Journal: Neural Regeneration Research

Article Title: Microglial depletion impairs glial scar formation and aggravates inflammation partly by inhibiting STAT3 phosphorylation in astrocytes after spinal cord injury

doi: 10.4103/1673-5374.357912

Figure Lengend Snippet: PLX3397 attenuates astrocytic scar formation and increases the inflammatory response following SCI. (A) Representative images of astrocytic scars in sagittal sections of the spinal cord at 14 days after spinal cord injury (SCI). In SCI mice, astrocytes surrounding the lesion area packed tightly together as an astrocytic scar barrier, and this astrocytic front was less prominent in mice with PLX3397 treatment. Scale bars: 50 μm. (B) In the SCI mice, the spread of CD68 + inflammatory cells was restricted by a dense glial scar, whereas the boundary of the PLX3397-treated mice was disrupted, and a significant infiltration of CD68 + cells was observed. Scale bars: 50 μm. (C) aCasp3/NeuN costaining in sagittal sections indicating apoptotic neurons. Scale bars: 50 μm. (D) PLX3397 treatment increased the lesion size (calculated as GFAP-negative staining area). Microglia elimination increased the concentration of the proinflammatory cytokines (E) TNF-α, (F) IL-6, and (G) IL-1β at 14 days post-injury (dpi). (H) The proportion of NeuN + /aCasp3 + cells. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance with Tukey’s post hoc test for E–G; unpaired t -test for D and H). Data are expressed as the mean ± SD. The experiments were repeated six times. aCasp3: Activated caspase-3; Ctrl: control; DAPI: 4′,6-diamidino-2-phenylindole; IL: interleukin; NeuN: neuronal nuclei; PLX3397: colony-stimulating factor 1 receptor inhibitor; SCI: spinal cord injury; TNF-α: tumor necrosis factor-α.

Article Snippet: Sections were deparaffinized and rehydrated and then incubated with the primary antibodies mouse anti-GFAP (1:1000, Millipore, Cat# MAB360, RRID: AB_11212597), rabbit anti-GFAP (1:1000, Sigma, Cat# SAB4501162, RRID: AB_10746077), rabbit anti-ionized calcium binding adaptor molecule 1 (Iba1; 1:1000, Abcam, Cat# ab153696, RRID: AB_2889406), mouse anti-neuronal nuclei (NeuN; 1:1000, Millipore, Cat# MAB377, RRID: AB_2298772), mouse anti-CD68 (1:200, Millipore, Cat# MAB1435), rabbit anti-activated caspase-3 (aCasp3; 1:100, Cell Signaling Technology, Cat# 9661, RRID: AB_2341188), mouse anti-Olig2 (1:1000, Millipore, Cat# MABN50, RRID: AB_10807410) and rabbit anti-pStat3 (1:200, Cell Signaling Technology, Cat# 9145, RRID: AB_2491009) at 4°C overnight.

Techniques: Negative Staining, Concentration Assay

Journal: Nutrients

Article Title: Re-Visiting Antioxidant Therapy in Murine Advanced Atherosclerosis with Brussels Chicory, a Typical Vegetable in Mediterranean Diets

doi: 10.3390/nu15040832

Figure Lengend Snippet: Brussels chicory consumption reduces plaque apoptosis without affecting the content of macrophages and smooth muscle cells in aortic root plaques in Apoe −/− mice. ( A – C ) Representative immunohistochemistry images of cleaved CASP3 (( A ), left panel), CD68 (( B ), left panel), and ACTA2 (( C ), left panel) in aortic root sections and quantification of the percentage of cleaved CASP3 (( A ), right panel), CD68 (( B ), right panel), and ACTA2 positive cells (( C ), right panel) in aortic root plaques. Scale bar, 100 μm. Data are presented as means ± SD. n = 15. * Significantly different from the control group ( p < 0.05). Scale bars: 100 μm. ACTA2, actinα2; Apoe, apolipoprotein E; Chicory, Brussels chicory; NS, nonsignificant ( p ≥ 0.05).

Article Snippet: Primary antibodies used were rat polyclonal anti-CD68 (14-0681-82, Invitrogen, Waltham, MA, USA), rabbit polyclonal anti-actin α 2 (ACTA2; PA5-85070, Invitrogen), and rabbit polyclonal anti-cleaved CASP3 (9664, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Immunohistochemistry

Journal: Toxics

Article Title: L-Ascorbic Acid 2-Phosphate Attenuates Methylmercury-Induced Apoptosis by Inhibiting Reactive Oxygen Species Accumulation and DNA Damage in Human SH-SY5Y Cells

