Journal: Precision Clinical Medicine

Article Title: Agrimol B inhibits pancreatic ductal adenocarcinoma by induction of lethal mitophagy through decreasing mitochondrial transcription termination factor 3

doi: 10.1093/pcmedi/pbag009

Figure Lengend Snippet: Agrimol B blocks autophagic flux in PDAC cells. (A, B) Western blot analysis of P62 and CTSD in PANC-1 and AsPC-1 cells treated with Agrimol B for 24 h. (C, E, F) Immunofluorescence analysis of RFP-GFP-LC3 after PANC-1 and AsPC-1 cells were transfected with RFP-GFP-LC3 for 48 h, followed by treatment with or without Agrimol B for another 24 h. Scale bars, 10 μm. (D, G-L) Immunofluorescence analysis of the colocalization of endogenous LC3 with LAMP2 after treatment with Agrimol B or rapamycin for 24 h in PANC-1 and AsPC-1 cells. Scale bars, 10 μm. (M-O) Immunofluorescence analysis of LC3 in PDAC cells treated with or without Agrimol B in the presence or absence of HCQ. Scale bars, 10 μm.

Article Snippet: An anti-microtubule-associated protein light chain 3 (LC3) antibody (NB100-2220) was purchased from Novus, while anti-MTERF3 (EM1701-29) and lysosomal associated membrane protein 2 (LAMP2) (M1603-5) antibodies were purchased from HuaBio.

Techniques: Western Blot, Immunofluorescence, Transfection

Journal: eLife

Article Title: Extracellular vesicle-mediated release of bis(monoacylglycerol)phosphate is regulated by LRRK2 and glucocerebrosidase activity

doi: 10.7554/eLife.106330

Figure Lengend Snippet: ( A ) Confocal microscopy of endogenous BMP (yellow) and LAMP2 (magenta) immunofluorescence in wild-type (WT) and R1441G LRRK2 MEFs. Scale bar: 20 µm. Quantification of vesicular BMP intensity ( B ) and LAMP2 relative intensity ( C ) per cell area. Colored dots represent mean value from four independent experiments, and violin plots show the distribution of individual cell data. Significance determined by two-tailed paired t -test **p < 0.01, ***p < 0.001. ( D ) Representative transmission electron microscopy (TEM) images of multivesicular endosomes (MVE) from WT and R1441G LRRK2 MEFs. MVB periphery highlighted in yellow. Scale bar: 250 nm. ( E ) MVE area (µm 2 ) quantification in WT and R1441G LRRK2 mutant cells. Colored dots represent mean values from 3 independent experiments and violin plots show the distribution of individual cell data (35–45 cells/group). ( F ) Quantification of intraluminal vesicles (ILVs) per MVE in WT and R1441G LRRK2 MEF cells. The number of ILVs per MVE is binned in three groups and plotted as a percentage of MVE from the total population of each experiment independently. Data from 3 independent experiments (mean ± SEM). Significance determined by two-tailed unpaired t -test ( E ) and ordinary two-way ANOVA, uncorrected Fisher’s LSD ( F ). *p < 0.05, ****p < 0.0001. Figure 1—source data 1. IF images in panel A. Figure 1—source data 2. TEM images in panel D. Figure 1—source data 3. Plotted values in panels B, C, E, F.

Article Snippet: Antibody , anti-mouse LAMP2, clone GL2A7 (Rat monoclonal) , Developmental Studies Hybridoma Bank , GL2A7; RRID: AB_2281134 , IF (1:500) WB (1:1000).

Techniques: Confocal Microscopy, Immunofluorescence, Two Tailed Test, Transmission Assay, Electron Microscopy, Mutagenesis

Journal: eLife

Article Title: Extracellular vesicle-mediated release of bis(monoacylglycerol)phosphate is regulated by LRRK2 and glucocerebrosidase activity

doi: 10.7554/eLife.106330

Figure Lengend Snippet: ( A ) Whole cell lysates (WCL) from wild-type (WT) and R1441G LRRK2 mouse embryonic fibroblast (MEF) cells treated with 200 nM MLi-2 for 24 hr were analyzed by immunoblotting. Representative images of LAMP2, phospho-Rab10, and α-Tubulin levels are shown. Molecular weight marker mobility is shown in kDa. ( B ) Flow cytometry measurement of GCase activity using PFB-FDGlu fluorescent GCase substrate in WT and R1441G LRRK2 mutant MEF cells treated with 300 µM conduritol β-epoxide (CBE) for 24 hr. WCL and isolated EVs from WT and R1441G LRRK2 mutant MEF cells treated with 200 nM MLi-2 ( C ) or 300 µM CBE ( G ) for 48 hr were analyzed by immunoblotting. Representative images of LAMP2, Flotillin-1, and α-Tubulin levels are shown. Molecular weight marker mobility is shown in kDa. Immunoblots for LAMP2 and Flotillin-1 in EV fractions required longer exposure times to visualize clear signals across all conditions. Quantification of LAMP2 and Flotillin-1 levels relative to R1441G LRRK2 MEF cells in WCL ( D , H ) and isolated EVs ( E , F , I, J ). Data from 6 to 8 independent experiments (mean ± SEM). Significance determined by Kruskal–Wallis test followed by an uncorrected Dunn’s post hoc test compared to R1441G LRRK2 control *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. Figure 2—source data 1. Uncropped blots. Figure 2—source data 2. Annotated uncropped blots. Figure 2—source data 3. Plotted values in panels D–F, H–J.

