Figure 3. HMGB1 restores mitochondrial functions.

1. The accumulation of MitoTracker Deep Red, a far red-fluorescent dye indicator of the mitochondrial membrane potential, in the mitochondria of mutant Atxn1(86Q)-DsRed-transfected HeLa cells was reduced by co-expression of HMGB1-GFP.
2. A knock-down of HMGB1 by two types of HMGB1-siRNA (HMGB1_A, HMGB1_B) decreased the number of mitochondria with the normal membrane potential (stained red with JC-1) and increased the number of mitochondria with an abnormal membrane potential (stained green with JC-1). NC: negative control siRNA. Right panels show HMGB1 signals in the transfected cells.
3. Expression levels of HMGB1 in the transfected cells used in (B) were confirmed by Western blot analysis.
4. Mitochondrial enzyme histochemical analysis revealed reduction of succinate dehydrogenase (SDH) and cytochrome oxidase (COX) activity in Purkinje cells of Atxn1-KI mice. The reduced activity of these enzymes was restored in Atxn1-KI;HMGB1 mice.
5. FACS analysis with the well-characterized potentiometric fluorescent dye tetramethylrhodamine methyl ester (TMRM) to quantify the changes of the mitochondrial membrane potential and mitochondrial permeability transition induced by HMGB1. The upper left panel shows experimental procedure, and the upper right panel shows parameters in the following graphs. Non-specific negative control siRNA did not affect TMRM signals, whereas siRNA-A and siRNA-B against HMGB1 substantially reduced a part of transfected HeLa cells. The siRNAs that were used for this analysis were similar to those in Supplementary Fig S8, where suppression of HMGB1 by these siRNAs was confirmed. The results from three sets of independent transfection experiments indicated that the mitochondrial membrane potential and mitochondrial permeability transition were changed by the deficiency in HMGB1.

Source data are available online for this figure.