Conversely, a good fit would suggest the assumptions are valid and no critical elements have been overlooked. the specificity of the PI inhibitor for the chymotrypsin-like activity of the proteasome. Data was normalized to the proteasome activity in lysates from cells transfected having a pcDNA control vector. Experiments were performed in triplicate. Error bars represent standard deviation of the mean.(0.28 MB TIF) pcbi.1000944.s001.tif (277K) GUID:?703CD9CC-FEDB-49F3-B48F-A9B5996E341E Number S2: Distribution of polyQ monomers, oligomers and inclusion bodies. Simulation output from 3 runs of the model showing that the size of the oligomeric pool remains constant even when inclusions form.(1.26 MB TIF) pcbi.1000944.s002.tif (1.2M) GUID:?0BD9875D-4976-45D9-A2B2-C8C7E64EDCB2 Text S1: Magic size details.(0.14 MB DOC) pcbi.1000944.s003.doc (139K) GUID:?A5992219-7818-4BEA-A1B5-E6289A65E4EF Video S1: Time-lapse imaging of IB formation and UPS dysfunction in U87MG cells transfected with HttQ103YFP-pIRES-mRFPu. Live cell imaging was initiated at 24 hours post-transfection and images were acquired every 10 minutes. Cells were visualized under white light, and filters that detect YFP or RFP. The brightfield and fluorescence emanating from your YFP and RFP channels were merged to create a movie file. Images were acquired using a 10 objective for a total of 24 hours. At the beginning of the movie HttQ103 is indicated throughout the cell. At 36 hours (the half way point) many cells have formed an inclusion body and have Hyodeoxycholic acid notable accumulation of the reddish reporter protein (indicative of proteasome inhibition). Note that based on their morphology many cells with IB look like viable at the end of the movie.(5.79 MB MOV) pcbi.1000944.s004.mov (5.5M) GUID:?CBEC53DB-F5FC-4282-9BF8-E24DB6ECAFCF Video S2: Time-lapse imaging of IB formation and UPS dysfunction in U87MG cells transfected with HttQ103YFP-pIRES-mRFPu. Live cell imaging was initiated at 24 hours post-transfection and images acquired every 10 minutes. Cells were visualized using filters that detect YFP or RFP. Fluorescent images were merged to create a movie file. Images were acquired using a 10 objective for a total of 24 hours. At the beginning of the movie HttQ103 is indicated throughout the cell. At 36 hours (the half way point) many cells have formed an inclusion body and have notable accumulation of the reddish reporter protein (indicative of proteasome inhibition). Note that based on their morphology many cells with IB look like viable at the end of the movie.(5.63 MB MOV) pcbi.1000944.s005.mov (5.3M) GUID:?2AB0F02C-B8E1-4D50-AD46-F3C68930E98E Video S3: Time-lapse imaging of U87MG cells transfected with HttQ103YFP-pIRES-mRFPu treated with PI 24 hours post-transfection showing an increase of IB formation and UPS dysfunction. Cells were visualized under white light, and filters that detect YFP or RFP. The brightfield and fluorescence emanating from your YFP and RFP channels Hyodeoxycholic acid were merged to create a movie file. Images were acquired every 10 minutes using a 10 objective for a total of 24 hours. The movie shows quick and progressive accumulation of the mRFPu reporter protein (reddish colour) after the addition of PI. This coincides with the formation of IB at an earlier time point (30 hours). At the conclusion of the movie there are considerably more Hyodeoxycholic acid IB and cell death events as compared to untreated cells (video clips S1 and S2).(6.83 MB MOV) pcbi.1000944.s006.mov (6.5M) GUID:?B133B2F9-5CC6-4885-B8F0-E6C0F262A364 Video S4: Time-lapse imaging of U87MG cells expressing HttQ103YFP-pIRES-mRFPu treated with PI 24 hours post-transfection. Cells were visualized using filters that detect YFP or RFP. Fluorescence emanating from your YFP and RFP channels was merged to create a movie which shows an increase of IB formation and UPS dysfunction. Images acquired every 10 minutes using a 10 objective for a total of 24 hours. The movie shows quick and progressive accumulation of the mRFPu reporter protein (reddish colour) after the addition of PI. This coincides with the formation of IB at an earlier time point (30 hours). At the conclusion of the movie there are considerably more IB and cell death events as compared to untreated cells (video clips S1 and S2).(4.26 MB MOV) pcbi.1000944.s007.mov (4.0M) GUID:?8ACFDABE-3FAE-43E0-AC9D-1438F59AF7CA Video S5: Time-lapse imaging of U87MG cells transfected with HttQ103YFP-pIRES-mRFPu treated with BSO 24 hours post-transfection. Treated cells displayed an increase in UPS dysfunction without IB formation. Cells were visualized under white light and filters that detect YFP or RFP. The brightfield and fluorescence emanating from your YFP and DGKH RFP channels was merged to create a movie file. Images were acquired every 10 minutes using a 10 objective for a total of 24 hours. Cells treated with BSO display a rapid and progressive build up of the mRFPu reporter protein (reddish.