Fig. 5: MYCT1 controls endocytosis and environmental sensing in human HSPCs.
From: MYCT1 controls environmental sensing in human haematopoietic stem cells
a–d, Evaluating the effects of MYCT1 KD and OE on endocytosis. a,b, Quantification (left) and representative FACS histogram (right) of low-molecular-weight (10 kDa) fluorescent dextran (a) or ECgreen (b) internalized over 30 min in control, MYCT1 KD or OE CB HSPCs 72 h after transduction. n = 5 replicates from 3 independent experiments, mean ± s.e.m., two-tailed t-test. c, Quantification of internalized fluorescent dextran in CB HSPC and HPC subsets over time in culture. n = 4 (d0), n = 6 (d5), n = 12 (d10 and d15) replicates from 4 independent experiments and n = 3 from 3 independent experiments (16 h), mean ± s.e.m., two-tailed Mann–Whitney test. d, Quantification (left) and representative histogram (right) of internalized fluorescent dextran in control or MYCT1 OE CB HSPCs (CD34+CD90+) cultured for 10–12 days. n = 9 from 4 experiments, mean ± s.e.m., two-tailed t-test. e–g, Evaluating CB HSPCs with different endocytosis levels. e, Experimental outline for scRNA-seq of CB HSPCs (CD34+CD38–CD90+EPCR+) with low, medium or high levels of endocytosis after 4 days in culture. f,g, Dot plots depicting MYCT1 expression (f) or the scores for MYCT1-associated functional categories (Fig. 2) (g) in HLF+ HSCs. h, KIT internalization in response to SCF stimulation by FACS in control or MYCT1 KD CB HSPCs (CD34+CD38–EPCR+). n = 3 experiments, mean ± s.e.m., two-way analysis of variance. i, Western blot (left) and quantification (right) of phospho-AKT (pAKT) in control, MYCT1 KD or OE CB HSPCs 72 h after transduction. n = 3 experiments, mean ± s.e.m., two-tailed paired t-test. GAPDH is a sample processing control. For gel source data, see Supplementary Fig. 1. j, Dot plots for signalling scores (scRNA-seq) in control, MYCT1 KD or OE cells 72 h after transduction, uncultured and day 4 cultured CB HLF+ HSCs, and HSCs with low, medium or high dextran internalization. AKT+ and AKT– indicate positive regulators and negative regulators, respectively, of AKT signalling. For a, b and d, ΔMFI is between the GFP+ and GFP– cells within the same sample.
