Journal: PLoS ONE

Article Title: Truncated mutants of beta-glucosidase 2 (GBA2) are localized in the mitochondrial matrix and cause mitochondrial fragmentation

doi: 10.1371/journal.pone.0233856

Figure Lengend Snippet: (A) Colocalization of various forms of GBA2 with mitochondrial markers TOMM20 or cytochrome c, expressed as the Manders M1 coefficient (data indicate means + SD; n = 3; 10 cells were analyzed per experiment). (B and C) Distribution of mitochondrial morphologies among cells expressing various forms of GBA2 under the control of (B) the CMV promoter and (C) the MSCV LTR. Data indicate means + SD; at least 250 cells were scored in each of three independent experiments. Statistical comparisons between GBA2-WT and GBA2 mutants by (A) one- or (B and C) two-way ANOVA: *, P < 0.0001. Statistical comparisons between the same forms of GBA2 expressed under different promoters (CMV v MSCV): $, P = 0.0002; #, P < 0.0001.

Article Snippet: Rabbit anti- DYKDDDDK (FLAG) mAb was used at 1:2000 (Cell Signaling 2368S), mouse anti-TOMM20 mAb at 1:250 (BD Transduction Laboratories 612278), mouse anti-cytochrome c mAb at 1:1000 (BD Bioscience 556432), and anti-TST (StrepMAB-Classic, IBA 2-1507-001) at 1:1500.

Techniques: Expressing

Journal: Cells

Article Title: Follicle-Stimulating Hormone Alleviates Ovarian Aging by Modulating Mitophagy- and Glycophagy-Based Energy Metabolism in Hens

doi: 10.3390/cells11203270

Figure Lengend Snippet: Activation of the AMPK signaling pathway is responsible for FSH-induced mitophagy in senescent GCs. The SYF-GCs of D280 hens were treated with D-gal (200 mM) for 24 h and were induced to become premature senescent GCs. Subsequently, 0.01 IU/mL of FSH was added and stimulated the premature senescent GCs for 24 h. ( A ) Transmission electron micrograph of mitophagosomes (red arrows) in the senescent GCs with FSH treatment. Swollen mitochondria were observed in the D-gal group, whereas this damage was alleviated after FSH addition. Scale bars: 1 μm and 500 nm. ( B ) GCs were collected for JC-1 staining to observe the healthy mitochondria (uptaking red fluorescent aggregates of JC-1) and defective mitochondria (uptaking green fluorescent monomeric JC-1). ( C , D ) Western blot and quantitative analyses of LC3-II and TOMM20 expression in normal, D-gal, D-gal + FSH, FSH alone-treated GCs. ( E ) Western blot and quantitative analyses of AMPK phosphorylation in GCs (normal, D-gal, D-gal + FSH, FSH). ( F , G ) Colocalization of mitophagic markers (LC3 and TOMM20) in GCs treated as indicated above. CC: Compound C. Scale bar: 5 μm. ( H , I ) Western blot analysis of p-AMPK/AMPK, LC3-II, TOMM20 proteins in normal, D-gal, D-gal + FSH, D-gal + FSH +Compound C (AMPK inhibitor), Compound C groups. ( J ) ATP levels of GCs that were treated as indicated above ( H ). Different lowercase letters indicate significant differences ( p < 0.05).

Article Snippet: The membranes were blocked in 5% milk with Tris-buffered saline Tween-20 (TBST PH 7.4) for 1 h at room temperature, then probed overnight at 4 °C with the following primary antibodies: GLUT1 (1:500, HUABIO), PFKFB2/PFK2 (1:500, ER1915-04, HUABIO), LDHA (1:1000, HUABIO), SDHA (1:500, ET1703-40, HUABIO), Caspase-3 (1:500, ER1802-42, HUABIO), PCNA (1:500, R1306-5, HUABIO), CDK2 (1:500, R1309-3, HUABIO), CCND1 (1:500, RE6025, HUABIO), TOMM20 (1:1000, ET1609-25, HUABIO), AKT1/2/3 (1:500, ET1609-51, HUABIO), Phospho-AKT1 (Ser7473) (1:500, ET1607-73, HUABIO), GSK3 beta (1:500, ET1607-7, HUABIO), Phospho-GSK3 beta (Ser 9) (1:500, ET1607-60, HUABIO), HK2 (1:1000, ABclonal), AMPKa1/AMPKa2 (1:1000, A17290, ABclonal), Phospho-AMPKa1/AMPKa2-T183/T172 (1:1000, AP1171, ABclonal), GABARAPL1 (1:1000, A7790, ABclonal), LC3 (1:500, Novus), STBD1 (1:500, 11842-1-AP, Proteintech, Rosemont, IL, USA), cleaved Caspase-3 (Asp175) (1:500, 9664, Cell Signaling Technology, Beverly, MA, USA), and β-actin (1:5000, EM21002, HUABIO).

