Figure 4S100a8 is significantly up-regulated in Rps14 haploinsufficient bone marrows, is regulated by p53 induction, and is necessary for the erythroid differentiation defect
(a) Immunohistochemical staining of S100a8 in bone marrows from Mx1Cre+ and Rps14−/+Mx1Cre+ mice 8 weeks after the induction of the Rps14 excision with poly(I:C); representative pictures are shown (n=5). Scale bar 100µm. (b) Quantification of S100a8 in lineage-negative bone marrow cells by quantitative real-time PCR (mean±SD, n=5; *p<0.05). Data are normalized to expression in Mx1Cre+ control cells 8 weeks post poly(I:C). Western blot on protein lysates from Mx1Cre+ and Rps14−/+Mx1Cre+ lineage-negative bone marrow cells. Data are representative of 3 independent experiments. (c) Mean fluorescence intensity (MFI) in CD11b−Gr1− erythroid progenitor populations characterized by CD71 and Ter119 expression (RI-RIV). (mean±SD, n=5; *p<0.05). Mean fluorescence intensity (MFI) of S100a8 expression in Gr1+CD11+ granulocytes, Gr1−CD11b+ monocytes and F4/80+ macrophages (mean±SD, n=5; *p<0.05). (d) Co-immunofluorescent staining of F4/80 (green), p53 (dark blue) and S100a8 (red) on cytospins of Rps14−/+Mx1Cre+ and Mx1Cre+ bone marrow cells 12 weeks after the induction of the Rps14 excision with poly(I:C). Inserts highlight areas of magnification shown in the lower panel. Scale bar: 20µm. Data are representative of 3 independent experiments. (e) Percentage of p53 and S100a8 co-expressing cells in RI-III erythroid progenitor cells from Rps14−/+Mx1Cre+ and Mx1Cre+ mice 12 weeks after the induction of the Rps14 excision with poly(I:C), (mean±SD, n=5; *p<0.05). (f) Representative histograms showing S100a8 expression in the RIII erythroid progenitor cell population and mean fluorescence intensity (MFI) of S100a8 expression in F4/80+ macrophages and RIII erythroblasts in Mx1Cre+, p53−/−, Rps14−/+Mx1Cre+ and Rps14−/+p53−/−Mx1Cre+ mice 6 days after induction of hemolysis with Phenylhydrazine (mean±SD, n=5; *p<0.05; *p*<0.001). (g) Fluorescence intensity normalized to background signals of inflammatory cytokines in bone marrows from Mx1Cre+ and Rps14−/+Mx1Cre+ mice 12 weeks after the induction of the Rps14 excision with poly(I:C). Log10 scale. (mean±SD, n=4; **p<0.001). (h) Ckit+ HSPCs were transduced with lentiviral shRNAs (n=5 each) targeting Tnfα, S100a8 and Tlr4 and a luc control. Transduced cells were selected with puromycin and induced to undergo erythroid differentiation for 5 days in vitro. The frequency of RIV erythroid progenitor populations in the culture is shown (mean±SD, **p<0.001). Circles represent the median of three replicates for each individual shRNA (n=5). The mean of all shRNAs targeting a given gene is shown with a grey bar or red bar. (i) Representative histogram presentation and mean fluorescence intensity (MFI) of p53 expression in the RIII population of HSPCs transduced with shRNA targeting Tnfα, S100a8 and Tlr4 and luc control after 5 days of erythroid differentiation in vitro. (mean±SD, **p<0.001, n=5). Circles represent the median of three replicates for each individual shRNA (n=5). The mean of all shRNAs targeting a given gene is shown with a grey bar or red bar. (j) Representative histograms in F4/80+ macrophages and in the RIII population and mean fluorescence intensity (MFI) of S100a8 expression in macrophages of HSPCs transduced with shRNA targeting Tnfα, S100a8 and Tlr4 and luc control after 5 days of erythroid differentiation in vitro. (mean±SD, **p<0.05, **p<0.001, n=5). Circles represent the median of three replicates for each individual shRNA (n=5). The mean of all shRNAs targeting a given gene is shown with a grey bar or red bar. Unpaired two-sided t-test (b, c, e; h-j) or multiple group comparison by analysis of variance with posthoc Tukey correction (f) were applied for statistical analysis.