Supplementary Figure 4: N-Ras^G12D but not FLT3-ITD or Tet2 haploinsufficiency leads to HSC protection from ER stress-induced apoptosis.

a, Experimental scheme of the assay for results depicted in Figure 2a-e, b, Summary of the percentage of Annexin V positive cells in HSCs purified from 8-10 weeks old FLT3-ITD knock-in mice (FLT3-ITD/+) and sex-matched littermate control mice (+/+) and treated 18 hours of 0.6 μg/ml Tunicamycin(Tm) and 0.2 μM Thapsigargin (Tg) (n=3-5 biological replicates in 3 independent experiments). c, Summary of apoptosis analysis of HSCs from Mx1-cre^-; Tet2^fl/+ (+/+) or Mx1-cre⁺; Tet2^fl/+ (fl/+) mice at least 2 weeks after 6 dose of pIpC injection, and treated with 18 hours of 0.6 μg/ml Tunicamycin(Tm) and 0.2 μM Thapsigargin (Tg) (n=3 biological replicates in 1 independent experiments). d, Experimental scheme of chimerism maintenance experiment for Figure 2f. 0.5 million bone marrow cells from Mx1-cre⁺; Nras^G12D/+ (G12D/+) or control CD45.2 mice (2 weeks after pIpC injection) were transplanted with 0.5 million competitor (CD45.1) bone marrow cells into recipient mice. Six weeks after transplantation, transplant recipients were injected with 2 mg/kg LPS for 3 doses every other day and HSCs chimerism was analyzed 2-4 weeks after LPS injections. e, Experimental scheme of analysis in Col1α1-H2B-GFP; Rosa26-M2-rtTA mice. Mice were treated with doxycycline for 6 weeks to label HSCs with GFP, after which doxycycline water was removed to allow HSCs to dilute GFP signal upon cell division. GFP^high and GFP^low HSCs (Gating strategy to separate GFP^high and GFP^low HSCs is shown on the bottom right) were purified after 18 weeks off doxycycline and were treated with Tg or Tm for 18 hours. Data represent mean±s.d. for panels b and c. Two-sided student t-test was used for statistical analysis.