SCENITH ribosome activity in BM compartments with Ras pathway lesions. (A) SCENITH analysis of protein synthesis levels at the ribosome. Incorporated puro is quantitatively measured. (B) Comparisons of SCENITH in Ki67– and Ki67+ fractions in total wild-type BM. Numbers in the histograms indicate the mean fluorescent intensity (MFI) for the anti-puro antibody signal. (C) Representative histograms of SCENITH analysis for KRASG12D/Mx1CRE and RoLoRiG/Mx1CRE total BM, compared with wild-type. Numbers in the histograms indicate MFI for the anti-puro antibody signal. Representative example of at least 3 independent biological experiments. (D) Percentage relative SCENITH signal for RoLoRiG/Mx1CRE (n = 5), compared with wild-type (n = 3). ∗P < .05. In each individual experiment, comparing wild-type with aberrant Ras signals, wild-type control was arbitrarily set at 100%, allowing for comparisons across separate experiments. (E) For KRASG12D/Mx1CRE (n = 3) with 3 different wild-type controls, in which we used a Alexa488-coupled anti-puro antibody. (F) Bar graphs of fractions of puro-low and puro-high total BM cells for RoLoRiG/Mx1CRE and wild-type, setting the wild-type arbitrarily at 0.2 following the gating depicted in supplemental Figure 11A. ∗P < .05. (G) As in panel F, but for KRASG12D/Mx1CRE and wild-type total BM. Panels B-C are representative examples of at least 3 independent biological experiments. 2DG, 2-deoxy-D-glucose, glycolysis inhibitor; FACS, fluorescence-activated cell sorter; mRNA, messenger RNA; ns, not significant; OG, oligomycin, inhibiting adenosine triphosphate synthase; puro, puromycin.
Figure 4.

SCENITH ribosome activity in BM compartments with Ras pathway lesions. (A) SCENITH analysis of protein synthesis levels at the ribosome. Incorporated puro is quantitatively measured. (B) Comparisons of SCENITH in Ki67– and Ki67+ fractions in total wild-type BM. Numbers in the histograms indicate the mean fluorescent intensity (MFI) for the anti-puro antibody signal. (C) Representative histograms of SCENITH analysis for KRASG12D/Mx1CRE and RoLoRiG/Mx1CRE total BM, compared with wild-type. Numbers in the histograms indicate MFI for the anti-puro antibody signal. Representative example of at least 3 independent biological experiments. (D) Percentage relative SCENITH signal for RoLoRiG/Mx1CRE (n = 5), compared with wild-type (n = 3). ∗P < .05. In each individual experiment, comparing wild-type with aberrant Ras signals, wild-type control was arbitrarily set at 100%, allowing for comparisons across separate experiments. (E) For KRASG12D/Mx1CRE (n = 3) with 3 different wild-type controls, in which we used a Alexa488-coupled anti-puro antibody. (F) Bar graphs of fractions of puro-low and puro-high total BM cells for RoLoRiG/Mx1CRE and wild-type, setting the wild-type arbitrarily at 0.2 following the gating depicted in supplemental Figure 11A. ∗P < .05. (G) As in panel F, but for KRASG12D/Mx1CRE and wild-type total BM. Panels B-C are representative examples of at least 3 independent biological experiments. 2DG, 2-deoxy-D-glucose, glycolysis inhibitor; FACS, fluorescence-activated cell sorter; mRNA, messenger RNA; ns, not significant; OG, oligomycin, inhibiting adenosine triphosphate synthase; puro, puromycin.

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