Additional file 4: of Inhibition of peripheral macrophages by nicotinic acetylcholine receptor agonists suppresses spinal microglial activation and neuropathic pain in mice with peripheral nerve injury

Figure S4. Suppressive effects of peripheral TC-2559 on upregulation of IRF5 in the SDH after PSL. Mice were subject to PSL or sham surgery. TC-2559 was administered (p.n., 20 nmol) on days 0, 1, 2, and 3, and the lumbar SDH (L4–5) was collected. Expression level of IRF5 protein on day 7 after PSL was analyzed by western blotting. Data are presented as the mean ± SEM; n = 5–6. **P < 0.01 versus sham. #P < 0.05 versus PSL/Veh. (TIFF 898 kb

Additional file 1: of Inhibition of peripheral macrophages by nicotinic acetylcholine receptor agonists suppresses spinal microglial activation and neuropathic pain in mice with peripheral nerve injury

Figure S1. Inhibition of interferon regulatory factor 5 (IRF5) upregulation in macrophages by TC-2559. Mice were subject to PSL or sham surgery. TC-2559 (20 nmol) and DHβE (40 nmol) were perineurally (p.n.) administered on days 0, 1, 2, and 3, and the SCN was collected. (A) Time course of IL-1β mRNA expression in the SCN on days 1 to 14 after sham or PSL was analyzed by RT-qPCR. Data are presented as the mean ± SEM; n = 6–7. ***P < 0.001 versus Sham. (B) Representative micrographs of F4/80, IRF5, and merged images in the injured SCN on day 7 after PSL analyzed by immunohistochemistry are presented. Scale bars = 20 μm. (TIFF 7197 kb

Additional file 3: of Inhibition of peripheral macrophages by nicotinic acetylcholine receptor agonists suppresses spinal microglial activation and neuropathic pain in mice with peripheral nerve injury

Figure S3. Upregulation of microglial molecules in the SDH after PSL. Mice were subject to PSL or sham surgery, and the L3–4 and L4–5 segments of lumbar SDH were separately collected. Expression levels of Iba1 and CD11b mRNA in each segment on day 7 after PSL were analyzed by RT-qPCR. Data are presented as the mean ± SEM; n = 5–6. **P < 0.01, *P < 0.05 versus sham. (TIFF 2362 kb

Additional file 2: of Inhibition of peripheral macrophages by nicotinic acetylcholine receptor agonists suppresses spinal microglial activation and neuropathic pain in mice with peripheral nerve injury

Figure S2. Improvement of mechanical allodynia after PSL by peripheral bupivacaine. Mice were subject to PSL or sham surgery, and bupivacaine (Bupi; 0.5% w/v) was p.n. administered once a day for 6 days (days 0–5). The 50% paw withdrawal mechanical threshold was assessed by the up-down method using the von Frey test. Improving effects of bupivacaine on PSL-induced mechanical allodynia on day 7 in the ipsilateral side are shown. Data are presented as the mean ± SEM; n = 6–7. ***P < 0.001 versus sham. #P < 0.05 versus PSL/Veh. (TIFF 1190 kb

The effect of TRAP1 on the expression levels of mitochondrial fusion proteins.

<p>A, Cells were fractionated into cytoplasm (cyto) and mitochondrial (mito) fractions, which were analyzed by western blotting using antibodies against mitofusin (Mfn), OPA1, TRAP1, HADHA (mitochondrial marker) and β-tubulin (cytoplasmic marker). B, Quantification of the expression levels of Mfn1/2 and OPA1. These data were obtained by densitometric analysis of each band on the western blots, and the data are expressed as the means ± s.e.m. of at least four independent experiments. β-tubulin (cyto) and HADHA (mito) were used as internal controls. One-way ANOVA showed no statistically significant difference between the groups (Mfn1, <i>P</i> = 0.414; Mfn2, <i>P</i> = 0.636; OPA1, <i>P</i> = 0.093).</p

Localization of TRAP1 in SH-SY5Y cells.

<p>A, SH-SY5Y cells were stained with anti-TRAP1 and anti-HADHA antibodies (mitochondrial marker) (a,d, TRAP1, green; b,e, HADHA, red; c,f, overlapping, yellow) and observed under a confocal microscope as described in the Materials and Methods. Upper panels show low magnification images. Lower panels show higher magnification images of part of each low magnification image (white windows). B, Equal amounts of lysates from cytoplasm (cyto) and mitochondria (mito) were analyzed by western blotting using anti-TRAP1 (upper panels), anti-HADHA (middle panel) or β-tubulin antibodies (lower panels). Scale bars, 20 µm (A, upper), 10 µm (A, lower).</p

The effect of transient TRAP1 KD on mitochondrial morphology in SH-SY5Y cells.

