Journal: eLife

Article Title: Ca2+-dependent release of synaptotagmin-1 from the SNARE complex on phosphatidylinositol 4,5-bisphosphate-containing membranes

doi: 10.7554/elife.57154

Figure Lengend Snippet: Figure 5. Analysis of interactions between Syt1 C2AB* and SNARE complexes anchored on nanodiscs using FRET. (A) Diagrams illustrating NDs, cisSC- NDs and transSC-NDs. Syntaxin-1 is in yellow, SNAP-25 in green and synaptobrevin in red. Although we used full-length syntaxin-1 to make the nanodisc-anchored SNARE complexes, the N-terminal region preceding the SNARE motif of syntaxin-1 is not shown for simplicity. (B) Fluorescence emission spectra of C2AB* (labeled with Alexa488 at residue 346) in the presence of increasing concentrations of cisSC-NDs (15% PS, 5% Rho-PE), in Figure 5 continued on next page

Article Snippet: Key resources table Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pGEX-KG-GSTSyt1_C2B Brewer et al., 2015 Protein expression plasmid for E. coli (rat synaptotagmin-1 C2AB domain, residues 271–421) Recombinant DNA reagent pGEX-KG-GSTSyt1_C2AB Araç et al., 2006 Protein expression plasmid for E. coli (rat synaptotagmin-1 C2AB domain, residues 140–421) Recombinant DNA reagent pMSP1E3D1 Addgene 20066 Protein expression plasmid for E. coli (MSP1E3D1) Chemical compound, drug DEUTERIUM OXIDE (D, 99.8%) Cambridge Isotope Laboratories, Inc DLM-4–99.8 Chemical compound, drug AMMONIUM CHLORIDE (15N, 99%) Cambridge Isotope Laboratories, Inc NLM-467 Chemical compound, drug D-GLUCOSE (1,2,3,4,5,6,6-D7, 97–98%) Cambridge Isotope Laboratories, Inc DLM-2062 Chemical compound, drug L-Methionine(methyl-13C) Sigma-aldrich 299146 Continued on next page Voleti et al. eLife 2020;9:e57154.

Techniques: Fluorescence, Labeling, Residue

Journal: EMBO Molecular Medicine

Article Title: Isoginkgetin antagonizes ALS pathologies in its animal and patient iPSC models via PINK1-Parkin-dependent mitophagy

doi: 10.1038/s44321-025-00323-2

Figure Lengend Snippet: ( A ) Effects of ISO (5 to 150 μM) on the egg laying rate, egg hatching rate (L1), L4 development rate, and the adulthood rate (Adult day 1) in the WT N2 nematodes. Toxicity tests revealed a safe concentration range of ISO between 0 and 75 μM ( n = 5; 10 worms for each biological repeats). ( B ) Representative images showing the LGG-1 and DCT-1 co-localization in DMSO or ISO treatment group on adult day 1. unc119 + neurons are marked with white dotted borders. Scale bar, 5 μm. ( C , D ) Statistical results of mt-Rosella ( C ) and LGG-1/DCT-1 ( D ) transgenic worms showed that 15 μM ISO activated neuronal mitophagy ( n = 30 neurons from 30 worms for each group). ( E ) UV-killed OP50 experiment showing that the protective effect of ISO on the motility of SOD1 G93A worms was independent of microbial metabolism ( n = 20 worms for each group). ( F ) Effect of ISO on pharyngeal pumping speed from adult day 2 to day 14 in WT (N2) and SOD1 G93A worms. The results showed that ISO did not restrict the nematode diet ( n = 30 worms for each group). Data are presented as the mean ± SD. Exact P values are reported in Appendix Table . One-way ANOVA followed by Dunnett’s multiple comparisons test in ( A , C , D , F ). Two-way ANOVA followed by Tukey’s multiple comparisons test in ( E ).

Article Snippet: SOD1 G93A transgenic mice that express a G93A mutant form of human SOD1 (B6SJL-Tg (SOD1*G93A)1Gur/J, JAX stock No. 002726) and C57BL/6J mice (JAX stock No. 000664) were purchased from the Jackson Laboratory.

Techniques: Concentration Assay, Transgenic Assay

Journal: EMBO Molecular Medicine

Article Title: Isoginkgetin antagonizes ALS pathologies in its animal and patient iPSC models via PINK1-Parkin-dependent mitophagy

doi: 10.1038/s44321-025-00323-2

Figure Lengend Snippet: ( A ) A representative image and schematic of unc-25 promoter expression in 26 GABAergic neurons, including 19 D-type MNs in the ventral cord (blue), 4 RMEs (purple), one AVL (red), and one RIS (green) in the head, and one DVB (red) in the tail. Scale bar, 100 μm. ( B ) Representative images showing the co-localization of LGG-1::DsRed and DCT-1::BFP in D-type MNs of N2 or SOD1 G93A transgenic worms. unc25 + D-type motor neurons are marked with white arrows. Scale bar, 1 μm. ( C ) Quantification of ( B ) showed lower basal mitophagy level in SOD1 G93A transgenic worms MNs than WT. ISO (15 μM) treatment induced mitophagy in MNs of SOD1 G93A transgenic worms. n = 30 D-type MNs from 30 worms for each group. ( D – I ) ISO treatment improved ALS-related phenotypes in SOD1 G93A worms, including ( D ) swimming ability, ( E ) paralysis, ( F , G ) MN loss (white square in ( F )), scale bar, 100 μm, ( H ) MN degeneration defined by loss of connections between MNs (red arrows in ( F )), and ( I ) lifespan. WT N2 worms as control in ( D , E , I ); WT N2 worms crossed with unc-25 ::GFP worms to visualize MNs as control in ( F – H ). n = 30 worms for each group. RNAi technology was used to knock down pink-1 and pdr-1 in worm MNs. TU3401 worms expressing SID-1 were crossed to each strain, ensuring gene knockdown within neurons. ( J ) Representative images of mt-Rosella indicated mitophagy in WT N2 worms and SOD1 G93A transgenic worms crossed with mt-Rosella worms and SID-1 worms. The effect of ISO on promoting neuronal mitophagy ( K ) and improving swimming ability ( L ) was inhibited by knockdown of pink-1 and pdr-1 . n = 30 worms for each group. Data are presented as the mean ± SD. Exact P values are reported in Appendix Table . One-way ANOVA followed by Dunnett’s multiple comparisons test in ( C , K , L ). Log-rank (Mantel–Cox) test in ( I ). Two-way ANOVA followed by Tukey’s multiple comparisons test in ( D , E , G , H ). .

Article Snippet: SOD1 G93A transgenic mice that express a G93A mutant form of human SOD1 (B6SJL-Tg (SOD1*G93A)1Gur/J, JAX stock No. 002726) and C57BL/6J mice (JAX stock No. 000664) were purchased from the Jackson Laboratory.

