Supplementary Figure 1:

(A) values for cross sectional area and solidity and the corresponding , including the number of cells analysed (n) and the number of biological replicates performed (N). (B, C) Percentage of the total explained by each principal component (blue bars) and cumulative contribution to the total variance (red points) for the corresponding principal component analysis in (B) Fig. 1E and (C) Fig.

S1D.

Supplementary Figure 2:

(A) Representative confocal images of a Rex1-GFP Gap43-mCherry cells as naïve

ES cells, T24 cells and T48 cells on laminin. One imaging plane at the interface with the substrate is shown. The nucleus is dyed using DAPI (blue), Gap-43 marks the cell membrane (grey). (B-E) Shape measurements of Rex1-GFP and Gap43-mCherry expressing naïve ES cells (pink; n=82, N=3), T24 cells (green; n=104, N=3) and T48 cells (blue; n=43, N=3) on laminin including all points. Showing (B) the cross-sectional area: 493 ± 164 µm2 (n=82) in ES cells to 760 ± 273 µm2 (n=104) in

T24 cells and 798 ± 301µm2 (n=43) in T48 cells (C) Median solidity: 0.814 ± 0.088

(n=82) in ES cells to 0.835 ± 0.090 (n=104) in T24 and 0.791 ± 0.123 (n=43) in T48 cells, (D) Principal Component Analysis (PCA) of the Fourier descriptors and (E) shape changes due to different values of the first and second principal component.

The reference shape is the average of all cells. The colour code marks the values of

PC 1 and PC 2. in (B and C) was assessed using a Mann-

Whitney U-test. (F) Mean values for cross sectional area and solidity and the corresponding standard deviation, including the number of cells analysed (n) and the number of biological replicates performed (N).

1 Supplementary Figure 3:

(A) Violin of the drift corrected mean instantaneous velocity of unconfined ES cells on gelatine coated plates (pink; 0.20 ± 0.06 µm/min; n=28, N=2), ES cells confined under a 10 µm roof (purple; 0.48 ± 0.44 µm/min; n=51, N=2) and ES cells confined under a 5 µm roof (violet; 1.12 ± 0.58 µm/min; n=18, N=2) including all data points.

Statistical significance was assessed using a Mann-Whitney U-test. (B) Drift corrected instantaneous velocity distribution of ES cells on gelatine coated plates (pink), ES cells confined under a 10 µm roof (purple) and ES cells confined under a 5 µm roof (violet).

(C) Drift corrected mean squared displacement for ES cells on gelatine coated plates

(pink), ES cells confined under a 10 µm roof (purple) and ES cells confined under a 5

µm roof (violet) including the mean . (D) Trajectories of unconfined ES cells on laminin coating, unconfined ES cells on gelatine coating (data is the same as in Fig. 2C) and confined ES cells under a 5 µm roof (data is the same as in Fig. 2C).

The first 60 minutes of each trajectory are shown and 9 representative trajectories for each condition. (E) Violin plot of the mean instantaneous velocity of unconfined ES cells on laminin coating (green; 0.37 ± 0.23 µm/min; n=129, N=2), unconfined ES cells on gelatine coating (pink; 0.20 ± 0.07 µm/min; n=28, N=2) and ES cells confined under a 5 µm roof (violet; 1.16 ± 0.60 µm/min; n=18, N=2) including all data points. Statistical significance was assessed using a Mann-Whitney U-test. (F) Velocity distribution of unconfined ES cells on laminin coating (green), unconfined ES cells on gelatine coating (pink) and ES cells confined under a 5 µm roof (violet). (G) Mean squared displacement for unconfined ES cells on laminin coating (green), unconfined ES cells on gelatine coating (pink) and ES cells confined under a 5 µm roof (violet) including the mean standard error. (H) Trajectories of confined ES cells under a 5 µm roof grown in N2B27 medium alone starting 1h before imaging was started. The first 60 minutes

2 of each trajectory are shown and 9 representative trajectories for each condition. (I)

Violin plot of the mean instantaneous velocity of ES cells confined under a 5 µm roof height (purple; 1.16 ± 0.60 µm/min; n=18, N=2) grown in N2B27 +2i +Lif and ES cells confined under a 5 µm roof height (orange; 0.97 ± 0.47 µm/min; n=42, N=2) grown in

N2B27 alone starting 1h prior imaging including all data points. Statistical significance was assessed using a Mann-Whitney U-test. (J) Instantaneous velocity distribution of

ES cells confined under a 5 µm roof grown in N2B27 +2i +Lif (purple) and ES cells confined under a 5 µm roof grown in N2B27 (orange). (K) Mean squared displacement for ES cells confined under a 5 µm roof grown in N2B27 +2i +Lif (purple) and ES cells confined under a 5 µm roof grown in N2B27 (orange) including the mean standard error.

