Journal: Nature cancer

Article Title: FoxM1 insufficiency hyperactivates Ect2-RhoA-mDia1 signaling to drive cancer

doi: 10.1038/s43018-020-00116-1

Figure Lengend Snippet: (a) Left: Images of indicated MEFs in metaphase stained with TRITC-Phalloidin. Right: Quantification of mitotic cortical actin intensity of the indicated MEFs (n = 12 independent MEF lines per group). (b) Incidence of slow centrosome movement in the indicated prophases treated Cytochalasin D (Cyto D; actin depolymerizer) or SMIFH2 (pan-Formin inhibitor) for 4 h. Significance is denoted for comparison between the DMSO and treatment groups within each genotype (n = 3 independent MEF lines per group). (c) Left: Images of the indicated MEFs expressing GFP-anillin (RhoA biosensor). Right: Quantification of GFP-anillin intensity at the cell cortex in the indicated MEFs (n = 5 independent MEF lines per group). (d) Quantification of cortical actin intensity in indicated MEFs treated with vehicle (Veh) or 1.5 μg/ml RhoA inhibitor (C3) for 4 h (n = 7 independent MEF lines per group). (e) Incidence of slow prophase centrosome movement and (f) Quantification of cells with non-perpendicular spindles in the indicated MEFs as in d (n = 7 independent MEF lines per group). (g) Western blot analysis of Foxm1−/− MEFs stably transduced with the indicated shRNAs. Western blots are representative of 5 independent MEF lines. (h-l) Foxm1−/− MEFs stably transduced with the indicated shRNAs and then analyzed for cortical actin intensity in metaphase (h), non-perpendicular mitotic spindles (i), slow centrosome movement in prophase, (j), chromosome segregation errors (k), and aneuploid metaphase spreads (l). n = 5 independent MEF lines per group in h-j and 3 independent MEF lines per group in k, l. Data represent mean ± s.e.m. Statistics: a-l one-way ANOVA with Sidak’s correction; Scale bar, 5 μm. See source file for original data and uncropped immunoblots.

Article Snippet: GFP-anillin (RhoA biosensor; Addgene #68026) was sub-cloned into pTSIN PGK-puro2 lentiviral vector.

Techniques: Staining, Expressing, Western Blot, Stable Transfection, Transduction

Journal: JCI Insight

Article Title: Polycystin-1 regulates ARHGAP35-dependent centrosomal RhoA activation and ROCK signaling

doi: 10.1172/jci.insight.135385

Figure Lengend Snippet: ( A ) GTP-RhoA was significantly increased in PKD1 cystic cell lines compared with control cells ( n = 4) using a Rhotekin-GTP pulldown assay. ( B ) GTP-RhoA was significantly increased in Pkd1- knockout kidneys compared with controls ( n = 3). ( C ) Phosphorylation of myosin light chain (pMLC), a major downstream target of ROCK, was significantly upregulated in isogenic PKD1 -null cells ( n = 3). ( D ) Expression of dominant negative RhoA (T19N) in PKD1 cells resulted in a significant increase in cilia length compared with dominant negative Cdc42 (N17) or Rac1 (N17) ( n = 3 independent experiments, N = 81 cells). ( E ) Active RhoA was localized using a GTP-RhoA biosensor (R-GBD) in control and PKD1 cells. In ciliated cells, active RhoA (GFP) was visualized at the cilia base (arrows) and was significantly increased in PKD1 cells ( n = 3 independent experiments, N = 22 cells). Insets show cilia under higher magnification (original magnification, ×1000). ( F ) Rapamycin-inducible centrosomal targeted expression (arrow) of constitutively active RhoA (Q63L) was associated with a significant decrease in cilia length in control cells compared with uninduced cells ( n = 3 independent experiments, N = 70 cells) * P < 0.05, ** P < 0.01, **** P < 0.0001. Significance determined by 2-tailed Student’s t test ( A–C and E ). Significance determined by 1-way ANOVA corrected (Dunnett) for multiple comparison ( D and F ). ADPKD, autosomal dominant polycystic kidney disease.

Article Snippet: The active RhoA biosensor GFP-rGBD (gift of William Bement, University of Wisconsin–Madison, Madison, Wisconsin, USA; Addgene plasmid 26732) ( ) was transfected into UCL93 or OX161 cells.

Techniques: Control, Knock-Out, Phospho-proteomics, Expressing, Dominant Negative Mutation, Comparison

Journal: JCI Insight

Article Title: Polycystin-1 regulates ARHGAP35-dependent centrosomal RhoA activation and ROCK signaling

doi: 10.1172/jci.insight.135385

Figure Lengend Snippet: A model showing how mutation of PC1 could lead to reduced cilia/centrosomal localization or retention of ARHGAP35. Two possible scenarios are shown: (a) trafficking and delivery of PC1 and ARHGAP35 in the same vesicles to the centrosome compartment and (b) retention of cleaved PC1 C-terminus (CT1) bound to centrosomal ARHGAP35. The RhoA-dependent kinase, ROCK, has been previously shown to be localized to centrosomes . Loss of centrosomal ARHGAP35 leads to the accumulation of centrosomal “active” GTP-RhoA, the activation of ROCK and its downstream effectors (e.g., pMLC), leading to increased actin polymerization and shorter cilia. It is plausible that the local increase in centrosomal ROCK activity could lead in turn to a cascade of ROCK activation, which spreads throughout the cell. PC1, polycystin-1; ADPKD, autosomal dominant polycystic kidney disease.

Article Snippet: The active RhoA biosensor GFP-rGBD (gift of William Bement, University of Wisconsin–Madison, Madison, Wisconsin, USA; Addgene plasmid 26732) ( ) was transfected into UCL93 or OX161 cells.

Techniques: Mutagenesis, Activation Assay, Activity Assay