doi: 10.3390/toxics11020144

Figure Lengend Snippet: A cell damage model of MeHg was created by the treatment of SH-SY5Y cells with 0–4 μM MeHg at indicated time intervals. ( A ) MTT assay showing cell viability of SH-SY5Y cells treated with 0–4 μM MeHg for 6–48 h ( B ) The live cell workstation used to observe cell morphology of SH-SY5Y cells treated with 0–2 μM MeHg for 24 h. ( C ) Flow cytometry detection of apoptosis in 0–2 μM MeHg treated SH-SY5Y cells for 24 h. ( D ) GraphPad Prism 8.0.2 software was used to analyze the total apoptosis rate of SH-SY5Y cells treated with 0–2 μM MeHg for 24 h. Statistical significance was determined using ANOVA; p < 0.05. ( E ) Western blot analysis of the protein expression levels of PARP1, AIF, PAR, Cyt C, and cleaved Caspase-3 in SH-SY5Y cells treated with 0–2 μM MeHg for 24 h. ( F ) The Tanon-2500 fully automated digital gel image analysis system was used to analyze the net optical density of the Western blot strips. All values are represented as means ± SD ( n = 3). Superscript denotes a statistically significant difference between groups ( p < 0.05). Abbreviations: AIF, apoptosis-inducing factor; ANOVA, analysis of variance; Cyto C, cytochrome C; MeHg, methylmercury; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PAR, poly (adp-ribose); PARP1, poly (adp-ribose) polymerase 1; SH-SY5Y, neuroblastoma cells.

Article Snippet: The membranes were incubated in 5% non-fat milk powder (Anchor, Auckland City, New Zealand) at room temperature for 1 h. The membranes were then incubated with the following primary antibodies: PARP1 rabbit mAb (1:1000; CST), AIF rabbit mAb (1:1000, CST), Cleave Caspase-3 rabbit mAb (1:1000, CST), γH2AX rabbit mAb (1:1000, CST), Cyt C rabbit mAb (1:1000; CST), KU70 rabbit mAb (1:1000; CST), BRCA1 rabbit mAb (1:1000; CST), RAD51 rabbit mAb (1:1000; CST), GAPDH mouse mAb (1:5000; ProteinTech Group Inc.), HRP-conjugated AffiniPure Goat Anti-Rabbit IgG (H + L) (1:10,000; ProteinTech Group Inc., Wuhan East Lake New Technology Development Zone, China), and HRP-conjugated AffiniPure Goat Anti-Mouse IgG (H + L) (1:10,000; Protein-Tech Group Inc., Wuhan East Lake New Technology Development Zone, China).

Techniques: MTT Assay, Flow Cytometry, Software, Western Blot, Expressing

Journal: Toxics

Article Title: L-Ascorbic Acid 2-Phosphate Attenuates Methylmercury-Induced Apoptosis by Inhibiting Reactive Oxygen Species Accumulation and DNA Damage in Human SH-SY5Y Cells

doi: 10.3390/toxics11020144

Figure Lengend Snippet: MeHg induces DNA damage and participates in both NHEJ repair and induced apoptosis in SH-SY5Y cells ( A ) The alkaline comet assay detects cell trailing in 1 μM MeHg-treated SH-SY5Y cells from 0–24 h. The images were photographed with a Leica fluorescence microscope. ( B ) CASP software analyses of comet image and the Olive tail moment can be used to determine the extent of DNA damage. ( C ) Western blot analysis detected the protein expression levels of γH2AX, BRCA1, RAD51, KU70, PARP1, AIF, PAR, Cyt C, and cleaved Caspase-3 in SH-SY5Y cells treated with 1 μM MeHg from 0–24 h. ( D ) The Tanon-2500 fully automated digital gel image analysis system was used to analyze the net optical density of the Western blot strips. The mean was calculated using GraphPad Prism 8.0.2 software. The data shown represent the mean from one of three independent experiments. Statistical significance was determined using variance (ANOVA). All values are represented as means ± SD ( n = 3). Superscript denotes a statistically significant difference between groups ( p < 0.05). Abbreviations: BRCA1 and RAD51, homologous repair-associated repair proteins; HR, homologous recombination; KU70, non-homologous end joining-associated repair protein; NHEJ, nonhomologous end joining; γH2AX, phosphorylated histone H2AX.

Article Snippet: The membranes were incubated in 5% non-fat milk powder (Anchor, Auckland City, New Zealand) at room temperature for 1 h. The membranes were then incubated with the following primary antibodies: PARP1 rabbit mAb (1:1000; CST), AIF rabbit mAb (1:1000, CST), Cleave Caspase-3 rabbit mAb (1:1000, CST), γH2AX rabbit mAb (1:1000, CST), Cyt C rabbit mAb (1:1000; CST), KU70 rabbit mAb (1:1000; CST), BRCA1 rabbit mAb (1:1000; CST), RAD51 rabbit mAb (1:1000; CST), GAPDH mouse mAb (1:5000; ProteinTech Group Inc.), HRP-conjugated AffiniPure Goat Anti-Rabbit IgG (H + L) (1:10,000; ProteinTech Group Inc., Wuhan East Lake New Technology Development Zone, China), and HRP-conjugated AffiniPure Goat Anti-Mouse IgG (H + L) (1:10,000; Protein-Tech Group Inc., Wuhan East Lake New Technology Development Zone, China).