Supplementary Figure 4:

(A) Trajectories of unconfined ES cells on laminin coating (data is the same as in Fig.

S3D) and unconfined T24 cells on laminin coating. The first 60 minutes of each trajectory are shown and 9 representative trajectories for each condition. (B) Violin plot of the mean instantaneous velocity of unconfined ES cells on laminin (purple; 0.37

± 0.23 µm/min; n=129, N=3 and unconfined T24 cells on laminin (green; 0.68 ± 0.38

µm/min; n=136, N=3. Statistical significance was assessed using a Mann-Whitney U- test. (C) Instantaneous velocity distribution of unconfined ES cells on laminin (purple) and unconfined T24 cells on laminin (green). (D) Mean squared displacement for unconfined ES cells on laminin (purple) and unconfined T24 cells on laminin (green) including the mean standard error.

3 Supplementary Figure 5:

(A) Trajectories of unconfined T24 cells treated with DMSO, 100 µM CK666, 5 µM

Y27632 and 5 µM Blebbistatin on laminin coating. The first 60 minutes of each trajectory are shown and 9 representative trajectories for each condition. (B) Violin plot of the mean instantaneous velocity of unconfined T24 cells on laminin treated with

DMSO (light green; 0.73 ± 0.40 µm/min; (n=148, N=8), treated with 100 µM CK666

(dark green; 0.46 ± 0.26 µm/min; N=60, N=5), treated with 5 µM Y27632 (grey; 0.78 ±

0.43 µm/min, (n=53, N=3) and treated with 5 µM Blebbistatin (black; 1.00 ± 0.40

µm/min; (n=160, N=3) including all data points. Statistical significance was assessed using a Mann-Whitney U-test. (C) Instantaneous velocity distribution of unconfined

T24 cells on laminin coating treated with DMSO (light green), 100 µM CK666 (dark green), 5 µM Y27632 (grey) and 5 µM Blebbistatin (black). (D) Mean squared displacement for unconfined T24 cells on laminin coating treated with DMSO (light green), 100 µM CK666 (dark green), 5 µM Y27632 (grey) and 5 µM Blebbistatin (black) including the mean standard error.

Supplementary Table 1:

(A) Table of ribosome-depleted (total) RNA; RPKM = reads per kilobase of transcript per million mapped reads. Data taken from (Kalkan et al., 2017).

4 Supplementary Figure 1 A Cells Cross sectional area Solidity n N ES 157 ± 64 µm2 0.962 ± 0.046 65 3 T24 311 ± 306 µm2 0.803 ± 0.170 60 3 T48 360 ± 256 µm2 0.617 ± 0.136 31 3

B C Percentage of variation per PC Percentage of variation per PC for cells plated on gelatine for cells plated on laminin 100 100 90 90 80 80 individual individual 70 cumulative 70 cumulative 60 60 50 50 40 40 30 30 20 20 Contribution to variance (%) Contribution to variance (%) 10 10 0 0 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 PCs included PCs included Supplementary Figure 2 A B Cross sectional area ES cells on laminin T24 cells on laminin T48 cells on laminin p<0.0001 p=0.1217 1800

1600 p<0.0001 ] 2 1400

1200 DAPI Gap-43 10µm 1000 C Solidity D PCA of shape features 800 1 600

0.9 ES cells Cross section area [µm 35 0.8 T24 cells 400 30 T48 cells 0.7 25 200 0.6 20 0 0.5 15 Solidity 10 ES cells 0.4 T24 cells T48 cells 5 on laminin

0.3 PC 2 (26.1%) p=0.6303 p=0.0050 0 E 0.2 -5 PC 1 PC 2 10 p=0.0329 0.1 -10 5 -15 0 0 -10 0 10 20 30 -5 ES cells PC 1 (41.4%) T24 cells T48 cells -10 on laminin on laminin 10 µm F Cells Cross sectional area Solidity n N ES 493 ± 164 µm2 0.814 ± 0.088 82 3 T24 760 ± 273 µm2 0.835 ± 0.090 104 3 T48 798 ± 301 µm2 0.791 ± 0.123 43 3 Supplementary Figure 3 A Drift corrected B Drift corrected C Drift corrected Instantaneous velocity Velocity distribution MSD p<0.0001 3 180 ES cells unconf. p<0.0001 ES cells unconfined 2.5 160 ES cells 10 µm conf. 2.5 ES cells 10 µm confinement ES cells 5 µm conf. 140 2 0.0497 ES cells 5 µm confinement 2 120 1.5 100

