Figure 4.

Metabolic changes increase TET activity and preserve SSPCs

(A and B) Ratio of α-ketoglutarate (αKG) to succinate (suc) and of αKG to 2-hydroxyglutarate (2HG) in cells treated with vehicle (veh) or 10 μM AMA for 3 days (A, n = 15) or 3 weeks (B, n = 7).

(C) Immunoblot of HIF-1α with Lamin A/C as loading control in cells treated with veh or AMA for 3 days (n = 3). IOX2, an inhibitor of HIF prolyl hydroxylase 2, was used as positive control. Representative image is shown.

(D) Immunoblot of dimethylated lysine 9 of histone H3 (H3K9me2), trimethylated lysine 27 of histone H3 (H3K27me3), and dimethylated lysine 79 of histone H3 (H3K79me2), with total histone 3 (H3) and Ponceau red as loading control (n = 3). Representative image is shown.

(E) Dot blot assay of 5-hydroxymethylcytosine (5hmC) in cells treated with veh or AMA for 3 days, with methylene blue staining as loading control. Representative images of four experiments are shown.

(F) Percentage of 5hmC of total cytosine (n = 4).

(G) Dot blot assay of 5hmC in cells treated with veh or AMA in the presence or absence of 0.5 mM ascorbic acid (Asc A) with methylene blue staining as loading control. Representative images of three experiments are shown.

(H–J) Number of colonies formed (H, n = 8), and percentage of SSCs (I, n = 5) and pre-BCSPs (J, n = 5) in cells treated with veh or AMA in the presence or absence of Asc A.

(K and L) αKG, succinate, and 2HG levels (K, n = 4) and ratio of αKG to succinate and of αKG to 2HG (L, n = 4) in cells treated with veh or 500 nM atpenin A5 (Atp).

(M) Dot blot assay of 5hmC in veh- or Atp-treated cells with methylene blue staining as loading control. Representative images of four experiments are shown.

(N–P) Number of colonies formed (N, n = 6), and percentage of SSCs (O, n = 5) and pre-BCSPs (P, n = 5) in cells treated with veh (control) or Atp.

(Q) Tet1, Tet2, and Tet3 mRNA levels in periosteal cells (n = 9).

(R) Tet2 mRNA levels in control (Tet2^CO) or Tet2 knockdown (Tet2^KD) periosteal cells (n = 3).

(S) Dot blot assay of 5hmC in Tet2^CO and Tet2^KD cells with methylene blue staining as loading control. Representative images of three experiments are shown.

(T–V) Number of colonies formed (T, n = 6–7), and percentage of SSCs (U, n = 5–6) and pre-BCSPs (V, n = 5–6) in Tet2^CO and Tet2^KD cells.

Data are means ± SD. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001 versus veh (two-tailed Student’s t test). ^##p < 0.01 (one-way ANOVA), ^$p < 0.05 (two-way ANOVA).