Journal: Cell reports
Article Title: p120 RasGAP and ZO-2 are essential for Hippo signaling and tumor-suppressor function mediated by p190A RhoGAP
doi: 10.1016/j.celrep.2023.113486
Figure Lengend Snippet: (A) Immunofluorescence to detect YAP protein (red) in H661 cells with or without expression of p190A(WT) or p190A(Y2F), as well as in H661-p190A cells with KO of TJP2 or depleted of LATS1/2. Nuclei were labeled with DAPI (blue). (B) Quantification using CellProfiler (github.com) of nuclear/cytoplasmic YAP from the experiment illustrated in (A). The experiment was performed with three biological replicates and a minimum of 912 cells sampled from each condition. Data are presented as mean ± SEM. Statistical testing was performed using pairwise Student’s t test as indicated by brackets. (C) Immunofluorescence to detect BrdU-positive nuclei in H661 cells with or without expression of p190A(WT) or p190A(Y2F), as well as in H661-p190A cells with KO of TJP2 or depleted of LATS1/2. Nuclei were labeled with DAPI (blue). (D) Quantification using CellProfiler (github.com) of BrdU-positive nuclei from the experiment illustrated in (C). The experiment was performed with three biological replicates and a minimum of 626 cells sampled from each condition. Data are presented as mean ± SD (n = 3). Statistical testing was performed using pairwise Student’s t test as indicated by brackets. (E) CDK6 transcript levels in H661 cells with or without expression of p190A(WT) or p190A(Y2F), as well as in H661-p190A cells with KO of TJP1 or TJP2. (F) Western blotting to detect CDK6, pRB(S780), pRB(S807/811), total RB, and β-actin in whole-cell lysates from H661 cells with or without expression of p190A(WT) or p190A(Y2F), as well as in H661-p190A cells with KO of TJP1 or TJP2. (G) Quantification of pRB(S780) and pRB(S807/811) levels by densitometry of western blots as shown in (F). Data are presented as mean ± SD. Statistical testing was performed using pairwise Student’s t test as indicated by brackets (n = 3). (H) Western blotting to detect p190A, ZO-2, YAP, cyclin A, and β-actin in whole-cell lysates from H661 cells with or without expression of p190A(WT) or p190A(Y2F) ± YAP-KD, as well as in H661-p190A cells with KO of TJP2 ± YAP-KD. (I) Growth curves for control and H661 cells reconstituted with p190A(WT) or p190A(Y2F) ± YAP-KD, as well as in H661-p190A cells with KO of TJP2 ± YAP-KD. 5 × 105 cells were seeded per well of a 6-well plate in triplicate and propagated for the number of days indicated with a change of medium every 2 days. Cell number was quantified manually. Data are presented as mean ± SD (n = 3). Statistical testing was performed using pairwise Student’s t test as indicated by brackets. (J) Western blotting to detect cyclin A and β-actin in whole-cell lysates from H661 cells with or without expression of p190A(WT) or p190A(Y2F) ± verteporfin, as well as in H661-p190A cells with KO of TJP2 ± verteporfin. Cells were treated with 3 μM verteporfin for 48 h at 37°C. (K) Quantification of cyclin A levels by densitometry of western blots as shown in (J). Data are presented as mean ± SD. Statistical testing was performed using pairwise Student’s t test as indicated by brackets (n = 3).
Article Snippet: pRB(S780) , rabbit monoclonal , Cell Signaling Technologies , 8180T.
Techniques: Immunofluorescence, Expressing, Labeling, Western Blot
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