Techniques: Alkaline Single Cell Gel Electrophoresis, Fluorescence, Microscopy, Software, Western Blot, Expressing, Homologous Recombination, Non-Homologous End Joining

Journal: Toxics

Article Title: L-Ascorbic Acid 2-Phosphate Attenuates Methylmercury-Induced Apoptosis by Inhibiting Reactive Oxygen Species Accumulation and DNA Damage in Human SH-SY5Y Cells

doi: 10.3390/toxics11020144

Figure Lengend Snippet: AA2P regulation of the PARP1/AIF and Caspase-3 pathways during MeHg-induced apoptosis of SH-SY5Y cells. ( A ) Flow cytometry detection of apoptosis in SH-SY5Y cells co-treated with 300 μM AA2P and 1 μM MeHg for 12 h. ( B ) GraphPad Prism 8 software analysis of the apoptosis rate of SH-SY5Y cells co-treated with 300 μM AA2P and 1 μM MeHg for 12 h. Statistical significance was determined using ANOVA, p < 0.05. ( C ) Flow cytometry detection of apoptosis in SH-SY5Y cells co-treated with 300 μM AA2P and 1 μM MeHg for 24 h. ( D ) GraphPad Prism 8.0.2 software analysis of the apoptosis rate of SH-SY5Y cells co-treated with 300 μM AA2P and 1μM MeHg for 24 h. Statistical significance was determined using ANOVA, p < 0.05. ( E ) Western blot analysis of PARP1, PAR, AIF, Cyt C, and cleaved Caspase-3 in SH-SY5Y cells co-treated with 300 μM AA2P and 1 μM MeHg for 24 h. ( F ) The Tanon-2500 fully automated digital gel image analysis system was used to analyze the net optical density of the Western blot strips. All values are represented as means ± SD ( n = 3).

Article Snippet: The membranes were incubated in 5% non-fat milk powder (Anchor, Auckland City, New Zealand) at room temperature for 1 h. The membranes were then incubated with the following primary antibodies: PARP1 rabbit mAb (1:1000; CST), AIF rabbit mAb (1:1000, CST), Cleave Caspase-3 rabbit mAb (1:1000, CST), γH2AX rabbit mAb (1:1000, CST), Cyt C rabbit mAb (1:1000; CST), KU70 rabbit mAb (1:1000; CST), BRCA1 rabbit mAb (1:1000; CST), RAD51 rabbit mAb (1:1000; CST), GAPDH mouse mAb (1:5000; ProteinTech Group Inc.), HRP-conjugated AffiniPure Goat Anti-Rabbit IgG (H + L) (1:10,000; ProteinTech Group Inc., Wuhan East Lake New Technology Development Zone, China), and HRP-conjugated AffiniPure Goat Anti-Mouse IgG (H + L) (1:10,000; Protein-Tech Group Inc., Wuhan East Lake New Technology Development Zone, China).

Techniques: Flow Cytometry, Software, Western Blot

Journal: Frontiers in Oncology

Article Title: Pancreatic cancer derived 3D organoids as a clinical tool to evaluate the treatment response

doi: 10.3389/fonc.2022.1072774

Figure Lengend Snippet: Expression of caspase 3 an apoptotic marker in vivo tumor tissues and organoids. (A) , untreated control and after treating with 100 µM 3-BP (C) ; Tumor organoids untreated (B) , and after treating with 100 µM 3-BP antitumor drug (D) .

Article Snippet: The blocking reagent was removed, and the tumor tissues and organoids were incubated with either: 1) a primary antibody anti-mouse vimentin (Biolegend, USA) in a 1:100 dilution, or 2) with primary antibody anti-rabbit cleaved caspase-3 (Cell Signaling Technology; Danvers, MA) in a 1:300 dilution in a cold room overnight.

Techniques: Expressing, Marker, In Vivo

Journal: Neural Development

Article Title: Lrig1 expression identifies quiescent stem cells in the ventricular-subventricular zone from postnatal development to adulthood and limits their persistent hyperproliferation

doi: 10.1186/s13064-022-00169-1

Figure Lengend Snippet: Lrig1 knock-out resulted in persistent hyperproliferation in the lateral wall even in old mice. A - C KI-67, ASCL1, or DCX immunoreactivity in 1 year and 3 month-old mice. Scale bar, 100 μm. D - E Graphs of KI-67+ and ASCL1+ cell counts. Mean ± standard deviation. Student’s t test. F-H KI-67, ASCL1, or DCX immunoreactivity in 2 years and 4 month-old mice. Scale bar, 100 μm. I-J Graphs of KI-67+ and ASCL1+ cell counts. Mean ± standard deviation. Student’s t test. K-N KI-67, ASCL1, DCX, or cleaved CASP3 immunoreactivity in 1 year and 8 month-old mice. Scale bar, 100 μm

Article Snippet: ASCL1 (MASH1) mouse monoclonal clone 24B72D11.1 BD Biosciences 556604 1:500 β-CATENIN mouse monoclonal clone 14/Beta-catenin BD Biosciences 610153 1:500 BrdU mouse monoclonal clone MoBU-1 BioLegend 317902 1:500 Cleaved CASP3 rabbit polyclonal Cell Signaling Technology 9661 T 1:200 DCX guinea pig polyclonal Milipore AB2253 1:5000 GFAP chicken polyclonal BioLegend (Covance) 829401 1:500 GFP chicken polyclonal Rockland Immunochemicals 600–901-215 1:500 KI-67 rat monoclonal clone SolA15 Invitrogen 14–5698-82 1:500 RFP goat polyclonal Rockland Immunochemicals 200–101-379 1:500 RFP rabbit polyclonal Rockland Immunochemicals 600–401-379 1:500 S100 rabbit polyclonal Invitrogen PA5–16257 (product discontinued) 1:500 VCAM1 rat monoclonal clone 429 (MVCAM.A) BD Biosciences 553330 1:200

