The dose response curves for WT and <i>Ndufs4</i> KO mice for LORR after intraperitoneal propofol.

<p>Dose-response curves were generated using the percentage of mice that presented LORR at the measured concentrations (n = 5–7 per group for each injection dose). The values for the KO animals were significantly different from those for WT (p<0.01).</p

EC₅₀s for isoflurane (ISO) and halothane (HAL) to cause immobility in response to tail pinch.

<p>Dark bars show the EC₅₀s for wild-type (WT) mice (n = 10, ISO; n = 6, HAL); open bars show the values for the <i>Ndufs4</i> KO mice (n = 10, ISO; n = 6, HAL). Error bars show the standard deviations. The values for the KO animals were significantly different from those for WT with a p<0.01.</p

Additional file 1 of Interleukin-6-elicited chronic neuroinflammation may decrease survival but is not sufficient to drive disease progression in a mouse model of Leigh syndrome

Additional file 1: Supplementary Figure 1. Microgliosis and astrogliosis at the late stage divided by sex. Related with Figures 4 and 5. (a) Quantification of IBA-1 fluorescence intensity in the VN, cerebellum, OB, cortex, and CA1, as well as the number of IBA-1+ cells in the cortex, and CA1 of the different genotypes. (b)GFAP mean fluorescence intensity quantification in the VN, cerebellum, OB, cortex, and CA1. ● Ndufs4 effect p≤ 0.05,  ★ GF-IL6 effect p≤ 0.05, ♦ p≤ 0.05 interaction between both factors. Supplementary Figure 2. Microgliosis and astrogliosis at the mid stage divided by sex. Related with Figure 6. (a) Quantification of IBA-1 fluorescence intensity in the VN, cerebellum, OB, cortex, and CA1, as well as the number of IBA-1+ cells in the cortex, and CA1 of the different genotypes. (b) GFAP mean fluorescence intensity quantification in the VN, cerebellum, OB, cortex, and CA1. ● Ndufs4 effect p≤ 0.05,  ★ GF-IL6 effect p≤ 0.05, ♦ p≤ 0.05 interaction between both factors

Knockout mice exhibit impaired locomotor learning.

<p>(<i>A</i>). Rotarod performance by control (n = 12) and knockout (n = 11) animals across days; two-way, repeated-measures ANOVA: genotype effect <i>F</i>(1, 21) = 70.5, <i>P</i><0.001; day effect <i>F</i>(8, 168) = 1.63, <i>P</i> = 0.12; genotype x day effect <i>F</i>(8, 168) = 2.37, <i>P</i> = 0.02 (#<i>P</i><0.05); *<i>P</i><0.05, **<i>P</i><0.01, **<i>P</i><0.001 compared to trial 1 within genotype. (<i>B</i>) Latency to fall during a wire-grip test in control (n = 12) and knockout (n = 11) animals; <i>P</i> = 0.08 by unpaired t test.</p

Knockout mice are smaller than normal but have normal spontaneous locomotion, and intact exploratory behavior.

<p><i>(A)</i> Spontaneous locomotion by control (n = 10) and knockout (n = 11) animals; light cycle, <i>P</i> = 0.85; dark cycle, <i>P</i> = 0.93; total, <i>P</i> = 0.98 by unpaired t tests. (<i>B</i>). Body weight of 8-week-old animals; control (n = 15) and knockout (n = 12) males, <i>P</i><0.0001; control (n = 11) and knockout (n = 10) females, <i>P</i> = 0.002 by unpaired t tests; **<i>P</i><0.01; ***<i>P</i><0.0001. (<i>C</i>) Locomotor activity of control (n = 10) and knockout (n = 5) animals in an open field; <i>P</i> = 0.17 by unpaired t test. (<i>D</i>) Fraction of time spent in the center and periphery of the open field by control (n = 10) and knockout (n = 5) animals; <i>P</i> = 0.08 by unpaired t tests. (<i>E</i>) Number of open-field center crossings by control (n = 10) and knockout (n = 5) animals; <i>P</i> = 0.28 by unpaired t test.</p

LH induces ribosomal protein translation.

<p>(A) Microarray cluster containing the probes that showed increased polysome-association in Cyp17iCre: RiboTag mice after 1 h and 4 h of LH administration. All ribosomal proteins and elongation/initiation factors present in the cluster are listed. Statistical analysis was performed using One-way Analysis of Variance (ANOVA) with Newman-Keuls multiple comparison post-hoc test (p<0.01 <i>vs</i> acyline). (B) Pie chart representing the percentage of probes for ribosomal proteins contained in (A). (C) Western blot analysis of phospho-S6 (S235/236) levels in testis lysates after acyline and LH treatment (D) Immunohistochemistry for phospho-S6 ribosomal protein (Ser 235/236) in testis sections of saline, acyline, or acyline+LH-treated mice. Arrows point to the interstitial spaces, where Leydig cells are located. (E) Representative polysome profiles of untreated, LH-treated or rapamycin+LH-treated MA-10 cells. Fractions 1–3 contain the cytosolic fraction, 4–5 the monosome fraction, and fractions 6–12 contain the polysome fraction. Cells were serum-starved overnight and treated for 30 min with Rapamycin (20 nM) or vehicle prior to the addition of LH (0.2 u/ml) for 1 h.</p

Aquaporin 2 is actively translated in Leydig cells after LH stimulation.

