1. Supplementary Figure 1 Sensitive Resistant Cell Count Propidium Iodide
G2/M: %
S: %
SubG1: 3.66 %
G1/S: 1.35
HT-29 CONTROL 50
100
150
200
250
Propidium Iodide
SW480 CONTROL
G1: %
G2/M: 9.9 %
S: 19.6 %
SubG1: 1.53 %
G1/S: 3.52
300
500
700
900
HCT-15 CONTROL
G1: 68.3 %
G2/M: 9.64 %
S: %
SubG1: 6.2 %
G1/:S: 4.31
400
DLD-1 CONTROL
G1: %
G2/M: %
S: %
SubG1: 1.85 %
G1/S: 2.34
HT-29 AZD 24 h
G1: %
G2/M: %
S: %
SubG1: 4.15 %
G1/: 3.08
DLD-1 AZD 24 h
G1: %
G2/M: %
S: %
SubG1: 2.87 %
G1/S: 2.48
HCT-15 AZD 24 h
G1: %
G2/M: 7.73 %
S: %
SubG1: 4.72 %
G1/: 3.07
SW480 AZD 24 h
G1: 89.4%
G2/M: 3.88 %
S: 3.56 %
SubG1: 3.16 %
G1/S: 25.11
750
1000
1250
Sensitive
Resistant
Cell Count
Cell Count
Supplementary Figure 1

2. Supplementary Figure 2 HGUE-C-1 DLD-1 p-S6K S6K p-RPS6 RPS6 b-actin
Control 24h
500nM AZD
50nM BEZ
AZD+BEZ
p-S6K
S6K
p-RPS6
RPS6
b-actin
HGUE-C-1
DLD-1
Supplementary Figure 2

3. Supplementary Figure 3 *** ** 100% 91.72% 81.63% 63.17% 81.71% 83.2%
59.58%
91.75%
62.64%
51.77%
81.9%
80.14%
60.35% **
Supplementary Figure 3

4. HCT-15
HGUE-C-1
DLD-1 * ** NS
% Proliferation
Supplementary Figure 4

5. A B C
Supplementary Figure 5

6. Supplementary Figure 6 ** *** * * ** *** ** ** ** HCT-15 HGUE-C-1
DLD-1 **
*** * * **
*** ** ** **
Supplementary Figure 6

7. Supplementary Table 1 SW620 24h 48h 72h Control AZD G1/S SW480 HT-29
3.2±0.04
117.8±0.14
3.7±0.06
43.4±0.37
4.9±0.13
12.4±0.05
SW480
4.4±0.12
19.7±0.25
5.8±0.10
14.3±0.08
6.8±0.06
11.7±0.22
HT-29
3.1±0.30
7.1±0.17
4.0±0.15
12.9±0.09
5.6±0.04
9.8±0.06
LS 174T
3.0±0.05
5.1±0.03
2.5±0.08
6.1±0.07
4.0±0.03
5.5±0.05
HCT-15
2.5±0.04
4.5±0.08
6.9±0.07
6.0±0.33
9.5±0.11
9.1±0.06
HGUE-C-1
7.1±0.05
6.9±0.05
6.3±0.07
6.0±0.04
6.0±0.03
6.0±0.02
Caco-2
2.5±0.01
3.3±0.08
3.3±0.04
4.3±0.12
4.8±0.12
5.3±0.02
DLD-1
3.0±0.24
3.5±0.23
4.6±0.29
4.5±0.24
5.7±0.24
6.0±0.25
Supplementary Table 1

8. LEGENDS TO SUPPLEMENTARY FIGURES AND TABLES
Supplementary Figure 1. Effect of Selumetinib on cell cycle distribution. Sensitive HT-29 and SW-480 and resistant HCT-15 and DLD-1 CRC cancer cell lines were treated with DMSO (vehicle) or 1 µM Selumetinib (AZD) for 24 hours and cell cycle distribution was determined by flow cytometry, as described in Materials and Methods. Representative cell cycle histograms are shown .
Supplementary Figure 2. Effect on S6K and RPS6 phosphorylation levels. HGUE-C-1 and DLD-1 cells were treated with DMSO, AZD (500 nM), BEZ (50nM), or a combination of both for 24 hours and cell extracts were analysed by Western blot using the appropriate antibodies.
Supplementary Figure 3. Effect of Selumetinib (AZD) and Rapamycin (RAP) on resistant cell lines. Cells were treated with DMSO (Control), AZD (500 nM), RAP (1 nM), or a combination of both (AZD+RAP) for 72 hours. Then, cell proliferation was measured using the MTT method, as described in Materials and Methods. Values represent mean +/- SEM of at least three independent experiments performed in sextuplicate. Difference between means was non-significant (N.S.), or statistically significant *(p<0.05) **(p<0.01), ***(p<0.001), Student’s t test.

9. Supplementary Figure 4. Effect of Selumetinib (AZD) and PF (PF) on cell proliferation of resistant cell lines. Cells were treated with DMSO (Control), AZD (1, 5 or 10 mM), PF (30 mM), or a combination of both (AZD+PF) for 72 hours. Then, cell proliferation was measured using the MTT method. Values represent mean +/- SEM of at least three separate experiments performed in sextuplicate. Difference between means was non-significant (N.S.), or statistically significant **(p< 0.01), Student’s t-test.
Supplementary Figure 5. Interaction between Selumetinib (AZD) and PF (PF) on resistant cell lines. HCT-15 (A), HGUE-C-1 (B) and DLD-1 (C) cells were treated with AZD (0, 0.5, 1, 5 or 10 mM), in the absence or presence of PF (30 mM) for 72 hours. Then, cell proliferation was measured using the MTT method, as described in Materials and Methods. Curve for AZD alone, experimental curve for AZD plus PF and theoretical (E bliss) curve for AZD plus PF are represented. In all graphs, results represent the average of three independent experiments and error bars are the S.E.M. Each experiment was performed in sextuplicate (n = 6).
Supplementary Figure 6. Effect of Selumetinib (AZD) and PF (PF) on cell cycle of resistant cell lines. Cells were treated with DMSO (Control), AZD (1, 5 or 10 mM), PF (30 mM), or a combination of both (AZD+PF) for 24 hours and cell cycle was analysed by flow cytometry. SubG1 phase is represented, as described in Materials and Methods. Values represent mean +/- SEM of at least three independent experiments. Difference between means was statistically significant *(p<0.05), **(p<0.01), ***(p<0.001), Student’s t-test.

10. Supplementary Table 1. Effect of Selumetinib on cell cycle distribution. Sensitive and resistant CRC cell lines were treated with DMSO or 1 µM Selumetinib (AZD) for 24, 48 and 72 hours and cell cycle distribution was determined by flow cytometry, as described in Materials and Methods, The G1/S ratio is represented as the average of at least three separate experiments.