Journal: bioRxiv

Article Title: Reduced glycolysis links resting zone chondrocyte proliferation in the growth plate

doi: 10.1101/2023.01.18.524550

Figure Lengend Snippet: (A) Reduced cytosolic citrate and acetyl-CoA (Ac-CoA) levels in Mir140UGCG mice. Ac-CoA and citrate levels were determined in primary rib chondrocytes from 10-day-old mice. (B) Immunofluorescence of indicated acetylated histones in the spheno-occipital growth plate. Dotted boxes indicate the resting zone. Insets are magnified views of the resting zone. MIr1430UGCG mice show reduced histone acetylation. (C) Immunoblot analysis for H3K27 acetylation. (D) Immunofluorescence assessment of H3K27 acetylation in the tibial growth plate of control and Ldha cKO mice. Insets are magnified views of dotted boxes. (E) Quantification of H3K27 acetylation (H3K27Ac) in D . H3K27Ac-positive cells were counted in the resting zone and proliferating zone and normalized to the total number of cells per area. Ldha cKO show reduced numbers of chondrocytes positive for H3K27Ac. (F) Immunoblot quantification of acetylation of indicated histones in primary rib chondrocytes. Ldha cKO shows reduced histone acetylation.

Article Snippet: Immunofluorescence was performed according to the protocol described in the data sheets of the anti-acetylated histone 3 lysine 27 (H3K27Ac) antibody (# 8173, Cell Signaling Technology) and anti-acetylate histone 3 lysine 9 (H3K9Ac) antibody (#9649, Cell Signaling Technology).

Techniques: Immunofluorescence, Western Blot

Journal: bioRxiv

Article Title: Reduced glycolysis links resting zone chondrocyte proliferation in the growth plate

doi: 10.1101/2023.01.18.524550

Figure Lengend Snippet: (A) Acly cKO mice show shortening and bowing of the tibia ( Top) . Mice are perinatally lethal. H/E-stained sections of the tibia at P 0.5. The bottom panels show magnified views of proliferating chondrocytes in the boxed areas. The flat morphology of the columnar chondrocytes is lost in cKO mice. (B) Expansion of the resting zone chondrocytes in the spheno-occipital growth plate in E18.5 Acly cKO mice. Left , H/E-stained ( Top ) and EdU-stained ( Bottom ) sections. Boxed areas indicate the resting zone. Right , The number of resting chondrocytes is increased in the Acly cKO growth plate. (C) Reduced histone 3 acetylation in primary rib chondrocytes from E18.5 Acly cKO mouse embryos. Whole-cell lysates from an overnight culture of primary rib chondrocytes were subjected to immunoblot analysis. Acly cKO chondrocytes show reduced acetylation of H3K27 and H3K9. (D). Differential phosphorylation of AMPK between Acly cKO and Ldh compound cKO chondrocytes. The level of p-AMPK is unchanged in Acly cKO, whereas it is upregulated in Ldh compound cKO cells, suggesting ATP deficiency in Ldh compound cKO, but not in Acly cKO chondrocytes.

Article Snippet: Immunofluorescence was performed according to the protocol described in the data sheets of the anti-acetylated histone 3 lysine 27 (H3K27Ac) antibody (# 8173, Cell Signaling Technology) and anti-acetylate histone 3 lysine 9 (H3K9Ac) antibody (#9649, Cell Signaling Technology).

Techniques: Staining, Western Blot