Rig-I deletion protects the HSC niche function of BMSCs under ATRA treatment. (A) qPCR analysis of HSC niche factors in NRF2-knockdown Rig-I^+/+ or Rig-I^−/− BMSCs treated with vehicle or ATRA, as indicated; n = 3 biologically independent replicates. (B) Schematic of experimental design for in vivo HSC niche function after ATRA treatment in Rig-I^+/+ or Rig-I^−/− mice. Wild-type CD45.2⁺ mice, pretreated with vehicle or ATRA, consecutively 6 times, were lethally irradiated and transplanted with 1 × 10⁶ CD45.1⁺ BMNCs for recovery. After an 8-week recovery, 1 × 10⁶ recovered CD45.1⁺ BMNCs were mixed with 2 × 10⁵ fresh CD45.2⁺ BMNCs for competitive reconstitution analysis. (C-D) Absolute numbers of donor-derived HSPCs (C) and cell cycle analysis for donor-derived HSCs (D) in Rig-I^+/+ or Rig-I^−/− recipient mice after the 8-week recovery. (E) Engraftment analysis of total engrafted CD45.1⁺ donor cells (Total), B cells (B220⁺), T cells (CD3⁺), and myeloid cells (Gr1⁺Mac1⁺) recovered from Rig-I^+/+ recipients or Rig-I^−/− recipients at indicated weeks after transplantation; n = 6 donors per group and n = 11–12 recipient mice per group. (F) Schematic of experimental design for co-transplantation of BMNCs with BMSCs derived from Rig-I^+/+ and Rig-I^−/− mice pretreated with ATRA or control vehicle. (G) Absolute numbers of donor-derived HSPCs in recipients on 8-week recovery after BMNC transplantation and BMSC co-transplantation as indicated. (H) The recovery of hematopoietic mononuclear cells in the peripheral blood of co-transplanted recipients, as indicated. Error bars indicated mean ± SD. Data were analyzed using 2-way (A, C-E, G, and H) ANOVA followed by Dunnett’s test multiple comparisons. ^‡P < .05; ^‡‡P < .01; ^‡‡‡P < .001; ^‡‡‡‡P < .0001.