Techniques: Expressing, Transgenic Assay, Control, Knockdown

Journal: EMBO Molecular Medicine

Article Title: Isoginkgetin antagonizes ALS pathologies in its animal and patient iPSC models via PINK1-Parkin-dependent mitophagy

doi: 10.1038/s44321-025-00323-2

Figure Lengend Snippet: ( A ) Schematic of intranasal administration. Magnified schematic showing the transport of Nano-ISO and Nano-ISO#ICG from olfactory filaments across the cribriform plate into the olfactory bulb and release into the brain. ( B ) Representative IVIS images of the distribution of Nano-ISO#ICG in the brain after a single intranasal administration. ( C ) ISO concentrations in mice within 120 h detected by the UPLC-MS/MS assay. ( D ) The onset of tremor in SOD1 G93A mice was delayed in the Nano-ISO group, n = 12 mice in the Veh group, n = 12 mice in the Nano-ISO group, and n = 9 mice in the Riluzole group. ( E ) The hanging time of the animals in the Nano-ISO group after 4 and 5 weeks of drug administration was longer than that animals in the Veh group. ( F ) Nano-ISO prolonged the lifespan of SOD1 G93A mice compared to the Veh treatment. ( G ) Representative images of ChAT staining showing lumbar MNs of wild-type (WT) mice, Veh-treated SOD1 G93A mice, and Nano-ISO-treated SOD1 G93A mice. The ventral horn of the spinal cord is marked with white dotted lines. Scale bars, 200 μm. ( H ) Quantification of ( G ) showed that the number of ChAT + MNs was significantly increased in the Nano-ISO group. Each point represents the average value of ten images for each mouse, n = 5 mice/group. ( I ) Representative images of microglia and astrocyte activation revealed by Iba-I staining and GFAP staining in WT, Veh group, and Nano-ISO group. Scale bars, 50 μm. ( J ) Quantification of ( I ) showed microglia activation was inhibited in the Nano-ISO group. Each point represents the average value of 10 images for each mouse, n = 5 mice/group. ( K ) Quantification of ( I ) showed astrocyte activation was inhibited in the Nano-ISO group. Each point represents the average value of ten images for each mouse, n = 5 mice/group. ( L ) Representative images of MTCO2 and LAMP2 co-localization levels in lumbar ChAT + MNs. ChAT + motor neurons are marked with white dotted borders. Scale bars, 5 μm. ( M ) Quantification of ( L ) MTCO2 and LAMP2 co-localization using Pearson’s correlation coefficient showed that ISO induced mitophagy in lumbar ChAT + MNs. Each point represents the average value of 10 MNs for each mouse, n = 5 mice/group. ( N ) Representative images of pSer65-Ub protein expression in lumber ChAT + MNs. ChAT + motor neurons are marked with white dotted borders. Scale bars, 5 μm. ( O ) Quantification of ( N ) showed that ISO increased pSer65-Ub protein expression in MNs. Each point represents the average value of ten MNs for each mouse, n = 5 mice/group. ( P ) The ultrastructure of mitochondria was observed using EM. ISO improved mitochondrial morphology (green arrow) and induced mitolysosome-like structures (red arrow) in lumbar MNs. Scale bars, 0.5 μm. Data are presented as the mean ± SD. Exact P values are reported in Appendix Table . One-way ANOVA followed by Dunnett’s multiple comparisons test in ( E , H , J , K , M , O ). Log-rank (Mantel–Cox) test in ( D , F ). .

Article Snippet: SOD1 G93A transgenic mice that express a G93A mutant form of human SOD1 (B6SJL-Tg (SOD1*G93A)1Gur/J, JAX stock No. 002726) and C57BL/6J mice (JAX stock No. 000664) were purchased from the Jackson Laboratory.

Techniques: Tandem Mass Spectroscopy, Staining, Activation Assay, Expressing

Journal: EMBO Molecular Medicine

Article Title: Isoginkgetin antagonizes ALS pathologies in its animal and patient iPSC models via PINK1-Parkin-dependent mitophagy

doi: 10.1038/s44321-025-00323-2

Figure Lengend Snippet: ( A ) Schematic diagram of electrophysiological test in mice. ( B – D ) Statistical results of the CMAP amplitude of gastrocnemius muscle by stimulating sciatic nerve in SOD1 G93A mouse and WT group on Day 50 ( B ), Day 60 ( C ), and Day 90 ( D ). The maximum CMAP amplitude was noted to decrease significantly in SOD1 G93A mice on day 60, n = 5 mice/group on day 50; n = 6 mice for WT and n = 7 mice for G93A on day 60; n = 3 mice/group on day 90. ( E ) Representative images of microglia and astrocyte activation revealed by Iba-I staining and GFAP staining. Scale bar, 25 μm. ( F ) Representative images of ChAT and NeuN staining. White arrows indicate motor neurons with shrunken cell bodies. Scale bar, 25 μm. ( G , H ) Statistical results of Iba-I staining and GFAP staining in ( E ) demonstrating that both the microglia and astrocyte activation were detected in the ventral horn of lumbar spinal cord of SOD1 G93A mice on day 60. Each point represents the average value of 10 images for each mouse, n = 5 mice/group. ( I , J ) Statistical results of the number of MNs by NeuN staining and ChAT staining. No loss of MNs was detected in the ventral horn of lumbar spinal cord of SOD1 G93A mice on day 60. Each point represents the average value of 10 images for each mouse, n = 5 mice/group. ( K ) Schematic diagram of the exploration of Nano-ISO therapeutic effects in SOD1 G93A mice. Data are presented as the mean ± SD. Exact P values are reported in Appendix Table . Unpaired t test.

Article Snippet: SOD1 G93A transgenic mice that express a G93A mutant form of human SOD1 (B6SJL-Tg (SOD1*G93A)1Gur/J, JAX stock No. 002726) and C57BL/6J mice (JAX stock No. 000664) were purchased from the Jackson Laboratory.

Techniques: Activation Assay, Staining

Journal: The Journal of Neuroscience

Article Title: Microarray Analysis of the Cellular Pathways Involved in the Adaptation to and Progression of Motor Neuron Injury in the SOD1 G93A Mouse Model of Familial ALS

doi: 10.1523/JNEUROSCI.1470-07.2007

Figure Lengend Snippet: Genes significantly altered at 60 d in motorneurons expressing the G93A SOD1

Article Snippet: Murine SOD1 G93A transgenic mouse model. Transgenic mice B6SJL-Tg (SOD1-G93A) 1 Gur/J high copy number were backcrossed onto C57BL/6J Ola-Hsd (Harlan UK, Bicester, UK) for >20 generations to eliminate the SJL dysferlin mutation ( Bittner et al., 1999 ) and to achieve a homogeneous genetic background more suitable for microarray studies.

Techniques: Expressing, Membrane, Sequencing, Activity Assay, Binding Assay, Protein Binding, Translocation Assay, Ubiquitin Proteomics, Virus

Journal: The Journal of Neuroscience

Article Title: Microarray Analysis of the Cellular Pathways Involved in the Adaptation to and Progression of Motor Neuron Injury in the SOD1 G93A Mouse Model of Familial ALS

doi: 10.1523/JNEUROSCI.1470-07.2007

Figure Lengend Snippet: Q-PCR results. A, Significant upregulation of necdin at 60 d (p = 0.04), which is downregulated at the late stage of the disease (p = 0.003). B, Consistent upregulation of cyclin I throughout the pathology (60 d, p = 0.05; 90 d, p = 0.1; 120 d, p = 0.03). C, Consistent downregulation of plexin domain containing 1 (60 d, p = 0.02; 90 d, p = 0.003; 120 d, p = 0.001). Error bars indicate SD. Tg, Transgenic; N-Tg, nontransgenic. *p ≤ 0.05; **p ≤ 0.01.