Techniques: Activation Assay, Transmission Assay, Staining, Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: PBLD promotes IRF3 mediated the type I interferon (IFN-I) response and apoptosis to inhibit viral replication

doi: 10.1038/s41419-024-07083-w

Figure Lengend Snippet: A HeLa cells were transfected with Flag-PBLD or pCMV-Flag for 24 h, and then the cells were infected with BPIV3 (MOI = 1) for the indicated time. The expression of intrinsic mitochondrial apoptosis proteins was determined by western blot analysis. B , C HeLa cells were transfected with pCMV-Flag, pCMV-HA-IRF3, siIRF3, or siNC for 24 h, and then the cells were infected with BPIV3 (MOI = 1) for the indicated time. The expression of intrinsic mitochondrial apoptosis proteins was determined by western blot analysis. D Flag-PBLD HeLa cell lines were treated with siIRF3 or scrambled siRNA (siNC) for 24 h, and then infected with BPIV3 (MOI = 1) for the indicated time. The expression of Bax, Bcl 2 , Cytc, and cleaved PARP proteins were analyzed by western blotting. E Mitochondrial isolation and sub-cellular localization assay for IRF3 affected by PBLD during BPIV3 infection. The cells were transfected with Flag-PBLD or vector for 24 h. Following BPIV3 (MOI = 1) infection for 24 h, cytosolic and mitochondrial proteins were prepared, and the expression of IRF3 was determined by western blot analysis. β-actin and TOMM20 were used for loading control of cytosolic and mitochondrial protein, respectively. F Mapping the ubiquitination sites of IRF3. G – J pCMV-Flag or pCMV-Flag-PBLD in combination of pCMV-HA-IRF3, pCMV-HA-IRF3(S385/386 A) and pCMV-HA-IRF3(S396A) plasmids were transfected into IRF3 knockout HeLa cell lines and then infected with BPIV3 (MOI = 1) or SeV (100HAU/mL) for 24 h, respectively. The expression of cleaved PARP apoptosis protein was verified by western blotting. The gray intensity of the bands in ( A – E and G – J ) from three independent experiments were analyzed using ImageJ software. Significance was determined by two-way ANOVA in ( A – E ), one-way ANOVA in ( G – J ). Ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Rabbit polyclonal antibody to TOMM20 (#AF5206) was obtained from Affinity Biosciences.

Techniques: Transfection, Infection, Expressing, Western Blot, Isolation, Plasmid Preparation, Control, Knock-Out, Software

Journal: Cell Death & Disease

Article Title: PBLD promotes IRF3 mediated the type I interferon (IFN-I) response and apoptosis to inhibit viral replication

doi: 10.1038/s41419-024-07083-w

Figure Lengend Snippet: A Interaction between IRF3 and Puma was analyzed by co-immunoprecipitation. HeLa cells expressing HA-IRF3 were exposed to BPIV3 infection for 24 h, and then immunoprecipitation of IRF3 was performed, and the presence of Puma and Noxa proteins was assessed by western blot analysis. B The effect of PBLD on the binding between IRF3 and Puma was examined by co-immunoprecipitation analysis. HeLa cells were cotransfected with HA-IRF3 and different doses (1.2 μg, 2.4 μg) of PBLD for 24 h, and then infected with BPIV3 for another 24 h. After that, the cell lysates were immunoprecipitated with HA antibody, and the presence of Puma was detected by western blot analysis. C PBLD facilitates the interaction between IRF3 and Puma within the mitochondria as demonstrated by western blot analysis. HeLa cells were transfected with Flag-PBLD and infected with BPIV3 for 24 h. The mitochondrial and cytosolic fractions were isolated and analyzed by western blot for the indicated proteins. β-actin and TOMM20 function as the loading control for cytosolic proteins and mitochondrial proteins, respectively. D PBLD promoted the localization of IRF3 at the mitochondria and was examined in Puma knockout cell lines. E The effect of PBLD on the mitochondrial localization of Puma was detected in IRF3 knockout cell lines. F Wild-type IRF3 (HA-IRF3) and IRF3 mutants: HA-IRF3(K313/315 N) interacted with Puma was analyzed by co-immunoprecipitation in the context of PBLD. G – J Knockout of Puma attenuated IRF3- or PBLD-induced apoptosis during BPIV3 or VSV infection. Puma knockout cell lines were transfected with HA-IRF3 or Flag-PBLD, and infected with BPIV3 at a MOI of 1 for the indicated time. Then, apoptosis-associated proteins in these cells were determined by western blotting. The gray intensity of the bands in ( A – J ) from three independent experiments was analyzed using ImageJ software. Significance was determined by Student’s t -test in ( A ), one-way ANOVA in ( B ), two-way ANOVA in ( C – J ). Ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Rabbit polyclonal antibody to TOMM20 (#AF5206) was obtained from Affinity Biosciences.

Techniques: Immunoprecipitation, Expressing, Infection, Western Blot, Binding Assay, Transfection, Isolation, Control, Knock-Out, Software