 Supplementary Figure 1 Technique validation. (a) Detection of GFP-N-terminal, FLAG-C-terminal, and FLAG-N-terminal tagged proteins. All GFP-tagged fusion proteins, but only 20 of the 25 FLAG-C-terminal tagged and only 22 of the 25 FLAG-N-terminal tagged proteins were detected using mass cytometry. (b) HEK293T cells overexpressing GFP-GFP, FLAG-C-terminal-GFP, and FLAG-N-terminal-GFP fusion proteins were co-stained with anti- GFP and anti-flag antibodies. The fusion protein FLAG-C-terminal-GFP was detected by the anti-gfp antibody but not by the anti- FLAG antibody. This indicates that in certain contexts the FLAG tag is not accessible to the anti-flag antibody. The FLAG epitope may be masked due to protein folding or by the denaturation process that is part of our experimental protocol.

2 Supplementary Figure 2 GFP-tagged POIs have normal localization. HEK293T cells that overexpressed the GFP-tagged POIs used in this study were imaged with confocal microscopy. For each POI, the main panel shows the image in a given z-depth; the bottom panel and the side panel shows x-z and y-z cross-sectional images, respectively. POI-GFP subcellular localization was determined by overlapping with two control stains: Hoechst for the nucleus and Alexa Fluor 647 carboxylic acid succinimidyl ester indicating the cell outline. The POI-GFP localization was verified by comparison with information of the UniProt subcellular localization database (Supplementary Table 4).

3 Supplementary Figure 3 GFP tag does not disrupt catalytic activities of POIs. (a-d) Catalytic activities of GFP-tagged POIs were compared with FLAG-C-terminal and FLAG-N-terminal tagged POIs. The examples shown here indicate that the GFP tag did not alter signaling relationships or signaling dynamics after EGF stimulation (the complete dataset with comparison of all constructs used in this study is shown in Supplementary Dataset 1). (e) Heat map showing abundancedependent signaling relationship strengths from overexpressed POIs with three different tags as determined by BP-R 2 analysis (Supplementary Figure 10 and Methods). Measured markers showing at least one strong relationship in any of the conditions were included in the heat map. Strong relationships were detected independently of tag. BP-R 2 values slightly vary for the 3 tags, due to the antibody accessibility and differences in transfection efficiencies (Supplementary Figure 1).

4 Supplementary Figure 4 Total protein antibody staining of HEK293T cells overexpressing a GFP-tagged POI. (a) HEK293T cells transfected with KRAS-GFP, HRAS-GFP, MEK1-GFP, ERK2-GFP, AKT1-GFP, GSK3 -GFP, or S6-GFP for 18 h were stained with anti-total POI and anti-gfp antibodies. A linear regression analysis for each pair was performed in the original scale. R 2 ranges from 0.74 to 0.88, indicating the total POI is linearly correlated with GFP and that the POI overexpression does not alter the expression of the endogenous POI. (b) The same cells were stained with nine antibodies to quantify total protein as well as with a GFP antibody. Median ion counts for all measured markers are shown. Overexpression of a POI-GFP for 18 h does not cause notable changes in the measured network nodes. (c) ERK2-GFP transfected HEK293T cells and the untransfected control with or without EGF stimulation were stained for total-erk and phospho-erk (Thr202/Tyr204). The dynamic range in the overexpression condition allows observation of abundance-dependent signaling relationships. With total ERK staining, the same signaling relationships as shown in the Supplementary Figure 2b is recapitulated, verifying GFP as an indicator of POI expression level.

5 Supplementary Figure 5 Comparison of mass cytometry and flow cytometry (FACS). HEK293T cells were transfected with the FLAG-GFP overexpression vector. With flow cytometry, cells were gated into GFP low, medium, and high populations with the gating strategy shown in the left panel. With mass cytometry, each of the three sorted populations was measured independently to determine the gating windows. Unsorted cells were then assessed by the mass cytometry. The maximum difference in population percentage between mass cytometry and flow cytometry was less than 3%.