Article Snippet: Murine SOD1 G93A transgenic mouse model. Transgenic mice B6SJL-Tg (SOD1-G93A) 1 Gur/J high copy number were backcrossed onto C57BL/6J Ola-Hsd (Harlan UK, Bicester, UK) for >20 generations to eliminate the SJL dysferlin mutation ( Bittner et al., 1999 ) and to achieve a homogeneous genetic background more suitable for microarray studies.

Techniques: Transgenic Assay

Journal: The Journal of Neuroscience

Article Title: Microarray Analysis of the Cellular Pathways Involved in the Adaptation to and Progression of Motor Neuron Injury in the SOD1 G93A Mouse Model of Familial ALS

doi: 10.1523/JNEUROSCI.1470-07.2007

Figure Lengend Snippet: Genes significanlty altered at 90 d in motorneurons expressing the G93A SOD1

Article Snippet: Murine SOD1 G93A transgenic mouse model. Transgenic mice B6SJL-Tg (SOD1-G93A) 1 Gur/J high copy number were backcrossed onto C57BL/6J Ola-Hsd (Harlan UK, Bicester, UK) for >20 generations to eliminate the SJL dysferlin mutation ( Bittner et al., 1999 ) and to achieve a homogeneous genetic background more suitable for microarray studies.

Techniques: Expressing, Variant Assay, Activity Assay, Membrane

Journal: The Journal of Neuroscience

Article Title: Microarray Analysis of the Cellular Pathways Involved in the Adaptation to and Progression of Motor Neuron Injury in the SOD1 G93A Mouse Model of Familial ALS

doi: 10.1523/JNEUROSCI.1470-07.2007

Figure Lengend Snippet: Genes significantly altered at 120 d in motorneurons expressing the G93A SOD1

Article Snippet: Murine SOD1 G93A transgenic mouse model. Transgenic mice B6SJL-Tg (SOD1-G93A) 1 Gur/J high copy number were backcrossed onto C57BL/6J Ola-Hsd (Harlan UK, Bicester, UK) for >20 generations to eliminate the SJL dysferlin mutation ( Bittner et al., 1999 ) and to achieve a homogeneous genetic background more suitable for microarray studies.

Techniques: Expressing, Binding Assay, Sequencing, Activity Assay, Protein Binding, Transformation Assay, Ubiquitin Proteomics, Activation Assay, Membrane

Journal: The Journal of Neuroscience

Article Title: Microarray Analysis of the Cellular Pathways Involved in the Adaptation to and Progression of Motor Neuron Injury in the SOD1 G93A Mouse Model of Familial ALS

doi: 10.1523/JNEUROSCI.1470-07.2007

Figure Lengend Snippet: A, Motor neurons isolated from G93A mice at 60 d show upregulation in several classes of genes delineating what is likely to be happening in these cells under stress. There is upregulation of the transcriptional machinery, along with upregulation of translation-related ribosomal and folding proteins, as the motor neuron attempts to compensate for the ongoing cellular stress. All these mechanisms require ATP, provoking a massive increase in the work load of mitochondria, leading to upregulation of carbohydrate metabolism and respiratory chain activity, which in turn causes increased ROS production. The observed imbalance among the subunits forming the ATP synthase complex, shown by downregulation of the δ subunit, will generate additional oxidative stress, with consequent production of oxidized proteins. These are then ubiquitinated and targeted for proteasomal degradation. B, Over time, the accumulation of damaged proteins and ROS is likely to cause a general collapse in cellular functioning, leading to downregulation of the compensatory pathways previously activated and leaving the cell with decreased energy and protein turnover. The cell increases protein degradation functions, with activation of the lysosomal machinery. Production and secretion of some subunits of the complement cascade are important signals of cellular stress for neighboring cells. The final abortive attempt at survival comes through activation of the cell cycle, with upregulation of cyclin L1 (involved in the transition from the quiescent state, G0, to the first phase of the cell cycle, G1) and cyclins D2 and E2 (involved in the progression of the cell cycle through G1 phase). The upregulation of cyclin I suggests that motor neurons are trying to exercise negative control on the transition between the G1 and S phase to prevent the abnormal progression through the cell cycle. Atf4, Activating transcription factor 4; Atp5a1, ATP synthase F1 complex α1 subunit; Atp5d, ATP synthase F1 complex δ subunit; Cct4, chaperonin subunit 4; Ctsz, cathepsin-Z; Eef1, eukaryotic translation elongation factor 1; Eif3, eukaryotic translation initiation factor 3; Hsp, heat shock protein; Lyz, lysozyme; Lzp-s, P-lysozyme structural; Mdh1, malate dehydrogenase 1; Ndn, necdin; Psmc6, proteasome 26S; Rpl, ribosomal protein L; Sdha, succinate dehydrogenase complex subunit A; Taf9, transcription activator factor 9; Tcerg1, transcription elongation regulator 1 (CA150); Tgfb1i4, transforming growth factor β1-induced transcript 4; Ube1c, ubiquitin-activating enzyme E1C; Uble1b, ubiquitin-like activating enzyme E1B; Usp36, ubiquitin-specific preotease 36; H+, hydrogen ion; TCA, tricarboxylic acid.

Article Snippet: Murine SOD1 G93A transgenic mouse model. Transgenic mice B6SJL-Tg (SOD1-G93A) 1 Gur/J high copy number were backcrossed onto C57BL/6J Ola-Hsd (Harlan UK, Bicester, UK) for >20 generations to eliminate the SJL dysferlin mutation ( Bittner et al., 1999 ) and to achieve a homogeneous genetic background more suitable for microarray studies.

Techniques: Isolation, Activity Assay, Activation Assay, Negative Control, Ubiquitin Proteomics

Journal: International Journal of Molecular Sciences

Article Title: Progressive Mitochondrial SOD1 G93A Accumulation Causes Severe Structural, Metabolic and Functional Aberrations through OPA1 Down-Regulation in a Mouse Model of Amyotrophic Lateral Sclerosis

doi: 10.3390/ijms22158194

Figure Lengend Snippet: Mitochondrial damage in chromaffin cells of SOD1 G93A mice. ( A ) Transmission electron microscopy images showing a representative example of an orthodox mitochondria in CCs from control mice (WT) at both time points, with a circular, elliptical, tubular or branched shape: P30 ( Aa , Ab ) and P120 ( Ac , Ad ). Note the abundance of dense core granules containing catecholamines all around the cytosol, a hallmark of this cell type. WT mitochondria have fairly regular spaced lamellar cristae. ( B ) Microphotographs from SOD1 G93A mouse, showing the different mitochondrial alterations identified at both stages: mitochondrial sprouting of translucent vesicles (( Ba ); black arrow), mitochondrial vacuoles derived from the matrix or cristae (( Bb , Bc ); white arrow), mitochondrial outer membrane expansion (( Bd ); double white arrow), cristae swelling (( B ), white arrow head), disruption of the mitochondrial membrane (( Bb,Bc ); double black arrow), mitochondrial matrix dilatation (( Be – Bg ), black pad #) and mitochondrial degradation (M) by mitophagy (( Bh ), degenerating organelles are marked with an white asterisk *). All images were obtained from adrenal gland tissue from three different mice per group.

