Journal: Animals : an open access journal from MDPI

Article Title: Mucosal Exosome Proteomics of Hybrid Grouper Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂ Infected by Pseudomonas plecoglossicida .

doi: 10.3390/ani14233401

Figure Lengend Snippet: Figure 1. Characterization of exosomes from the mucus of the hybrid grouper Epinephelus fuscoguttatus♀× E. lanceolatus♂. (A) Transmission electron microscopy observation of exosomes. Field of view = 100 nm; (B) Western blotting of exosomes using HSP70, TSG101, CD81, CD63 and GAPDH; (C) NTA analysis of exosomes showing the particle size distributions and concentrations.

Article Snippet: Themembraneswere blockedwith 5% skimmilk (0.75 g powdered milk + 15 mL PBS) at 37 ◦C for 1–2 h. After washing with 1× PBST (1000 mL 1× PBS + 1mLTween‐20) three times, themembraneswere incubatedwith primary antibodies (CD81 antibody (66866‐1‐Ig): 1:500; CD63 antibody (25682‐1‐AP): 1:1000; HSP70 antibody (10995‐1‐AP): 1:2000; TSG101 antibody (28283‐1‐AP): 1:1000; GAPDH antibody (D110016‐ 0200): 1:10,000; PROTEINTECH, USA) overnight at 4 ◦C.

Techniques: Transmission Assay, Electron Microscopy, Western Blot

Journal: International immunopharmacology

Article Title: Allogeneic platelet lysate activates the SIRT1-PINK1/Parkin pathway: A promising approach for improving mitochondrial function in an in vitro model of intervertebral disc degeneration.

doi: 10.1016/j.intimp.2024.113700

Figure Lengend Snippet: Fig. 2. Complete blood and P-PRP. Platelet and leukocyte counts. A, TGF-β, PDGF, IL-1β, and TNF-α levels in C-G. ap < 0.05 compared with whole blood. All the results were derived from three independent experiments and are shown as the means ± SDs.

Article Snippet: Primary antibodies against the following proteins were used at a 1:1000 dilution rate: β-actin (Affinity, USA, AF7018), IL-1β (Biosynthesis Biotechnology, bs-6319R), NLRP3 (Biosynthesis Biotechnology, China, bs-23723R), VDAC (Biosynthesis Biotechnology, bs-23683R), SIRT1 (Biosynthesis Biotechnology, bs-2257R), ASC (Biosynthesis Biotechnology, bs-6741R), MFN2 (Biosynthesis Biotechnology, bs-23685R), AGC (Biosynthesis Biotechnology, bs-1695R), PINK1 (Biosynthesis Biotechnology, #6946S), Parkin (Proteintech, USA, 14060-1-AP), Caspase-1 (Proteintech, 22915-1-AP), cleaved-Caspase-1(Affinity, USA, AF4022) and GSDMD-N (Biosynthesis Biotechnology, bs-23685R).

Techniques: Derivative Assay

Journal: International immunopharmacology

Article Title: Allogeneic platelet lysate activates the SIRT1-PINK1/Parkin pathway: A promising approach for improving mitochondrial function in an in vitro model of intervertebral disc degeneration.

doi: 10.1016/j.intimp.2024.113700

Figure Lengend Snippet: Fig. 3. NPC isolation and culture. A Immunofluorescence staining showing collagen II in NPCs. Scale bar = 50 μm and Scale bar = 100 μm. B, C Trypan Blue staining showing cell apoptosis among the different groups. Scale bar = 100 μm. ap < 0.05 versus the control group, bp < 0.05 versus the IL-1β group, and cp < 0.05 versus the APL group. All results were derived from three independent experiments and are shown as the mean ± SD.

Article Snippet: Primary antibodies against the following proteins were used at a 1:1000 dilution rate: β-actin (Affinity, USA, AF7018), IL-1β (Biosynthesis Biotechnology, bs-6319R), NLRP3 (Biosynthesis Biotechnology, China, bs-23723R), VDAC (Biosynthesis Biotechnology, bs-23683R), SIRT1 (Biosynthesis Biotechnology, bs-2257R), ASC (Biosynthesis Biotechnology, bs-6741R), MFN2 (Biosynthesis Biotechnology, bs-23685R), AGC (Biosynthesis Biotechnology, bs-1695R), PINK1 (Biosynthesis Biotechnology, #6946S), Parkin (Proteintech, USA, 14060-1-AP), Caspase-1 (Proteintech, 22915-1-AP), cleaved-Caspase-1(Affinity, USA, AF4022) and GSDMD-N (Biosynthesis Biotechnology, bs-23685R).