6 Supplementary Figure 6 Comparison of EGF stimulations in starved (FBS is absent) and non-starved (FBS is present) cell culture conditions. HEK293T cells were stimulated with EGF with or without FBS over a 1-h time course. In the non-starved condition basal signaling states of the major MAPK/ERK or AKT pathway components were higher than in starved conditions, but these elevated levels did not affect the signaling responses to the EGF stimulation. Mean value of each sample is shown with circle. Standard deviation is indicated by shaded area.

7 Supplementary Figure 7 TrypLE treatment time course. HEK293T cells were treated with TrypLE for 30 s, 1 min, 2 min, or 4 min with or without EGF stimulation for 5 min (time from EGF addition to PFA crosslinking). Within the first 2-min TrypLE treatment (i.e., the time after which we quenched cells in all experiments), only phosphorylation of Ser167/170 on MARCKS varied relatively. Mean value of each sample is shown with circle. Standard deviation is indicated by shaded area.

8 Supplementary Figure 8 Live imaging of GFP fluorescence at 18 to 19 h after HEK293T cells were transfected with a FLAG-GFP construct. Quantification of the GFP intensity showed a slight increase of 5.4% over the 1-h time course. There was a fluctuation in total GFP signal, indicating that the 5.4% increase is most likely attributable to technical variability of the measurement. The analysis of signaling relationships in our study was performed based on a binning strategy on arcsinh transformed GFP ion counts (mass cytometry). Thus, the measured change will not significantly affect the binning over the time course. Standard deviation is indicated by shaded area.

9 Supplementary Figure 9 Abundance-dependent signaling analyses performed in individual experiment replicates are highly reproducible. (a) Different batches of HEK293T cells were transfected with JNK1-GFP, P38 -GFP, PDK1-GFP, or p90rsk-gfp constructs, stained, and analyzed by mass cytometry on three different days. Highly consistent signaling responses were observed among the three individual experiment replicates. Panels (b) and (c) show analyses of representative phosphorylation sites in the MAPK/ERK, AKT, stress pathways, and the STAT5 protein in cells in which (b) KRAS G12V -GFP and (c) MEK1 DD -GFP was overexpressed. Panels (d) and (e) show all relationships that passed the BP-R 2 threshold (see Methods for details) for the (d) KRAS G12V -GFP and (e) MEK1 DD -GFP overexpression experiments.

10 Supplementary Figure 10 Binned pseudo R 2 (BP-R 2 ) analysis. (a) BP-R 2 analysis considers deviation from bin median versus the global mean of bin medians. (b) Examples of BP-R 2 and Spearman correlation of bin medians values. The top left and top right plots show examples of positive and negative Spearman correlations of bin medians. The top left and bottom left plots show replicates of the same overexpression condition and how a (supposedly) increased noisiness affects the BP-R 2 values. The bottom right plot shows a complex signaling relationship with the corresponding BP-R 2 value. The BP-R 2 metric detects complex arbitrary relationship (bottom right). (c) Density distribution of the median BP-R 2 values for the 700 POI-GFP-marker relationships from the negative controls (FLAG-GFP, untransfected) and the 3500 POI-GFP-marker relationships of the signaling node overexpression conditions. Cutoff for strong signaling relationships were determined at a median BP-R 2 value of 0.11, the highest median BP-R 2 of the negative controls.

11 Supplementary Figure 11 Benchmark of BP-R 2 against other methods used to identify relationships in mass cytometry data. (a) Venn diagram of strong relationships detected by BP-R 2, Spearman correlation, and DREMI in our dataset using the same cutoff - the 99 percentile of the BP-R 2 / Spearman correlation / DREMI score in the control groups (FLAG-GFP overexpression and the untransfected cells). BP-R 2 outperforms the other two measures. (b) BP-R 2, Spearman correlation, and DREMI measurements of signaling relationship strength between p-erk1/2 and overexpressed ERK2-GFP. BP-R 2 is suitable for analyzing non-monotonic signaling relationships and outperforms the other two measures in representing actual signaling activation status.

12 Supplementary Figure 12 Analysis of signal spill over among mass channels. (a) Strategy to exclude spill over among mass channels. When strong signaling relationships as determined by BP-R 2 were identified (measured phosphorylation of p70s6k in the p90rsk-gfp overexpression is shown here as a selected example), all other potentially affected channels (details in Methods) were evaluated for spillover that might have led to a high BP-R 2 value. Using an experimental spillover filter (b), spillover-affected relationships were discarded. Here three groups of antibody stains were performed simultaneously: First, all antibodies; second, all antibodies except for the one that potentially causes spillover; third, only the antibody that potentially causes spillover. If spillover induced background was over 10% of the actual ion counts, the channel was discarded from the analysis.