Article Snippet: Ultrathin sections of adrenal glands were etched in 0.1 N HCl for 5 min, rinsed three times for 5 min each in Tris buffered saline (TBS: 20 mM TRIS, 140 mM NaCl, and 2.7 mM KCl, pH 8.0), treated with blocking buffer (0.1% gelatin, 1% normal goat serum (NGS) and 0.3% Triton X-100 in TBS) for 30 min, incubated for 2 h at room temperature with the primary antibody (anti-hSOD1 G93A , 1:500; Medimabs, C4F6, Montréal, QC, Canada).

Techniques: Transmission Assay, Electron Microscopy, Control, Derivative Assay, Membrane, Disruption

Journal: International Journal of Molecular Sciences

Article Title: Progressive Mitochondrial SOD1 G93A Accumulation Causes Severe Structural, Metabolic and Functional Aberrations through OPA1 Down-Regulation in a Mouse Model of Amyotrophic Lateral Sclerosis

doi: 10.3390/ijms22158194

Figure Lengend Snippet: Changes in mitochondrial number and size serves as evidence for the dynamic impairment in chromaffin cells of SOD1 G93A mice. TEM images (5000×) obtained from the adrenal medulla of WT (left) and SOD1 G93A (right) mice. Each image is a cross section of a CC at presymptomatic P30 ( A , B ) or symptomatic P120 stages ( C , D ). ( A’ – D’ ) are magnifications of a cytoplasmic area with a high density of mitochondria (inset in each image). Note the internal alterations in the mitochondria from SOD1 G93A mice compared with the WT at both ages. ( E ) Average number of mitochondria in CCs normalized to the area (μm 2 ) of cytosol (the number of mitochondria counted and pictures analyzed are shown in parentheses: * p < 0.05 and ** p < 0.01 comparing WT with SOD1 G93A at the same age). ( F ) bar graph summarizing the average data of the mitochondrial area (μm 2 ): * p < 0.05; ** p < 0.01. Data are the mean ± SEM. The analyses were performed from at least three adrenal medulla from three different mice per group. # p < 0.05 comparing the effect of aging in the WT group and in the SOD1 G93A group. Statistical analyses were performed with two-way ANOVA with Šídák’s multiple comparison post hoc test.

Article Snippet: Ultrathin sections of adrenal glands were etched in 0.1 N HCl for 5 min, rinsed three times for 5 min each in Tris buffered saline (TBS: 20 mM TRIS, 140 mM NaCl, and 2.7 mM KCl, pH 8.0), treated with blocking buffer (0.1% gelatin, 1% normal goat serum (NGS) and 0.3% Triton X-100 in TBS) for 30 min, incubated for 2 h at room temperature with the primary antibody (anti-hSOD1 G93A , 1:500; Medimabs, C4F6, Montréal, QC, Canada).

Techniques: Comparison

Journal: International Journal of Molecular Sciences

Article Title: Progressive Mitochondrial SOD1 G93A Accumulation Causes Severe Structural, Metabolic and Functional Aberrations through OPA1 Down-Regulation in a Mouse Model of Amyotrophic Lateral Sclerosis

doi: 10.3390/ijms22158194

Figure Lengend Snippet: Mitochondrial cristae swelling in SOD1 G93A chromaffin cells. ( A ) TEM images (40,000×) acquired from adrenal medullae tissue from WT and SOD1 G93A mice showing independent mitochondria at presymptomatic (P30, ( a , b )) or symptomatic stages (P120, ( c , d )). Next to each picture there is a representative drawing of the mitochondria analyzed, achieved by manually selecting the inner and outer mitochondrial membranes and the cristae. ( B ) Bar graph depicting the average number of cristae per μm 2 of mitochondria (between 200 and 300 cristae were measured per group). ( C ) Quantitative analysis of the mean cristae size (μm 2 ). The mitochondrial area filled by cristae is represented as a percentage in ( D ). Data represent the mean ± SEM of the number of cristae and mitochondria (in parentheses) from at least three mice. The statistical analysis was performed using a two-way ANOVA with Šídák’s multiple comparison post hoc test: * p < 0.05; *** p < 0.001 vs. WT at the same stage. ### p < 0.001 comparing the effect of aging in the WT and SOD1 G93A groups (only in panel B).

Article Snippet: Ultrathin sections of adrenal glands were etched in 0.1 N HCl for 5 min, rinsed three times for 5 min each in Tris buffered saline (TBS: 20 mM TRIS, 140 mM NaCl, and 2.7 mM KCl, pH 8.0), treated with blocking buffer (0.1% gelatin, 1% normal goat serum (NGS) and 0.3% Triton X-100 in TBS) for 30 min, incubated for 2 h at room temperature with the primary antibody (anti-hSOD1 G93A , 1:500; Medimabs, C4F6, Montréal, QC, Canada).

Techniques: Comparison

Journal: International Journal of Molecular Sciences

Article Title: Progressive Mitochondrial SOD1 G93A Accumulation Causes Severe Structural, Metabolic and Functional Aberrations through OPA1 Down-Regulation in a Mouse Model of Amyotrophic Lateral Sclerosis

doi: 10.3390/ijms22158194

Figure Lengend Snippet: Widening of the cristae junctions in the mitochondria from SOD1 G93A mice chromaffin cells at both presymptomatic and symptomatic stages. ( A ) Cross section of mitochondria obtained by TEM from WT and SOD1 G93A mice at presymptomatic (P30) or symptomatic (P120) stages (40,000×). Magnifications of the cristae junctions observed are shown below each picture (see arrow) and at the bottom of the panel; a drawing of the mitochondrial external membrane and the cristae junction of each image can be found, with the cristae junction size in nm. ( B ) Bar plot of the average cristae junction size (in nm) in all groups. Data represent the mean ± SEM, and the statistical analysis was performed using the two-way ANOVA with Šídák’s multiple comparison post hoc test: ** p < 0.01 and *** p < 0.001 vs. WT at the same stage. Scale bar = 200 nm. There was no difference with respect to variable age.

Article Snippet: Ultrathin sections of adrenal glands were etched in 0.1 N HCl for 5 min, rinsed three times for 5 min each in Tris buffered saline (TBS: 20 mM TRIS, 140 mM NaCl, and 2.7 mM KCl, pH 8.0), treated with blocking buffer (0.1% gelatin, 1% normal goat serum (NGS) and 0.3% Triton X-100 in TBS) for 30 min, incubated for 2 h at room temperature with the primary antibody (anti-hSOD1 G93A , 1:500; Medimabs, C4F6, Montréal, QC, Canada).