Techniques: Knock-Out, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Protective Effects of Hexarelin and JMV2894 in a Human Neuroblastoma Cell Line Expressing the SOD1-G93A Mutated Protein

doi: 10.3390/ijms24020993

Figure Lengend Snippet: Caspase-3 and caspase-7 mRNA levels following incubation with increasing concentrations of H 2 O 2 . SH-SY5Y WT and SOD1 G93A cells were incubated with different concentrations of H 2 O 2 alone (0, 100, 150, 200 μM). Caspase-3 ( A ) and caspase-7 ( B ) mRNA levels were measured by RT-PCR and normalized for the respective β-actin mRNA levels. Data are expressed as mean ± SEM of 18 replicates obtained in 3 independent experiments ( n = 6 in each experiment). For SH-SY5Y WT: § p < 0.05 vs. CTRL; for SH-SY5Y SOD1 G93A * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. CTRL.

Article Snippet: After 3 washes in PBS-T, membranes were incubated with the primary antibody overnight at 4 °C (Anti-cleaved caspase-3 (Asp175) (5A1E) rabbit antibody, #9664, Cell Signaling Technology, 1:1000; anti-cleaved caspase-7 (Asp198) rabbit antibody, #9491, Cell Signaling Technology, 1:1000; anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) rabbit antibody, #9101, Cell Signaling Technology, 1:1000; anti-p44/42 MAPK (Erk1/2) rabbit antibody, #4695, Cell Signaling Technology, 1:1000; anti-Phospho-p38 MAPK (Thr180/Tyr182) rabbit antibody, #4511, Cell Signaling Technology, 1:1000; anti-p38 MAPK rabbit antibody, #9212, Cell Signaling Technology, 1:1000; anti-Phospho-Akt rabbit antibody, #4060, Cell Signaling Technology, 1:2000; anti-Akt rabbit antibody, #4685, Cell Signaling Technology, 1:1000; anti-actin rabbit antibody, #A2066, Sigma Aldrich, 1:2500), and then with a peroxidase-coupled goat anti-rabbit IgG (#31460, Thermo Scientific, 1:5000) for 1 h at room temperature.

Techniques: Incubation, Reverse Transcription Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Protective Effects of Hexarelin and JMV2894 in a Human Neuroblastoma Cell Line Expressing the SOD1-G93A Mutated Protein

doi: 10.3390/ijms24020993

Figure Lengend Snippet: Caspase-3 and caspase-7 mRNA levels following incubation with H 2 O 2 and GHS. The SH-SY5Y SOD1 G93A were also co-incubated with 1 µM GHS and 150 µM H 2 O 2 for 24 h. Caspase-3 ( A ) and caspase-7 ( B ). The mRNA levels were measured by RT–PCR and normalized for the respective β-actin mRNA levels. Data are expressed as mean ± SEM of 18 replicates obtained in 3 independent experiments ( n = 6 in each experiment). * p < 0.05, and *** p < 0.001 vs. CTRL; ° p < 0.05 vs. H 2 O 2 .

Article Snippet: After 3 washes in PBS-T, membranes were incubated with the primary antibody overnight at 4 °C (Anti-cleaved caspase-3 (Asp175) (5A1E) rabbit antibody, #9664, Cell Signaling Technology, 1:1000; anti-cleaved caspase-7 (Asp198) rabbit antibody, #9491, Cell Signaling Technology, 1:1000; anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) rabbit antibody, #9101, Cell Signaling Technology, 1:1000; anti-p44/42 MAPK (Erk1/2) rabbit antibody, #4695, Cell Signaling Technology, 1:1000; anti-Phospho-p38 MAPK (Thr180/Tyr182) rabbit antibody, #4511, Cell Signaling Technology, 1:1000; anti-p38 MAPK rabbit antibody, #9212, Cell Signaling Technology, 1:1000; anti-Phospho-Akt rabbit antibody, #4060, Cell Signaling Technology, 1:2000; anti-Akt rabbit antibody, #4685, Cell Signaling Technology, 1:1000; anti-actin rabbit antibody, #A2066, Sigma Aldrich, 1:2500), and then with a peroxidase-coupled goat anti-rabbit IgG (#31460, Thermo Scientific, 1:5000) for 1 h at room temperature.

Techniques: Incubation, Reverse Transcription Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Protective Effects of Hexarelin and JMV2894 in a Human Neuroblastoma Cell Line Expressing the SOD1-G93A Mutated Protein

doi: 10.3390/ijms24020993

Figure Lengend Snippet: GHS inhibits apoptotic pathway through caspases inactivation. The SH-SY5Y SOD1 G93A cells were treated with H 2 O 2 alone, GHS alone, or with a combination of GHS and H 2 O 2 for 24 h. Western blot assays were used to measure levels of ( A ) cleaved caspase-3/β-actin, and ( B ) cleaved caspase-7/β-actin. All assays were performed in at least 3 independent experiments ( n = 3). Statistical significance: ** p < 0.01, and *** p < 0.001 vs. CTRL; °° p < 0.01 vs. H 2 O 2 .