13 Supplementary Figure 13 Median intensities and BP-R 2 analysis for all experimental conditions. (a) Heat map of median intensities of all measured markers at 0, 5, 15, 30, and 60 min post-egf stimulation in all overexpression conditions (Table 1). Data visualized as log 2 of the ratio of the median signals divided by the mean of median signals of the FLAG-GFP controls at time point 0. (b) Heat map of BP-R 2 values of all measured markers versus GFP signals at 0, 5, 15, 30, and 60 min post- EGF stimulation in all overexpression conditions.

14 Supplementary Figure 14 Heat map of consistent and robust examples for overexpression-induced phosphorylation site abundance peak time changes after EGF stimulation for each of the three replicates.

15 Supplementary Figure 15 Post-transcriptional constraint analysis of overexpressed POIs. (a) Coefficient of variation (CV) was computed for each strong signaling relationship that had a BP-R 2 value above 0.11 (i.e., a strong signaling relationship), and CVs were plotted against BP-R 2. No correlation was observed. (b) Overexpression ranges (median value of GFP in Bin10 minus the median value of GFP in Bin1) calculated for all POIs. (c) Maximum amplitudes of phosphorylation sites were independent of the level of overexpression of the POIs.

16 Supplementary Table 1: Antibody panel Isotope Antigen Immunogen Sup plier Clone Staining Concen tration [μg/ml] Gene ID UniProt Entry La139 p- CREB/AT F1 p-ser133 of CREB/pSer6 3 of ATF1 BD J CREB1 ATF1 P16220 P18846 Pr141 p-stat5 p-tyr694 BD 47/Stat5 2 STAT5A P42229 Nd142 p-shp2 p-tyr580 CST D66F10 4 PTPN11 Q06124 Nd143 p-fak p-tyr397 CST Polyclonal 2 PTK2 Q05397 Nd144 p-mek1/2 p-ser221 CST 166F8 2 MAP2K1 MAP2K2 Q02750 P36507 Nd145 p- MAPKAP K2 p-thr334 CST 27B7 1 MAPKAP K2 P49137 Nd146 p-p70s6k p-thr389 CST 1A5 1 RPS6KB1 P23443 Sm147 p-mkk3 p-thr222 Assa y Biote chnol ogy Polyclonal 2 MAP2K3 P46734 Nd148 p-stat1 p-ser727 CST Polyclonal 1 STAT1 P42224 Sm149 p-p53* p-ser15 CST 16G8 1 TP53 P04637 Nd150 p-nf B p-ser529 BD K RELA Q04206 Eu151 p-p38 p-thr180/p- Tyr182 BD 36/p38 2 MAPK14 MAPK11 MAPK12 MAPK13 Q16539 Q15759 P53778 O15264 Sm152 p-ampk p-thr172 CST 40H9 1.5 PRKAA Q13131 Eu153 p-akt p-ser473 CST D9E 0.5 AKT1 AKT2 AKT3 P31749 P31751 Q9Y243 1

17 Sm154 p-erk1/2 p-thr202/p- Tyr-204 BD 20A 1 MAPK3 MAPK1 P27361 P28482 Gd155 p- MARCKS p- Ser167/170 CST D13E4 2 MARCKS P29966 Gd156 cyclin B1 Recombinant human cyclin B1 BD GNS CCNB1 P14635 Gd158 p-gsk3 p-ser9 CST D85E GSK3B P49841 Tb159 p- SMAD1/5 p- Ser463/465 CST 41D10 4 SMAD1 SMAD5 Q15797 Q99717 Gd160 p-mkk3/6 p-ser189 of MKK3/p- Ser207 of MKK6 CST D8E9 0.5 MAP2K3 MAP2K6 P46734 P52564 Dy161 p-pdk1 p-ser241 BD J PDPK1 O15530 Dy162 p- BTK/ITK p-tyr551 of BTK/p- Tyr551 of ITK BD 24a/BTK 1 BTK ITK Q06187 Q08881 Dy163 p- p90rsk p-ser380 CST D5D8 1 RPS6KA1 RPS6KA2 RPS6KA3 Q15418 Q15349 P51812 Dy164 p- SMAD2/3 p- Ser465/467 of SMAD2/p- Ser423/425 of SMAD3 CST D27F4 2 SMAD2 SMAD3 Q15796 P84022 Ho165 -catenin Nonphospho Ser33/37/Thr 41 CST D13A1 0.5 CTNNB1 P35222 Er166 p-stat3 p-tyr705 BD 4/P-STAT3 1 STAT3 P40763 Er167 p-jnk p- Thr183/Tyr1 85 CST G9 2 MAPK8 MAPK9 MAPK10 P45983 P45984 P

