Fig 2: Effects of modulating RhoC expression on the formation of F‐actin in DDCs. Representative confocal microscopy images shows localization of F‐actin in DDCs transduced with LV‐NS (A,B), LV‐138 (C,D), LV‐138 + RhoC (E,F), LV‐ShRNA‐RhoC (G,H), or LV‐RhoC (I,J) after 48 hours in osteogenic medium. Yellow‐boxed areas in (A), (C), (E), (G), and (I) are shown at higher magnification in (B), (D), (F), (H), and (J), respectively. Scale bars in (A), (C), (E), (G), and (I) = 200 μm; scale bars in (B), (D), (F), (H), and (J) = 50 μm.

Fig 3: Effects of loss of RhoA, RhoB and RhoC on endothelial barrier function. (A) Western blot analysis of whole cell lysates collected from HUVECs 72 hours after transfection with siNT, siRhoA, siRhoB or siRhoC. Representative blots of 2 experiments are shown. Tubulin is included as loading control. (B) Effect of loss of RhoA, RhoB and RhoC on basal endothelial barrier function. (C) Basal endothelial barrier function at t = 72 hours. (D) Absolute endothelial resistance attributable to cell-cell adhesion (Rb) of RhoA, RhoB and RhoC knockdown cells. (E) Rb at t = 72 hours. (F) Absolute endothelial resistance attributable to cell-matrix adhesion (α) of RhoA, RhoB and RhoC knockdown cells. (E) α at t = 72 hours. All data represent average values (line graphs, representing barrier formation, Rb or α from medium change at 16 hours after transfection (t = 0) until the end of the experiment) or mean ± SEM (bar graphs) of N = 5 experiments performed in triplicates. **p < 0.01 Dunnett's post-hoc analysis of one-way ANOVA. siNT, non-targeting siRNA.

Fig 4: RhoC overexpression rescues miR‐138‐induced inhibition of osteogenesis. Alizarin Red stained cultures at day 14 of dedifferentiated chondrocyte (DDC) osteogenic induction shows enhanced mineralization induced by cotransduction of cells with LV‐138 and LV‐RhoC (A). Additional controls (RhoC knockdown alone; LV‐Sh‐RhoC and RhoC overexpression alone; LV‐RhoC) were also included (A). Representative micro‐CT images of human decalcified, decellularized bone scaffolds seeded with DDCs transduced with either LV‐NS, LV‐138 + LV‐CTL (empty virus to control for LV‐RhoC overexpression) or LV‐138 + LV‐RhoC (B). Quantification of bone volume relative to total volume and BMD is shown in panels (C) and (D), respectively. BV/TV and BMD data are expressed ± SD; n = 4. *p < 0.05; **p < 0.01. Scale bars in (B) = 1 mm.

Fig 5: Reduced level of Lis1 specifically activates RhoC in brain. (A) Abnormal increase in RhoC NDU in Lis1fl/- cortex. Ubiquitin binding domain pull down from Lis1fl/+ or Lis1fl/- brain is increased in mono-, di- and tri-ubiquitinated RhoC when Lis1 protein is reduced. Graph quantifies results from n = 5 biologically independent experiments, **P = 0.0037. Data are shown as the mean ± s.d. (B) Elevated RhoC NDU disrupts its interaction of total RhoC with RhoGDIa in vivo. RhoGDIa immunoprecipitation from Lis1fl/+ or Lis1fl/- brain shows decreased RhoC binding, while RhoA and Rac3 remain unchanged. Graph quantifies results from n = 3 biologically independent experiments done in duplicate, *P < 0.0312, ns = not significant with P = 0.4375 (RhoA) and P < 0.9999 (Rac3). Data are shown as the mean ± s.d. (C) RhoC, but not RhoA knock down by shRNAi rescues cell spreading impairment in Lis1fl/- neonatal astrocytes. Immunostaining of Lis1fl/+ or Lis1fl/- astrocytes transduced with a GFP-expressing lentivirus to knock down RhoA or RhoC for tGFP (anti-tGFP, orange). Golgi apparatus (GM-130, white) and DNA (DAPI, blue). GFP staining is used to define cell area (shown in the black and white mask of transduced cells) measured in arbitrary units (AU) in Image J. Control = scrambled shRNA transduced cells. Scale bar, 10 µm. Graph represents cell area measurements from three independent experiments. Data are presented as individual data points from all three biological replicates in dark gray (Lis1fl/+) or light gray (Lis1fl/–) circles and their mean ± s.d. as triangles color coded by experiment. *P < 0.05, **P < 0.01, ***P < 0.001. Where indicated, statistical significances were obtained using a two-tailed paired t-test for amount of ubiquitinated RhoC (A) and cell spreading assay (C), or non-parametric, two-tailed Wilcoxon matched-rank signed test for the RhoGDIa IP (B).