Article Snippet: Antibody , anti-mouse LAMP2, clone GL2A7 (Rat monoclonal) , Developmental Studies Hybridoma Bank , GL2A7; RRID: AB_2281134 , IF (1:500) WB (1:1000).

Techniques: Western Blot, Molecular Weight, Marker, Flow Cytometry, Activity Assay, Mutagenesis, Isolation, Control

Journal: eLife

Article Title: Extracellular vesicle-mediated release of bis(monoacylglycerol)phosphate is regulated by LRRK2 and glucocerebrosidase activity

doi: 10.7554/eLife.106330

Figure Lengend Snippet: ( A–C ) No significant differences in EV release between MLi-2/conduritol β-epoxide (CBE)-treated and untreated wild-type (WT) MEF cells. Quantification of LAMP2 and Flotillin-1 levels relative to WT control MEF cells in whole cell lysates (WCL) ( A ) and isolated EVs ( B–C ). Data from 7 to 8 independent experiments (mean ± SEM). Significance determined by ordinary one-way ANOVA, uncorrected Fisher’s LSD. ( D–E ) Characterization of WT or R1441G LRRK2 MEF-derived purified EVs by nanoparticle tracking analysis (NTA). ( D ) Representative plots of EV size distribution in each indicated condition. ( E ) Yield comparison of MEF-derived EVs from each indicated condition determined by NTA. Each colored dot represents an independent experiment. Figure 2—figure supplement 1—source data 1. Plotted values in panels A–C, E.

Article Snippet: Antibody , anti-mouse LAMP2, clone GL2A7 (Rat monoclonal) , Developmental Studies Hybridoma Bank , GL2A7; RRID: AB_2281134 , IF (1:500) WB (1:1000).

Techniques: Control, Isolation, Derivative Assay, Purification, Comparison

Journal: eLife

Article Title: Extracellular vesicle-mediated release of bis(monoacylglycerol)phosphate is regulated by LRRK2 and glucocerebrosidase activity

doi: 10.7554/eLife.106330

Figure Lengend Snippet: ( A ) Whole cell lysates (WCL) and isolated EVs from wild-type (WT) mouse embryonic fibroblast (MEF) cells treated with 10 µM GW4869 or 10 nM bafilomycin-A1 (B-A1) for 24 hr were analyzed by immunoblotting. Representative immunoblots of LAMP2, Flotillin-1, and α-Tubulin are shown. Molecular weight marker mobility is shown in kDa. Ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) determination of EV-associated di-18:1-BMP normalized to protein content from WT MEF cells treated with 10 µM GW4869 ( B ) or 10 nM B-A1 ( C ) for 24 hr. Data shown as fold change relative to WT control MEF cells. Only BMP isoforms that were detected are shown. Data from 6 independent experiments (mean ± SEM). Significance determined by two-tailed unpaired t -test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Quantitation of BMP isoforms normalized to protein content in WCL from MEF WT cells treated with 10 µM GW4869 ( D ) or 10 nM B-A1 ( E ) for 24 hr. Data shown as fold change relative to WT control MEF cells. Only BMP isoforms that were detected are shown. Data from 3 independent experiments (mean ± SEM). Significance determined by two-tailed unpaired t -test; *p < 0.05, **p < 0.01, ***p < 0.001. Figure 6—source data 1. Uncropped blots. Figure 6—source data 2. Annotated uncropped blots. Figure 6—source data 3. Plotted values in panels B–E.

Article Snippet: Antibody , anti-mouse LAMP2, clone GL2A7 (Rat monoclonal) , Developmental Studies Hybridoma Bank , GL2A7; RRID: AB_2281134 , IF (1:500) WB (1:1000).

Techniques: Isolation, Western Blot, Molecular Weight, Marker, Liquid Chromatography, Mass Spectrometry, Tandem Mass Spectroscopy, Control, Two Tailed Test, Quantitation Assay

Journal: eLife

Article Title: Extracellular vesicle-mediated release of bis(monoacylglycerol)phosphate is regulated by LRRK2 and glucocerebrosidase activity

doi: 10.7554/eLife.106330

Figure Lengend Snippet: Whole cell lysates (WCL) and isolated EVs from R1441G LRRK2 MEF cells treated with 10 µM GW4869 or 10 nM bafilomycin-A1 for 24 h were analyzed by immunoblotting. Representative images of LAMP2, Flotillin-1, and α-Tubulin levels are shown. Molecular weight marker mobility is shown in kDa. Figure 6—figure supplement 1—source data 1. Uncropped blots. Figure 6—figure supplement 1—source data 2. Annotated uncropped blots.

Article Snippet: Antibody , anti-mouse LAMP2, clone GL2A7 (Rat monoclonal) , Developmental Studies Hybridoma Bank , GL2A7; RRID: AB_2281134 , IF (1:500) WB (1:1000).

Techniques: Isolation, Western Blot, Molecular Weight, Marker