Journal: Genes

Article Title: Next-Generation Sequencing Identifies Novel PMPCA Variants in Patients with Late-Onset Dominant Optic Atrophy

doi: 10.3390/genes13071202

Figure Lengend Snippet: Mitochondrial dynamic and PMPCA distribution studies of fibroblasts from control and PMPCA mutated patients. ( A ): Representative fluorescent images of mitochondrial network structure overlaid on phase-contrast (on the left) showed a mitochondrial network hyperconnection in PMPCA fibroblasts. Mitochondrial volume (in purple on black background in the middle) was assessed using the Mitotracker Green fluorescent signal by Imaris software and color-coded on the right. The inset illustrates the classification code. To present the changes in mitochondrial morphology in patients’ cells, types of mitochondria were classified into 5 groups according to mitochondrial length: blobs < 1 μm; fragmented < 5 μm; tubular < 10 μm; filamentous < 20 μm; mitochondrial network > 20 um. Bar graphs show the distribution of the mitochondrial population of Control, P1 and P2. Mean ± SEM. Scale bar: 10 μm. ( B ): Western blots (left) against PMPCA, PMPCB, OPA1 and citrate synthase (CS) on control (C1) and two patients’ (P1 and P2) fibroblasts reveal decreased levels of PMPCA and equal levels of PMPCB and OPA1 in the pathological conditions, as shown on the histogram (right). ( C ): Enzymatic activities of the respiratory complexes (CI to CV) from the control and the PMPCA mutated fibroblast strains related to the citrate synthase (CS) enzymatic activity, did not reveal a significant difference between control and mutated fibroblasts. Biochemical data were generated using the two-tailed paired t -test. Results are Mean ± S.E.M. from four independent experiments. ( D ): Single-molecule localization microscopy dSTORM was used to analyze PMPCA distribution, correlated to total internal reflection fluorescence TIRF microscopy for mitochondrial staining. Using Imaris 8.0 ® software, the dSTORM PMPCA immunofluorescence signal was used to quantify their mitochondrial surface protein distribution.

Article Snippet: Respiratory chain enzymatic activities and western blots were assessed as described [ , ], using the following antibodies: PMPCA (Novus Biologicals, CO, USA, #NBP1-89126; 56kD), PMPCB (Novus Biologicals, #NBP1-92120; 56kD); OPA1 (Abcam, Cambridge, England, ab42364; 95 and 85kD); Citrate Synthase (Abcam ab96600; 52kD).

Techniques: Control, Software, Western Blot, Activity Assay, Generated, Two Tailed Test, Microscopy, Fluorescence, Staining, Immunofluorescence

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Role of IL-6-IL-27 Complex in Host Antiviral Immune Response.

doi: 10.4049/jimmunol.2100179

Figure Lengend Snippet: FIGURE 1. Interactions between the subunits of IL-6 and IL-27 in vivo and in vitro. (AC) CoIP and immunoblotting of serum samples were performed with the indicated Abs. IL-6 interacted with IL-27A (A), IL-6 interacted with EBi3 (B), and IL-27A interacted with EBi3 (C). (DG) HEK293T cells were transfected with the indicated plasmids for 24 h. CoIP and immunoblotting were performed with the indicated Abs. IL-6-3HA interacted with IL-27A-V5 (D), IL-6-3HA interacted with EBi3-Myc (E), IL-27A-V5 interacted with EBi3-Myc (F), and IL-6-3HA interacted with IL-27-V5 (G). (H) Colocalization of IL-6 and IL-27 was detected by confocal microscopy. A549 cells were transfected with IL-6-eGFP-KDEL and IL-27-DsRed-KDEL for 24 h, and the nuclei were stained with DAPI before observation using confocal microscopy. All experiments were repeated at least three times with consistent results.