18 Er168 p-plc 2 p-tyr759 BD K PLCG2 P16885 Tm169 GFP TLR9-GFP expressed cell line BioL egen d FM264G 0.1 GFP Er170 p-hh3 p-ser28 BD HTA H3F3A P68431 Yb171 p-s6 p- Ser235/Ser2 36 BD N RPS6 P62753 Yb172 cleaved PARP A peptide correspondin g to the N- terminus of the cleavage site (Asp 214) of human PARP BD F PARP1 P09874 Yb172 cleaved caspase3 A synthetic peptide correspondin g to aminoterminal residues adjacent to Asp175 of human caspase-3 CST 5A1E 1 CASP3 P42574 Yb173 p-mtor p-ser2448 CST D9C2 2 MTOR P42345 Yb174 E- cadherin Cytoplasmic domain BD 36/E- Cadherin 2 CDH1 P12830 Lu175 p-rb p-807/811 CST D20B12 1 RB1 P06400 Yb176 p-4ebp1 p-thr37/46 CST 236B EIF4EBP1 Q13541 *p-p53 (Ser15) antibody was only used in experiments with mutant (KRAS G12V -GFP and MEK1 DD - GFP) overexpression. 3

19 Supplementary Table 2: Phosphorylation site regulatory table (information from SIGNOR) Antigen Immunogen Regulatory Protein Modification Type p-creb/atf1 p-ser133 of CREB AKT2 AKT3 CAMK4 MAPKAPK2 PRKACA RPS6KA1 RPS6KA4 RPS6KA5 TSSK4 p-ser63 of ATF1 CDK3 CSNK2A1 RPS6KA5 p-stat5 p-tyr694 FLT3 JAK2 JAK3 LCK SRC p-shp2 p-tyr580 N/A p-fak p-tyr397 MET PTK2 SRC p-mek1/2 p-ser221 ARAF BRAF MAP3K1 MAP3K8 PDPK1 RAF1 p-mapkapk2 p-thr334 MAPK14 MAPK1 p-p70s6k p-thr389 N/A p-mkk3 p-thr222 N/A p-stat1 p-ser727 CAMK2B 4

20 IKBKE PRKCD MAPK14 p-p53* p-ser15 PPM1D (phosphatase) AMPK ATM ATR CDK5 CHEK2 DYRK1A ERK1/2 MAPK14 MAPK1 MAPK3 NUAK1 PRKAA1 PRKDC SMG1 STK11 TP53RK CHEK1 p-nf B p-ser529 CSN1S1 CSNK2A1 p-p38 p-thr180/p-tyr182 MAP2K3 MAP2K4 MAP2K6 RET MAP2K3 Inhibition p-ampk p-thr172 N/A p-akt p-ser473 PHLPP1(phosphatase) PHLPP2(phosphatase) PPP2R4(phosphatase) PTPRF(phosphatase) CRTC2 ILK LRRK2 MTOR PDPK1 PIK3CA Inhibition Inhibition Inhibition Inhibition 5

