Journal: bioRxiv

Article Title: MITOCHONDRIAL ANTIVIRAL PATHWAYS CONTROL ANTI-HIV RESPONSES AND ISCHEMIC STROKE OUTCOMES VIA THE RIG-1 SIGNALING AND INNATE IMMUNITY MECHANISMS

doi: 10.1101/2024.06.07.598027

Figure Lengend Snippet: Pericytes were transfected with the PCMV3-OCLN vector and HIV-1 or mock-infected as in . FIS1 (A) , MFF (C) , MFN2 (E) , and OPA1 (G) mRNA and protein levels were measured by q-PCR and Western Blotting. n= 4-8 per group. In addition, age- and sex-matched ocln −/− , ocln +/− , and ocln +/+ mice (n=4-11 animals per group) were EcoHIV or mock-infected and RNA was extracted from isolated microvessels. FIS1 (B) , MFF (D) , MFN2 (F) and OPA1 (H) mRNA expression levels were measured by q-PCR. Graphs indicate the mean ± SD from three independent experiments. Individual animal data points are marked by blue (ocln +/+ ), green (ocln +/− ), and red (ocln −/− ) dots. ****p < 0.0001, ***p = 0.0002, **p = 0.003, *p < 0.0449.

Article Snippet: Gene amplification was achieved by employing TaqMan Gene Expression Assays and the following primers (all from Thermo Fisher Scientific): human-ISG15:Hs01921425_s1; human-IFIT1: Hs03027069_s1; human-MX1:Hs00895608_m1; human-MX2: Hs01550814_m1; human-HERC5:Hs00180943_m1; human-IRF7: Hs01287244_cn; human-IRF3: Hs01510490_cn; human-RIG-1: Hs01061433_m1; human-USP18:Hs00276441_m1; human-IFIH1: Hs00223420_m1; human-TRAF3: Hs00936781_m1; human-MAVS: Hs00920075_m1; human-MFN2: Hs00208382_m1; human-OPA1: Hs01047019_m1; human-FIS1: Hs00211420_m1; human-MIFF: Hs00697394_g1; human-PPARGC1α: Hs00173304_m1; human-CALCOCO2: Hs00977443_m1; human-P62: Hs01061917_g1; human-TJP1/ZO-1: Hs01551867_m1; human-OCLN: Hs05465837_g1; human-BCL2: Hs00608023_m1; human-BAX: Hs00180269_m1 and mouse-INFα5: Mm00833976; mouse-INFβ1:Mm00439552; mouse-INFɣ: Mm01168134; mouse-ISG15: Mm01705338; mouse-IFIT1: Mm07295796; mouse-RIG-1: Mm00487934; mouse-IFIH1: Mm00459183; mouse-FIS1: Mm00481580; mouse-BAX: Mm00432051; mouse-MFF: Mm00030720_cn; mouse-OPA1: Mm00451274_cn; mouse-BCL2: Mm00529770_cn; mouse-MAVS: Mm00523170_m1; mouse-TRAF3: Mm00385377_cn; and mouse-MFN2: Mm00500120.

Techniques: Transfection, Plasmid Preparation, Infection, Western Blot, Isolation, Expressing

Journal: Ecotoxicology and environmental safety

Article Title: Endoplasmic reticulum stress mediated by ROS participates in cadmium exposure-induced MC3T3-E1 cell apoptosis.

doi: 10.1016/j.ecoenv.2023.114517

Figure Lengend Snippet: Fig. 3. Cd exposure induced cellular Mitochondrial dysfunction. (A) The protein expression of Mfn2, OPA1, Drp1, Fis1, GRP78, Bax, Bcl2, and cleaved caspase 3 was detected by Western blot with GADPH as the loading control in MC3T3-E1 cells treated with Cd for 24 h. (B) The associated protein expression was analyzed using Image J software. (C) cells were cultured with 5, 10, and 20 μM Cd for 24 h. The Drp1, GRP78, and Bcl2 expressions were detected by Immunofluorescence. (D) Dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent staining showed that Cd activated the overproduction of cellular ROS. (E) The effect of Cd on the ultrastructural structure in MC3T3-E1 was observed. Blue arrows indicated the Endoplasmic reticulum, M indicated Mtochondria, and N indicated the cell nucleus. (F) Effects of Cd on the levels of CAT, TAC, and MDA in MC3T3-E1 cells. The values presented are the mean ± SD of three independent experiments. *P-value < 0.05, * *P-value < 0.01 relative to the control group.

Article Snippet: After the proteins were transferred into NC membranes, the NC membranes were blocked with 5% skimmed milk for 1 h. In addition, the NC membranes were incubated with the primary antibody Drp1 (1:2000, Wanleibio), Fis1 (1:2000, Proteintech), OPA1(1:2000, Abbkine), Mfn2(1:2000, Abbkine), GRP78 (1:2000, Wanleibio), CHOP (1:1000, Wanleibio), ATF4 (1:1500, Wanleibio), P-EIF2a(1:1000,Cell signaling), ATF6 (1:1000, Wanleibio), IRE1-a (1:2000, Wanleibio), P-IRE1a (1:1000, Wanleibio), PERK (1:1000, Wanleibio), P-PERK (1:500, Wanleibio), Nrf2 (1:2000, Affinity), NQO1 (1:2000, ABclonal), Bax (1:1000, Wanleibio, China), Bcl2 (1:500, Wanleibio), Cleaved Caspase-3 (1:2000, Affinity) and GADPH (1:2000, ABclonal), Lamin B (1:2000, Affinity) at 4 ◦C overnight.

Techniques: Expressing, Western Blot, Control, Software, Cell Culture, Immunofluorescence, Staining

Journal: Cells

Article Title: Adenosine A2a Receptor Stimulation Mitigates Periodontitis and Is Mitoprotective in Gingival Fibroblasts Promoting Cellular Resilience

doi: 10.3390/cells14161266

Figure Lengend Snippet: Mitoprotective effect of adenosine A2aR stimulation is due to improved mitochondrial quality control through increased mitochondrial fusion. ( A – F ) MRNA levels of Mitofusins 1 and 2 ( Mfn1/2 ) and Fission 1 ( Fis1 ) in murine gingival fibroblasts pretreated with CGS21680 (10 µM) and stimulated with Tnf-α at 5 ng/mL for ( A – C ) 3 h or ( D – F ) 6 h. Immunoblots showing protein expressions of mitochondrial quality control targets pro-fusion ( G ) Mfn2 and ( H ) Opa1 and pro-fission ( I ) pDrp1 and Drp1, with their respective densitometry analysis relative to β-Tubulin as a loading control. Data are presented as means ± S.D.s. (* p < 0.05; ** p < 0.01).

Article Snippet: The following inventoried Taqman assays for quantitative PCR were used: murine Mfn1 (Mm00612599_m1), Mfn2 (Mm00500120_m1), and Fis1 (Mm00481580_m) in a 10 μL final volume with TaqMan Fast Advanced Master Mix in a StepOne Plus Real-Time PCR system (Life Technologies, Carlsbad, CA, USA).

Techniques: Control, Western Blot