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. Author manuscript; available in PMC: 2020 May 1.
Published in final edited form as: Nature. 2019 Jan 16;565(7740):511–515. doi: 10.1038/s41586-019-0874-3

Extended Data Figure 8. Inhibition of pyruvate uptake does not affect collagen or bone properties.

Extended Data Figure 8.

(a) Intracellular α-ketoglutarate (αKG) levels in cultured chondrocytes, with or without treatment with an inhibitor of monocarboxylate transporter 2 (MCT2i) (n=3 biologically independent samples). (b) P-AMPKT172 and AMPK immunoblot with quantification of p-AMPKT172 to AMPK ratio in cultured chondrocytes treated with MCT2i. Representative images of 3 independent experiments are shown. (c) Tibia length of mice treated with MCT2i (n=5 mice). (d) Collagen synthesis in cultured chondrocytes, with or without MCT2i treatment (n=4 biologically independent samples). (e) BiP, cleaved (c)ATF6 and β-actin immunoblot in cultured chondrocytes treated with MCT2i. Representative images of 3 independent experiments are shown. (f) Hydroxyproline (OH-Pro) content in neonatal growth plates of mice treated with MCT2i (n=5 biologically independent samples). (g) Safranin O staining of the tibia of mice treated with MCT2i, and quantification of the percentage Safranin O (SafO) positive matrix relative to bone volume (BV) (n=5 mice). (h) Type II collagen (COL2) immunostaining of the tibia of mice treated with MCT2i, with quantification of the percentage COL2-positive matrix (green) relative to bone volume (n=5 mice). GP is growth plate, PS is primary spongiosa, arrowheads indicate COL2 cartilage remnants. (i) 3D microCT models of the tibial metaphysis of mice treated with MCT2i, and quantification of trabecular bone volume (TBV) (n=5 mice). Data are means ± SEM in (a-b, d-e), or means ± SD in (c, f-i). #p<0.05 (ANOVA), §p<0.05 vs. Phd2chon+-veh (ANOVA). Exact p values: Phd2chon+-veh vs. Phd2chon--veh 0.00002 (a), 0.015 (b), 0.00001 (c), 0.00011 (d), 0.012 (BiP; e), 0.008 (cATF6; e), 0.00012 (f), 0.00013 (g), 0.00001 (h) or 0.00001 (i); Phd2chon+-MCT2i vs. Phd2chon--MCT2i 0.0003 (a), 0.000002 (c), 0.005 (d) or 0.0003 (f); Phd2chon+-veh vs. Phd2chon--MCT2i 0.02 (b), 0.004 (BiP; e), 0.002 (cATF6; e), 0.00007 (g), 0.0001 (h) or 0.00002 (i). Scale bars in (g) and (h) are 250 μm.