Fig 2: Expression of IL-1 pathway genes are upregulated in MPN.a Expression of IL-1R pathway gene signatures is tested for enrichment by Gene Set Enrichment Analysis (GSEA) in peripheral blood CD34+ HSPCs from PMF patients (n = 23) and bone marrow CD34+ HSPCs from normal controls (n = 15). Comparisons with p-value <0.05 and FDR q-value <0.05 were considered significant. Analysis of publicly available dataset19. Affymetrix data were dowloaded as normalized expression levels from Gene Expression Omnibus database (GSE53482)19 using the GEOquery package (R, Vienna, Austria. https://www.R-project.org/). The normalization of the expression data was checked by boxplot representation. Gene Set Enrichment Analysis (GSEA) was performed with the GSEAv4.1.0 software (Broad Institute). All gene sets were obtained from GSEA website (https://www.gsea-msigdb.org). Enrichment map was used for visualization of the GSEA results. Normalized Enrichment score (NES) and False discovery rate (FDR) p-values were applied after a 10,000 gene set permutations. b Heatmap representation of expression levels of IL-1R pathway genes in CD34+ HSPCs from PMF patients (n = 23) and normal controls (n = 15). Analysis of publicly available dataset19. c Heatmap representation of the differential expression of IL-1 signaling pathway genes in hematopoietic stem cells (HSC; Lin-Sca1+cKit+CD48-CD150+), megakaryocyte-erythroid precursors (MEP or pre-MegE; Lin-Sca1-cKit+CD41-CD16-105-CD150+) and megakaryocyte progenitors (MkP; Lin-Sca1-cKit+CD41+CD150+) between VF (n = 3) and WT (n = 3) mice is shown (left). Barcode plots showing custom Gene Set Enrichment Analysis (GSEA) of the Biocarta IL-1R pathway in HSC, pre-MegE (or MEP) and MkP from VF vs WT (right). Source data are provided as a Source Data file.

Fig 3: Heterozygous loss of Rps12 in the hematopoietic cells impairs translation in the hematopoietic stem and progenitor cells and leads to pancytopenia.(A) Schematic representation of the time-course analysis testing the effect of the heterozygous loss of Rps12 activity in hematopoietic stem cells (HSCs). (B–D) Analysis of Rps12flox/+; Tal1-Cre-ERT (Rps12flox/+) and Rps12flox/+(CNTRL) at 12 weeks post tamoxifen treatment (TAM): (B) Peripheral blood cell analysis, (C) quantification of bone marrow cellularity and absolute cell count of bone marrow populations per femur, (D) representative flow histograms, and quantification of the median OPP intensity in bone marrow populations. (E) Peripheral blood cell analysis at 14 weeks and (F) quantification of bone marrow cellularity and absolute cell count of bone marrow populations per femur. (G) Schematic summary of the phenotypic analysis of mouse models. Immunophenotypic populations were defined as follows: MPROG: Lin-cKit+Sca1-, LSK: Lin-cKit+Sca1+; MPP3,2: Lin-cKit+Sca1+Flk2-CD48+, HSCs: Lin-cKit+Sca1+Flk2-CD48-. Statistical analysis: quantifications represent mean +/- SEM, shown as the error bars. Significance was determined using the Welch’s t-test *p=0.05, **p=0.01. At each time point n (Rps12flox/+)=5 and n (CNTRL)=5. Schematics created with BioRender. Figure 8—source data 1.Conditional KO hematopoietic populations.

Fig 4: Hematopoietic stem cells (HSCs) and other hematopoietic progenitors have altered cycling, global translation levels, and apoptosis in Rps12KO/+ bone marrow.(A) Representative flow cytometry gating of HSCs (Flk2-CD48-LSK) cell cycle stages (G0, G1, S/G2/M) distribution determined by DNA (Hoechst) and Ki67 levels. (B) Cell cycle stages distribution in HSCs and in indicated progenitor populations. Asterisks correspond to p values assessing significant differences in each cell cycle stage between Rps12KO/+ and Rps12+/+ mice (6- to 8-week-old littermates, +/+ n=4, and KO/+ n=3). (C) Representative flow cytometry histogram showing OPP intensity in RpS12KO/+ (green) and Rps12+/+ (gray) HSCs. (D, E) Median o-propargyl puromycin (OPP) intensity of the indicated bone marrow populations (6- to 8-week-old littermates, +/+ n=4, and KO/+ n=3). This analysis was repeated in 6- to 7-month-old mice with similar results. (F) Representative images of bone marrow cytospins showing the high number of apoptotic cells (arrows) in Rps12KO/+ samples. (G) Representative flow cytometry gating of LIN- population showing apoptotic populations as determined by AnexinV and PI staining. (H, I) Percentage of apoptotic (AnnexinV+) cells in LSK (Lin-cKit+Sca1+) and Myeloid progenitor (MPROG; Lin-cKit+Sca1-) populations (6- to 8-weeks-old littermates, +/+ n=4, and KO/+ n=3). Statistical analysis: quantifications represent mean +/- SEM, shown as the error bars, two-way ANOVA (B–F), and unpaired t-tests were performed to establish significance among populations between genotypes *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figure 6—source data 1.Hematopoietic population cycling, apoptosis, and translation.