<p>A, Cells were analyzed by immunofluorescence microscopy using anti-HADHA antibody. High magnification images of a part of each photograph are shown in the white windows (normal, untreated; control, scrambled siRNA; siRNA, siRNA to TRAP1). B, Percentages of cells with the indicated mitochondrial morphologies in normal SH-SY5Y cells, control and TRAP1 KD cells. C, Cells were fractionated into mitochondrial (mito) fractions and blotted with antibodies against TRAP1, Drp1, Mff, fis1, Mfn1/2, OPA1, HADHA and β-tubulin. D, Quantification of the results shown in panel C. Data are expressed as the means ± s.e.m. of at least three independent experiments. The conventions are the same as those shown in Fig. 3. The asterisks on each bar indicate statistical significance (TRAP1: <i>P</i> = 0.025, Drp1: <i>P</i> = 0.012, Mff: <i>P</i> = 0.001, Fis1: <i>P</i> = 0.614, Mfn1: <i>P</i> = 0.764, Mfn2: <i>P</i> = 0.432, OPA1: <i>P</i> = 0.343, vs. control, Welch’s t-test, *<i>P</i><0.05, **<i>P</i><0.01). Scale bar, 10 µm (A).</p

Transient TRAP1 KD induces abnormal mitochondrial morphology and a strong decrease in Drp1 level in KNS cells.

<p>A, Cells were stained with an anti-HADHA antibody and analyzed by immunofluorescence microscopy. High magnification images of a part of each photograph are shown in the white windows (control, scrambled siRNA; siRNA, siRNA to TRAP1). B, Percentages of cells with the indicated mitochondrial morphologies in control and TRAP1 KD cells. C, Mitochondrial fractions (mito) from each cell group were blotted with antibodies against TRAP1, Drp1, Mff, fis1, Mfn1/2, OPA1, HADHA and β-tubulin. D, Quantification of the results shown in panel C. The data are expressed as the means ± s.e.m. of at least three independent experiments. The conventions are the same as those shown in Fig. 3. The asterisks on each bar indicate statistical significance (TRAP1: <i>P</i> = 0.030, Drp1: <i>P</i> = 0.778, Mff: <i>P</i> = 0.030, Fis1: <i>P</i> = 0.355, Mfn1: <i>P</i> = 0.286, Mfn2: <i>P</i> = 0.173, OPA1: <i>P</i> = 0.199, vs. control, Welch’s t-test, *<i>P</i><0.05). Scale bar, 20 µm (A).</p

The effect of TRAP1 on the expression levels of mitochondrial fission proteins.

<p>A, A, The expression levels of Drp1, Mff and Fis1 were analyzed by western blotting. B, Quantification of the expression levels of Drp1, Mff and Fis1. The conventions are the same as those shown in Fig. 3. The asterisks on each bar indicate statistical significance between the group and normal SH-SY5Y cells (Drp1 (cyto): one-way ANOVA, <i>P</i> = 2.02×10⁻⁵, post hoc Games-Howell test, *<i>P</i><0.05, **<i>P</i><0.01; Mff: one-way ANOVA, <i>P</i> = 1.53×10⁻⁶, post hoc Games-Howell test, *<i>P</i><0.05, **<i>P</i><0.01; Fis1: one-way ANOVA, <i>P</i> = 0.684). C, Mitochondrial fractions from cells were blotted with antibodies against TRAP1, MiD51/MIEF1, HADHA and β-tubulin. D, Quantification of the expression level of MiD51/MIEF1. Data are expressed as the means ± s.e.m. of seven independent experiments. Welch’s t-test showed no statistical significance (<i>P</i> = 0.863).</p

The effect of Drp1 and Mff overexpression on mitochondrial morphology.

<p>A, Mitochondrial fractions (mito) from cells infected with GFP, Mff291, Mff342 or HA-Drp1 were blotted with antibodies against TRAP1, Drp1, Mff, HADHA and β-tubulin. B, Cells treated as in A were analyzed by immunofluorescence microscopy using anti-HADHA antibody. C, Percentages of cells with the indicated mitochondrial morphology in B. Data were collected from three independent experiments and represent means ± s.e.m. Scale bar, 10 µm (B).</p