Journal: Cell

Article Title: LAP2 Proteins Chaperone GLI1 Movement Between Lamina and Chromatin to Regulate Transcription

doi: 10.1016/j.cell.2018.10.054

Figure Lengend Snippet: (A) BASU-GLI1 vicinal labeling in ASZ followed by streptavidin pulldown ± CRT0329868 (CRT) (+biotin, +CRT, 5hr). (B) APEX2-GLI1 vicinal labeling in ASZ followed by streptavidin pulldown ± PSI. (C) Co-IP of FLAG-GLI1 in ASZ ± PSI followed by immunoblot. (D) PLA between total GLI1 and LAP2α (top) or LAP2β (bottom) in ASZ treated with indicated inhibitors for 2hr (scale bar=20μm, n=10 fields, ANOVA). (E) PLA between total GLI1 and LAP2α (left) or LAP2β (right) in 1º human BCCs treated with vorinostat ex vivo (scale bar= 66μm, n=10 fields, ANOVA). (F) Co-IP of in vitro translated HA-GLI1 zinc-finger domain (HA-GLI1ZF) from WCE. Inputs in Figure S5B. (G) LAP2-binding mutants mapped onto GLI1:DNA crystal structure (pdb:2GLI). Mutations which inhibit (red) or are permissive of (grey) LAP2 binding are illustrated as spheres. Co-IP in Figure S5C. (H) Co-IP of HA-GLI1WT/T296E transfected into HEK293T followed by immunoblot of endogenous LAP2. Inputs in Figure S5D. (I) qRT-PCR of GLI1 and GAPDH following transfection of GLI1WT/T296E into NIH3T3 (n=9, ANOVA). Associated immunoblot in Figure S5E. (J) Co-IP of full length GLI1 (GLI1 FL) or zinc-finger domain GLI1 (GLI1 ZF) with recombinant LAP2 constant region (−/+ indicate the addition of LAP2 peptide). Input in Figure S5F. (K) Co-IP of wheat germ cell extract in vitro translated HA-GLI1 with chemically synthesized biotin-LEM-like (residues 5–48), biotin-LEM (residues 109–153), or biotin-scrambled LEM-like domains. Associated inputs and saturation binding experiment in Figure S5G and S5H. (L) Co-IP of FLAG-GLI1 co-transfected into HEK293T with a gradient of LAP2α, followed by immunoblot for total LAP2 (n=3). Input and reciprocal IP in Figures S5I and S5J. (M) GLI1 transfected in HEK293T (top, cellular IP) or in vitro translated and incubated in WCE (bottom three, in vitro IP) with indicated mutations/truncations co-IP with associated epitope tag. IP washed over a gradient of high salt conditions prior to immunoblot. Complex strength=−slope(x)−1−slope(α)−1 Error bars represent standard error, error bars omitted when smaller than the width of associated data point symbol, ns=not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See also Figure S5.

Article Snippet: Curve fitting for FRAP analysis and saturation binding was also performed in GraphPad PRISM 6.

Techniques: Labeling, Co-Immunoprecipitation Assay, Western Blot, Ex Vivo, In Vitro, Binding Assay, Transfection, Quantitative RT-PCR, Recombinant, Synthesized, Incubation

Journal: Chromosoma

Article Title: The effect of the lamin A and its mutants on nuclear structure, cell proliferation, protein stability, and mobility in embryonic cells

doi: 10.1007/s00412-016-0610-9

Figure Lengend Snippet: Analysis of HEK 293 sublines stably expressing EGFP alone and lamin A and its mutants D446V and Δ50 (progerin). a Confocal microscopy shows that all lamin A variants localize within the NE. Protein aggregates are much smaller than during transient transfection studies due to the lower level of exogenous proteins; however, some foci are still clearly visible. Progerin causes NE lobulations and the strongest deformation of the nuclear shape. b A proliferation comparison of the sublines was performed with the SRB assay 5 days after seeding. EGFP and lamin A expression significantly inhibits proliferation relative to the control, suggesting that the general effect of introducing exogenous protein may have a major role in this process. D446V and Δ50 abolished this effect, so they were able to increase the proliferation rate in embryonic cells. n/s not significant. c A comparison of protein mobility was performed using FRAP analysis. EGFP alone is very mobile: it immediately refilled the bleached area. By contrast, wild-type lamin A, progerin, and D446V proteins did not replace bleached lamin A. It shows that mutations did not significantly change protein mobility within the NE. Measurements were performed for up to 120 seconds. The insert nucleus picture shows the typical bleached area with a red circle (the merge of the EGFP and transmission light channels)

Article Snippet: FRAP analysis software (Zeiss LSM 510 software – ZEN 2008) was used for the experimental setup and data collection (pre-bleach imaging, laser pulse, recovery imaging, collection of data, etc.).

Techniques: Stable Transfection, Expressing, Confocal Microscopy, Transfection, Comparison, Sulforhodamine B Assay, Control, Transmission Assay

Journal: Cancers

Article Title: Characterization of Adenomatous Polyposis Coli Protein Dynamics and Localization at the Centrosome

doi: 10.3390/cancers8050047

Figure Lengend Snippet: APC full-length and mutant dynamics at the centrosome are slowed by nocodazole treatment. ( A ) pAPC-FL-GFP and pAPC1-1309-GFP ( green ) were each co-transfected with pRFP-PCNT-C241 (red) into HeLa cells. APC-GFP was analysed for dynamic recruitment at the centrosome by FRAP in the presence and absence of 33 µM nocodazole. The effect of γ-tubulin on APC dynamics was also tested where FRAP was performed after depletion with γ-tubulin siRNA. ( B ) Fluorescence recovery curves were plotted as shown for APC-FL, indicating relative rates of recovery and equilibration (plateau) at the centrosome for up to 100 s after bleaching. The presence of nocodazole ( black dashed line ) significantly reduced the rate of recovery of APC-FL-GFP compared to that of untreated cells ( blue line ) ( n = 20–30). This was also indicated by comparison of T 1/2 values for the fast recovery pools (T = 0–40 s) ( p < 0.0001), and extrapolated retention levels, calculated from the recovery curve data using Graph Pad Prism software as above (see ). ( C ) The dynamic exchange profile of APC1-1309 at the centrosome +/− nocodazole ( black dotted line ) showed a small difference in the dynamic rate of recruitment compared to untreated cells ( p = 0.0447). There was a small but significant difference in T 1/2 value; however, no change in retention after nocodazole treatment. ( D ) Fluorescence recovery curves are shown for APC1-1309 for siCTRL ( red ) and γ-tubulin siRNA ( green ) transfected cells ( n = 9–10). Confirmation of γ-tubulin knockdown was by Western blot, and vinculin was used as loading control. Column graph shows the T 1/2 of the fluorescence recovery over 40 s, which was significantly increased after the knockdown of γ-tubulin ( p = 0.0194). No significant change in the maximum recovery (retention) was detected. (*, p < 0.05; ****, p < 0.0001).

Article Snippet: The FRAP assay data was analysed in GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA, USA) to determine the rate of fluorescence recovery over time, and the relative size of the dynamic and immobile pools of APC ( B–D).

Techniques: Mutagenesis, Transfection, Fluorescence, Comparison, Software, Knockdown, Western Blot, Control