Fig 2: Deregulation of the Hif1a pathway and the cartilage markers in miR-140 mutant chondrocytes.a, (top) Sequence alignments between miR-140-5p-G and its putative binding sites in the coding sequence of mouse and human Hif1a. (bottom) Representative images from two independent experiments of Western blot analysis of Hif1a protein expression in primary rib chondrocytes at the normoxic condition. Hif1a protein was detected by Western blot analysis using anti-Hif1a antibody (Novus Biologicals NB100-449). Bar graphs represent mean (n = 2 animals).b, Validation of the Hif1a target site. HEK293T cells were transfected with wild-type or mutant mouse Hif1a expression plasmids and pri-miRNA expression plasmids, and subjected to Western blot analysis. Representative images from two independent experiments are shown. A mutation of the target site is shown in panel (a).c, Relative expression of Hif1a and its target genes was determined by qRT-PCR in primary rib chondrocytes isolated from P7 mice. Results represent mean ± s.e.m. (n = 3 animals). Statistical significance was assessed using one-way ANOVA and post hoc Tukey-Kramer test. P values indicate comparison versus WT.d, Expression of lysyl oxidases in P7 primary rib chondrocytes assessed by RNA-seq. Results represent mean ± s.e.m. (n = 3 animals). Statistical significance was assessed using one-way ANOVA and post hoc Tukey-Kramer test. P values indicate comparison versus WT.e, Reduced collagen crosslinking in miR-140G/G rib cartilage cells. Rib cartilage of 4 week-old mice was subjected to collagen crosslink analysis by mass spectrometry. Hydroxylysyl pyridinoline and lysyl pyridinoline were quantified. Results represent mean ± s.e.m. (n = 4 animals). Statistical significance was assessed using one-way ANOVA and post hoc Tukey-Kramer test. P values indicate comparison versus WT.f, RNA expression of cartilage markers. The x axis and y axis indicate miR-140 genotypes and normalized expression (reads per kilo base per million mapped reads, RPKM), respectively. Each dot represents the expression level in an individual sample (n = 3 animals), and the thick bars indicate the mean value. Statistical significance was assessed using one-way ANOVA and post hoc Tukey-Kramer test. Connector lines at the top of each panel indicate significant changes in expression (P < 0.05).

Fig 3: hMRP8 Hif-1a KO mice are protected from DSS-induced colitis. (A-D) Body weight change (A), a photograph of the rectal area (B), disease activity score (C) and colon length (D) of hMRP8 WT or Hif-1a KO mice fed with water for 4 days or 5% DSS for 5 days. Data in A, C and D are mean±s.e.m. for n=5 mice per group. **P<0.01 and ***P<0.001, determined by two-way (C) or one-way (D) ANOVA. (E,F) H&E staining of the colon is also shown for mice fed with water (E) or 5% DSS (F). Black arrows in F indicate the thickened muscularis mucosa observed in WT mice; red arrowhead indicates inflammatory cells. Scale bars: 100 µm.

Fig 4: Signalling regulation by PRC2 in chondrocytes.PRC2 is necessary for normal skeletal growth. Derepression of PRC2 target genes causes aberrant overactivation of multiple signalling systems and reduces Hif1a expression. Overactivation of Wnt and TGF-ß signalling pathways is primarily responsible for the skeletal abnormalities of mice with Eed-deficient chondrocytes.

Fig 5: Stabilization of HIF proteins in myeloid cells does not influence EIU at peak disease.Flow cytometric analyses of (A) absolute cell numbers and (B) proportions of various myeloid subsets infiltrated in the eye 18 hours after EIU induction in animals with myeloid cells deficient in Vhl (stabilization of Hif1a and Epas1), Hif1a & Vhl (stabilization of Epas1) and Epas1 & Vhl (stabilization of Hif1a) with their floxed littermate controls. Myeloid cell populations are defined using standard gating strategy. N? = neutrophils; Mo/M?-monocyte/macrophages. Graphs show mean ± SD; n = 10–12 injected eyes per group; >3 independent experiments.