Expression of apoptosis-related proteins before and after IITT+F treatment.

<p>Antibody array analysis of apoptosis-related proteins in ESCs of (A) untreated, non-differentiated (St-T1, red bar; KdS1, blue bar) and decidualized (dSt-T1, bright red bar; dKdS1, bright blue bar) ESCs, n = 4 ± SEM, *p<0.05 Sdc-1 wildtype vs. Sdc-1 kd cells, #p<0.05 non-differentiated vs. decidualized cells. (B) Antibody Array with protein from IITT+F treated, non-differentiated (St-T1, red bar; KdS1, blue bar) and decidualized (dSt-T1, bright red bar; dKdS1, bright blue bar) ESCs. Pixel density is given as mean±SEM of n = 4 independent experiments, *p<0.05 Sdc-1 wildtype vs. Sdc-1 kd cells, #p<0.05 non-differentiated vs. decidualized cells, ✝p<0.05 untreated vs. IITT+F treated.</p

Quantification of active Caspase-3 in ESCs treated with embryonic stimuli.

<p>Non-differentiated (St-T1, red bar; KdS1, blue bar) and decidualized (dSt-T1, bright red bar; dKdS1, bright blue bar) ESCs were treated with IITT and F for 24h individually or in combination as indicated and the amount of active Caspase-3 was analyzed in ng/ml and displayed as mean±SEM of n = 3 independent experiments; *p<0.05 Sdc-1 wildtype vs. Sdc-1 kd cells, #p<0.05 non-differentiated vs. decidualized cells, ✝p<0.05 untreated controls vs. treated cells.</p

Induction of FasR expression after IITT treatment.

<p>Fold change of FasR mRNA in non-differentiated (St-T1, red bar; KdS1, blue bar) and decidualized (dSt-T1, bright red bar; dKdS1, bright blue bar) ESCs after treatment with IITT 24h. 2^-ΔΔCt is are given as mean±SEM of n = 5 independent experiments, ✝p<0.05 untreated vs. IITT treated.</p

Loss of membrane asymmetry after IITT+F treatment.

<p>Non-differentiated (St-T1, KdS1) and decidualized (dSt-T1, dKdS1) ESCs were treatmed with IITT+F and loss of membrane asymmetry was visualized with Annexin V FITC staining (green), n = 3; (A)–(D) untreated controls: (A) St-T1, (B) KdS1, (C) dSt-T1, (D) dKdS1. (E)-(H) IITT+F treated: (E) St-T1, (F) KdS1, (G) dSt-T1, (H) dKdS1; blue nuclei are stained with Hoechst 33342. Scale bars indicate 100μm.</p

Activation of the pro-apoptotic JNK pathway after IITT and F treatment.

<p>Western blot analysis of pJNK and JNK in non-differentiated (St-T1, first line; KdS1, second line) and decidualized (dSt-T1, third line; dKdS1, fourth line) ESCs after treatments with F 15min, IITT 15min and IITT 24h + F 15min vs. untreated controls. (A) Representative blot of pJNK (46/54kDa), JNK (46/54kDa) and β-Actin (42kDa) as loading control. (b) Pixel densitiy evaluation of pJNK normalized to JNK is given as mean±SEM of n = 6 independent experiments, *p<0.05 wildtype vs. Sdc-1 kd cells, ✝p<0.05 untreated control vs. treated.</p

Activation of the pro-survival NFκB pathway after IITT and F treatment.

<p>Western blot analysis of NFκB members p65 and IκBα in non-differentiated (St-T1, first line; KdS1, second line) and decidualized (dSt-T1, third line; dKdS1, fourth line) ESCs after treatments with F 15min, IITT 15min and IITT 24h + F 15min vs. untreated controls. (A) Representative blot of pp65 (65kDa), p65 (65kDa) and β-Actin (42kDa) as loading control. (B) Pixel densitiy evaluation of pp65 normalized to p65 is given as mean±SEM of n = 6 independent experiments, *p<0.05 wildtype vs. Sdc-1 kd cells, #p<0.05 undifferentiated vs. decidualized cells, ✝p<0.05 untreated control vs. treated. (C) Representative blot of IκBα (39kDa) and β-Actin (42kDa) as loading control. (D) Pixel densitiy evaluation of IκBα normalized to β-Actin is given as mean±SEM of n = 6 independent experiments, *p<0.05 wildtype vs. Sdc-1 kd cells, #p<0.05 undifferentiated vs. decidualized cells, ✝p<0.05 untreated control vs. treated.</p

Activation of the pro-survival protein Akt after IITT and F treatment.

<p>Western blot analysis of pAkt and Akt in non-differentiated (St-T1, first line; KdS1, second line) and decidualized (dSt-T1, third line; dKdS1, fourth line) ESCs after treatments with F 15min, IITT 15min and IITT 24h + F 15min vs. untreated controls. (A) Representative blot of pAkt (60kDa), Akt (60kDa) and β-Actin (42kDa) as loading control. (B) Pixel densitiy evaluation of pAkt normalized to Akt is given as mean±SEM of n = 6 independent experiments, *p<0.05 wildtype vs. Sdc-1 kd cells, #p<0.05 undifferentiated vs. decidualized cells, ✝p<0.05 untreated control vs. treated.</p

Investigation of the extrinsic and intrinsic apoptosis pathway after IITT+F treatment.

<p>Analysis of Caspase-8 and -9 activation of treated cells vs. untreated controls in non-differentiated (St-T1, red bar; KdS1, blue bar) and decidualized dSt-T1, bright red bar; dKdS1, bright blue bar) ESCs after treatment with IITT+F. Untreated controls were assigned being 1 and enzymatic activity of caspases after treatment was determined as fold induction vs. controls and given as mean±SEM of n = 3 independent experiments, *p<0.05 Sdc-1 wildtype vs. Sdc-1 kd cells, ✝p<0.05 untreated controls vs. treated.</p