Fig 1: P-Rex1 deficiency reduces the S1P-stimulated activation of Rac1 and basal Rac3 activity in PC12-S1PR1 cells. (A) P-Rex1 is required for the S1P-stimulated activation of Rac1 in PC12-S1PR1 cells. Wild-type and P-Rex1-deficient PC12-S1PR1 cells were serum-starved overnight and stimulated with 5 nM S1P for the indicated periods of time at 37 °C prior to cell lysis and isolation of active, GTP-bound Rac by Pak-CRIB pulldown assay. Top panels are representative Western blots showing the level of active, GTP-loaded Rac1 compared to total Rac1 in 1% of the cell lysate, processed on the same film. Bottom panel shows the quantification by densitometric analysis. Data are from six independent experiments and are presented as the mean ± SEM. Statistical analysis involved two-way ANOVA with Sidak’s multiple comparisons test. (B) P-Rex1 is required for basal Rac3 activity in PC12-S1PR1 cells. The same cell lysates as in (A) were analysed for Rac3 activity. Top panels are representative Western blots showing active, GTP-loaded Rac3 from Pak-CRIB pulldown assay compared to total Rac3 level in 1% of the cell lysate, processed on the same film. Bottom panels show quantification by densitometric analysis. Data are from three independent experiments and are presented as the mean ± SEM. Statistical analysis involved two-way ANOVA with Sidak’s multiple comparisons test; ‘# ns’ denotes no significant differences between S1P-stimulated and control conditions within each genotype. (C) Rac3 antibody specificity. Wild-type PC12-S1PR1 cells were treated with the indicated doses of Rac3 siRNA or control siRNA, and total cell lysates were Western blotted for endogenous Rac1 and Rac3; Coomassie staining of the membrane was used as a loading control.
Fig 2: 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).
Fig 3: Validation of the expression of hub genes in vivo and in vitro. A: Hub genes mRNA expression in db/db and db/m mice by reverse transcription quantitative real-time PCR (RT-qPCR). In comparison to db/m mice, H2-T24 and Rac3 showed a reduction in expression in db/db mice, whereas Tfrc demonstrated an elevation in expression in db/db mice; B: Palmitic acid (PA) was used as a type 2 diabetes mellitus model in vitro. Hub genes mRNA expression in PA and negative control (NC) groups by RT-qPCR. H2-T24 and Rac3 showed a reduction in expression in the PA group, whereas Tfrc demonstrated an elevation in expression in the PA group; C: Protein levels of RAC3 and TFRC in db/db and db/m mice by western blotting. Each lane represents an independent mouse hippocampus; D: Analysis of relative protein levels in db/db and db/m mice. In comparison to db/m mice, Rac3 showed a reduction in expression in db/db mice, whereas Tfrc demonstrated an elevation in expression in db/db mice; E: Protein levels of RAC3 and TFRC in PA and NC groups by western blotting. Each lane represents an independent experiment of cell lysate; F: Analysis of relative protein levels in PA and NC group. Rac3 showed a reduction in expression in the PA group, whereas Tfrc demonstrated an elevation in expression in the PA group; G: Protein expression of RAC3 and TFRC in db/db and db/m mice by immunohistochemistry; H: Analysis of average OD in db/db and db/m mice. In comparison to db/m mice, Rac3 showed a reduction in expression in db/db mice, whereas Tfrc demonstrated an elevation in expression in db/db mice. Data are expressed as the mean ± SEM (n = 3). aP < 0.05; bP < 0.01; cP < 0.001. NC: Negative control; PA: Palmitic acid.
Fig 4: Evaluation of the receiver operating characteristic. A-C: In GSE125387, the receiver operating characteristic (ROC) curve for each candidate gene (H2-T24, Rac3, and Tfrc) was analyzed; D: The levels of expression for the three genes in GSE125387; E-G: In GSE152539, the ROC curve for each candidate gene (H2-T24, Rac3, and Tfrc) is displayed; H: The levels of expression for the three genes in GSE152539 were also confirmed. aP < 0.05. AUC: Area under the curve; 95%CI: 95% confidence interval; TPR: True-positive rate; FPR: False-positive rate.
Fig 5: Utilizing machine learning to evaluate potential diagnostic biomarkers in candidate screening. A and B: Screening of biomarkers in the Lasso model. For diagnosis, the most appropriate gene count (n = 4) is the one that corresponds to the lowest point on the curve; C and D: The error is displayed by the random forest algorithm. The importance score is used to rank genes; E: The Venn diagram illustrates that the above two algorithms have identified three potential diagnostic genes: H2-T24, Rac3 and Tfrc.
Supplier Page from Abcam for Anti-RAC3 antibody [EPR6680]