Figure 6. In vitro, B55alpha is upregulated in a density dependent manner and needed to protect HUVECs against activation of the endogenous apoptosis pathway.

(A,B) Analysis of B55α expression in HUVECs cultured at different densities, showing an upregulation in a density dependent manner on RNA (A) and protein level (B).

(C-E) Analysis of living ECs after KD of B55α showing a drastic reduction of living HUVECs

(C) and simultaneously an upregulation of apoptosis markers as analyzed by FACS (C) and WB (E).

(F-H) Immunostainings of B55α-KD HUVECs treated with pan-Caspase inhibitors (qVD or zVAD) for Phalloiding (green), the apoptosis marker cleaved Caspase3 (red) and Hoechst (Scale bar: 100µm) (F) and quantitative analysis thereof (G-H).

(I) Schematic representation and timeline of the experiments depicted in (J) and (K).

(J,K) Analysis of living HUVECs after KD of B55α showing a stronger induction of apoptosis in dense HUVECs, mimicking a more quiescent-like phenotype.

(L) Results showing that chemical inhibition of PP2A phosphatase activity recapitulates the reduction of living HUVECs in dense conditions, while barely affecting sparse HUVECs.

All statistical analysis was conducted on a minimum of 3 samples per group and are representative from 3 independent repetitions.

P values are *p<0,05; **p<0,001; ***p<0,001; ****p<0,0001.

Graphs show standard error of the mean (SEM).