ragb q99l Search Results


prk5 gfp ragb  (Addgene inc)
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(A,B) High content microscopy (HCM) quantification and image analysis of the effect of Hexa KO on lysotracker intensity. (>500 primary objects examined per well; minimum number of wells, 9). Masks; white: ORF3a-mCherry expressing cells, algorithm-defined cell boundaries; green masks: computer-identified lysotracker dots). p values were determined using GraphPad prism (n=9) ANOVA. Images, a detail from a large database of machine-collected and computer-processed images. Scale bar 10 µm. (C,D) HCM quantification and image analysis of the effect of Hexa KO on ORF3a induced Gal3 puncta formation. (>500 primary objects examined per well; minimum number of wells, 8). Pseudo colours: Red, ORF3a-mCherry; green, Gal3. p values, (n=8) ANOVA. Scale bar 5 µm. (E) HCM analysis of the effect of ORF3a on Gal3 and LAMP1 colocalization in HeLa WT and Hexa KO cells. (>500 primary objects examined per well; minimum number of wells, 6). p values were determined using GraphPad prism (n=6) ANOVA (n=6) ANOVA. (F) Representative micrographs showing the effect of ORF3a expression on Gal3 and LAMP1 colocalization in HeLa WT and Hexa KO cells. Scale bar 5 µm. Pseudo colours: Red, LAMP1; green, Gal3. ORF3a-mCherry is removed from images to avoid confusion. (G,H) HCM analysis of effect of Hexa KO on ORF3a-mCherry induced nuclear translocation of TFEB (>500 primary objects/cells examined per well; minimum number of wells,10). p values were determined using GraphPad prism (n=10) ANOVA. Scale bar 10 µm . (I) Western blot analysis of the effect of ORF3a-mCherry expression on mTOR target p70S6K, in HeLa WT and Hexa KO cells. (J,K) HCM quantifications and micrograph of the effect of ORF3a-mCherry expression on mTOR puncta formation in HeLa WT and Hexa KO cells. Data, means ± SEM, p values were determined using GraphPad prism (n=6) ANOVA; HCM, >500 cells counted per well; minimum number of valid wells 6). Scale bar 5 µm. (L,M) HCM quantifications and micrograph of the effect of ORF3a-mCherry expression on colocalization between mTOR and LAMP1 in HeLa WT and Hexa KO cells. Data, means ± SEM, p values were determined using GraphPad prism (n=6) ANOVA; HCM, >500 cells counted per well; minimum number of valid wells 6. Scale bar 5 µm. Pseudo colours: Red, mTOR; blue, mCherry; green, LAMP1. (N) Co-IP analysis of the effect of ORF3a expression on interaction between <t>FLAG-RagB</t> and endogenous mTOR. (O) Co-IP analysis of the effect of ORF3a expression on interaction between FLAG-TFEB and endogenous mTOR. (P) HCM quantification of the effect of RagB <t>Q99L</t> expression on ORF3a mediated inhibition of lysotracker puncta. Data, means ± SEM, p values were determined using GraphPad prism (n=6) ANOVA; HCM, >500 cells counted per well; minimum number of valid wells 6. (Q) HCM quantification of the effect of RagB Q99L expression on ORF3a mediated formation of Gal3 puncta. Data, means ± SEM, p values were determined using GraphPad prism (n=6) ANOVA; HCM, >500 cells counted per well; minimum number of valid wells 6.
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(A,B) High content microscopy (HCM) quantification and image analysis of the effect of Hexa KO on lysotracker intensity. (>500 primary objects examined per well; minimum number of wells, 9). Masks; white: ORF3a-mCherry expressing cells, algorithm-defined cell boundaries; green masks: computer-identified lysotracker dots). p values were determined using GraphPad prism (n=9) ANOVA. Images, a detail from a large database of machine-collected and computer-processed images. Scale bar 10 µm. (C,D) HCM quantification and image analysis of the effect of Hexa KO on ORF3a induced Gal3 puncta formation. (>500 primary objects examined per well; minimum number of wells, 8). Pseudo colours: Red, ORF3a-mCherry; green, Gal3. p values, (n=8) ANOVA. Scale bar 5 µm. (E) HCM analysis of the effect of ORF3a on Gal3 and LAMP1 colocalization in HeLa WT and Hexa KO cells. (>500 primary objects examined per well; minimum number of wells, 6). p values were determined using GraphPad prism (n=6) ANOVA (n=6) ANOVA. (F) Representative micrographs showing the effect of ORF3a expression on Gal3 and LAMP1 colocalization in HeLa WT and Hexa KO cells. Scale bar 5 µm. Pseudo colours: Red, LAMP1; green, Gal3. ORF3a-mCherry is removed from images to avoid confusion. (G,H) HCM analysis of effect of Hexa KO on ORF3a-mCherry induced nuclear translocation of TFEB (>500 primary objects/cells examined per well; minimum number of wells,10). p values were determined using GraphPad prism (n=10) ANOVA. Scale bar 10 µm . (I) Western blot analysis of the effect of ORF3a-mCherry expression on mTOR target p70S6K, in HeLa WT and Hexa KO cells. (J,K) HCM quantifications and micrograph of the effect of ORF3a-mCherry expression on mTOR puncta formation in HeLa WT and Hexa KO cells. Data, means ± SEM, p values were determined using GraphPad prism (n=6) ANOVA; HCM, >500 cells counted per well; minimum number of valid wells 6). Scale bar 5 µm. (L,M) HCM quantifications and micrograph of the effect of ORF3a-mCherry expression on colocalization between mTOR and LAMP1 in HeLa WT and Hexa KO cells. Data, means ± SEM, p values were determined using GraphPad prism (n=6) ANOVA; HCM, >500 cells counted per well; minimum number of valid wells 6. Scale bar 5 µm. Pseudo colours: Red, mTOR; blue, mCherry; green, LAMP1. (N) Co-IP analysis of the effect of ORF3a expression on interaction between FLAG-RagB and endogenous mTOR. (O) Co-IP analysis of the effect of ORF3a expression on interaction between FLAG-TFEB and endogenous mTOR. (P) HCM quantification of the effect of RagB Q99L expression on ORF3a mediated inhibition of lysotracker puncta. Data, means ± SEM, p values were determined using GraphPad prism (n=6) ANOVA; HCM, >500 cells counted per well; minimum number of valid wells 6. (Q) HCM quantification of the effect of RagB Q99L expression on ORF3a mediated formation of Gal3 puncta. Data, means ± SEM, p values were determined using GraphPad prism (n=6) ANOVA; HCM, >500 cells counted per well; minimum number of valid wells 6.

