Fig 2: Serine 73 phosphorylation of MITF affects MITF-PDH interaction during mast cell stimulation. (A), IP of MITF in IgE-DNP activated RBL cell lysates in the presence and absence of ERK1/2 inhibitor. Cell lysates were pre-treated with U0126 as described in Figure 2. Non-sensitized cells are presented as Control (Q: Quiescent). For IP anti-MITF antibody was used and for Western blot analysis, anti-pS73-MITF, anti-MITF, anti-PDHA antibodies were used. (B), Densitometry of PDHA/MITF levels in RBL cells after IP experiment. A two-tailed Student t-test was performed for all the western blots. Results represent means ± SEMs (n = 4, ** p ≤ 0.01; * p ≤ 0.05; ns p > 0.05).

Fig 3: Expression and phosphorylation of PDHA1 subunit after the action of the PDHC inhibitors administered at 0.1 mmol/kg. (A) Protein levels of PDHA1. (B) Levels of PDHA1 phosphorylation at Ser293 residue. (C) Levels of PDHA1 phosphorylation, normalized to the protein expression in the same samples. Data are presented as % from the indicated parameters in the control (Ctrl) animals. Significance of the treatment factors and differences between experimental groups are estimated and shown in the same way as in Figure 2.

Fig 4: Nigrostriatal region-specific increase of mitochondrial proteins by metforminA. Representative Western blot of mitochondrial marker proteins (SDHA, PDHA, VDAC, and HSP60) in total protein lysates from SH-SY5Y cells treated with different doses (0.1, 0.25, 0.5 mM) of metformin for 48hr. b-actin was used as an internal loading control. B. Quantification of relative mitochondrial protein levels in SH-SY5Y cells according to treatment and normalized to -actin (n = 3 per group). C. Cartoon of experimental plan. Drinking water containing PBS or metformin was supplied for 14 days. D. Mass spectrometric analysis confirmed brain-penetrated metformin (n = 3 per group). E. Representative Western blots of mitochondrial marker proteins in the indicated brain subregions (SN, STR, and CTX) from mice treated with PBS or different doses of metformin for 7 days. b-actin was used as an internal loading control. F. Quantification of relative mitochondrial protein levels in brain subregions from the indicated experimental mouse groups. Band intensities of each protein were normalized to b-actin and compared to the PBS group (n = 3 per group). Quantified data are expressed as the mean ± SEM. Statistical significance was determined by unpaired two-tailed Student's t test or ANOVA test with Tukey post-hoc analysis, *p < 0.05, **p< 0.01 and ***p < 0.001.

Fig 5: Low villous SDHB expression reduces succinate accumulation and correlates with RSA.a Western blot analysis of glycolysis and citrate cycle enzymes in villi from individuals with RSA or normal controls (n = 6 persons/group). Right panel: quantitative western blot results of six samples per group. b mRNA expression of glycolytic and citrate cycle genes in villi from individuals with RSA and normal controls (n = 6 persons/group). Exact P values in b are listed below: HK1 0.800, GPI 0.326, TPI 0.369, PGK1 0.202, ENO1 0.145, PKM2 0.685, 6PGD 0.715, TKT 0.914, PDHA1 0.691, CS 0.841, IDH1 0.546, IDH2 0.801, IDH3A 0.969, OGDH 0.163, SUCLA2 0.816, SDHA 0.00134, SDHB 0.00243, SDHC 0.000233, FH 0.222, and MDH2 0.938. c Relative SDH activities in SDHB-overexpressing JEG3 and HTR8 cells and control cells (n = 5 independent experiments). The SDHB overexpression efficiency is shown in the right panel. d Succinate levels in SDHB-overexpressing JEG3 and HTR8 cells and control cells (n = 5 independent experiments). e Relative SDH activities in SDHB-knockdown JEG3 and HTR8 cells and control cells (n = 5 independent experiments). The SDHB-knockdown efficiency is shown in the right panel. f Succinate levels in SDHB-knockdown JEG3 and HTR8 cells and control cells (n = 5 independent experiments). g The relative SDH activities in villi and decidua tissues from both RSA patients and normal controls (n = 10 persons/group). a–g Data represent the mean ± standard error; ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ns not significant. Two-tailed unpaired Student’s t tests in a–g. Source data are provided as a Source Data file.