Techniques: Membrane, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Progressive Mitochondrial SOD1 G93A Accumulation Causes Severe Structural, Metabolic and Functional Aberrations through OPA1 Down-Regulation in a Mouse Model of Amyotrophic Lateral Sclerosis

doi: 10.3390/ijms22158194

Figure Lengend Snippet: Chromaffin cells from SOD1 G93A mice have impaired mitochondrial membrane potential. The m ψ was measured in CCs with TMRE. ( A ) A 3D representation of the fluorescence intensity from a single chromaffin cell loaded with TMRE (25 nM). The images on the right show the basal fluorescence at the beginning of the experiments in a CC from WT ( top ) and SOD1 G93A ( bottom ) mice. Note that every peak represents the ability of each mitochondrion to load the fluorescent probe based on its m ψ. The images on the left show the change in the m ψ elicited by the release of the fluorescent probe from the mitochondria induced by the addition of FCCP. ( B , D ) The graph represents the average TMRE fluorescence intensity of single cells from P30 ( B ) and P120 ( D ) mice following a protocol of 30 s basal recording, 30s with a FCCP depolarizing pulse and 1 min washout (2 s intervals between data acquisition). The images of the CCs at specific points of the protocol are depicted in a – c . ( C , E ) Bar graphs showing the average variation in m ψ in CCs from P30 and P120 mice after depolarization with FCCP. The data represent the mean ± SEM. The number of cells analyzed and cell cultures obtained from different animal are shown in parentheses. Statistical analyses were performed using a Student´s t test: * p < 0.05; *** p < 0.001 vs. WT.

Article Snippet: Ultrathin sections of adrenal glands were etched in 0.1 N HCl for 5 min, rinsed three times for 5 min each in Tris buffered saline (TBS: 20 mM TRIS, 140 mM NaCl, and 2.7 mM KCl, pH 8.0), treated with blocking buffer (0.1% gelatin, 1% normal goat serum (NGS) and 0.3% Triton X-100 in TBS) for 30 min, incubated for 2 h at room temperature with the primary antibody (anti-hSOD1 G93A , 1:500; Medimabs, C4F6, Montréal, QC, Canada).

Techniques: Membrane, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Progressive Mitochondrial SOD1 G93A Accumulation Causes Severe Structural, Metabolic and Functional Aberrations through OPA1 Down-Regulation in a Mouse Model of Amyotrophic Lateral Sclerosis

doi: 10.3390/ijms22158194

Figure Lengend Snippet: More reactive oxygen species were produced by the chromaffin cells of SOD1 G93A mice. The cellular redox balance in CCs was evaluated over 30 min under basal conditions using the DCH 2 FDA fluorescent dye. ( A , B ) are 3D reconstructions of single fluorescent dye-loaded CCs at the beginning (t = 0 min, t 0 ) and end of the experiment (t = 30 min, t 30 ). Each peak represents the fluorescent intensity, which increases continuously over time due to cell activity and is therefore more intense at the end of the experiment (t 30 ). ( C , D ) represent the average fluorescent intensity in single cells over the 30 min period, acquiring images at 1 min intervals. ( E , G ), bar graphs quantifying the increase in fluorescence from t 0 to t 30 in cells from P30 and P120 mice, respectively. The average ratio of ROS production, calculated as the slope of the fluorescence increase, is shown in ( F , H ). Data represent the mean ± SEM of the number of cells shown in parentheses from four cultures of the same number of animals. The statistical analysis was performed using a Student´s t test: * p < 0.05; ** p < 0.01; *** p < 0.001 vs. WT.

Article Snippet: Ultrathin sections of adrenal glands were etched in 0.1 N HCl for 5 min, rinsed three times for 5 min each in Tris buffered saline (TBS: 20 mM TRIS, 140 mM NaCl, and 2.7 mM KCl, pH 8.0), treated with blocking buffer (0.1% gelatin, 1% normal goat serum (NGS) and 0.3% Triton X-100 in TBS) for 30 min, incubated for 2 h at room temperature with the primary antibody (anti-hSOD1 G93A , 1:500; Medimabs, C4F6, Montréal, QC, Canada).

Techniques: Produced, Activity Assay, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Progressive Mitochondrial SOD1 G93A Accumulation Causes Severe Structural, Metabolic and Functional Aberrations through OPA1 Down-Regulation in a Mouse Model of Amyotrophic Lateral Sclerosis

doi: 10.3390/ijms22158194

Figure Lengend Snippet: Bioenergetic impairment in chromaffin cells of SOD1 G93A mice. The bioenergetic profile of oxygen consumption was analyzed in primary CC cultures from WT (blue line) and SOD1 G93A (red line) mice using a seahorse XFp analyzer. The mitochondria stress test was performed on CCs from P30 ( A ) and P120 ( B ) mice, analyzing the oxygen consumption rate (OCR) during the application of drugs targeting the mitochondria: oligomycin, FCCP and rotenone/antimycin A. This procedure allowed us to extract the following parameters in ( C ): ( a ), basal respiration; ( b ), proton leak; ( c ), maximal respiration, ( d ), spare respiratory capacity; ( e ), non-mitochondrial oxygen consumption; ( f ), ATP production; ( g ), percentage coupling efficiency and ( h ); percentage spare respiratory capacity. The bar graphs represent the mean ± SEM of measurements from at least three different cultures, and the statistical analysis was performed using a two-way ANOVA with Šídák’s multiple comparison post hoc test: * p < 0.05; ** p < 0.01; *** p < 0.001 vs. WT at the same age. ## p < 0.01 comparing the effect of aging in the WT or SOD1 G93A group (panel Cc, Cd, Ce, Ch). These parameters are summarized in the .

Article Snippet: Ultrathin sections of adrenal glands were etched in 0.1 N HCl for 5 min, rinsed three times for 5 min each in Tris buffered saline (TBS: 20 mM TRIS, 140 mM NaCl, and 2.7 mM KCl, pH 8.0), treated with blocking buffer (0.1% gelatin, 1% normal goat serum (NGS) and 0.3% Triton X-100 in TBS) for 30 min, incubated for 2 h at room temperature with the primary antibody (anti-hSOD1 G93A , 1:500; Medimabs, C4F6, Montréal, QC, Canada).

Techniques: Comparison

Journal: International Journal of Molecular Sciences

Article Title: Progressive Mitochondrial SOD1 G93A Accumulation Causes Severe Structural, Metabolic and Functional Aberrations through OPA1 Down-Regulation in a Mouse Model of Amyotrophic Lateral Sclerosis

doi: 10.3390/ijms22158194

Figure Lengend Snippet: Bioenergetic parameters obtained by the Seahorse method.

