Journal: Physiological Genomics

Article Title: Human gene copy number spectra analysis in congenital heart malformations

doi: 10.1152/physiolgenomics.00013.2012

Figure Lengend Snippet: Known CHD risk genes

Article Snippet: TFAP2B , TRANSCRIPTION FACTOR AP2-BETA , 6p12.3 , 50894397 , 28888 , Hs01355864_cn , NS, S , 601601 , 10802654.

Techniques: Retroviral, Sequencing, Virus, Reverse Transcription

Journal: iScience

Article Title: Mild hypothermia promotes neuronal differentiation of human neural stem cells via RBM3-SOX11 signaling pathway

doi: 10.1016/j.isci.2024.109435

Figure Lengend Snippet:

Article Snippet: The protein concentration was determined using the BCA protein assay kit (Sangon, C503021), and an equal amount of protein was loaded and separated using SDS-PAGE Bis-Tris Protein Gel (Vazyme, E304/305) with SDS running buffer.

Techniques: Virus, Recombinant, Protease Inhibitor, Bicinchoninic Acid Protein Assay, cDNA Synthesis, Reporter Assay, RNA Sequencing, Sequencing, Software

Journal: Neural regeneration research

Article Title: CHCHD2 Thr61Ile mutation impairs F1F0-ATPase assembly in in vitro and in vivo models of Parkinson's disease.

doi: 10.4103/1673-5374.378010

Figure Lengend Snippet: Figure 2 ｜Cellular ATP levels and F1F0-ATPase activity in isolated mitochondria in a in SH-SY5Y cell model of Parkinson’s disease. (A, B) Flag-vector: LV-Flag-vector (Ubi-MCS-3FLAG-SV40-puromycin vector)-transfected SH-SY5Y cells cultured in MEM-F12 medium; Flag-vector + MPP+: LV-Flag-vector- transfected SH-SY5Y cells treated with MPP+ (500 μM for 24 hours); CHCHD2-Flag + MPP+: LV-CHCHD2-Flag-transfected SH-SY5Y cells treated with MPP+ (500 μM for 24 hours); CHCHD2-T61I + MPP+: LV-CHCHD2-T61I-Flag-transfected SH-SY5Y cells treated with MPP+ (500 μM for 24 hours). (A) Detection of ATP levels in control or MPP+-treated SH-SY5Y cells stably transfected with empty vector or constructs encoding WT or T61I-mutant CHCHD2. (B) Effect of WT or T61I-mutant CHCHD2 on ATP synthase specific activity in control or MPP+-treated cells. (C) Effect of WT or T61I-mutant CHCHD2 on ATP synthase specific activity in AVV-transfected mice with or without MPTP treatment. CHCHD2-Flag + MPTP: AVV-CHCHD2-Flag–transfected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks); CHCHD2-T61I + MPTP: AVV-CHCHD2-T61I–transfected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks); Flag-vector: AVV-Flag (CMV- betaGlobin-MCS-3Flag-SV40 polyA (GV411) vector)-transfected mice treated with normal saline (twice a week, for 5 weeks); Flag-vector + MPTP: AVV-Flag-transfected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks). Data are expressed as the mean ± SD (n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). ATP: Adenosine triphosphate; MPP+: 1-methyl-4-phenylpyridinium; MPTP: 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine.

Article Snippet: Detection of mitochondrial permeability transition pore opening Opening of the mitochondrial permeability transition pore (mPTP) was detected by the calcein-AM/cobalt assay following the manufacturer’s instructions (C2009S, Beyotime Biotechnology).

Techniques: Activity Assay, Isolation, Plasmid Preparation, Transfection, Cell Culture, Control, Stable Transfection, Construct, Mutagenesis, Saline

Journal: Neural regeneration research

Article Title: CHCHD2 Thr61Ile mutation impairs F1F0-ATPase assembly in in vitro and in vivo models of Parkinson's disease.

doi: 10.4103/1673-5374.378010

Figure Lengend Snippet: Figure 3 ｜CHCHD2 T61I mutation promotes mitochondrial permeability transition pore (mPTP) opening in SH-SY5Y cells. CHCHD2-Flag: LV-CHCHD2-Flag-transfected SH-SY5Y cells; CHCHD2-T61I: LV-CHCHD2-T61I-Flag-transfected SH-SY5Y cells; Flag-vector: LV-Flag-vector (Ubi-MCS-3FLAG-SV40-puromycin vector)-transfected SH-SY5Y cells. (A) Calcein-AM and CoCl2 were administered to live cells expressing WT or T61I-mutant CHCHD2, and fluorescence intensity was detected by flow cytometry. (B) Effect of WT or the T61I-mutant CHCHD2 on mPTP opening as determined by detecting mitochondrial membrane potential using JC-1 fluorescent probes (white squares) in control or MPP+-treated SH-SY5Y cells. The fluorescence intensity of intracellular JC-1 aggregates (red) in the MPP+ + CHCHD2-Flag group was greater than that in the MPP+ + Flag-vector group. The CHCHD2-T61I group showed an increased amount of JC-1 monomers (green). Scale bars: 10 μm. (C) The mean fluorescence intensity of JC-1 aggregates or monomers in control or MPP+-treated SH-SY5Y cells expressing WT or T61I-mutant CHCHD2. Data are expressed as the mean ± SD (n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). Calcein-AM: Calcein acetoxymethyl ester; MPP+: 1-methyl-4-phenylpyridinium; mPTP: mitochondrial permeability transition pore; WT: wild type.

Article Snippet: Detection of mitochondrial permeability transition pore opening Opening of the mitochondrial permeability transition pore (mPTP) was detected by the calcein-AM/cobalt assay following the manufacturer’s instructions (C2009S, Beyotime Biotechnology).

