SCENITH analysis of the Lin– and LSK BM compartments. (A-B) Bar graphs representing relative SCENITH signals in Lin– cells from (A) RoLoRiG/Mx1CRE BM (n = 5) and wild-type (n = 3), or (B) KRASG12D/Mx1CRE (n = 3) and wild-type (n = 3) BM. Wild-type control was arbitrarily set at 100% for both (A) and (B), ∗P < .05. (C-D) Fractions of puromycin-low Lin– cells for the indicated genotypes, setting wild-type arbitrarily at 0.2, as in Figure 4F. (E) Representative spectral flow dot plots of cKit+ and SCA-1+ LSK cells and their frequency. (F) Representative SCENITH histograms of LSK cells from the indicated mouse models. Large numbers depict the MFI for the anti-puromycin antibody signal, small numbers the fraction of cells with low or high puromycin signal. Panels E-F are representative examples of at least 3 independent biological experiments. (G-H) Bar graphs representing relative SCENITH signals in LSK cells from RoLoRiG/Mx1CRE (n = 5) and wild-type (n = 3) (G) or KRASG12D/Mx1CRE (n = 3) and wild-type (n = 3) BM (H). Wild-type control was arbitrarily set at 100%, ∗P < .05. (I-J) Bar graphs of fractions of puromycin-high and puromycin-low LSK cells for KRASG12D/Mx1CRE and wild-type, setting the wild-type arbitrarily at 0.2. ∗P < .05. (K) Relative frequency of HSCs and relative SCENITH signals in these HSCs for RoLoRiG/Mx1CRE and wild-type mice. Wild-type control HSC frequency was arbitrarily set at 20% (supplemental Figure 11E-F), with a relative SCENITH signal of 100%. ∗P < .05. (L) As in panel K, but comparing KRASG12D/Mx1CRE (n = 3) HSCs and against 3 other wild-type HSCs. Note that both the fraction of KRASG12D/Mx1CRE HSCs in the LSK compartment and the LSK fraction itself were reduced in KRASG12D/Mx1CRE mice (supplemental Figure 11E-F). ∗∗P < .01. ns, not significant.
Figure 5.

SCENITH analysis of the Lin– and LSK BM compartments. (A-B) Bar graphs representing relative SCENITH signals in Lin– cells from (A) RoLoRiG/Mx1CRE BM (n = 5) and wild-type (n = 3), or (B) KRASG12D/Mx1CRE (n = 3) and wild-type (n = 3) BM. Wild-type control was arbitrarily set at 100% for both (A) and (B), ∗P < .05. (C-D) Fractions of puromycin-low Lin– cells for the indicated genotypes, setting wild-type arbitrarily at 0.2, as in Figure 4F. (E) Representative spectral flow dot plots of cKit+ and SCA-1+ LSK cells and their frequency. (F) Representative SCENITH histograms of LSK cells from the indicated mouse models. Large numbers depict the MFI for the anti-puromycin antibody signal, small numbers the fraction of cells with low or high puromycin signal. Panels E-F are representative examples of at least 3 independent biological experiments. (G-H) Bar graphs representing relative SCENITH signals in LSK cells from RoLoRiG/Mx1CRE (n = 5) and wild-type (n = 3) (G) or KRASG12D/Mx1CRE (n = 3) and wild-type (n = 3) BM (H). Wild-type control was arbitrarily set at 100%, ∗P < .05. (I-J) Bar graphs of fractions of puromycin-high and puromycin-low LSK cells for KRASG12D/Mx1CRE and wild-type, setting the wild-type arbitrarily at 0.2. ∗P < .05. (K) Relative frequency of HSCs and relative SCENITH signals in these HSCs for RoLoRiG/Mx1CRE and wild-type mice. Wild-type control HSC frequency was arbitrarily set at 20% (supplemental Figure 11E-F), with a relative SCENITH signal of 100%. ∗P < .05. (L) As in panel K, but comparing KRASG12D/Mx1CRE (n = 3) HSCs and against 3 other wild-type HSCs. Note that both the fraction of KRASG12D/Mx1CRE HSCs in the LSK compartment and the LSK fraction itself were reduced in KRASG12D/Mx1CRE mice (supplemental Figure 11E-F). ∗∗P < .01. ns, not significant.

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