Article Snippet: After 3 washes in PBS-T, membranes were incubated with the primary antibody overnight at 4 °C (Anti-cleaved caspase-3 (Asp175) (5A1E) rabbit antibody, #9664, Cell Signaling Technology, 1:1000; anti-cleaved caspase-7 (Asp198) rabbit antibody, #9491, Cell Signaling Technology, 1:1000; anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) rabbit antibody, #9101, Cell Signaling Technology, 1:1000; anti-p44/42 MAPK (Erk1/2) rabbit antibody, #4695, Cell Signaling Technology, 1:1000; anti-Phospho-p38 MAPK (Thr180/Tyr182) rabbit antibody, #4511, Cell Signaling Technology, 1:1000; anti-p38 MAPK rabbit antibody, #9212, Cell Signaling Technology, 1:1000; anti-Phospho-Akt rabbit antibody, #4060, Cell Signaling Technology, 1:2000; anti-Akt rabbit antibody, #4685, Cell Signaling Technology, 1:1000; anti-actin rabbit antibody, #A2066, Sigma Aldrich, 1:2500), and then with a peroxidase-coupled goat anti-rabbit IgG (#31460, Thermo Scientific, 1:5000) for 1 h at room temperature.

Techniques: Western Blot

Journal: Oncology Letters

Article Title: Effect of integrin α7 on cell proliferation, invasion, apoptosis and the PI3K/AKT pathway, and its association with clinicopathological features in endometrial cancer

doi: 10.3892/ol.2022.13612

Figure Lengend Snippet: Antibodies applied in western blotting.

Article Snippet: Cleaved caspase 3 rabbit mAb , Cell Signaling Technology, Inc. , #9664 , 1:1,000.

Techniques: Western Blot

Journal: Cell Death & Disease

Article Title: Restoring mitochondrial cardiolipin homeostasis reduces cell death and promotes recovery after spinal cord injury

doi: 10.1038/s41419-022-05369-5

Figure Lengend Snippet: A – C Biomarkers of apoptosis following SCI. Time course of cytochrome c release ( A ), Smac/DIABLO release ( B ), and active caspase-3 expression ( C ) following SCI. * P < 0.05, ** P < 0.01 vs sham (One-way ANOVA, Dunnett post-test, n = 4 rats/group). Data represent mean ± s.e.m. Cyt. c cytochrome c, Smac/D. Smac/DIABLO, A. casp.-3 active casepase-3. D – I Lipidomic analysis after SCI. Lipid extracts of spinal cord from sham, 3 h, and 24 h SCI rats were prepared by using the methyl-tert-butyl ether (MTBE) extraction. D , E Expanded negative-ion ESI mass spectra of rat spinal cord lipid extracts obtained using a Q Exactive™ Hybrid Quadrupole-Orbitrap mass spectrometer. The asterisks indicate the identified CL plus-one isotopologues, which were characteristic of the doubly charged CL molecular species and were used to quantify individual CL molecular species. Each spectrum is displayed after being normalized to the intensity of internal standard 1’, 3’-bis [1, 2-dimyristoyl-sn-glycero-3-phospho]- sn -glycerol (tetra14:0 CL, [CL-2H + 1] 2- = 619.91791) ion. The x -axis is mass-to-charge ratio ( m/z ). A significant decrease in CL was detected at 3 and 24 h post-injury ( D – F ) while a corresponding increase in lyso-CL and ratio of lyso-CL/CL was observed at 24 h post-injury ( G , H ). 4-HNE, a marker of lipid peroxidation, was significantly increased at 3 and 24 h after SCI ( I ). Data represent mean ± s.e.m. * P < 0.05, ** P < 0.01 (One-way ANOVA, Tukey’s multiple comparisons test, n = 4 rats/groups). CL cardiolipin, Lyso-CL lyso-cardiolipin, 4-HNE 4-hydroxy Nonenal.

Article Snippet: The primary antibodies included rabbit polyclonal anti-caspase-3 antibody (Cat# 9664, cleaved, 1:1000, 0.04 µg/ml, Cell Signaling Technology, Boston, MA), mouse anti-cytochrome c antibody (Cat# 556433, 1:300, 1.67 µg/ml, 7H8.2C12, BD Pharmingen, San Jose, CA), mouse anti-Smac/DIABLO antibody (Cat# 612246, 1:1000, 0.25 µg/ml, BD Transduction Laboratories, San Jose, CA), rabbit anti-PARP-1 (Cat# 9542, 1:500, 0.0976 µg/ml, Cell Signaling Technology, Boston, MA), rabbit anti-Bcl-2 (Cat# sc-492, 1:100, 2 µg/ml, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Bax (Cat# sc-526, 1:100, 2 µg/ml, Santa Cruz Biotechnology, Santa Cruz, CA), and mouse anti-β-tubulin antibody (Cat# 5293, 1:1000, 4.7 µg/ml, Sigma, St. Louis, MO).