Article Snippet: Abs against V5 tag (66007-1-Ig), EBi3 (12371-1-AP), myxovirus resistance A (MxA; 13750-1-AP), MAVS (14341-1-AP), TGFb-activated kinase 1 (12330-2-AP), IRF3 (11312-1-AP), NF-kB p65 (10745- 1-AP), NF-kB p50 (14220-1-AP), Lamin A/C (10298-1-AP), GAPDH (60004-1-Ig), and b-actin (60008-1-Ig) were purchased from ProteinTech Group (Wuhan, China).

Techniques: In Vivo, In Vitro, Western Blot, Transfection, Confocal Microscopy, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Role of IL-6-IL-27 Complex in Host Antiviral Immune Response.

doi: 10.4049/jimmunol.2100179

Figure Lengend Snippet: FIGURE 2. Construction of the IL-6IL-27 complex and its antiviral activity. (A) A schematic of the IL-6IL-27 construction via flexible self-cleaved linker is shown. Immunoblotting analysis of IL-6, EBi3, IL-27A, and b-actin of HEK293T cells transfected with empty vector pcDNA3.1(1) or IL-6IL-27 complex expression plasmids for 24 h. (B) A549 cells were transfected with indicated plasmids for 24 h followed by H1N1 infection (MOI = 1) for 12 h. The NP-specific mRNA, cRNA, and vRNA levels were measured using QRT-PCR. (C) RD cells were transfected with the indicated plasmids for 24 h fol- lowed by EV71 infection (MOI = 1) for 24 h. The VP1 mRNA levels were measured using QRT-PCR. (D) Vero cells were (Figure legend continues)

Article Snippet: Abs against V5 tag (66007-1-Ig), EBi3 (12371-1-AP), myxovirus resistance A (MxA; 13750-1-AP), MAVS (14341-1-AP), TGFb-activated kinase 1 (12330-2-AP), IRF3 (11312-1-AP), NF-kB p65 (10745- 1-AP), NF-kB p50 (14220-1-AP), Lamin A/C (10298-1-AP), GAPDH (60004-1-Ig), and b-actin (60008-1-Ig) were purchased from ProteinTech Group (Wuhan, China).

Techniques: Activity Assay, Western Blot, Transfection, Plasmid Preparation, Expressing, Infection, Quantitative RT-PCR

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Role of IL-6-IL-27 Complex in Host Antiviral Immune Response.

doi: 10.4049/jimmunol.2100179

Figure Lengend Snippet: FIGURE 5. The IL-6IL-27 complex interacts with MAVS. (AC) A549 cells were transfected with increasing amounts of the indicated expression plas- mids (wedge; 100, 200, and 300 ng) of the IL-6IL-27 complex expression plasmid and luciferase reporter plasmids containing the IFN-b (A), NF-kB (B), and ISRE (C) promoter for 24 h followed by VSV infection (MOI = 1) for 24 h. Reporter assays were performed. pRL-TK was used as an internal control. (DI) HEK293T cells were transfected with the indicated plasmids for 24 h. CoIP and immunoblotting were performed with the indicated Abs. IL-6-3HA interacted with Flag-MAVS (D), IL-27A-V5 interacted with Flag-MAVS (E), EBi3-Myc interacted with Flag-MAVS (F), IL-6-HA interacted with Flag- TBK1 (G), IL-27A-V5 interacted with Flag-TBK1 (H), and EBi3-Myc interacted with Flag-TBK1 (I). All experiments were repeated at least three times with consistent results.

Article Snippet: Abs against V5 tag (66007-1-Ig), EBi3 (12371-1-AP), myxovirus resistance A (MxA; 13750-1-AP), MAVS (14341-1-AP), TGFb-activated kinase 1 (12330-2-AP), IRF3 (11312-1-AP), NF-kB p65 (10745- 1-AP), NF-kB p50 (14220-1-AP), Lamin A/C (10298-1-AP), GAPDH (60004-1-Ig), and b-actin (60008-1-Ig) were purchased from ProteinTech Group (Wuhan, China).

Techniques: Transfection, Expressing, Plasmid Preparation, Luciferase, Infection, Control, Western Blot