Fig. 3.

The sulfur oxidation pathway is required for persulfide-mediated mitochondrial activation in Hepa1c1c7 cells.

(A and C) MMP was analyzed by using JC-10 fluorescence imaging for Hepa1c1c7 cells with Cars2, Sqor, Ethe1 and Suox knockdown (A) and GSSSG treatment and/or Sqor knockdown (C). Representative images from 5 to 7 images of randomly selected areas per experiment from three independent experiments (left) and relative red and green fluorescence ratios (right) are shown. Scale bars correspond to 50 μm. (B and D) OCR analysis. A representative OCR respirogram from three independent experiments is shown (left). Respiration parameters were calculated from the results of three independent experiments (right). (E) ADP/ATP ratio measurement. Relative ratios are shown.

At 24 h after siRNA transfection, the culture medium was replaced with 200 μM cystine-containing fresh medium (A and B) and 200 μM cystine-containing fresh medium with vehicle (water) or 10 μM GSSSG (C and D). Analyses were conducted 24 h after the medium change. The data are presented as the mean ± S.D. of three independent experiments. *α < 0.05 and **α < 0.01 compared with the control cells (A and B) and with GSSSG-treated control cells (D and E), determined using confidence interval estimation. An unpaired Student's t-test was conducted for comparison of GSSSG-treated cells with and without Sqor knockdown (C). *p < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)