Fig 1: RNA aberrant expression detection and validation.(a) Aberrantly expressed genes (Hochberg corrected P value<0.05 and |Z-score|>3) for each patient fibroblasts. (b) Gene-wise RNA expression volcano plot of nominal P values (-log10 P value) against Z-scores of the patient #35791 compared against all other fibroblasts. Z-scores with absolute value >5 are plotted at ±5, respectively. (c) Same as (b) for patient #73804. (d) Sample-wise RNA expression is ranked for the genes TIMMDC1 (top) and MGST1 (bottom). Samples with aberrant expression for the corresponding gene are highlighted in red (#35791, #66744, and #73804). (e) Gene-wise comparison of RNA and protein fold changes of patient #35791 compared to the average across the fibroblast cell lines of all other patients. Subunits of the mitochondrial respiratory chain complex I are highlighted (red squares). Reliably detected proteins that were not detected in this sample are shown separately with their corresponding RNA fold changes (points below solid horizontal line). (f) Western blot of TIMMDC1, NDUFA13, NDUFB3 and NDUFB8 protein in three fibroblast cell lines without (#62346, #91324, NHDF) and three with a variant in TIMMDC1 (#35791, #66744 and #96687), and fibroblasts re-expressing TIMMDC1 (‘-T’) (#35791-T, #66744-T and #96687-T). UQCRC2 was used as loading control. CI, complex I subunit; CIII, complex III subunit; MW, molecular weight. (g) Blue native PAGE blot of the control fibroblasts re-expressing TIMMDC1 (NHDF-T), the control fibroblasts (NHDF), patient fibroblasts (#96687) and patient fibroblast re-expressing TIMMDC1 (#96687-T). Immunodecoration for complex I and complex III was performed using NDUFB8 and UQCRC2 antibodies, respectively. CI, complex I subunit; CIII, complex III subunit.
Fig 2: Detecting and quantifying mitochondrial protein expression in Purkinje cells. Quadruple immunofluorescence of cerebellar Purkinje cells against Glutamic acid decarboxylase (GAD-65/67), synaptophysin (SY-38), complex IV subunit 4 (COX4) and complex 1 alpha subcomplex subunit 13 (NDUFA13). GAD-65/67 is used to detect GABAergic cell bodies and inhibitory synapses. Combined with SY- 38 indicates the convergence of inhibitory presynaptic terminals. COX4 is a mitochondrial mass marker, and NDUFA13 protein is used to detect complex I deficiency. Control Purkinje cells demonstrate co-localization of COX4 and NDUFA13 protein, indicating intact complex I expression, while NDUFA13 protein is significantly reduced in patient Purkinje cells (complex I-deficient). Purkinje cells in the three examples shown above (Patients 1, 8 and 11) exhibit variably decreased NDUFA13 protein relative to the amount of COX4 present. Scale bar: 7 µm.
Fig 3: Correlation between NMDAS score for cerebellar ataxia and NDUFA13 protein expression in inhibitory presynaptic terminals. More severe ataxia scores are related to lower z scores for NDUFA13 expression (Spearman's rho = -0.60, P value = 0.0493).
Fig 4: Determination of Grim-19 expression in decidual macrophages of RSA patients and the NP group, and the expression of autophagy related proteins. (A) The expression of Grim-19, Beclin1, LC3B II/I and BNIP3 in decidual macrophages of RSA patients (n = 10) and the NP group (n = 8) was examined through western blot analysis. GAPDH served as a loading control. (B) Quantification of Grim-19, Beclin1, LC3B II/I and BNIP3 expression through relative densities. Data are shown as the mean ± SD. ***p < 0.001, **p < 0.01, *p < 0.05.
Fig 5: Effects of metformin (1 mM) on the expression of AMPK, p-AMPK, and mitochondrial biogenesis genes under high glucose conditions. Cells were divided into four groups: i) Con, normal medium; ii) Man, 33.3 mM Man; iii) HG, 33.3 mM glucose; and iv) HG + Met, 33.3 mM glucose pretreated with 1 mM Met. (A) Protein expression levels of p-AMPK and AMPK were (A) determined by western blotting and (B) the ratio of p-AMPK/AMPK was semi-quantified. mRNA expression levels of (C) NDUFA1, NDUFA2, NDUFA13, Mn-SOD, (D) PGC-1a, NRF1 and NRF2 were detected via reverse transcription-quantitative PCR. *P<0.05 vs. Con; #P<0.05 vs. HG. AMPK, AMP-activated protein kinase; p, phosphorylated; Man, mannitol; HG, high glucose; Met, metformin; NDUF, NADH: Ubiquinone oxidoreductase subunit; Mn-SOD, manganese superoxide dismutase; PGC-1a, peroxisome proliferator-activated receptor-? coactivator-1a; NRF, nuclear respiratory factor; Con, control.
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