Techniques: Mutagenesis, Permeability, Transfection, Plasmid Preparation, Expressing, Fluorescence, Flow Cytometry, Membrane, Control

Journal: Neural regeneration research

Article Title: CHCHD2 Thr61Ile mutation impairs F1F0-ATPase assembly in in vitro and in vivo models of Parkinson's disease.

doi: 10.4103/1673-5374.378010

Figure Lengend Snippet: Figure 5 ｜CHCHD2 T61I mutation aggravates movement deficits and nigral DA neuron function in a mouse model of PD. (A) Experimental design. AAV-CHCHD2, AAV-CHCHD2 T61I, or AAV-Vector was stereotaxically injected into the substantia nigra pars compacta, and 4 weeks later mice were injected intraperitoneally with normal saline or MPTP for 5 weeks. One day after the last MPTP/normal saline injection, behavioral tests were performed, and then the mice were sacrificed. Flag-vector: AVV-Flag(CMV-betaGlobin-MCS-3Flag-SV40 polyA (GV411) vector)-transfected mice treated with normal saline (twice a week, for 5 weeks); Flag-vector+MPTP: AVV-Flag– transfected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks); CHCHD2-Flag+MPTP: AVV-CHCHD2-Flag–infected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks); CHCHD2-T61I+MPTP: AVV-CHCHD2-T61I–transfected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks). (B) The grasping test was used to examine grip strength. (C) The pole-climbing test was used to examine bradykinesia. (D) The rotarod test was used to examine motor coordination. (E) Representative immunohistochemical staining of TH-positive neurons in the substantia nigra (SN). There were fewer TH-positive neurons in the SN in the AAV-CHCHD2-T61I + MPTP group than in the AAV-Flag-vector + MPTP and AAV-CHCHD2-Flag + MPTP groups. Scale bars: 800 μm. (F) Quantification of the TH-positive cells shown in E. (G) Representative images of double-immunofluorescent staining for CHCHD2-Flag (green, Alexa Fluor 488) and TH (red, Alexa Fluor 555) (white squares) in the SN. There were significantly fewer TH-positive neurons in the SN in the AAV-CHCHD2-T61I + MPTP group than in the AAV-Flag-vector + MPTP and AAV-CHCHD2-Flag + MPTP groups. Scale bars: 50 μm. (H) Quantification of TH mean fluorescence in the SN as shown in G. (I) Representative immunoblot for TH in the SN. (J) TH expression in the SN. Results are expressed as the mean ± SD (n ≥ 6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). ACTB: β-Actin; AAV: adeno-associated virus; DA: dopaminergic; DAPI: 4′,6-diamidino-2-phenylindole; MPTP: 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine; TH: tyrosine hydroxylase; SN: substantia nigra.

Article Snippet: Detection of mitochondrial permeability transition pore opening Opening of the mitochondrial permeability transition pore (mPTP) was detected by the calcein-AM/cobalt assay following the manufacturer’s instructions (C2009S, Beyotime Biotechnology).

Techniques: Mutagenesis, Plasmid Preparation, Injection, Saline, Transfection, Infection, Immunohistochemical staining, Staining, Fluorescence, Western Blot, Expressing, Virus

Journal: Neural regeneration research

Article Title: CHCHD2 Thr61Ile mutation impairs F1F0-ATPase assembly in in vitro and in vivo models of Parkinson's disease.

doi: 10.4103/1673-5374.378010

Figure Lengend Snippet: Figure 8 ｜CHCHD2 modulates OSCP expression in MPP+-/MPTP-induced PD models. (A, B) Flag-vector: LV-Flag-vector (Ubi-MCS-3FLAG-SV40-puromycin vector)-transfected SH-SY5Y cells cultured in MEM-F12 medium; Flag-vector+MPP+: LV-Flag-vector–transfected SH- SY5Y cells treated with MPP+ (500 μM for 24 hours); CHCHD2-Flag+MPP+: LV-CHCHD2-Flag–transfected SH-SY5Y cells treated with MPP+ (500 μM for 24 hours); CHCHD2-T61I + MPP+: LV-CHCHD2-T61I-Flag–transfected SH-SY5Y cells treated with MPP+ (500 μM for 24 hours). (A) Representative immunoblot of OSCP, CHCHD2, and ACTB in MPP+-treated SH-SY5Y cells. (B) Quantification of relative OSCP expression shown in A. (C, D) Flag-vector: AVV-Flag(CMV-betaGlobin-MCS-3Flag-SV40 polyA (GV411) vector)-transfected mice treated with normal saline (twice a week, for 5 weeks); Flag-vector + MPTP: AVV-Flag-infected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks); CHCHD2-Flag + MPTP: AVV-CHCHD2-Flag- infected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks); CHCHD2-T61I + MPTP: AVV-CHCHD2-T61I–transfected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks). (C) Representative immunoblot of OSCP, CHCHD2, and ACTB in control or MPTP-treated mice. (D) Quantification of relative OSCP expression shown in C. Data normalized to SH-SY5Y cells overexpressing Flag-vector or C57BL/6J mice transfected with AAV-Flag are expressed as the mean ± SD (n = 3 independent experiments for cells, n = 6 for animals). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). ACTB: β-Actin; MPP+: 1-methyl-4-phenylpyridinium; MPTP: 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine; OSCP: oligomycin sensitivity conferral protein.

Article Snippet: Detection of mitochondrial permeability transition pore opening Opening of the mitochondrial permeability transition pore (mPTP) was detected by the calcein-AM/cobalt assay following the manufacturer’s instructions (C2009S, Beyotime Biotechnology).

Techniques: Expressing, Plasmid Preparation, Transfection, Cell Culture, Western Blot, Saline, Infection, Control