1.5 δ [µm² ] 1 80 [µm/min] p(v) [min/µm] 1 60 0.5 40 0.5 0 20 0 0 unconf. 0 1 2 3 4 5 0 5 10 15 20 5 µm conf. 10 µm conf. v [µm/min] Δt [min] D ES cells unconf. on laminin ES cells unconf. on gelatine ES cells 5 µm confinement E Instantaneous velocity p<0.0001 2.5 50 50 50 from 2 Fig. 2D

1.5 } 0 0 0 p<0.0001 y [µm] y [µm] y [µm] 1 [µm/min] -50 -50 -50 0.5 Same data as in Fig. 2C Same data as in Fig. 2C -50 0 50 -50 0 50 -50 0 50 0 x [µm] x [µm] x [µm] laminingelatine 5 µm conf. F Velocity distribution G MSD H ES cells 5 µm conf. 1h N2B27 unconf. laminin 60 3 unconf. gelatine from unconf. laminin 250 5 µm conf. } Fig. 2F 50 50 2.5 unconf. gelatine from 200 40 2 5 µm conf. } Fig. 2E 150 0 1.5 30 y [µm] 100 t [min] 1 δ [µm² ] 20 p(v) [min/µm] 0.5 50 -50 10 0 0 0 0 1 2 3 0 5 10 15 20 -50 0 50 v [µm/min] Δt [min] x [µm]

I Instantaneous velocity J Velocity distribution K MSD p=0.2422 ES cells 5 µm 2i + LIF 0.8 ES cells 5 µm 2i + LIF 250 from Fig. 2F 2.5 0.7 from Fig. 2E ES cells 5 µm 1h N2B27 2 0.6 ES cells 5 µm 1h N2B27 200 1.5 0.5 150 1 0.4 δ [µm² ]

[µm/min] 0.3 100 0.5 p(v) [min/µm] 0.2 0 50 0.1 2i + LIF 0 0 1h N2B27 from Fig. 2D 0 1 2 3 4 5 0 2 4 6 8 10 12 14 16 18 20 ES cells 5 µm unconf. v [µm/min] Δt [min] Supplementary Figure 4 A ES cells unconfined T24 cells unconfined B Instantaneous velocity on laminin on laminin p<0.0001 60

50 50 50 2.5

40 2 0 0 30 y [µm] y [µm] t [min] 20 1.5 -50 -50 10

Data as shown in Fig. S3D [µm/min] 0 1 -50 0 50 -50 0 50 x [µm] x [µm] 0.5 C Velocity distribution D MSD

3 0 ES cells laminin, from Fig. S3F 200 ES cells laminin, from Fig. S3G T24 cells laminin T24 cells laminin 2.5 ES cells laminin 150 from Fig. S3ET24 cells laminin 2

1.5 δ [µm² ] 100 p(v) [min/µm]

1 50 0.5

0 0 0 0.5 1 1.5 2 2.5 3 0 5 10 15 20 v [µm/min] Δt [min] Supplementary Figure 5 A T24 cells unconfined on laminin + DMSO + 100 µM CK666 + 5 µM Y27632 + 5 µM Blebbistatin 60 50 50 50 50 50 40 0 0 0 0 30 y [µm] t [min] y [µm] y [µm] y [µm] 20

-50 -50 -50 -50 10 0 -50 0 50 -50 0 50 -50 0 50 -50 0 50 x [µm] x [µm] x [µm] x [µm] B Instantaneous velocity C Velocity distribution D MSD p<0.0001 p=0.3749 1.6 350 1.4 2 p<0.0001 + DMSO 300 + DMSO 1.2 + 100 µM CK666 + 100 µM CK666 250 1.5 1 + 5 µM Y27632 + 5 µM Y27632 + 5 µM Blebbistatin 200 + 5 µM Blebbistatin

0.8 δ [µm² ] 1 p(v) [min/µm] 150 0.6 [µm/min] 100 0.5 0.4 0.2 50 0 0 0 0 0.5 1 1.5 2 2.5 3 0 5 10 15 20 DMSO v [µm/min] Δt [min] 100 µM CK6665 µM Y27632 5 µM Blebbistatin Supplementary Table 1 A