Cd135

Single cell resuspended FL or BM mononuclear cells underwent red cell lysis treatment before staining with cKIT at 1:100 concentration (BioLegend 105819), SCA1 (eBioscience 25-5981-81), CD135 (BioLegend), CD48 (BioLegend 103422), and CD150 (BioLegend 115925) antibodies, as well as the lineage antibodies: B220 (BD 553090), GR1 (BioLegend 108408), CD3 (BD 555275), CD4 (BD 553653), CD5 (BD 553023), and TER119 (BD 553673) at the manufacturer’s recommended concentrations. Cells were stained for 30 min on ice, protected from light. Blocking and washing buffer contained 2%FBS/ PBS. For viability, dead cell exclusion staining using DAPI (Thermo 62248, 1μg/ml) was included at a concentration of 1 μg/ml. Flow cytometric analysis was performed using FACS Canto2 and LSR2 instruments (BD Biosciences). For intracellular staining, FL or BM mononuclear cells were stained with surface markers and fixed with 2% PFA for 15 minutes, permeabilized with 0.5% saponin and stained with anti-p53, anti-p53S15 and anti-Cdc7. Samples were acquired with a LSR2 (Becton-Dickinson) and data were analyzed using FlowJo (10.6.1) software to quantify mean fluorescent intensity (MFI). Reagent Supplementaary Table 1 for details.

Before flow cytometric analysis of primary mouse tissues (i.e., BM and spleen) red blood cells were lysed with ammonium chloride (Stemcell technologies). Dead cells were excluded with Draq7 (1:1600, Biostatus, Shepshed, United Kingdom) or Fixable Viability Dye eFluor^® 780 (1:1000, eBioscience, San Diego, CA, USA). For cell cycle analysis, cells were fixated and permeabilized using 1.6% formaldehyde (Merck) and 95% ethanol (CCS Healthcare AB) and stained with Ki-67 (1:40, clone REA183) (Miltenyi) and DAPI (1:50, Biolegend, San Diego, CA, USA). For immunophenotyping, the following antibodies were used: Ly-6G/Ly-6C/Gr-1 (1:800, clone RB6-8C5), CD11b (1:800, clone M1/70), CD45RA (1:800, clone RA3-6B2), CD3ε (1:800, clone 145-2C11), CD16/32 (1:200, clone 92), CD34 (1:20, clone MEC14.7), CD135 (1:200, clone A2F10), Ly-6A/Ly-6E/Sca-1 (1:100, clone D7), CD117 (1:100, clone ACK2), CD127/IL-7R (1:200, clone A7R34) and a lineage cocktail (1:50, CD3, clone 17A2; Ly-6G/Ly-6GC/Gr-1, clone RB6-8C5; CD11b, clone M1/70; CD45RA/B220, clone RA3-6B2; Ter-119, clone Ter-119), all from Biolegend. Flow cytometric analysis and fluorescence-activating cell sorting were performed on FACS Aria IIu or FACS Aria Fusion (BD, Franklin Lakes, NJ, USA). Data analysis was performed using the FlowJo software (FlowJo, LLC, Ashland, OR, USA).