Article Snippet: Ultrathin sections of adrenal glands were etched in 0.1 N HCl for 5 min, rinsed three times for 5 min each in Tris buffered saline (TBS: 20 mM TRIS, 140 mM NaCl, and 2.7 mM KCl, pH 8.0), treated with blocking buffer (0.1% gelatin, 1% normal goat serum (NGS) and 0.3% Triton X-100 in TBS) for 30 min, incubated for 2 h at room temperature with the primary antibody (anti-hSOD1 G93A , 1:500; Medimabs, C4F6, Montréal, QC, Canada).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Progressive Mitochondrial SOD1 G93A Accumulation Causes Severe Structural, Metabolic and Functional Aberrations through OPA1 Down-Regulation in a Mouse Model of Amyotrophic Lateral Sclerosis

doi: 10.3390/ijms22158194

Figure Lengend Snippet: Human SOD1 G93A expression in mouse chromaffin cells. Human SOD1 G93A expression was explored in CCs by immunofluorescence and in Western blots. ( A ) Confocal images of two isolated CCs obtained from WT and SOD1 G93A mice at presymptomatic stages (P30). ( B ) Confocal images obtained at the symptomatic (P120) stage. The cytoskeleton was labeled with Alexa Fluor 568 phalloidin (red), the nucleus with DAPI (blue) and the mutated protein with an anti-human SOD1 G93A antibody C4F6 (green). ( C ) Bar graph depicting the mean fluorescence for hSOD1 G93A measured in at least three P30 and P120 CC cultures. Data in the graph reflect the epifluorescence intensity, and the insets above each bar show the immunofluorescence from a representative cell: *** p < 0.001 vs. WT at same age; ### p < 0.001 vs. SOD1 G93A P30. ( D ) Immunoblot showing hSOD1 G93A expression in adrenal gland medulla, using β-actin as a loading control. ( E ) Quantitative analysis of the mutated protein expression calculated as the hSOD1 G93A /β-actin ratio: ** p < 0.01. The bar graph represents the mean ± SEM of at least five experiments, and statistical analysis was performed using an unpaired Student´s t test.

Article Snippet: Ultrathin sections of adrenal glands were etched in 0.1 N HCl for 5 min, rinsed three times for 5 min each in Tris buffered saline (TBS: 20 mM TRIS, 140 mM NaCl, and 2.7 mM KCl, pH 8.0), treated with blocking buffer (0.1% gelatin, 1% normal goat serum (NGS) and 0.3% Triton X-100 in TBS) for 30 min, incubated for 2 h at room temperature with the primary antibody (anti-hSOD1 G93A , 1:500; Medimabs, C4F6, Montréal, QC, Canada).

Techniques: Expressing, Immunofluorescence, Western Blot, Isolation, Labeling, Fluorescence, Control

Journal: International Journal of Molecular Sciences

Article Title: Progressive Mitochondrial SOD1 G93A Accumulation Causes Severe Structural, Metabolic and Functional Aberrations through OPA1 Down-Regulation in a Mouse Model of Amyotrophic Lateral Sclerosis

doi: 10.3390/ijms22158194

Figure Lengend Snippet: Human SOD1 G93A -mitochondria colocalization in chromaffin cells during disease instauration: Confocal cross-section of a fixed CC labeled with MitoTracker Red, the anti-human SOD1 G93A antibody (green) and DAPI (Blue) at P30 ( A ) and P120 ( B ). Colocalization areas of the red and green signal are seen in yellow. The individual signals within the white inset are shown above the merge image, in which hSOD1 G93A and MitoTracker colocalization is highlighted in white. ( C ) The bar graph shows the hSOD1 G93A -mitochondrial colocalization normalized to the mitochondrial signal: *** p < 0.001; Mann–Whitney test. Data are the means ± SEM of the number of cells from two cultures shown in parentheses.

Article Snippet: Ultrathin sections of adrenal glands were etched in 0.1 N HCl for 5 min, rinsed three times for 5 min each in Tris buffered saline (TBS: 20 mM TRIS, 140 mM NaCl, and 2.7 mM KCl, pH 8.0), treated with blocking buffer (0.1% gelatin, 1% normal goat serum (NGS) and 0.3% Triton X-100 in TBS) for 30 min, incubated for 2 h at room temperature with the primary antibody (anti-hSOD1 G93A , 1:500; Medimabs, C4F6, Montréal, QC, Canada).

Techniques: Labeling, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Progressive Mitochondrial SOD1 G93A Accumulation Causes Severe Structural, Metabolic and Functional Aberrations through OPA1 Down-Regulation in a Mouse Model of Amyotrophic Lateral Sclerosis

doi: 10.3390/ijms22158194

Figure Lengend Snippet: Subcellular localization of the human SOD1 G93A . TEM images showing the distribution of human SOD1 G93A recognized by the C4F6 antibody and a secondary antibody conjugated to 10 nm gold particles. The gold particles were detected in different mitochondrial compartments (intermembrane space, cristae and mitochondrial matrix), both at P30 ( A , B ) and P120 ( C , D ). The human SOD1 G93A protein also accumulates in other organelles, such as ( E ) the smooth endoplasmic reticulum (ER), which shows a large accumulation, and ( F ), the nucleus (N). The human SOD1 G93A was also found in the cytosol, lysosomes, peroxisomes and different chromaffin granules (images not shown). Gold particles are indicated by white arrows, while the larger accumulations are signaled with a white asterisk ( A , C ). Magnification of the selected areas in each picture are represented as ( a’ – f’ ). Scale bar = 500 nm.

Article Snippet: Ultrathin sections of adrenal glands were etched in 0.1 N HCl for 5 min, rinsed three times for 5 min each in Tris buffered saline (TBS: 20 mM TRIS, 140 mM NaCl, and 2.7 mM KCl, pH 8.0), treated with blocking buffer (0.1% gelatin, 1% normal goat serum (NGS) and 0.3% Triton X-100 in TBS) for 30 min, incubated for 2 h at room temperature with the primary antibody (anti-hSOD1 G93A , 1:500; Medimabs, C4F6, Montréal, QC, Canada).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Progressive Mitochondrial SOD1 G93A Accumulation Causes Severe Structural, Metabolic and Functional Aberrations through OPA1 Down-Regulation in a Mouse Model of Amyotrophic Lateral Sclerosis

doi: 10.3390/ijms22158194

Figure Lengend Snippet: Optic atrophy protein 1 (OPA1) expression is down-regulated in the SOD1 G93A mice adrenal medulla from presymptomatic stages, with changes in its cleavage with aging. ( A ) OPA1 expression was analyzed in Western blots of total protein extracted from the adrenal medulla of WT and SOD1 G93A mice at P30 and P120. ( B ) Densitometry of the total OPA1 normalized to β-actin ( n = 4, two-way ANOVA with Šídák’s multiple comparison post hoc test, * p < 0.05 relative to WT at same stage). ( C ) Densitometry of the OPA1 bands in A. The first peak of the densitometry represents the long OPA1 isoform (L-OPA1), while the second peak represents the short OPA1 isoform (S-OPA1). ( D ) Bar-chart quantifying the ratio of the OPA1 isoforms ratio measured as the short isoform relative to the long isoform: ### p < 0.001 relative to the data at P30.

Article Snippet: Ultrathin sections of adrenal glands were etched in 0.1 N HCl for 5 min, rinsed three times for 5 min each in Tris buffered saline (TBS: 20 mM TRIS, 140 mM NaCl, and 2.7 mM KCl, pH 8.0), treated with blocking buffer (0.1% gelatin, 1% normal goat serum (NGS) and 0.3% Triton X-100 in TBS) for 30 min, incubated for 2 h at room temperature with the primary antibody (anti-hSOD1 G93A , 1:500; Medimabs, C4F6, Montréal, QC, Canada).

