Journal: EBioMedicine
Article Title: KIBRA regulates amyloid β metabolism by controlling extracellular vesicles secretion
doi: 10.1016/j.ebiom.2022.103980
Figure Lengend Snippet: Inhibition of lysosome function in KIBRA knockout cells rescues secretion of EVs harboring APP-CTFβ/Aβ. ( a ) WB analysis of WCLs in KIBRA-KD and CTRL cells overexpressing APP swe , and treated with 20 nM bafilomycinA1 (BafA1) for 12 h, when indicated, and WCLs were blotted for Alix, CD63, and CD9. ( b ) Representative NTA traces of EVs derived from KIBRA-KD and CTRL cells overexpressing APP swe , and treated with BafA1. Right panel represents quantification of NTA of three independent experiments. ( c ) Human Aβ40 and Aβ42 levels measured by Simoa analysis from small EVs purified from sucrose step gradient centrifugation from equal number of KIBRA-KD and CTRL cells overexpressing APP swe, and treated with BafA1. ( n = 3, from three independent experiments). ( d ) WB analysis of EVs secretion in KIBRA-KD and CTRL cells overexpressing APP swe , and treated with 20 nM bafilomycinA1 (BafA1) for 12 h, when indicated. EVs were blotted for Alix, CD63, CD9, and APP-CTFβ. ( e ) Quantification of EVs protein levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe , and treated with lysosome inhibitor BafA1 in three independent experiments. ( f ) Quantification of APP-CTFβ levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe , and treated with lysosome inhibitor BafA1 in three independent experiments. ( g and h ) WB analysis of EVs secretion in KIBRA-KD and CTRL cells overexpressing APP swe transfected with control small interfering RNAs (siRNAs) or siRNAs targeting Rab7. WCLs ( g ) and EVs ( h ) were blotted for Alix, CD63, CD9, Rab7, and APP-CTFβ. ( i ) Quantification of EVs protein levels (Alix, CD63, and CD9) in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe , and transfected with siRNAs in four independent experiments. ( j ) Quantification of APP-CTFβ levels in the small EVs obtained from KIBRA-KD and CTRL cells overexpressing APP swe , and transfected with siRNAs in three independent experiments. ( k ) Representative NTA traces of EVs derived from KIBRA-KD and CTRL cells overexpressing APP swe , and treated with siRNAs targeting Rab7. Right panel represents quantification of NTA of three independent experiments. ( l ) Human Aβ40 and Aβ42 levels measured by Simoa analysis from small EVs purified from sucrose step gradient centrifugation from equal numbers of KIBRA-KD and CTRL cells overexpressing APP swe , and treated with siRNAs in three independent experiments. The quantification results were plotted as dot plots, showing the mean ± SE. * P < 0.05, ⁎⁎ P < 0.01, ⁎⁎⁎ P < 0.001, ⁎⁎⁎⁎ P < 0.0001, n.s., not significant ( P > 0.05) as determined by one-way ANOVA followed by Tukey's post hoc comparisons tests.
Article Snippet: The primary antibodies were obtained from the following sources: mouse monoclonal anti-KIBRA (clone 2A5, provided by Prof Jixin Dong's lab), as previously described ; mouse monoclonal anti-Alix (Cell Signaling Technology Cat# 2171; Research Resource Identifiers [RRID]: AB_2299455, Danvers, MA, USA; 1:1000 in western blot [WB]); rabbit monoclonal anti-CD63 (Abcam Cat# ab217345; RRID: AB_2754982, Cambridge, UK; 1:1000 in WB and 1:100 in immunofluorescence [IF]); rabbit polyclonal anti-CD63 (Abcam Cat# ab216130, RRID not available; 1:25 in IF); mouse monoclonal anti-APP C1/6.1 C-terminal fragment (CTFs) (Biolegend Cat# 802803, RRID: AB_2715853, San Diego, CA, USA; 1:2000 in WB); rabbit monoclonal anti-BACE1 (Abcam Cat# ab108394, RRID: AB_10861218; 1:1000 in WB); Alexa Fluor 594 anti-4G8 (Biolegend Cat# 800715, RRID: AB_2721291; 1:100 in IF); mouse monoclonal anti-4G8 (Biolegend Cat# 800712, RRID: AB_2734548; 1:400 in IF); mouse monoclonal anti-6E10 (Biolegend Cat# 803004, RRID: AB_2715854; 1:1000 in WB); rabbit polyclonal anti-calnexin (Abcam Cat# ab22595, RRID: AB_2069006; 1:1000 in WB); rabbit monoclonal anti-CD9 (Abcam Cat# ab92726, RRID: AB_10561589; 1:1000 in WB); rabbit monoclonal anti-Cathepsin D (Abcam Cat# ab75852, RRID: AB_1523267; 1:1000 in WB and 1:100 in IF); rabbit monoclonal anti-Rab7 (Abcam Cat# ab137029, RRID: AB_2629474; 1:1000 in WB); rabbit polyclonal anti-oligomer A11 (Thermo Fisher Scientific Cat# AHB0052, RRID: AB_2536236; Waltham, MA, USA; 1:1000 in Dot blots); rat monoclonal anti-LAMP2 (Abcam Cat# ab13524, RRID: AB_2134736; 1:1000 in WB and 1:100 in IF); rabbit monoclonal anti-beta III Tubulin (Abcam Cat# ab18207; RRID: AB_ 444319; 1:300 in IF); rabbit monoclonal anti-NeuN (Abcam Cat# ab177487, RRID: AB_2532109; 1:200 in IF).
Techniques: Inhibition, Knock-Out, Derivative Assay, Purification, Gradient Centrifugation, Transfection