<p>(A) qRT-PCR analysis of <i>Aqp2</i> mRNA in the IPs of Cyp17iCre: RiboTag mice treated with saline, acyline, acyline+LH for 1 h and acyline+LH for 4 h (n = 4, from two independent experiments). Statistical analysis was performed using One-way Analysis of Variance (ANOVA) with Newman-Keuls multiple comparison post-hoc test. # p<0.05 <i>vs</i> saline; <b>***</b> p<0.001 <i>vs</i> acyline. Enrichment (IP <i>versus</i> input ratio) for <i>Aqp2</i> in saline-treated animals was 17.9±1.7 by qRT-PCR analysis. Data are the mean±SEM. (B) Western blot analysis of aquaporin 2 in testis lysates of mice treated with saline, acyline, acyline+LH for 1 h and acyline+LH for 4 h. Membrane was reprobed with an anti-beta-actin antibody to confirm equal loading of the samples. (C) Immunohistochemistry for aquaporin 2 in testis sections of mice treated with saline, acyline and acyline+LH for 4 h. Black arrows show staining in Leydig cells and white arrows show staining in germ cells. (D) qRT-PCR analysis of <i>Aqp2</i> mRNA in MA-10 cells after LH stimulation (0.2 u/ml). Statistical analysis was performed using One-way Analysis of Variance (ANOVA) with Newman-Keuls multiple comparison post-hoc test. <b>***</b> p<0.001 <i>vs</i> 0 h. (E) Western blot analysis of aquaporin 2 in MA-10 cell lysates after LH exposure (0.2 u/ml) for different time points.</p

Knockout mice fail to learn a simple (FR1) operant task.

<p>(<i>A</i>) Lever presses during operant conditioning sessions by control (n = 10) and knockout (n = 5) animals across days; two-way, repeated-measures ANOVA: genotype effect <i>F</i>(1, 13) = 15.72, <i>P</i> = 0.002; day effect <i>F</i>(6, 78) = 10.90, <i>P</i><0.001; genotype x day effect <i>F</i>(6,78) = 12.96, <i>P</i><0.001 (###<i>P</i><0.001); **<i>P</i><0.01, ***<i>P</i><0.001 compared to session 1 within genotype. (<i>B</i>) Latency to first lever press during operant conditioning sessions by control (n = 10) and knockout (n = 5) animals across days; two-way repeated measures ANOVA: genotype effect <i>F</i>(1, 13) = 38.75, <i>P</i><0.001; day effect <i>F</i>(6, 78) = 2.26, <i>P</i> = 0.04; genotype x day effect <i>F</i>(6,78) = 8.04, <i>P</i><0.001 (###<i>P</i><0.001); **<i>P</i><0.01, ***<i>P</i><0.001 compared to session 1 within genotype. (<i>C</i>) Total number of head entries during operant conditioning sessions by control (n = 10) and knockout (n = 5) animals across days; two-way repeated measures ANOVA: genotype effect <i>F</i>(1, 13) = 10.14, <i>P</i> = 0.007; day effect <i>F</i>(6, 78) = 1.93, <i>P</i> = 0.08; genotype x day effect <i>F</i>(6,78) = 2.25, <i>P</i> = 0.04 (#<i>P</i><0.05); *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 compared to session 1 within genotype.</p

Knockout mice that selectively lack NMDAR in striatal MSNs have abnormal MSN morphology.

<p><i>(A</i>) GPR88-CreGFP is expressed selectively in the striatum of knockout animals. A DAPI counterstain was performed. (<i>B</i>) NR1 Western blot of striatal (str) and cortical (cx) homogenates from control and knockout animals. (<i>C)</i> Representative micrographs and tracings of MSNs from control and knockout animals (20 µm scale bar). (<i>D)</i> Cell body size of MSNs in control and knockout mice (n = 18 neurons per genotype); <i>P</i> = 0.009 by unpaired t test; **<i>P</i><0.01. (<i>E)</i> Total MSN dendrite length in control and knockout mice (n = 18 neurons per genotype); <i>P</i> = 0.002 by unpaired t test; **<i>P</i><0.01. (<i>F)</i> Number of dendrites by dendrite order in control and knockout mice (n = 18 neurons per genotype); *<i>P</i><0.05 by unpaired t test. Diagram in (<i>F</i>) depicts the definition of dendrite order.</p

<i>In vivo</i> Leydig cell translational profile after 1 h of LH administration.

<p>(A) Cartoon shows the two experimental groups compared by microarray analysis. (B) Venn diagrams showing the number of mRNAs regulated after 1 h of LH administration (1.5 fold or higher) when compared to the acyline group in the IPs of Cyp17iCre: RiboTag mice by microarray analysis. Values in the intersection are the number of transcripts regulated by LH and enriched (IP/input >2 in untreated mice) in Leydig cells. GO analysis was performed on the transcripts that showed a fold increase of 1.5 or higher after 1 h of LH stimulation. Transcription factor activity (C) and cell cycle (D) were identified as significant categories. Heat maps show the regulation by microarray analysis of the transcripts included in these GO categories. AdjP: Adjusted p-value. Some transcripts (shown in red) were further confirmed by qRT-PCR because of their novelty or significance (E–F and G). Graphs show qRT-PCR confirmation for <i>Nr4a1</i>, <i>Egr1</i> (E–F) and <i>Rgs2</i> (G) in the IPs of Cyp17iCre: RiboTag mice treated with saline, acyline, acyline+LH for 1 h and acyline+LH for 4 h (n = 4, from two independent experiments). Statistical analysis was performed using One-way Analysis of Variance (ANOVA) with Newman-Keuls multiple comparison post-hoc test. <b>***</b> p<0.001, <b>**</b> p<0.01 and <b>*</b> p<0.05 <i>vs</i> acyline. IP <i>versus</i> input ratio (Enrichment) was assessed by qRT-PCR and shows the abundance of the transcript in Leydig cells. Data are the mean±SEM.</p