a blot #2 b blot #3 · cells and spheroids were harvested and western blot analysis of diﬀerent...

A Blot #2 2D 3D 2D 3D 2D 3D 2D 3D 2D 3D 2D 3D ERK1/2 rpS6 α Tubulin p70 S6K 4E-BP1 p-Akt S473 l.e. Akt p-PRAS40 T246 l.e. α Tubulin E-Cadherin l.e. p-ERK1/2 T202/Y204 s.e. p-ERK1/2 T202/Y204 l.e. p-Akt T308 p-p70 S6K T389 l.e. p-rpS6 S240/244 p-p70 S6K T389 s.e. E-Cadherin s.e. p-PRAS40 T246 s.e. p-Akt S473 s.e. 2D 3D 2D 3D 2D 3D 2D 3D 2D 3D 2D 3D Blot #3 ERK1/2 rpS6 α Tubulin p70 S6K 4E-BP1 Akt p-PRAS40 T246 l.e. α Tubulin E-Cadherin l.e. p-ERK1/2 T202/Y204 s.e. p-ERK1/2 T202/Y204 l.e. p-Akt T308 p-p70 S6K T389 l.e. p-rpS6 S240/244 l.e. p-p70 S6K T389 s.e. E-Cadherin s.e. p-PRAS40 T246 s.e. p-Akt S473 s.e. GAPDH GAPDH p-rpS6 S240/244 s.e. p-PKC (pan) β II S660 B Suppor(ng Figure SF1 (ﬁgshare) – Analysis of PI3K/AKT/mTOR- and RAS/RAF/MAPK-singaling in 2D vs 3D cell culture. The colon cancer cells (LS174T, HT29, SW620, HCT116, Caco2 and DLD-1) were cultured in 2D and 3D for 48h. Cells and spheroids were harvested and Western blot analysis of diﬀerent proteins of the PI3K/AKT/mTOR- and RAS/RAF/MAPK-pathway was carried out. Detec_on of phosphorylated proteins is indicated by p- prior to the protein name and the phosphorylated amino acid is indicated behind the protein name. α-tubulin and GAPDH served as loading control. Two independent biological replicates are shown (A,B).

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A Blot#2

ERK1/2

rpS6

αTubulin

p70S6K

4E-BP1

p-AktS473l.e.

Akt

p-PRAS40T246l.e.

αTubulin

E-Cadherinl.e.p-ERK1/2T202/Y204s.e.p-ERK1/2T202/Y204l.e.

p-AktT308

p-p70S6KT389l.e.

p-rpS6S240/244

p-p70S6KT389s.e.

E-Cadherins.e.

p-PRAS40T246s.e.

p-AktS473s.e.

Blot#3

ERK1/2

rpS6

αTubulin

p70S6K

4E-BP1

Akt

p-PRAS40T246l.e.

αTubulin

E-Cadherinl.e.

p-ERK1/2T202/Y204s.e.p-ERK1/2T202/Y204l.e.

p-AktT308

p-p70S6KT389l.e.

p-rpS6S240/244l.e.

p-p70S6KT389s.e.

E-Cadherins.e.

p-PRAS40T246s.e.

p-AktS473s.e.

GAPDH

p-rpS6S240/244s.e.

p-PKC(pan)βIIS660

Suppor(ngFigureSF1(figshare)–AnalysisofPI3K/AKT/mTOR-andRAS/RAF/MAPK-singalingin2Dvs3D cell culture. The colon cancer cells (LS174T,HT29, SW620,HCT116,Caco2andDLD-1)werecultured in 2D and 3D for 48h. Cells and spheroids were harvested andWestern blot analysis ofdifferentproteinsof thePI3K/AKT/mTOR-andRAS/RAF/MAPK-pathwaywascarriedout.Detec_onof phosphorylated proteins is indicated by p- prior to the protein name and the phosphorylatedaminoacid is indicatedbehind theproteinname.α-tubulin andGAPDHservedas loading control.Twoindependentbiologicalreplicatesareshown(A,B).

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FIGURE 1 A p38 EV Sh MAPK 14 HCT116-TP53KO GAPDH EV Sh MAPK 14bis Sh MAPK 12 Sh MAPK 13 EV Sh MAPK 11 B 0.0 0.5 1.0 1.5 2.0 2.5 Sh LUC Sh MAPK 14 Sh MAPK

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