Fig. 2: PCH-associated mutations of MINPP1 are deleterious for protein function. | Nature Communications

Fig. 2: PCH-associated mutations of MINPP1 are deleterious for protein function.

From: MINPP1 prevents intracellular accumulation of the chelator inositol hexakisphosphate and is mutated in Pontocerebellar Hypoplasia

Fig. 2

a Schematic representation of inositol phosphate cycle. myo-inositol (Inositol); phosphatidylinositol (PI); phosphatidylinositol phosphate (PIP); phosphatidylinositol 4,5-bisphosphate (PIP2); diacylglycerol (DAG); phospholipase C (PLC); inositol phosphate (IP); inositol bisphosphate (IP2); inositol 1,4,5-trisphosphate (IP3); inositol 1,3,4,5-tetrakisphosphate (IP4); inositol pentakisphosphate (IP5); inositol hexakisphosphate (IP6); inositol-polyphosphate multikinase (IPMK); I(1,4,5)P3 3-Kinase (IP3-3K); inositol-pentakisphosphate 2-kinase (IPPK); multiple inositol-polyphosphate phosphatase 1 (MINPP1). b, c Western blot analysis of MINPP1 level in patient fibroblasts and HEK293 cells with β-Actin shown as loading control. Patient fibroblasts CerID-30-1 and CerID-30-2 (b) and MINPP1−/− HEK293 clones (c) show absent MINPP1. The uncropped blots are provided as a source data file and are representative of two independent experiments. d Assessment of cell proliferation by MTT assay. For each clone, MTT absorbance was measured 3, 24, and 48 h post-seeding. Values represent the mean ± s.d. of triplicate determinations from four replicates (n = 4, two-tailed student’s t-test, **p ≤ 0.01 and ***p ≤ 0.001. At 24 h, Ctrl vs. MINPP1−/−: p = 0.0018; at 48 h Ctrl vs. MINPP1−/−: p = 0.0009). e MINPP1−/− HEK293 cells were transiently transfected with plasmids encoding empty vector, wild type, Y53D or E486K variant MINPP1. To assess the cell proliferation rate, MTT assay was performed 48 h post-nucleofection. The data are presented as mean percentage relative to control (Ctrl) ± s.d., with triplicate determinations from four replicates. The normalization was done with 3 h MTT assay data (n = 4, one-way ANOVA, Tukey’s post hoc test, **p ≤ 0.01 and ****p ≤ 0.0001. Ctrl vs. MINPP1−/−: p < 0.0001; MINPP1−/− vs. MINPP1−/− + MINPP1: p = 0.0029; MINPP1−/− + MINPP1 vs. MINPP1−/− + E486K: p = 0.0014; MINPP1−/− + MINPP1 vs. MINPP1−/− + Y53D: p = 0.0080). For (d) and (e), source data are provided as a source data file.

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