Journal: bioRxiv

Article Title: SARS-CoV-2 protein ORF3a induces Atg8ylation of lysosomal membranes

doi: 10.1101/2024.09.12.612614

Figure Lengend Snippet: (A,B) High content microscopy (HCM) quantification and image analysis of the effect of Hexa KO on lysotracker intensity. (>500 primary objects examined per well; minimum number of wells, 9). Masks; white: ORF3a-mCherry expressing cells, algorithm-defined cell boundaries; green masks: computer-identified lysotracker dots). p values were determined using GraphPad prism (n=9) ANOVA. Images, a detail from a large database of machine-collected and computer-processed images. Scale bar 10 µm. (C,D) HCM quantification and image analysis of the effect of Hexa KO on ORF3a induced Gal3 puncta formation. (>500 primary objects examined per well; minimum number of wells, 8). Pseudo colours: Red, ORF3a-mCherry; green, Gal3. p values, (n=8) ANOVA. Scale bar 5 µm. (E) HCM analysis of the effect of ORF3a on Gal3 and LAMP1 colocalization in HeLa WT and Hexa KO cells. (>500 primary objects examined per well; minimum number of wells, 6). p values were determined using GraphPad prism (n=6) ANOVA (n=6) ANOVA. (F) Representative micrographs showing the effect of ORF3a expression on Gal3 and LAMP1 colocalization in HeLa WT and Hexa KO cells. Scale bar 5 µm. Pseudo colours: Red, LAMP1; green, Gal3. ORF3a-mCherry is removed from images to avoid confusion. (G,H) HCM analysis of effect of Hexa KO on ORF3a-mCherry induced nuclear translocation of TFEB (>500 primary objects/cells examined per well; minimum number of wells,10). p values were determined using GraphPad prism (n=10) ANOVA. Scale bar 10 µm . (I) Western blot analysis of the effect of ORF3a-mCherry expression on mTOR target p70S6K, in HeLa WT and Hexa KO cells. (J,K) HCM quantifications and micrograph of the effect of ORF3a-mCherry expression on mTOR puncta formation in HeLa WT and Hexa KO cells. Data, means ± SEM, p values were determined using GraphPad prism (n=6) ANOVA; HCM, >500 cells counted per well; minimum number of valid wells 6). Scale bar 5 µm. (L,M) HCM quantifications and micrograph of the effect of ORF3a-mCherry expression on colocalization between mTOR and LAMP1 in HeLa WT and Hexa KO cells. Data, means ± SEM, p values were determined using GraphPad prism (n=6) ANOVA; HCM, >500 cells counted per well; minimum number of valid wells 6. Scale bar 5 µm. Pseudo colours: Red, mTOR; blue, mCherry; green, LAMP1. (N) Co-IP analysis of the effect of ORF3a expression on interaction between FLAG-RagB and endogenous mTOR. (O) Co-IP analysis of the effect of ORF3a expression on interaction between FLAG-TFEB and endogenous mTOR. (P) HCM quantification of the effect of RagB Q99L expression on ORF3a mediated inhibition of lysotracker puncta. Data, means ± SEM, p values were determined using GraphPad prism (n=6) ANOVA; HCM, >500 cells counted per well; minimum number of valid wells 6. (Q) HCM quantification of the effect of RagB Q99L expression on ORF3a mediated formation of Gal3 puncta. Data, means ± SEM, p values were determined using GraphPad prism (n=6) ANOVA; HCM, >500 cells counted per well; minimum number of valid wells 6.

Article Snippet: Plasmids related to SARS-CoV-2 ORF3a-mCherry (#165138), ORF3a-EGFP (#165121), NSP6-mCherry (#165133), NSP4-mCherry (#165132), NSP3-mCherry (#165131), GFP-Gal-3 (#73080), px459 sgFIP200 (#175024), px459 sgAtg5 (#175023), psPAX2 (#12260), pCMV-VSV-G (#8454), pRK5 Flag RagB (#112755), pRK5 GFP RagB Q99L(#112749), and Flag pLJM1 RagB 99L(#19315) were acquired from Addgene.

Techniques: Microscopy, Expressing, Translocation Assay, Western Blot, Co-Immunoprecipitation Assay, Inhibition