Techniques: Expressing, Mass Spectrometry, Marker

Journal: Cell Death & Disease

Article Title: Restoring mitochondrial cardiolipin homeostasis reduces cell death and promotes recovery after spinal cord injury

doi: 10.1038/s41419-022-05369-5

Figure Lengend Snippet: A , B Cultured spinal cord neurons were treated with the designated concentrations of oxidized CL (OXCL) and non-oxidized CL for 24 h. Neuronal cell death was measured by LDH, a stable cytoplasmic enzyme that is present in all cells but only released when the plasma membrane is damaged. MTT assay was used to determine the cell viability and mitochondrial activity, since tetrazolium is reduced to formazan by mitochondrial dehydrogenase activity. The oxidized CL induced spinal cord neuronal death ( A ) as measured by LDH release and mitochondrial dysfunction ( B ) as measured by MTT in a dose-dependent manner. ** P < 0.01 (Two-way ANOVA, Tukey’s multiple comparisons test). Data represent the mean ± s.e.m. from 3 independent experiments. C – H The spinal neuronal cultures were exposed to rotenone (125 nM) or H 2 O 2 (50 μM) either in the absence or presence of 10 μM XJB-5-131 (XJB) for 24 h. XJB was added 30 min before rotenone or H 2 O 2 administration, and the culture medium was removed for LDH at 24 h after oxidative stress. XJB-5-131 reversed rotenone or H 2 O 2 -induced CL loss ( C , F ), mitochondrial dysfunction ( D , G ), and neuronal death ( E , H ). * P < 0.05, ** P < 0.01 (One-way ANOVA, Tukey post hoc test, n = 6/group). Data represent the mean ± s.e.m. from 3 independent experiments. I – P Effects of CL synthase (CLS) siRNA on primary spinal neuronal death in vitro. Cultured spinal cord neurons were transfected with CLS1 siRNA (r) for 24 h. I CL content was assayed using acridine orange 10-nonyl bromide (NAO, Invitrogen). Knocking down CL synthase, the key enzyme of de novo CL biosynthesis, with CLS1 siRNA significantly decreased CL. J – O Activated caspase-3/7 was examined using CellEvent™ Caspase-3/7 Green Detection Reagent (Invitrogen). Apoptotic cells with activated caspase-3/7 showed bright green nuclei (arrows). Bar = 100 μm. P Bar graph showed that CLS1 siRNA significantly induced neuronal apoptosis, evidenced by increased number of activated caspase-3/7 cells. ** P < 0.01 (Student t -test, n = 6/group). Data represent the mean ± s.e.m. from 3 independent experiments. Q – S Effects of exogenous CL on mitochondrial dysfunction and spinal neuronal death in the scratch injury model in vitro. Cultured spinal cord neurons were pre-incubated with CL liposomes for 30 min before scratch injury. Q Scratch injury-induced neuronal death was significantly reversed by exogenous CL in a dose-dependent manner. R , S Exogenous CL (100 µM) also significantly reversed scratch injury-induced CL loss ( R ) and decreased mitochondrial membrane potential (MMP) ( S ). Veh, vehicle; * P < 0.05, ** P < 0.01 (One-way ANOVA, Tukey’s multiple comparisons test, n = 6/group). Data represent the mean ± s.e.m. from 3 independent experiments.

Article Snippet: The primary antibodies included rabbit polyclonal anti-caspase-3 antibody (Cat# 9664, cleaved, 1:1000, 0.04 µg/ml, Cell Signaling Technology, Boston, MA), mouse anti-cytochrome c antibody (Cat# 556433, 1:300, 1.67 µg/ml, 7H8.2C12, BD Pharmingen, San Jose, CA), mouse anti-Smac/DIABLO antibody (Cat# 612246, 1:1000, 0.25 µg/ml, BD Transduction Laboratories, San Jose, CA), rabbit anti-PARP-1 (Cat# 9542, 1:500, 0.0976 µg/ml, Cell Signaling Technology, Boston, MA), rabbit anti-Bcl-2 (Cat# sc-492, 1:100, 2 µg/ml, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Bax (Cat# sc-526, 1:100, 2 µg/ml, Santa Cruz Biotechnology, Santa Cruz, CA), and mouse anti-β-tubulin antibody (Cat# 5293, 1:1000, 4.7 µg/ml, Sigma, St. Louis, MO).

Techniques: Cell Culture, MTT Assay, Activity Assay, In Vitro, Transfection, Incubation

Journal: Cell Death & Disease

Article Title: Restoring mitochondrial cardiolipin homeostasis reduces cell death and promotes recovery after spinal cord injury

