Fig 2: Cartilaginous Idh1-KI; Gys1 deletion rescues some aspects of the Idh1-KI phenotype and is sufficient to reduce enchondroma-like cartilage lesions in the Idh1-KI growth plate(A) Alizarin red and Alcian blue whole-mount skeleton of Idh1LSL/wt (n = 3), Idh1LSL/wt; Gys1fl/fl (n = 5), and littermate controls (n = 20) at E18.5.(B) Percent mineralization of humerus and femur bones of Idh1LSL/wt (n = 3), Idh1LSL/wt; Gys1fl/fl (n = 5), and littermate control (n = 20) mice. Two-way ANOVA confirms significant statistical difference in percent mineralization in humerus and femur between genotypes (F(6,175) = 21.76, p < 0.0001). Tukey’s multiple comparisons test = p < 0.05, an asterisk (*) indicates that significant p values are shown.(C) Collagen 10 (COLX) staining of Idh1LSL/wt (n = 3), Idh1LSL/wt; Gys1fl/fl (n = 5), and littermate control (n = 6) growth plates.(D) COLX stained length measurements of Idh1LSL/wt, Idh1LSL/wt; Gys1fl/fl, and littermate control growth plates. One-way ANOVA confirms significant statistical difference in COLX height between three genotypes (F(2,12) = 6.175, p < 0.0143). Tukey’s multiple comparisons test = p < 0.05, an asterisk (*) indicates that significant p values are shown.(E) Alcian blue staining of Idh1LSL/wt (n = 3), Idh1LSL/wt; Gys1fl/fl (n = 5), and littermate control (n = 5) tibia growth plates at E18.5.(F) PAS staining of Idh1LSL/wt (n = 3), Idh1LSL/wt; Gys1fl/fl (n = 5), and littermate control (n = 5) growth plates.(G) Representative Safranin O staining of 5-month-old postnatal femur murine growth plates of specified genotypes. Enchondroma-like lesions are highlighted in black boxes. Representative Col2a1Cre-ERT2; Idh1LSL/wt (n = 7) femur growth plate displays 5 lesions, whereas representative Col2a1Cre-ERT2; Idh1LSL/wt; Gys1fl/fl (n = 7) growth plate displays 2 lesions.(H) Quantification of lesions in femur growth plates show that Col2a1Cre-ERT2; Idh1LSL/wt; Gys1fl/fl (n = 7) animals display less lesions compared with Col2a1Cre-ERT2; Idh1LSL/wt (n = 7) animals. Each data point represents 1 animal.(I) Total lesion burden is reduced in Col2a1Cre-ERT2; Idh1LSL/wt; Gys1fl/fl animals. Each data point represents 1 animal (n = 7).(J and K) Distribution of (J) volumes of enchondroma-like lesions and (K) widths of lesions. p value = Student’s t test p < 0.05, an asterisk (*) indicates that significant p values are shown. Means and error bars representing standard deviations are shown. Scale bars: 5,000 µm in blue, 100 µm in black, and 100 µm in white.

Fig 3: Immunoreactivity for IDH1 R132H in IDH1-muted gliomas including anaplastic astrocytoma (AST-19; A,B), oligodendroglioma (AST-18; C,D), and secondary glioblastoma (GLB-9; E,F) as stained separately with MRQ-67 (A,C,E) and H09 (B,D,F) on serial FFPE tissue sections. All of the tumor cells were labelled in a cytoplasmic and nuclear pattern, but blood vessels (arrow) were not. UltraView, visualized with DAB, and counterstained with hematoxylin. Scale bar = 100 µm.

Fig 4: Binding of the antibodies MRQ-67 (A,C) and H09 (B,D) with endogenous IDH1 R132H mutant, but not with the IDH1 wt protein, as demonstrated on cell pellet samples of cell lines BT142 mut/- (A,B) and HepG2 (C,D) by IHC. UltraView, visualized with DAB, and counterstained with hematoxylin. Scale bar = 100 µm.

Fig 5: Liquid-phase binding of the antibodies MRQ-67 (A,B) and H09 (C,D) to wild-type (wt) and mutant IDH1 (A,C) and IDH2 peptides (B,D), as measured by ELISA and expressed as optical densities at 450 nm (OD450).