MAPK14CA Induces Apoptosis and Autophagy in HCT116-TP53KO Cells

HCT116-TP53KO Sh MAPK 14 Sh MAPK 14bis Sh MAPK 12 Sh MAPK 13 Sh MAPK 11 EV EV EV EV p38 GAPDH 2.5 2.0 1.5 IC50 (nM) 1.0 0.5 0.0 Sh MAPK 14 Sh MAPK 14bis Sh MAPK 11 Sh MAPK 12 Sh MAPK 13 Sh LUC HCT116-TP53KO HCT116-TP53KO HCT116-TP53KO NT SN38 pp38 MAPK11 CA MAPK12 CA MAPK14 CA MAPK13CA GAPDH MAPK11 CA MAPK12 CA MAPK14 CA MAPK13CA HA p ATF2 tubulin HCT116-TP53KO 3.5 3.0 2.5 2.0 IC50 (nM) 1.5 1.0 0.5 0.0 MAPK 14 CA MAPK 11 CA MAPK 12 CA MAPK 13 CA EV FIGURE 1 A B Kinase Assay C D E F

HCT116-TP53KO EV HCT116-TP53KO-MAPK14CA 120 100 80 Doubling Time (hours) 60 40 20 0 EV MAPK14-CA HCT116-TP53KO 120 HCT116-TP53KO-EV 20 100 HCT116-TP53KO-MAPK14CA HCT116-TP53 KO 80 15 60 10 % APOPTOSIS % living cells 40 20 5 0 D7 D14 D21 D28 0 MAPK14 CA EV HCT116-TP53KO-EV HCT116-TP53KO-MAPK14CA 5 4 3 Blue cell number/field 2 1 0 HCT116- HCT116- TP53KO- MAPK14CA TP53KO -EV FIGURE 2 A B 4 3 Cell number (106) 2 1 0 0 2 4 6 8 10 12 Days C D E

HCT116-TP53KO-EV HCT116-TP53KO-MAPK14CA S 37.1% S 49.4% G0 G1 28.8% G0 G1 36.8% G2/M 19.3% G2/M 18.4% % of BrdU stained cells 120 HCT116-TP53KO 1.2 HCT116-TP53KO-EV 100 MAPK14CA 1.0 80 D7 D14 D21 D28 EV 0.8 60 p21 p21 Arbitrary RNA units 0.6 EV D7 D14 D21 D28 HCT116-TP53KO-MAPK14CA 40 HA 0.4 20 HCT116-TP53KO-Shp21-MAPK14CA 0.2 90 Actin 0 0.0 80 0 5 10 15 20 25 30 70 MAPK14CA 60 HCT116-TP53KO HCT116-TP53KO-MAPK14CA 50 40 HCT116-TP53KO-ShLuc- MAPK14CA shLUC shp21 30 p21 20 % of BrdU stained cells 10 GAPDH 0 0 5 10 15 20 25 30 HCT116-TP53KO -ShLuc-MAPK14CA HCT116-TP53KO -Shp21-MAPK14CA S 46.1% S 25.8% G0 G1 20.9% G0 G1 45.6% G2/M 23.16% G2/M 28.9% FIGURE 3 B A D C E G F

HCT116-TP53KO MAPK14CA HCT116-TP53KO D7 D14 D21 D28 EV EV MAPK14CA LC3 Actin H1299 MAPK14CA GFP-LC3 labelling EV D7 D14 D21 LC3 Tubulin HCT116-TP53KO EV MAPK14CA 500 nm 500 nm EV MAPK14CA HCT116-TP53 KO 6 5 4 Autophagosome number/cell 3 2 1 0 EV MAPK14CA HCT116-TP53 KO HCT116-TP53KO-MAPK14CA shLUC shp21 GFP-LC3 spots/cell LC3 MAPK14 CA EV GAPDH HCT116-TP53KO FIGURE 4 B A C D E 10- 8 - 6 - 4 - 2 - 0 -

HCT116-TP53KO (J14) HCT116-TP53KO EV MAPK14CA MAPK14CA NT 2H 4H 8H NT 2H 4H 8H EV D7 D14 D21 D28 p62 p62 GAPDH 2 160 Actin 1 120 p62 level (arbitrary unit) 80 0 100 EV 80 D7 D14 D21 D28 40 Cell viability (%) 60 p62 level (arbitrary unit) 0 40 nt 2h 4h 8h nt 2h 4h 8h EV MAPK14CA 20 0 HCT116-TP53KO MAPK14CA siCT siCT siAtg 5 siAtg 7 siAtg 5 siAtg 7 HCT116-TP53KO HCT116-TP53KO 3 EV MAPK14CA D14 2.5 HCT116-TP53KO EV MAPK14CA D14 2 siRNA LC3-II level (arbitrary unit) HCT116-TP53KO 1.5 LC3 Actin siCT D2 D4 1 0.5 Atg5 0 NT 3MA Baf NT Baf 3MA NT 3MA Baf NT 3MA Baf Atg7 EV MAPK14CA D14 Actin HCT116-TP53KO 100 80 Cell viability (%) 60 40 20 0 EV MAPK14CA D14 HCT116-TP53KO FIGURE 5 B A C E D NT 3MA Baf NT 3MA Baf

LC3 labelling HCT116 TP53KO SW480 Cytotoxic Assay LC3 4 3 2 1 0 7 NT SN NT SN 6 Actin NT LC3 5 SN38 IC50 (nM) Actin 4 LC3 spots/cell siCT siAtg5 siAtg7 3 2 1 0 HCT116-TP53KO NT SN38 SN38 HCT116-TP53KO HCT116-TP53 KO Cytotoxic Assay Clonogenic Assay 1.6 HCT116-TP53KO 1.2 SN+ Baf SN+ 3MA SN38 IC50 (nM) 0.8 SN38 8 0.4 LC3 6 0 siCT siATG5 siATG7 4 SN38 IC50 (nM) GAPDH HCT116-TP53KO 2 0 siCT siAtg5 siAtg7 SW480 120 100 80 % living cells 60 40 20 0 NT SN38 SN38+3MA HCT116-TP53KO FIGURE 6 B A D C E F G

HCT116-TP53KO-shMAPK14 HCT116-TP53KO-shLUC NT SN SN+3MA NT SN SN+3MA LC3 MAPK14 Actin B HCT116-TP53KO siCT siATG5 NT SN38 siATG7 shMAPK 12 shMAPK 13 shMAPK 12 shMAPK 13 shLUC shMAPK 11 shLUC shMAPK 11 LC3 GAPDH 3 SN38 SN38+3MA 2 SN38 IC50 (nM) 1 0 HCT116-TP53KO ShLuc HCT116-TP53KO ShMAPK14 3 2 SN38 IC50 (nM) 1 0 HCT116-TP53KO ShLuc HCT116-TP53KO ShMAPK14 FIGURE 7 A C D

MAPK14CA Induces Apoptosis and Autophagy in HCT116-TP53KO Cells

MAPK14CA Induces Apoptosis and Autophagy in HCT116-TP53KO Cells

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