Fig 5. FAM173B and mitochondrial function.

MitoTrackerRedCMXROS fluorescence, as a measure of mitochondrial potential, 48 hours after (A) mFam173b knockdown (MM-ODN n = 14; mFAM173b-AS n = 16 wells) or (B) hFAM173B overexpression (n = 7 wells) in N2A cells. (C) ΔTMRM fluorescence in HSV-mediated hFAM173B expression in cultured primary sensory neurons increased (n = 100–145 cells, 7 cultures). (D) HSV-mediated hFAM173B expression in cultured primary sensory neurons increased ROS production (DHE) after vehicle or 6 hours stimulation with 100 ng/ml TNFα (n = 90–130 cells, 9 cultures). (E–F) In vivo expression of hFAM173B in sensory neurons with HSV-hFAM173B prior to intraplantar carrageenan increased (E) DHE fluorescence intensity at day 5 (n = 7 mice) and (F) MitoTrackerRedCMH₂-XROS fluorescence intensity at day 3 (n = 9 mice) and day 6 (EV n = 4, hFAM173B n = 6 mice) in small-diameter neurons. (G) Intraperitoneal injection of the ROS scavenger PBN attenuated the hFAM173B-mediated prolongation of carrageenan-induced mechanical hypersensitivity (n = 5 mice; HSV-FAM173B + PBN n = 6 mice). Data are represented as mean ± SEM. * = P < 0.05; ** = P < 0.01. Statistical analyses were performed by unpaired two-tailed t tests (A-C, E/F), by one-way (D) or two-way (G) repeated measures ANOVA followed by a post-hoc Holm-Sidak multiple comparison test. For exemplar pictures of A, B, and F, see S3 Fig. Blue bars indicate mFam173b knockdown and red bars/lines hFAM173B overexpression. Underlying data can be found in S1 Data. DHE, dihydroethidium; EV, empty vector; HSV, herpes simplex virus; MM-ODN, mismatch ODN; N2A, Neuro2a; ODN, oligodeoxynucleotide; PBN, phenyl-N-t-butylnitrone; ROS, reactive oxygen species; SEM, standard error of the mean; TNFα, tumor necrosis factor alpha.