Techniques: Expressing, Western Blot, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Progressive Mitochondrial SOD1 G93A Accumulation Causes Severe Structural, Metabolic and Functional Aberrations through OPA1 Down-Regulation in a Mouse Model of Amyotrophic Lateral Sclerosis

doi: 10.3390/ijms22158194

Figure Lengend Snippet: Schematic representation of mitochondrial defects observed in the chromaffin cell of an SOD1 G93A mouse. The split-cell drawing represents our observations in the CC of WT mice ( upper part ) and the transgenic SOD1 G93A mice ( bottom ). In the cytoplasm of WT CC, there is one healthy mitochondrion with an inset for one cristae magnification ( right ). In the cristae is depicted the location of the OPA1 protein (both S and L isoforms, in blue), the complexes of the electronic transport chain (ETC) and F 1 F 0 -ATP synthase. The optimal management of the cristae junction size allows the proton gradient and the maintenance of m ψ. Otherwise, alterations observed in SOD1 G93A mice are represented in the bottom part by (1) greater number and smaller size mitochondria; (2) accumulation of the mutated protein hSOD1 G93A in the cytoplasm at all mitochondria levels and in the nucleus; (3) formation of hSOD1 G93A protein aggregates; (4) increased production of free radicals; (5) decrease of OPA1 gene expression; (6) increase of cristae junctions size; (7) diffusion of cristae components and ions towards the intermembrane space; (8) diffusion of big molecules and proteins as hSOD1 G93A inside cristae; (9) loss of m ψ with OXPHOS deficiency and (10) decrease in the ATP generation. The deficiencies here present lead to an autonomic nervous system impairment, with inability to overcome stressful events, defects in the bioenergetics profile and inefficient energy consumption. These alterations could be crucial for cell-specific and also patient survival, displaying novel pathological mechanisms and therapeutic targets to overcome ALS.

Article Snippet: Ultrathin sections of adrenal glands were etched in 0.1 N HCl for 5 min, rinsed three times for 5 min each in Tris buffered saline (TBS: 20 mM TRIS, 140 mM NaCl, and 2.7 mM KCl, pH 8.0), treated with blocking buffer (0.1% gelatin, 1% normal goat serum (NGS) and 0.3% Triton X-100 in TBS) for 30 min, incubated for 2 h at room temperature with the primary antibody (anti-hSOD1 G93A , 1:500; Medimabs, C4F6, Montréal, QC, Canada).

Techniques: Transgenic Assay, Gene Expression, Diffusion-based Assay, Biomarker Discovery

Journal: PLoS ONE

Article Title: EGFR Inhibitor Erlotinib Delays Disease Progression but Does Not Extend Survival in the SOD1 Mouse Model of ALS

doi: 10.1371/journal.pone.0062342

Figure Lengend Snippet: Kaplan-Meier survival plot showing the lifespan of the SOD1 Tg mice treated with vehicle (blue) or erlotinib (red). The lifespan of each mouse is represented by a black dot. All mice were included in the statistical analysis. Mice that were censored in the analysis because of a non-ALS death are represented by dots that are not accompanied by a lowering of the curve.

Article Snippet: SOD1 G93A high copy number transgenic mice (SOD1 Tg) and their non-transgenic littermates (SOD1 WT) were bred at Genentech.

Techniques:

Journal: PLoS ONE

Article Title: EGFR Inhibitor Erlotinib Delays Disease Progression but Does Not Extend Survival in the SOD1 Mouse Model of ALS

doi: 10.1371/journal.pone.0062342

Figure Lengend Snippet: Censoring information for survival study: animal n by cause of death/euthanasia for SOD1 Tg mice.

Article Snippet: SOD1 G93A high copy number transgenic mice (SOD1 Tg) and their non-transgenic littermates (SOD1 WT) were bred at Genentech.

Techniques: Injection

Journal: Stem Cells International

Article Title: Human Motor Neuron Progenitor Transplantation Leads to Endogenous Neuronal Sparing in 3 Models of Motor Neuron Loss

doi: 10.4061/2011/207230

Figure Lengend Snippet: hMNP transplantation spares endogenous neurons. (a) Comparison of NeuN positive cells in cranial ( n = 13 hMNP; n = 15 vehicle) and caudal ( n = 14 hMNP; n = 15 vehicle) transverse sections in SCI animals. (b) Comparison of ChAT positive cells in cranial ( n = 10 hMNP; n = 12 vehicle) and caudal ( n = 9 hMNP; n = 12 vehicle) transverse sections of ventral horns in SCI animals. (c) Comparison of NeuN positive cells in cranial transverse sections of SOD1 G93A animals ( n = 5 hMNP; n = 5 vehicle). (d) Comparison of NeuN positive cells in cranial ( n = 3 hMNP; n = 3 vehicle) and caudal ( n = 3 hMNP; n = 2 vehicle) transverse sections of Δ7SMN animals. NeuN stained histological sections of lumbar spinal cord from hMNP (e) and vehicle (f) injected into SOD1 G93A mice clearly show a difference in neuronal numbers. Data is expressed as mean ± standard error. * P < .05. Bar = 50 μ m.

Article Snippet: The SOD1 G93A mutant mice were supplied and bred for ALS-TDI by GTC Biotherapeutics.

Techniques: Transplantation Assay, Comparison, Staining, Injection

Journal: Frontiers in Physiology

Article Title: Effect of Mutated Cu, Zn Superoxide Dismutase (SOD1 G93A ) on Modulation of Transductional Pathway Mediated by M1 Muscarinic Receptor in SK-N-BE and NSC-34 Cells

doi: 10.3389/fphys.2018.00611

Figure Lengend Snippet: (A) Presence of muscarinic acetylcholine M1 receptor in human neuroblastoma SK-NBE, oligodendrocytes MO3-13 and neuroblastoma-spinal motor neuron fusion cells NSC-34 M1 receptor protein bands normalized to α-tubulin. Effect of SOD1 on ERK1-2 (B) and AKT (C) activation in NSC-34 cells incubated with 400 ng/ml of SOD1 for 10 and 30 min. The data represent the means ± SEM of three independent experiments relative to control obtained by densitometric analysis of P-ERK1-2 and P-AKT protein bands normalized to ERK1-2 and AKT, respectively. ∗ p < 0.05 vs. Ctr; ∗∗ p < 0.001 vs. Ctr; # p < 0.001 vs. SOD1 10 min.

Article Snippet: Mutated form, SOD1 G93A , was provided by the “Recombinant Protein Service” of PRIMM srl (Milano, Italy).

Techniques: Activation Assay, Incubation, Control

Journal: Frontiers in Physiology

Article Title: Effect of Mutated Cu, Zn Superoxide Dismutase (SOD1 G93A ) on Modulation of Transductional Pathway Mediated by M1 Muscarinic Receptor in SK-N-BE and NSC-34 Cells

doi: 10.3389/fphys.2018.00611

Figure Lengend Snippet: Activation of ERK1-2 and AKT trasductional pathways in presence of SOD1 wt and SOD1 G93A in SK-N-BE cells. Western blotting analysis of P-ERK (A) and P-AKT (B) in cells incubated with 400 ng/ml of SOD1 wt and SOD1 G93A for 10 and 30 min. The histograms show the mean values (+SE) evaluated by densitometric analysis of three independent experiments. The results were normalized to ERK1-2 and AKT respectively. ∗ p < 0.05 vs. Ctr; ∗∗ p < 0.001 vs. Ctr; # p < 0.05 vs. 10 min SOD1 wt ; ## p < 0.001 vs. 10 min SOD1 wt ; ς p < 0.05 vs. 30 min SOD1 wt .

