Supplementary Figure 1 Hehlgans et al. HCT-15 SW48 SW480 SW620 Suppl. Fig. 1. Phase contrast microscopy displays typical growth of three-dimensional cultured colorectal tumor cells at 6 days after plating in laminin-rich extracellular matrix (lrECM).

A SW620 B SW620 ** ** C SW620 ** ** ** Supplementary Figure 2 Hehlgans et al. A SW620 siRNA m co S X S+X Survivin XIAP β-actin B SW620 ** ** C SW620 1.00 ** m ** Surviving fraction 0.10 co S ** X S+X 0.01 2 4 6 Radiation dose [Gy] Suppl. Fig. 2. (A) Western blot confirmation of Survivin (S), XIAP (X) or Survivin and XIAP (S+X) knockdown. Mock (Roti-Fect) (m) or control siRNA (co) treated cells served as a control. Three-dimensional (3D) basal (B) and radiation survival (C) following single dose irradiation (0-6 Gy) of mock (m), control (co), Survivin (S), XIAP (X) or Survivin and XIAP (S+X) siRNA transfected cells (for all graphs means ± SD; n = 3; * P < 0.05; ** P < 0.01; Student’s t-test).

HCT-15 ** ** ** ** SW48 ** ** ** ** SW480 ** ** ** ** SW620 * ** ** ** Supplementary Figure 3 Hehlgans et al. HCT-15 ** ** ** ** SW48 ** ** ** ** SW480 ** ** ** ** SW620 * ** ** ** ** Suppl. Fig. 3. Densitometric analysis of Survivin and XIAP expression from Western blots after Survivin (S) and/or XIAP (X) knockdown in indicated 3D grown cell lines relative to control siRNA (co) transfected cells. Values were normalized to β-actin expression and mock (m) treated cells served as additional control (means ± SD; n = 4). Student’s t-test compared Survivin (S) and/or XIAP (X) knockdown conditions vs. non-specific control siRNA (co) transfected cells (* P < 0.05; ** P < 0.01).

A SW620 ** ** ** ** ** * ** B SW620 ** ** C SW620 ** ** ** Supplementary Figure 4 Hehlgans et al. A SW620 ** ** ** ** ** * ** B SW620 ** ** C SW620 ** ** ** Suppl. Fig. 4. (A) Cell cycle distribution of Survivin (S), XIAP (X) or Survivin and XIAP (S+X) knockdown and mock (m) or control siRNA (co) treated SW620 cells and (B) percentages of SubG1 fractions were analysed by flow cytometry. (C) Caspase 3/7 activity was measured 48 h after transfection and calculated relative to control siRNA transfected cells (for all graphs means ± SD; n = 3) Student’s t-test compared control siRNA (co) transfected cells vs. Survivin (S), XIAP (X) or Survivin and XIAP (S+X) knockdown cells (* P < 0.05; ** P < 0.01).

* * * * * ** ** ** ** * * * Supplementary Figure 5 Hehlgans et al. Suppl. Fig. 5. Exponential growth curves of Survivin (Surv.), XIAP or Survivin + XIAP (S+X) depleted and mock or control siRNA (co siRNA) transfected indicated cell lines. Knockdown cell cultures were plated and after 1, 2, 4 and 6 days cells were trypsinized and counted. Graphs display cell numbers relative to plated cells (n = 3; mean ± SD). Graphs were fitted using GraphPad Prism (GraphPad Software, La Jolla, USA). Student’s t-test compared knockdown cells and control siRNA treated cells (* P < 0.05; ** P < 0.01).

SW48 0 Gy 2 Gy 0 h 24 h 0 h 24 h co S X S+X Supplementary Figure 6 Hehlgans et al. SW48 0 Gy 2 Gy 0 h 24 h 0 h 24 h co S X S+X Suppl. Fig. 6. Photographs show a representative migration assay of Survivin (S), XIAP (X) or Survivin+XIAP (S+X) depleted and control (co) SW48 cells at 0 and 24 h after insert removal. Knockdown cells were plated in culture-inserts and irradiated with 0 or 2 Gy 24 h thereafter. Arrows indicate the cell-free gap at indicated time points.

A SW620 B SW620, 0 Gy ** ** ** ** ** C SW620, 2 Gy ** ** ** ** ** Supplementary Figure 7 Hehlgans et al. A SW620 B SW620, 0 Gy m co S X S+X ** ** ** ** ** C SW620, 2 Gy m co S X S+X ** ** ** ** ** Suppl. Fig. 7. (A) Relative migration of mock or 2 Gy irradiated SW620 cells as measured by a migration assay at indicated times. (B) Migration of 0 Gy or (C) 2 Gy irradiated mock (m), control (co), Survivin (S), XIAP (X) or Survivin/XIAP (S+X) siRNA treated cells relative to control siRNA (for all graphs means ± SD; n = 3; ** P < 0.01; Student’s t-test).

** * ** ** * ** SW620, 0 Gy SW620, 2 Gy Supplementary Figure 8 Hehlgans et al. SW620, 0 Gy SW620, 2 Gy ** * ** ** * ** 0 Gy 2 Gy Suppl. Fig. 8. SW620 cells were subjected to single Survivin (S) or XIAP (X) or Survivin/XIAP (S+X) double knockdown and plated in Matrigel invasion chambers. At 48 h after transfection, cells were irradiated (2 Gy) or left unirradiated (0 Gy) and serum-containing medium was added as a chemoattractant. Transmigration was calculated relative to mock (m) controls (means ± SD; n = 2; * P < 0.05; ** P < 0.01; Student’s t-test).

A SW620 B SW620 * ** C SW620 * * Supplementary Figure 9 Hehlgans et al. A SW620 siRNA m co S X S+X Survivin XIAP P-FAK Y397 FAK P-Akt1 S473 Akt1 P-ERK 1/2 ERK 1/2 P-GSK3β S9 GSK3β NF-B p65 β-actin B SW620 * ** C SW620 * * Suppl. Fig. 9. (A) Expression and/or phosphorylation of indicated proteins were analyzed in 3D grown mock (M), control siRNA (co), Survivin (S) and/or XIAP (X) siRNA transfected SW620 cells. Densitometric analysis of phosphorylation of Focal adhesion kinase FAK Y397 (B) or Akt1 S473 (C) after normalization to total protein and β-actin expression relative to control siRNA transfected cells (means ± SD; n = 3; * P < 0.05; ** P < 0.01; Student’s t-test).