Extended Data Fig. 9: SEL1L regulates MPL maturation.

a, Schematic of HSC (LSK CD150⁺CD48^-)-vasculature distance measurement in the BM. b, c, Representative 2D images (b) and quantification (c) of the distances between CFSE-labelled HSCs (green) and vascular structure (CD31⁺ and/or CD144⁺, magenta). 177 control (Ctrl) and 147 Sel1l^Vav-KO HSCs were analysed over n=3 independent experiments. Scale bar, 20 μm. d, Schematic for non-conditioned transplantation of donor cells into Ctrl or Sel1l^Mx1-KO congenic mice. e, The HSCs numbers in Ctrl and Sel1l^Mx1-KO mice 5 weeks after poly (I:C) injection. n = 5. f, Percentage of donor-derived (CD45.1⁺) cells in the PBMC of non-irradiated Ctrl or Sel1l^Mx1-KO recipient mice 16 weeks after transplantation. n = 4. g, h, Flow analysis of surface MPL expression in HSCs at different cell-cycle states. Representative flow cytometry plots (g) and quantification (h) are shown. n = 4. i, Quantification of surface c-Kit expression from 8-week-old Ctrl and Sel1l^Vav-ko HSCs. n = 5. j, RT-PCR analysis of Mpl mRNA in 8-week-old Ctrl or Sel1l^Vav-KO HSCs. Data are presented relative to Actb. Ctrl: n = 8; Sel1l^Vav: n = 7. k, Representative histogram and quantification of phospho-STAT5 (Tyr694) level from 30-week-old Ctrl or Sel1l^Vav-KO HSCs. n = 4. l, Quantification of p57 mRNA by qPCR in 8-week-old Ctrl or Sel1l^Vav-KO HSCs. n = 4. m, WB analysis of total MPL in HSCs from 8-week-old Ctrl and Sel1l^Vav-KO mice under native or denature conditions. n, Quantification of total MPL signal in Ctrl and Sel1l^Vav-KO HSCs from immunostaining experiment in Fig. 4f. Ctrl HSCs: n = 100; Sel1l^Vav HSCs: n = 250. o, Sequence alignment of MPL from indicated species with R257 (Human) highlighted in red. p, 293T cells were transfected with Myc-tagged wild-type MPL (WT-Myc), FLAG-tagged mutant MPL (Mut-FLAG) or 1:1-mixed WT and Mut MPL (WT-Myc + Mut-FLAG) for 48h. The expression of WT or Mutant MPL was quantified by qPCR primers specific to Myc or FLAG tag. n = 3 independent samples. q, WT or SEL1L^CRISPR-KO 293T cells were transfected with HA-tagged MPL and surface MPL expression was determined by flow cytometry 48h later. n = 3. r, WB analysis of HRD1 expression in WT, SEL1L^CRISPR-KO or HRD1^CRISPR-KO 293T cells. s, t, WT or HRD1^CRISPR-KO 293T cells were transfected with HA-tagged MPL and surface MPL or C-KIT expression were determined by flow cytometry 48h after transfection. n = 3. u-x, Expression of aggregation-prone mutant proAVP (G57S) (u) or POMC (C28F) (v) forms aggregates in WT 293T cells, but is not sufficient to inhibit ERAD activity (u, v, w) and thus does not reduce surface MPL expression (x). HRD1 autoubiquitination (u, v) and OS9 (OS9-1 and OS9-2) accumulation (u, v, w) were used as validated indicators for ERAD activity. The experiments for q and u-x are performed together, the MPL level in WT (r) and Mock (x) was from the same experiment. n=3. Results are shown as mean ± s.d. Two-tailed Student’s t-test (e, f, h-l, n, p, q, s, t, x), or two-sample Kolmogorov–Smirnov test (c) were used to assess statistical significance. ns, not significant. Statistical information and unprocessed blots are provided as source data.

Source data