Journal: bioRxiv

Article Title: The Legionella effector RidL promotes mitochondrial fragmentation through phosphorylation activation of the large GTPase Drp1

doi: 10.1101/2025.10.07.680855

Figure Lengend Snippet: ( A ) A549 cells depleted (48 h) by siRNA for Arf, Drp1, or Dnm1 were infected (MOI 10, 24 h) with GFP-producing L. pneumophila JR32 or Δ icmT (pNT28), and intracellular replication was assessed by GFP fluorescence using a microtiter plate reader. Graphs show the relative intracellular replication (normalized to GFP signal 1 h p.i.) and represent means + SEM of three independent experiments (Student’s t -test, *, p < 0.05; **, p < 0.01). ( B ) Mitochondria were isolated by differential centrifugation and sucrose density gradient ultracentrifugation from HeLa cells infected (MOI 100, 6 h) with GFP-producing L. pneumophila JR32, ΔicmT , ΔridL , or ΔridL _ ridL (pNT28 or pIF009). Western blot of RidL in cytoplasmic (cyt) and purified mitochondrial (mito) fractions; apoptosis-inducing factor (AIF) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a mitochondrial or cytoplasmic marker, respectively. The figure shown is representative for 3 independent experiments. ( C ) HEK293 cells were transfected (24 h) for ectopic production of GFP-RidL (RidL, pKB248), GFP-RidL 9-258 (RidL N , pKB249) or GFP-RidL 259-1167 (RidL C , pKB250), mitochondria were isolated, and the GFP fusion proteins were detected by anti-GFP Western blot in the cell lysate (lys) and purified mitochondrial (mito) fractions. The figure shown is representative for 3 independent experiments. ( D ) Quantification of (C). Signal intensities of GFP and AIF were calculated by Image J. Bars show relative GFP signal in pure mitochondria fractions. Means + SEM of 3 independent experiments are shown (Student’s t- test, **, p < 0.01). ( E, F ) Fluorescence micrographs of HeLa cells infected (MOI 25, 2 h) with mCerulean-producing L. pneumophila JR23, Δ icmT , Δ ridL or Δ ridL_ridL (pNP99 or pKB208). Mitochondria were visualized with MitoTracker Deep Red, and immuno-labelled with specific antibodies against ( E ) Drp1 or ( F ) Tom20 (green) to quantify the localization of these proteins to mitochondria. ( G ) Quantification of signal overlap in (E) and ( H ) quantification of signal overlap in (F) show 75 infected cells from three independent experiments (each dot is a cell; Student’s t -test, ****, p < 0.0001). The brightness of fluorescence signals was linearly increased in Image J for enhanced visibility (A, B); original signal intensities of the images were processed for quantification (C, D).

Article Snippet: The insert ( Dnm1 ) was amplified from pEGFP-N1-dynamin (Addgene #120313 ) using primers oKA288/oKA289 and Q5 Hot Start High-Fidelity DNA Polymerase (NEB) including Q5 High GC Enhancer.

Techniques: Infection, Fluorescence, Isolation, Centrifugation, Western Blot, Purification, Marker, Transfection

Journal: Frontiers in Synaptic Neuroscience

Article Title: Identification of Synaptic DGKθ Interactors That Stimulate DGKθ Activity

doi: 10.3389/fnsyn.2022.855673

Figure Lengend Snippet: Validation of candidate DGKθ-interacting proteins. (A) Streptavidin beads were used to isolate biotinylated proteins from DGKθ-GFP-APEX2 labeled cortical neuron lysates (DIV21), and biotinylation was confirmed biochemically for dynamin-1, synaptogyrin-1, Munc18-1, clathrin heavy chain 1, Syt1, CaMKIIα, Hsc70, and syntaxin-7. (B) DGKθ overexpressed in HEK cells is immunoprecipitated with a myc antibody. (C) DGKθ interacts with Hsc70, CaMKIIα, Syt1, synaptogyrin-1, dynamin-1, syntaxin-7, and Munc18-1 when both are overexpressed in HEK cells. Hsc70 interacts with DGKθ at its endogenous expression level. (D) Clathrin heavy chain 1 does not interact with DGKθ when overexpressed in HEK cells. All blots are representative of at least three separate experiments.

Article Snippet: Dynamin-1 , Origene , TP306284 , , .

Techniques: Labeling, Immunoprecipitation, Expressing

Journal: Frontiers in pharmacology

Article Title: Buyang Huanwu Decoction alleviates cerebral ischemic injury through modulating caveolin-1-mediated mitochondrial quality control.

doi: 10.3389/fphar.2023.1137609

Figure Lengend Snippet: FIGURE 5 BHD maintains mitochondrial dynamic balance in MCAO mice through Cav-1. (A–E) Western blot and quantitative analysis of DRP1, FIS1, MFN2 and OPA1 (n = 3). (F–I) RT‒qPCR and quantitative analysis of DRP1, FIS1, MFN2 and OPA1 (n = 6). All data are presented as the mean ± SD. **p < 0.01 vs. syngeneic sham group, #p < 0.05 and ##p < 0.01 vs. syngeneic model group, ▲p < 0.05 and ▲▲p < 0.01 vs. WT model group, ◇p < 0.05 and ◇◇p < 0.01 vs. WT BHD group.

Article Snippet: NeuN antibody (Proteintech, 26975-1-AP,Wuhan, China), dynamin 1-like (DRP1) antibody (Boster, A00556-2,Wuhan, Frontiers in Pharmacology frontiersin.org02 China), fission 1 (FIS1) (Boster, A01932-2,Wuhan, China), mitofusin 2 (MFN2) (Boster, BM4906,Wuhan, China), optic atrophy 1 (OPA1) (Boster, PB0773,Wuhan, China), PTEN induced putative kinase 1 (PINK1) (Proteintech, 23274-1- AP,Wuhan, China), PARKIN (Boster, PB9307,Wuhan, China), Beclin 1 (Boster, PB9076,Wuhan, China), microtubule-associated protein 1 light chain 3 beta (LC3) antibody (Proteintech, 14600-1- AP,Wuhan, China), sirtuin 1 (SIRT1) antibody (Boster, A000181,Wuhan, China), peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1α)Antibody (Proteintech, 66369-1-Ig,Wuhan, China), adenosine triphosphate (ATP) assay kit (Jiancheng, A095-1, Nanjing, China), ATPase assay kit (Jiancheng, A016-1, Nanjing, China), RNA Extraction Kit (Beyotime, R0026, Beijing, China), First Strand cDNA Synthesis Kit (Beijing Beyotime, D7178), Universal Genomic DNA Purification Mini Spin Kit (Beyotime, D0063, Beijing, China), SYBR Green qPCR Mix (Beyotime, product number D7265, Beijing, China), the PCR primers were synthesized by Sangon (Shanghai, China), citrate synthase (CS) assay kit (Solarbio, BC1060, Beijing, China), Nicotinamide adenine dinucleotide (NADH)- coenzyme Q reductase assay kit (Solarbio, BC0510, Beijing, China), succinate-coenzyme Q assay kit (Solarbio, BC3230, Beijing, China), coenzyme Q-cytochrome C assay kit (Solarbio, BC3240, Beijing, China), cytochrome C oxidase assay kit (Solarbio, BC0940, Beijing, China).

Techniques: Western Blot