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FIGURE 1 A B Kinase Assay C D E F HA tubulin p38 HCT116-TP53KO GAPDH
EV Sh MAPK 14 HCT116-TP53KO GAPDH 14bis 12 13 11 A 0.0 0.5 1.0 1.5 2.0 2.5 Sh LUC MAPK 14 14bis 11 12 13 IC50 (nM) HCT116-TP53KO B HA MAPK14 CA MAPK11 CA MAPK12 CA MAPK13CA tubulin HCT116-TP53KO Kinase Assay C D p ATF2 MAPK14 CA MAPK11 CA MAPK12 CA MAPK13CA 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 EV MAPK 14 CA 11 12 13 IC50 (nM) HCT116-TP53KO pp38 GAPDH HCT116-TP53KO NT SN38 E F
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FIGURE 2 A B C D E % living cells 4 Doubling Time (hours) 20 40 60 80
HCT116-TP53KO EV HCT116-TP53KO-MAPK14CA B 4 Doubling Time (hours) 20 40 60 80 100 120 EV MAPK14-CA HCT116-TP53KO 3 Cell number (106) 2 1 2 4 6 8 10 12 Days C HCT116-TP53KO-EV HCT116-TP53KO-MAPK14CA 20 40 60 80 100 120 D7 D14 D21 D28 % living cells D HCT116-TP53 KO 5 10 15 20 EV MAPK14 CA % APOPTOSIS HCT116-TP53KO-EV HCT116-TP53KO-MAPK14CA E 1 2 3 4 5 HCT116- TP53KO -EV TP53KO- MAPK14CA Blue cell number/field
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FIGURE 3 B A D C E G F HCT116-TP53KO-EV HCT116-TP53KO-MAPK14CA 20 40
S 49.4% G0 G1 28.8% G2/M 18.4% 37.1% 36.8% 19.3% 20 40 60 80 100 120 5 10 15 25 30 HCT116-TP53KO-EV HCT116-TP53KO-MAPK14CA % of BrdU stained cells p21 Actin EV D7 D14 D21 D28 MAPK14CA HCT116-TP53KO HA D 0.0 0.2 0.4 0.6 0.8 1.0 1.2 EV D7 D14 D21 D28 p21 Arbitrary RNA units MAPK14CA HCT116-TP53KO C p21 shLUC shp21 HCT116-TP53KO-MAPK14CA GAPDH E G F HCT116-TP53KO -ShLuc-MAPK14CA -Shp21-MAPK14CA S 25.8% G0 G1 45.6% G2/M 23.16% 46.1% 20.9% 28.9% 10 20 30 40 50 60 70 80 90 5 15 25 HCT116-TP53KO-ShLuc- MAPK14CA HCT116-TP53KO-Shp21-MAPK14CA % of BrdU stained cells
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FIGURE 4 A B C D E LC3 Actin EV D7 D14 D21 D28 MAPK14CA HCT116-TP53KO
Tubulin EV D D D21 MAPK14CA C 500 nm HCT116-TP53KO EV MAPK14CA Autophagosome number/cell MAPK14CA EV HCT116-TP53 KO 1 2 3 4 5 6 HCT116-TP53 KO MAPK14CA EV GFP-LC3 labelling D E 10- 8 - 6 - 4 - 2 - 0 - HCT116-TP53KO GFP-LC3 spots/cell EV MAPK14 CA HCT116-TP53KO-MAPK14CA GAPDH LC3 shLUC shp21
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FIGURE 5 B A C E D HCT116-TP53KO (J14) p62 NT 2H 4H 8H EV MAPK14CA
GAPDH p62 Actin EV D7 D14 D21 D28 MAPK14CA HCT116-TP53KO 40 80 120 160 nt 2h 4h 8h (arbitrary unit) p62 level EV HCT116-TP53KO MAPK14CA 1 2 EV D7 D14 D21 D28 (arbitrary unit) p62 level MAPK14CA HCT116-TP53KO HCT116-TP53KO EV MAPK14CA D14 0.5 1 1.5 2 2.5 3 NT 3MA Baf LC3-II level (arbitrary unit) C NT 3MA Baf NT Baf 3MA LC3 Actin EV HCT116-TP53KO MAPK14CA D14 E D HCT116-TP53KO EV MAPK14CA D14 Cell viability (%) 20 40 60 80 100 HCT116-TP53KO EV MAPK14CA D14 Cell viability (%) 20 40 60 80 100 siCT siAtg 5 siAtg 7 NT 3MA Baf NT 3MA Baf Atg7 Actin HCT116-TP53KO Atg5 siCT D D4 siRNA
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FIGURE 6 B A D C E F G NT SN HCT116 TP53KO LC3 Actin LC3 labelling
LC3 spots/cell 1 2 3 4 5 6 7 NT SN38 HCT116-TP53KO NT SN SW480 LC3 Actin D C SN38 SN+ Baf 3MA LC3 GAPDH HCT116-TP53KO siCT siAtg5 siAtg7 SW480 SN38 IC50 (nM) 2 4 6 8 Cytotoxic Assay E F G siCT siAtg5 siAtg7 4 3 2 1 HCT116-TP53KO SN38 IC50 (nM) Cytotoxic Assay 0.4 0.8 1.2 1.6 siCT siATG5 siATG7 HCT116-TP53KO SN38 IC50 (nM) Clonogenic Assay HCT116-TP53KO 20 40 60 80 100 120 NT SN38 SN38+3MA % living cells
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FIGURE 7 A B C D LC3 MAPK14 Actin NT SN SN+3MA NT SN SN+3MA
HCT116-TP53KO-shLUC HCT116-TP53KO-shMAPK14 B shLUC shMAPK 11 12 13 LC3 HCT116-TP53KO GAPDH NT SN38 C 1 2 3 HCT116-TP53KO ShLuc ShMAPK14 SN38 SN38+3MA SN38 IC50 (nM) D siCT siATG5 siATG7 1 2 3 HCT116-TP53KO ShLuc SN38 IC50 (nM) ShMAPK14
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