Fig. 6: Depletion of CALCRL sensitizes AML cells to chemotherapy in vivo.

a Experimental plan for assessing the consequence of CALCRL depletion on chemotherapy response in vivo. 2 × 10⁶ MOLM-14 expressing indicated inducible shRNAs were injected into the tail vein of NSG mice. Ten days later, when disease was established, mice were treated with 30 mg/kg/d AraC for 5 days. On day 18, a group of mice was kept to follow survival (d). The other group was sacrificed to assess human cell engraftment (b) and cell death (c). Human cells were also sorted by flow cytometry using mCD45.1⁻/hCD45⁺/hCD33⁺/AnnV⁻ markers and replated in culture dishes to assess protein expression (f), sensitivity to chemotherapeutic drugs (e), and to perform CALCRL depletion by shRNA (g, h). b Total leukemia burden measured using mCD45.1⁻/hCD45⁺/hCD33⁺/AnnV⁻ markers (unpaired t-test). c Graph shows the percentage of AnnV⁺ cells (unpaired t-test). d Mice survival monitoring (Log-rank (Mantel-Cox) test). e After replating, cells were treated with AraC or idarubicin ex vivo for 48 h. Cell viability was assessed by MTS assay and values were normalized to untreated condition (n = 1 in quadriplate). Curve fit to calculate IC50 was determined by log (inhibitor) vs response (three parameters). f Western blotting for CALCRL, RAD51, CHK1, BCL2, and β-ACTIN. Cellular extracts were collected 2 days after mice sacrifice. g, h Human cells from vehicle (g) or AraC (h) treated mice were transduced with indicated shRNA. After 4 days cells were treated with AraC or idarubicin for 48 h and cell viability was assessed by MTS assay (n = 1 in quadriplate). Curve fit to calculate IC50 was determined by log (inhibitor) vs response (three parameters) test. Data are mean ± s.e.m.