Techniques: Isolation, Immunofluorescence, Staining, Control, Derivative Assay

Journal: International immunopharmacology

Article Title: Allogeneic platelet lysate activates the SIRT1-PINK1/Parkin pathway: A promising approach for improving mitochondrial function in an in vitro model of intervertebral disc degeneration.

doi: 10.1016/j.intimp.2024.113700

Figure Lengend Snippet: Fig. 4a. APL inhibits mitochondrial and intracellular ROS levels by activating the SIRT1 signaling pathway, thereby activating mitochondrial autophagy. A Immunofluorescence staining showing that APL distinctly inhibits mitochondrial and intracellular ROS. B-G Western blotting showing that APL activates the SIRT1 signaling pathway. H-L RT-PCR showing that APL increases the mRNA expression of SIRT1 and MFN2 and suppresses that of PINK1/Parkin and VDCA1. (Bar = 100 μm) ap < 0.05 versus the control group, bp < 0.05 versus the IL-1β group, and cp < 0.05 versus the APL group. All results were derived from three independent experiments and are shown as the mean ± SD.

Article Snippet: Primary antibodies against the following proteins were used at a 1:1000 dilution rate: β-actin (Affinity, USA, AF7018), IL-1β (Biosynthesis Biotechnology, bs-6319R), NLRP3 (Biosynthesis Biotechnology, China, bs-23723R), VDAC (Biosynthesis Biotechnology, bs-23683R), SIRT1 (Biosynthesis Biotechnology, bs-2257R), ASC (Biosynthesis Biotechnology, bs-6741R), MFN2 (Biosynthesis Biotechnology, bs-23685R), AGC (Biosynthesis Biotechnology, bs-1695R), PINK1 (Biosynthesis Biotechnology, #6946S), Parkin (Proteintech, USA, 14060-1-AP), Caspase-1 (Proteintech, 22915-1-AP), cleaved-Caspase-1(Affinity, USA, AF4022) and GSDMD-N (Biosynthesis Biotechnology, bs-23685R).

Techniques: Immunofluorescence, Staining, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Derivative Assay

Journal: International immunopharmacology

Article Title: Allogeneic platelet lysate activates the SIRT1-PINK1/Parkin pathway: A promising approach for improving mitochondrial function in an in vitro model of intervertebral disc degeneration.

doi: 10.1016/j.intimp.2024.113700

Figure Lengend Snippet: Fig. 5. APL relieves mitochondrial oxidative stress and dysfunction induced by IL-1β. A, B Immunofluorescence staining showing that APL decreases mitochondrial permeability, whereas EX527 reverses the effects of APL. (Bar = 100 μm) ap < 0.05 versus the control group, bp < 0.05 versus the IL-1β group, and cp < 0.05 versus the APL group. C Transmission electron microscopy revealing that APL improves the morphology of IL-1β-induced mitochondria. EX527 can counteract or reverse this effect. All results were derived from three independent experiments and are shown as the mean ± SD.

Article Snippet: Primary antibodies against the following proteins were used at a 1:1000 dilution rate: β-actin (Affinity, USA, AF7018), IL-1β (Biosynthesis Biotechnology, bs-6319R), NLRP3 (Biosynthesis Biotechnology, China, bs-23723R), VDAC (Biosynthesis Biotechnology, bs-23683R), SIRT1 (Biosynthesis Biotechnology, bs-2257R), ASC (Biosynthesis Biotechnology, bs-6741R), MFN2 (Biosynthesis Biotechnology, bs-23685R), AGC (Biosynthesis Biotechnology, bs-1695R), PINK1 (Biosynthesis Biotechnology, #6946S), Parkin (Proteintech, USA, 14060-1-AP), Caspase-1 (Proteintech, 22915-1-AP), cleaved-Caspase-1(Affinity, USA, AF4022) and GSDMD-N (Biosynthesis Biotechnology, bs-23685R).

Techniques: Immunofluorescence, Staining, Permeability, Control, Transmission Assay, Electron Microscopy, Derivative Assay

Journal: International immunopharmacology

Article Title: Allogeneic platelet lysate activates the SIRT1-PINK1/Parkin pathway: A promising approach for improving mitochondrial function in an in vitro model of intervertebral disc degeneration.

doi: 10.1016/j.intimp.2024.113700

Figure Lengend Snippet: Fig. 6. APL attenuates IL-1β-induced NLRP3 inflammasome activation in NPCs. A, D Flow cytometry showing cell pyroptosis. B, E-H Protein levels of caspase-1, ASC, NLRP3, and GSDMD-N. I-K Gene expression of Caspase-1, ASC, and NLRP3. C, L-O Western Blotting showing the interaction between IL and 1β and NLRP3 inflammasome activation. ap < 0.05 versus the control group, bp < 0.05 versus the IL-1β group and cp < 0.05 versus the APL group. All results were derived from three independent experiments and are shown as the mean ± SD.

