Extended Data Fig. 12: Evaluation of MYCT1 moderated signalling responses in endothelial cell models.
From: MYCT1 controls environmental sensing in human haematopoietic stem cells
a-c Quantifying basal signalling responses by western blot in control and MYCT1 KD E4EC cultured with continuous presence of serum and cytokines. a Experimental outline. b,c Representative western blot (b) and quantification (c) of phospho-AKT and phospho-ERK in control and MYCT1 KD E4ECs. n = 3 independent experiments, mean±s.e.m, two-tailed ratio-paired t-test. d-g Determining the effect of MYCT1 KD on the signalling responses to cytokine stimulation by western blot in control or MYCT1 KD E4EC. d Experimental outline. e-g Representative western blot (e) and quantification of phospho-AKT and phospho-ERK in control and MYCT1 KD E4ECs after overnight starvation and subsequent stimulation with complete media (containing serum, FGF, EGF and IGF) for 30 min, 1 and 3 h (f). The quantifications under starvation conditions are also shown zoomed in (g). n = 4 (KD1) or n = 5 (KD2) independent experiments, mean±s.e.m, two-tailed ratio-paired t-test. P values in m from left to right for KD1: 0.0152 (pAKT), 0.0491, 0.0288 (pERK1); for KD2: 0.0211, 0.0359 (pAKT), 0.0442, 0.0045, 0.00515 (pERK1), 0.0095, 0.0267 (pERK2). h-k Determining the effect of MYCT1 KD on EGFR internalization and downstream signalling in response to EGF stimulation in non-immortalized HUVECs 72 h after transduction. h Experimental outline. i Quantification of relative membrane EGFR by FACS in starvation or in response to EGF stimulation for 5, 10, and 30 min. n = 3 independent experiments, mean±s.e.m, two-way ANOVA. j,k Representative western blot (j) and quantification (k) of phospho-AKT in control and MYCT1 KD HUVEC after overnight starvation and subsequent stimulation with EGF for 10 and 30 min. n = 4 independent experiments, mean±s.e.m, two-tailed paired t-test. l Model figure of MYCT1 regulated environmental sensing in HSC. For gel source data, see Supplementary Fig. 5. GAPDH is a loading control for AKT in b and j, and a sample processing control for pERK1/2 in b and all antibodies in e. Schematics in a,d,h,l were created with BioRender (https://www.biorender.com).