doi: 10.1038/s41419-022-05369-5

Figure Lengend Snippet: A Time course of mitochondrial cPLA 2 expression and activation after SCI. Data represent mean ± s.e.m., * P < 0.05, ** P < 0.01 vs sham (Two-way ANOVA, Tukey’s multiple comparisons test, n = 6 rats/group). B An electron microscopic image shows phosphorylated cPLA 2 (p-cPLA 2 ) immunopositive peroxidase reaction products in a neuronal dendrite in the rat spinal ventral horn. p-cPLA 2 immunoreactivity (IR) was observed to localize in mitochondria (*) including both the outer (yellow arrow) and inner (red arrows) mitochondrial membranes, and on the cell membrane (green arrow) at 3 days after SCI. Bar = 0.25 μm. C cPLA 2 activation-induced CL loss was reversed by AACOCF 3 , a cPLA 2 inhibitor. D SCI induced a decrease in the ratio of red/green in the MMP (Δψm) assay, which was reversed by AACOCF 3. ( E – H ) AACOCF 3 , a cPLA 2 inhibitor, reversed SCI-induced cytochrome c release ( E ), Smac/DIABLO release ( F ), active caspase-3 expression ( G ), and cleaved PAPRP expression ( H ). Data represent mean ± s.e.m. * P < 0.05, ** P < 0.01 (One-way ANOVA, Tukey’s multiple comparisons test, n = 6 rats/group). Cyt. c cytochrome c, Smac/D. Smac/DIABLO, A. casp-3 active caspase-3. I – M Oxidative stress induces cPLA 2 activation after SCI. I Immunohistochemistry shows a marked increase in p-cPLA 2 -expression, a marker of cPLA 2 activation, at the injury epicenter and 3 mm rostral to it after a single injection of H 2 O 2 into the T9 spinal cord. Right column images show that H 2 O 2 -induced p-cPLA 2 expression was substantially reversed by mepacrine (5 mg/kg), a PLA 2 inhibitor. The expression of p-cPLA 2 was found in neurons (arrows), swelling axons (open arrows), and glial cells (arrowheads). Bar, 100 μm. I – L Administration of XJB (10 mg/kg, i.p.) at 30 min post-injury significantly reduced SCI-induced mitochondrial cPLA 2 activation. J Representative images of phosphorylated cPLA 2 (p-cPLA 2 ), cPLA 2 , and VDAC expression. K Compiled results in a bar graph for the ratio of p-cPLA 2 /cPLA 2 expression. L Compiled results in a bar graph for the ratio of p-cPLA 2 /VDAC expression. M Compiled results in a bar graph for the ratio of cPLA 2 /VDAC expression. * P < 0.05, ** P < 0.01 (One-way ANOVA, Tukey’s multiple comparisons test, n = 6). Data represent the mean ± s.e.m.

Article Snippet: The primary antibodies included rabbit polyclonal anti-caspase-3 antibody (Cat# 9664, cleaved, 1:1000, 0.04 µg/ml, Cell Signaling Technology, Boston, MA), mouse anti-cytochrome c antibody (Cat# 556433, 1:300, 1.67 µg/ml, 7H8.2C12, BD Pharmingen, San Jose, CA), mouse anti-Smac/DIABLO antibody (Cat# 612246, 1:1000, 0.25 µg/ml, BD Transduction Laboratories, San Jose, CA), rabbit anti-PARP-1 (Cat# 9542, 1:500, 0.0976 µg/ml, Cell Signaling Technology, Boston, MA), rabbit anti-Bcl-2 (Cat# sc-492, 1:100, 2 µg/ml, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Bax (Cat# sc-526, 1:100, 2 µg/ml, Santa Cruz Biotechnology, Santa Cruz, CA), and mouse anti-β-tubulin antibody (Cat# 5293, 1:1000, 4.7 µg/ml, Sigma, St. Louis, MO).

Techniques: Expressing, Activation Assay, Immunohistochemistry, Marker, Injection

Journal: Cell Death & Disease

Article Title: Restoring mitochondrial cardiolipin homeostasis reduces cell death and promotes recovery after spinal cord injury

doi: 10.1038/s41419-022-05369-5

Figure Lengend Snippet: A cPLA 2 inhibitor AACOCF3 revised cPLA 2 activation-induced CL loss. ** P < 0.01 (One-way ANOVA, Tukey’s multiple comparisons test, n = 4–6). Data represent the mean ± s.e.m. from 3 independent culture experiments. B CL reduced cPLA 2 activation-induced neuronal death. CL liposomes were added 30 min before C-1-P (2 µM) treatment. ** P < 0.01; ns no significance. (One-way ANOVA, Tukey’s multiple comparisons test, n = 6). Data represent the mean ± s.e.m. from 3 independent culture experiments. C – H C-1-P induced mitochondrial membrane potential (MMP, Δψm) change measured with the cationic dye JC-1 in cultured spinal neurons. Vehicle-treated neurons showed strong J-aggregation (red). After C-1-P treatment, the majority of neurons showed green staining due to low Δψm. Bar, 50 µm. I Bar graph shows C-1-P induced a significant decrease in the ratio of red/green, indicating that activation of cPLA 2 induced mitochondrial dysfunction (** P < 0.01, Student t test, n = 3). J Cultured spinal cord neurons were treated with the designated concentrations of C-1-P or A23187 for 24 h. MTT assay revealed that both cPLA 2 activators, C-1-P and A23187, induced mitochondrial dysfunction and neuronal death in a dose-dependent manner. ** P < 0.01, ## P < 0.01 versus the vehicle control (One-way ANOVA, Tukey’s multiple comparisons test, n = 6–8). Data represent the mean ± s.e.m. from 3 independent experiments. K Importantly, mitochondrial dysfunction and neuronal death induced by C-1-P (2 µM) or A23187 (5 µM) were significantly reversed by AACOCF3 (15 µM), a cPLA 2 inhibitor. AACOCF3 was added 30 min before C-1-P or A23187 administration, and the culture cells were examined for MTT at 24 h after the activator treatment. ** P < 0.01 versus the vehicle control, ## P < 0.01 versus the C-1-P or A23187 group (Two-way ANOVA, Tukey’s multiple comparisons test, n = 7–8. Data represent the mean ± s.e.m. from 3 independent experiments. L – O cPLA 2 ablation protected against CL loss ( L ), mitochondrial dysfunction ( M ), and apoptosis ( N – O ) induced by SCI. L NAO-labeled cardiolipin expression at 1 day after SCI. M A ratio of red/green, indicating mitochondrial function. N Cytochrome c release at 1 day after SCI. O Expression of active caspase-3 at 1 day after SCI. Data represent mean ± s.e.m. * P < 0.05, ** P < 0.01 vs sham; ## P < 0.01 vs WT (Two-way ANOVA, Tukey’s multiple comparisons test, n = 6 mice/group).