Article Snippet: Mutated form, SOD1 G93A , was provided by the “Recombinant Protein Service” of PRIMM srl (Milano, Italy).

Techniques: Activation Assay, Western Blot, Incubation

Journal: Frontiers in Physiology

Article Title: Effect of Mutated Cu, Zn Superoxide Dismutase (SOD1 G93A ) on Modulation of Transductional Pathway Mediated by M1 Muscarinic Receptor in SK-N-BE and NSC-34 Cells

doi: 10.3389/fphys.2018.00611

Figure Lengend Snippet: Activation of PLC-PKC-ERK1-2-AKT-dependent transduction pathway in presence of SOD1 wt and SOD1 G93A in NSC-34 cells. Western Blotting analysis of the levels of P-ERK 1-2 (A) and P-AKT (B) in NSC-34 cells incubated with 400 ng/ml of SOD1 wt and with 400 ng/ml of SOD1 G93A for times of 10 and 30 min. The data represent the means ± SEM of three independent experiments relative to control obtained by densitometric analysis of P-ERK1-2 and P-AKT protein bands normalized to ERK1-2 and AKT, respectively. ∗ p < 0.05 vs. Ctr; ∗∗ p < 0.001 vs. Ctr; # p < 0.05 vs. 10 min SOD1 wt ; ## p < 0.001 vs. 10 min SOD1 wt ; ς p < 0.05 vs. 30 min SOD1 wt .

Article Snippet: Mutated form, SOD1 G93A , was provided by the “Recombinant Protein Service” of PRIMM srl (Milano, Italy).

Techniques: Activation Assay, Transduction, Western Blot, Incubation, Control

Journal: Frontiers in Physiology

Article Title: Effect of Mutated Cu, Zn Superoxide Dismutase (SOD1 G93A ) on Modulation of Transductional Pathway Mediated by M1 Muscarinic Receptor in SK-N-BE and NSC-34 Cells

doi: 10.3389/fphys.2018.00611

Figure Lengend Snippet: Activation of PLC-PKC-ERK 1-2-AKT-dependent transduction pathway in presence of SOD1 wt and SOD1 G93A . Western Blotting analysis of the levels of P-ERK 1-2 and P-AKT in SK-N-BE (A,B) and NSC-34 (C,D) cells incubated for 10 min with 400 ng/ml of SOD1 wt and with 400 ng/ml of SOD1 G93A in absence and in presence of Pirenzepine 10 μM for 5 min. The data represent the means ± SEM of three independent experiments relative to control obtained by densitometric analysis of P-ERK1/2 and P-AKT protein bands normalized to ERK1-2 and AKT respectively. ∗ p < 0.05 vs. Ctr; ∗∗ p < 0.01 vs. Ctr; ∗∗∗ p < 0.001 vs. Ctr; # p < 0.05 vs. SOD1 wt ; ## p < 0.001 vs. SOD1 wt ; ° p < 0.01 vs. SOD1 G93A .

Article Snippet: Mutated form, SOD1 G93A , was provided by the “Recombinant Protein Service” of PRIMM srl (Milano, Italy).

Techniques: Activation Assay, Transduction, Western Blot, Incubation, Control

Journal: Frontiers in Physiology

Article Title: Effect of Mutated Cu, Zn Superoxide Dismutase (SOD1 G93A ) on Modulation of Transductional Pathway Mediated by M1 Muscarinic Receptor in SK-N-BE and NSC-34 Cells

doi: 10.3389/fphys.2018.00611

Figure Lengend Snippet: Fluorimetric Determination of Reactive oxygen species (ROS) levels. SK-N-BE (A) and NSC-34 (B) semiconfluent cells were incubated for 18 h in medium containing 0.2% FBS and then for 30 min with 400 ng/ml SOD1 wt and mutated SOD1 G93A . To determine Reactive Oxygen Species (ROS) levels, SK-N-BE (A) and NSC-34 (B) cells were incubated with 10 μM of the ROS sensitive probe DCHF-DA and ROS levels were measured by fluorometric analysis. The graphs on the left show the mean ± SEM values ( n = 6) relative to control of the indicated time points. The histograms on the right show the values of 30 min point and the relative ANOVA analysis. ∗ p < 0.05 vs. Ctr; ∗∗ p < 0.001 vs. Ctr; # p < 0.001 vs. SOD wt .

Article Snippet: Mutated form, SOD1 G93A , was provided by the “Recombinant Protein Service” of PRIMM srl (Milano, Italy).

Techniques: Incubation, Control

Journal: Frontiers in Physiology

Article Title: Effect of Mutated Cu, Zn Superoxide Dismutase (SOD1 G93A ) on Modulation of Transductional Pathway Mediated by M1 Muscarinic Receptor in SK-N-BE and NSC-34 Cells

doi: 10.3389/fphys.2018.00611

Figure Lengend Snippet: Effect of SOD1 wt and mutated SOD1 G93A on intracellular calcium levels in neuroblastoma SK-N-BE cells (A) and in NSC-34 cells (B) . The data are compared with control and with carbachol-induced intracellular calcium increase (positive control). The graphs on the left show the mean ± SEM values ( n = 6) relative to control of the indicated time points. The histograms on the right show the values of 10 s point and the relative ANOVA analysis. ∗ p < 0.001 vs. Ctr; # p < 0.01 vs. SOD wt .

Article Snippet: Mutated form, SOD1 G93A , was provided by the “Recombinant Protein Service” of PRIMM srl (Milano, Italy).

Techniques: Control, Positive Control

Journal: Frontiers in Physiology

Article Title: Effect of Mutated Cu, Zn Superoxide Dismutase (SOD1 G93A ) on Modulation of Transductional Pathway Mediated by M1 Muscarinic Receptor in SK-N-BE and NSC-34 Cells

doi: 10.3389/fphys.2018.00611

Figure Lengend Snippet: SK-N-BE and NSC-34 semiconfluent cells were preincubated with 15 μM of pirenzepine or with 10 μM of BAPTA-AM for 30 and 5 min respectively. Then, the cells were treated for 4 h with 400 ng/ ml of SOD1 wt or mutated SOD1 G93A . Induction of apoptosis by SOD1 G93A on SK-N-BE (A) and NSC-34 (B) cells is showed by Western Blotting analysis of cleaved form of PARP-1 protein bands. Evaluation of cell viability by trypan blue assay in SK-N-BE and NSC-34 cells are showed in (C,D) , respectively; the data are reported as per cent variation compared to control. The data represent the means ± SEM relative to control obtained by densitometric analysis of cleaved PARP protein bands normalized to α-tubulin of three independent experiments. ∗ p < 0.005 vs. Ctr; # p < 0.005 vs. SOD G93A .

Article Snippet: Mutated form, SOD1 G93A , was provided by the “Recombinant Protein Service” of PRIMM srl (Milano, Italy).

Techniques: Western Blot, Control