21 TBK1 mtorc2 p-erk1/2 p-thr202/p-tyr-204 MAP2K1 MAP2K2 PPP2CA(phosphatase) DUSP3(phosphatase) LCK RET Inhibition Inhibition p-marcks p-ser167/170 N/A p-gsk3 p-ser9 MAP4K5 PRKACA PRKCA PRKCH PRKCZ RPS6KA1 AKT1 GSK3B p-smad1/5 p-ser463/465 BMPR1A BMPR1B BMPR2 p-mkk3/6 p-ser189 of MKK3/p-Ser207 of MKK6 TAOK2 p-pdk1 p-ser241 PDPK1 p-btk/itk p-tyr551 of BTK/p-Tyr551 of ITK BTK LYN p-p90rsk p-ser380 MAPK3 MAPK1 p-smad2/3 p-ser465/467 of SMAD2/p- Ser423/425 of SMAD3 TGFBR1 -catenin Non-phospho Ser33/37/Thr41 GSK3B/Axin/APC GSK3B JNK Inhibition Inhibition Inhibition p-stat3 p-tyr705 PTPN1 (phosphatase) PTPN2 (phosphatase) PTPRD (phosphatase) Inhibition Inhibition Inhibition 6

22 JAK1 JAK2 MAPK3 PKM p-jnk p-thr183/tyr185 MAP2K4 MAP2K7 p-plc 2 p-tyr759 BTK LCK p-s6 p-ser235/ser236 RPS6KA1 RPS6KB1 p-mtor p-ser2448 AKT1 RPS6KB1 p-4ebp1 p-thr37/46 MTOR mtorc1 GSK3B Inhibition Inhibition Inhibition 7

23 Supplementary Table 3: Antibody validation All antibodies used in this manuscript were thoroughly validated by the vendors. Many experiments done in the context of this manuscript also validate the antibodies: Antigen Validation method Validation data p-creb/atf1 Cell cycle stages p-stat5 SRC-GFP overexpression p-shp2 SRC-GFP overexpression p-fak SRC-GFP overexpression p-mek1/2 MEK1-GFP overexpression p-mapkapk2 JNK1-GFP overexpression 8

24 p-p70s6k p90rsk-gfp overexpression p-mkk3 GSK3 -GFP overexpression p-stat1 GSK3 -GFP overexpression p-nf B TNF stimulation p-p38 p38 -GFP overexpression p-ampk ASK1-GFP overexpression p-akt PI3K inhibition (GDC-0941) p-erk1/2 EGF stimulation 9

25 p-marcks Cell cycle stages cyclin B1 Cell cycle stage p-gsk3 GSK3 -GFP overexpression p-smad1/5 Vanadate stimulation p-mkk3/6 MKK6-GFP overexpression p-pdk1 PDK1-GFP overexpression p-btk/itk SRC-GFP overexpression p-p90rsk p90rsk-gfp overexpression 10

26 p-smad2/3 TGF treatment (in MCF10A cells) -catenin GSK3 -GFP overexpression p-stat3 SRC-GFP overexpression p-jnk JNK1-GFP overexpression p-plc 2 SRC-GFP overexpression GFP FACS sorted GFP overexpression cells (Supplementary Fig. 5) p-hh3 Cell cycle stage p-s6 PI3K inhibition (GDC-0941) 11

27 cleaved PARP/ cleaved caspase3 mtor inhibition (Rapamycin) cleaved PARP/ cleaved caspase3 p-mtor mtor inhibition (Rapamycin) p-mtor E-cadherin TGF treatment (in MCF10A cells) p-rb Cell cycle stage p-4ebp1 PI3K inhibition (GDC-0941) 12

28 Supplementary Table 4: Protein subcellular localization Protein UniProt subcellular localization Detected fluorescence localization of POI-GFPs Cell membrane Cytoplasm Nucleus Cell membrane and cytoplasm Nucleus KRAS AKT1 SRC p90rsk CRAF PDK1 p110 p38 MKK7 MKK6 JNK1 MEK1 S6 p70s6k SHP2 ASK1 BRAF ERK2 HRAS 13

29 GSK3 Supplementary Table 5: Literature list of POI-GFP used and validated in previous studies Overexpressed POI-GFP Literature SRC-GFP 1 5 PDK1-GFP 6 AKT1-GFP 7 11 GSK3 -GFP 12,13 MKK7-GFP 14 MKK6-GFP 15 p38 -GFP ERK2-GFP p90rsk-gfp 26,27 CRAF-GFP JNK1-GFP 31,32 p110 -GFP 33 BRAF-GFP ASK1-GFP 37 p70s6k-gfp 38,39 MEK1-GFP KRAS-GFP HRAS-GFP SHP2-GFP S6-GFP