Article Snippet: Primary antibodies against the following proteins were used at a 1:1000 dilution rate: β-actin (Affinity, USA, AF7018), IL-1β (Biosynthesis Biotechnology, bs-6319R), NLRP3 (Biosynthesis Biotechnology, China, bs-23723R), VDAC (Biosynthesis Biotechnology, bs-23683R), SIRT1 (Biosynthesis Biotechnology, bs-2257R), ASC (Biosynthesis Biotechnology, bs-6741R), MFN2 (Biosynthesis Biotechnology, bs-23685R), AGC (Biosynthesis Biotechnology, bs-1695R), PINK1 (Biosynthesis Biotechnology, #6946S), Parkin (Proteintech, USA, 14060-1-AP), Caspase-1 (Proteintech, 22915-1-AP), cleaved-Caspase-1(Affinity, USA, AF4022) and GSDMD-N (Biosynthesis Biotechnology, bs-23685R).

Techniques: Activation Assay, Flow Cytometry, Gene Expression, Western Blot, Control, Derivative Assay

Journal: International immunopharmacology

Article Title: Allogeneic platelet lysate activates the SIRT1-PINK1/Parkin pathway: A promising approach for improving mitochondrial function in an in vitro model of intervertebral disc degeneration.

doi: 10.1016/j.intimp.2024.113700

Figure Lengend Snippet: Fig. 7. Protective effect of APL in NPCs and the putative mechanism underlying IL-1β-induced pyroptosis. Through the SIRT1-PINK1/Parkin-mediated mitophagy signaling pathway, APL eliminates dysfunctional mitochondria, decreases mitochondrial ROS accumulation, and inhibits NLRP3-activated pyroptosis in NPCs (Drawn by Figdraw).

Article Snippet: Primary antibodies against the following proteins were used at a 1:1000 dilution rate: β-actin (Affinity, USA, AF7018), IL-1β (Biosynthesis Biotechnology, bs-6319R), NLRP3 (Biosynthesis Biotechnology, China, bs-23723R), VDAC (Biosynthesis Biotechnology, bs-23683R), SIRT1 (Biosynthesis Biotechnology, bs-2257R), ASC (Biosynthesis Biotechnology, bs-6741R), MFN2 (Biosynthesis Biotechnology, bs-23685R), AGC (Biosynthesis Biotechnology, bs-1695R), PINK1 (Biosynthesis Biotechnology, #6946S), Parkin (Proteintech, USA, 14060-1-AP), Caspase-1 (Proteintech, 22915-1-AP), cleaved-Caspase-1(Affinity, USA, AF4022) and GSDMD-N (Biosynthesis Biotechnology, bs-23685R).

Techniques:

Journal: Cell metabolism

Article Title: CRIF1 is essential for the synthesis and insertion of oxidative phosphorylation polypeptides in the mammalian mitochondrial membrane.

doi: 10.1016/j.cmet.2012.06.012

Figure Lengend Snippet: Figure 1. CRIF1 Transport into and Localization in Mitochondria (A) Schematic representation of CRIF1 domains based on the primary structure of CRIF1. (B) Immunofluorescence of endogenous CRIF1. HeLa cells were immunostained for CRIF1 (red) and Tom20 (green). Scale bar, 10 mm. (C) Confocal microscopy of HeLa cells transfected with GFP-tagged human CRIF1. CRIF1-GFP, GFP sequence introduced at the C terminus of CRIF1; GFP-CRIF1, GFP sequence introduced at the N terminus of CRIF1; CRIF1(MTS)-GFP, the MTS (aa 1–35) of CRIF1 was added to the GFP N terminus; CRIF1(DMTS)-GFP, GFP was added to the C terminus of MTS (1–15)-deleted CRIF1. Scale bar, 20 mm. (D) Western blot analysis following subcellular fractionation of MEFs. WCE, whole cell extract; Cyt, cytosolic fraction; Mt, mitochondrial fraction; Nuc, nuclear fraction. (E) Western blot analysis following subfractionation of mitochondria isolated from MEFs. OM, outer membrane; IMS, intermembrane space; IM, inner membrane; MA, matrix. (F) Mitochondrial protein import assay. Radiolabeled recombinant full-length human CRIF1 was incubated with isolated rat liver mitochondria. p, unprocessed precursor form; m, mature form.

Article Snippet: Immunocytochemical Studies The primary antibody used against endogenous CRIF1 was goat anti-human CRIF1 (Santa Cruz, sc-103443, sc-103445).