Article Snippet: The primary antibodies included rabbit polyclonal anti-caspase-3 antibody (Cat# 9664, cleaved, 1:1000, 0.04 µg/ml, Cell Signaling Technology, Boston, MA), mouse anti-cytochrome c antibody (Cat# 556433, 1:300, 1.67 µg/ml, 7H8.2C12, BD Pharmingen, San Jose, CA), mouse anti-Smac/DIABLO antibody (Cat# 612246, 1:1000, 0.25 µg/ml, BD Transduction Laboratories, San Jose, CA), rabbit anti-PARP-1 (Cat# 9542, 1:500, 0.0976 µg/ml, Cell Signaling Technology, Boston, MA), rabbit anti-Bcl-2 (Cat# sc-492, 1:100, 2 µg/ml, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Bax (Cat# sc-526, 1:100, 2 µg/ml, Santa Cruz Biotechnology, Santa Cruz, CA), and mouse anti-β-tubulin antibody (Cat# 5293, 1:1000, 4.7 µg/ml, Sigma, St. Louis, MO).

Techniques: Activation Assay, Cell Culture, Staining, MTT Assay, Labeling, Expressing

Journal: Cell Death & Disease

Article Title: Restoring mitochondrial cardiolipin homeostasis reduces cell death and promotes recovery after spinal cord injury

doi: 10.1038/s41419-022-05369-5

Figure Lengend Snippet: A – D lipidomic analysis at 1 day after SCI showed that XJB-5-131 (XJB, 10 mg/kg) attenuated SCI-induced mitochondrial 4-HNE expression ( A ), oxidized CL ( B ), CL loss ( C ), and lyso-CL ( D ) in adult rats. * P < 0.05, ** P < 0.01 (One-way ANOVA, Tukey’s multiple comparisons test, n = 4 rats/group). Data represent mean ± s.e.m. E – H XJB attenuated mitochondrial dysfunction ( E ), cytochrome c release ( F ), Smac/DIABLO release ( G ), and active caspase-3 expression ( H ) at 1 day after SCI in adult rats. Data represent mean ± s.e.m. * P < 0.05, ** P < 0.01 (One-way ANOVA, Tukey’s multiple comparisons test, n = 4–6 rats/group).

Article Snippet: The primary antibodies included rabbit polyclonal anti-caspase-3 antibody (Cat# 9664, cleaved, 1:1000, 0.04 µg/ml, Cell Signaling Technology, Boston, MA), mouse anti-cytochrome c antibody (Cat# 556433, 1:300, 1.67 µg/ml, 7H8.2C12, BD Pharmingen, San Jose, CA), mouse anti-Smac/DIABLO antibody (Cat# 612246, 1:1000, 0.25 µg/ml, BD Transduction Laboratories, San Jose, CA), rabbit anti-PARP-1 (Cat# 9542, 1:500, 0.0976 µg/ml, Cell Signaling Technology, Boston, MA), rabbit anti-Bcl-2 (Cat# sc-492, 1:100, 2 µg/ml, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Bax (Cat# sc-526, 1:100, 2 µg/ml, Santa Cruz Biotechnology, Santa Cruz, CA), and mouse anti-β-tubulin antibody (Cat# 5293, 1:1000, 4.7 µg/ml, Sigma, St. Louis, MO).

Techniques: Expressing

Journal: STAR Protocols

Article Title: Protocol for wireless deep brain stimulation in freely behaving mice with infrared light

doi: 10.1016/j.xpro.2022.101757

Figure Lengend Snippet: Dilution ratios of primary and secondary antibodies

Article Snippet: Rabbit anti-Cleaved Caspase-3 , Cell Signaling Technology , 9664.

Techniques:

Journal: STAR Protocols

Article Title: Protocol for wireless deep brain stimulation in freely behaving mice with infrared light

doi: 10.1016/j.xpro.2022.101757

Figure Lengend Snippet:

Article Snippet: Rabbit anti-Cleaved Caspase-3 , Cell Signaling Technology , 9664.

Techniques: Recombinant, Injection, Plasmid Preparation, Binding Assay, Microscopy, Electron Microscopy