30 Supplementary Table 6: Shortest directed path of identified signaling relationships Overexpre ssed POI Target Sign Shortest Directed Path (SIGNOR) Path (UniProt Entry) Nonsigned Signed GSK3 p-gsk P49841 AKT1 p-akt P31749 PDK1 p-pdpk O15530 p90rsk p- p90rsk Q15418 p38 p-p Q16539 MEK1 p- MEK1/ Q02750 ERK2 p-erk1/ P28482 MKK6 p- MKK3/ P52564 JNK1 p-jnk P45983 MEK1 p-gsk Q02750 P49841 PDK1 p- p70s6k PDK1 p- MEK1/ O15530 P O15530 Q

31 ASK1 p- MKK3/ Q99683 P46734 Q99683 P52564 O15530 P36507 SRC p-fak P12931 Q05397 AKT1 p-gsk P31749 P49841 SRC p-stat P12931 P42229 ASK1 p-p Q99683 Q16539 MKK6 p-p P52564 Q16539 P52564 Q15759 P52564 O15264 P52564 P53778 SRC p-stat P12931 P40763 AKT1 p- SMAD2/3 ERK2 p- SMAD1/ P31749 SIGNOR-C7 Q15796 P31749 SIGNOR-C7 P P28482 Q09472 Q15797 MEK1 p-4ebp Q02750 Q16539 Q13541 ASK1 p- MAPKAP K Q99683 Q16539 P49137 MEK1 p-stat Q02750 P27361 P42229 MKK6 p-stat P52564 Q16539 P42224 AKT1 p- p70s6k P31749 P42345 P23443 ASK1 p-jnk Q99683 O14733 P

32 Q99683 P52564 P45984 Q99683 P52564 P53779 SRC p-p P12931 P60953 Q16539 SRC p-erk1/ P12931 P29353 P27361 P12931 P29353 P28482 ASK1 p-stat Q99683 Q16539 P42224 p90rsk p- p70s6k Q15418 SIGNOR-C3 P23443 p110 p-p P42336 P63000 Q16539 p110 p-jnk P42336 P63000 P45983 P42336 P63000 P45984 ASK1 p- SMAD2/3 MEK1 p- SMAD2/ Q99683 Q16539 P Q02750 P28482 P29590 Q15796 Q02750 P28482 P29590 P84022 AKT1 p-4ebp P31749 Q15672 P68400 Q13541 GSK3 p-stat P49841 Q16539 P00533 P42224 GSK3 p-mkk P49841 Q16539 Q16584 P46734 AKT1 p- MKK3/ P31749 P04049 Q99683 P46734 P31749 P04049 Q99683 P52564 MKK6 p-stat P52564 Q16539 P04150 P42229 ASK1 p- SMAD1/ Q99683 Q16539 P49841 Q15797 PDK1 p-stat O15530 Q02750 P27361 P42229 HRAS p-jnk P01112 P42336 P63000 P

33 p110 p- SMAD1/ P42336 P31749 P12755 Q15797 P42336 P31749 P12755 Q99717 P01112 P42336 P63000 P45984 SHP2 p- SMAD1/ Q06124 P35221 P46937 Q15797 p70s6k p-s P23443 P45983 P04637 Q15418 P62753 PDK1 p-s O15530 P31749 SIGNOR-C3 P23443 P62753 ASK1 p-gsk Q99683 Q16539 P28562 P27361 P49841 HRAS p- SMAD2/ P01112 P48736 P31749 SIGNOR-C7 Q15796 P01112 P48736 P31749 SIGNOR-C7 P84022 p110 p- MKK3/ P42336 P31749 P04049 Q99683 P46734 P42336 P31749 P04049 Q99683 P52564 JNK1 p- MAPKAP K P45983 Q9UPT6 P45985 Q16539 P49137 JNK1 p-stat P45983 Q99640 P06493 P00533 P42224 p90rsk p-pdk Q15418 P03372 P27986 CID: O15530 GSK3 p-shp P49841 Q02156 P21802 Q8WU20 Q06124 ASK1 p- AMPK Q99683 Q16539 P49841 Q96RR4 Q

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