Techniques: Confocal Microscopy, Transfection, Sequencing, Western Blot, Fractionation, Isolation, Membrane, Recombinant, Incubation

Journal: Cell metabolism

Article Title: CRIF1 is essential for the synthesis and insertion of oxidative phosphorylation polypeptides in the mammalian mitochondrial membrane.

doi: 10.1016/j.cmet.2012.06.012

Figure Lengend Snippet: Figure 2. CRIF1 Deficiency Caused Neurodegeneration Associated with a Drastic Decrease of OXPHOS Subunits In Vivo (A and B) H&E (A) and TUNEL (B) staining of the hippocampus from 12-week-old mice. Hi, hippocampus; CA, cornu ammonis; DG, dentate gyrus. (C) BN-PAGE analysis of OXPHOS complexes. Mitochondria isolated from the cortex (Crx) or hippocampus (Hippo) were solubilized with n-dodecyl-b-D-mal- toside to assess the steady-state levels of OXPHOS complexes (*, ATP synthase subcomplex). (D) Western blot analysis of the indicated OXPHOS subunits. (E) Transmission electron microscopy of the hippocampal region. Black squares indicate the enlarged regions. Scale bar, 500 nm.

Article Snippet: Immunocytochemical Studies The primary antibody used against endogenous CRIF1 was goat anti-human CRIF1 (Santa Cruz, sc-103443, sc-103445).

Techniques: In Vivo, TUNEL Assay, Staining, Isolation, Western Blot, Transmission Assay, Electron Microscopy

Journal: Cell metabolism

Article Title: CRIF1 is essential for the synthesis and insertion of oxidative phosphorylation polypeptides in the mammalian mitochondrial membrane.

doi: 10.1016/j.cmet.2012.06.012

Figure Lengend Snippet: Figure 3. Marked OXPHOS Dysfunction and Loss of OXPHOS Subunits and Complexes in Crif1-Deficient Cells (A) Generation of Crif1-deficient (Crif1-/D) cells by infecting MEFs with a retrovirus carrying Cre-recombinase. The upper two panels show CRIF1 protein and the lower two panels show Crif1 mRNA. +/D, WT MEFs; -/D, Crif1-deficient MEFs. (B) Oxygen consumption rates measured using a Seahorse XF-24 flux analyzer. n = 4. Error bars indicate SD; **p < 0.01. (C) Ratio of red/green fluorescence of JC-1 as a measure of mitochondrial membrane potential. n = 3. Error bars indicate SD; **p < 0.01. (D) Fluorescence of MitoSox as a measure of mitochondrial ROS levels. n = 3. Error bars indicate SD; **p < 0.01. (E) ATP content. n = 5. Error bars indicate SD; **p < 0.01. (F) Measurement of the activities of mitochondrial OXPHOS complexes normalized to citrate synthase (CS) activity. n = 3. Error bars indicate SD; *p < 0.05, **p < 0.01. (G) Transmission electron microscopy shows differences in the appearance of mitochondria. Scale bar, 100 nm. (H) Relative mtDNA content. n = 3. (I and J) Western blot analysis of OXPHOS subunits encoded by nDNA (I) and mtDNA (J). (K and L) BN-PAGE analyses of OXPHOS complexes. Isolated mitochondria were solubilized with n-dodecyl-b-D-maltoside (K) or digitonin (L) to assess the steady-state levels of OXPHOS complexes and supercomplexes, respectively (*, ATP synthase subcomplex).

Article Snippet: Immunocytochemical Studies The primary antibody used against endogenous CRIF1 was goat anti-human CRIF1 (Santa Cruz, sc-103443, sc-103445).

Techniques: Membrane, Fluorescence, Activity Assay, Transmission Assay, Electron Microscopy, Western Blot, Isolation

Journal: Cell metabolism

Article Title: CRIF1 is essential for the synthesis and insertion of oxidative phosphorylation polypeptides in the mammalian mitochondrial membrane.

doi: 10.1016/j.cmet.2012.06.012

Figure Lengend Snippet: Figure 4. Impaired Mitochondrial Synthesis of OXPHOS Subunits in Crif1-Deficiency (A) Northern blot analysis of ribosomal RNA from nuclear and mitochondrial DNA. nDNA, nuclear DNA; mtDNA, mitochondrial DNA. +/D, WT MEFs; -/D, Crif1-deficient MEFs. (B) Western blot analysis of mitochondrial ribosomes fractions by density sucrose gradient sedimentation. (C) Pulse-chase labeling of newly synthesized OXPHOS subunits from mtDNA of MEFs. (D) Organelle translation assay using mitochondria isolated from MEFs. (E) Mitochondrial membrane flotation analysis of the organelle translation products. (F) Immunoprecipitation of CRIF1 and detection of nascent polypeptides from mitochondrial DNA.

Article Snippet: Immunocytochemical Studies The primary antibody used against endogenous CRIF1 was goat anti-human CRIF1 (Santa Cruz, sc-103443, sc-103445).

Techniques: Northern Blot, Western Blot, Sedimentation, Pulse Chase, Labeling, Synthesized, Isolation, Membrane, Immunoprecipitation