Fig 2: Depletion of RhoJ impairs the TNF signaling pathway. (A) Heatmap of the top 100 most differentially expressed genes (DEGs). The color change from blue to red represents the changing process from down-regulation to up-regulation. (B) A volcano plot of DEGs from RNA sequencing is shown. (C) GO biological pathway analysis of the down-regulated genes—cell signaling pathways. (D) List of the fold change of the selected down-regulated genes RhoJ, VEGFA and TNF-related genes NOD2, TRAF1, TNF and NFKBIA from RNA sequencing data. (E) qPCR validation of the selected genes. Data are shown as the mean ± SEM. Every experiment was repeated three times. ***p<0.001.

Fig 3: JNK/VEGFR2/RhoJ mediates activation of PAK and ERK pathways. (A) After HUVECs were treated with VEGF at different concentrations, cell lysates were subjected to immunoblotting to detect the activity of JNK and ERK, and (B) PAK2, PAK4. (C) The effect of SP600125 stimulation on VEGFR2 activation in HUVEC cells was detected by Western blotting. (D) Immunofluorescence staining for RhoJ, Pholloidin, and DAPI in HUVECs with or without treatment of VEGF or JNK inhibitor. Scale Bar 100 μm. (E) Quantification of fluorescence intensity of RhoJ and F-actin in (D). Scale bar, 100 μm. (F) the Cell Counting Kit-8 (CCK-8) assays of HUVECs after applying VEGF, SP600125, or vehicle. In all images, data are shown as the mean ± SEM. Every experiment was repeated for three times. *p<0.05; **p<0.01; ***p<0.001.

Fig 4: Overexpression of RhoJ promotes GBM progression and angiogenesis in the xenograft mouse model. (A) Representative images of RhoJ expression in the normal brain and grade II to grade IV glioma tissues by IHC staining, obtained at 200× magnification (upper) and 400× magnification (lower). All scale bars, 100 μm. (B) Semi-quantitative analysis results of RhoJ positive cells in different grades of glioma (WHO II-IV) and normal brain tissue samples by immunohistochemical (IHC) staining. (C) Representative tumor xenografts from U87 cells expressing plenti-RhoJ or plenti-con. (D) The xenograft tumor size was measured every 4 days before they were sacrificed. (E) The xenograft tumor weight was measured after sacrifice (mean ± SEM, n = 4). (F) IHC analyses of CD31, RhoJ, meosin and EpCAM in the xenograft tumor. scale bar, 100 μm. (G) Representative images of tumor xenografts for IF analysis of EpCAM and CD31. Scale bar, 100 μm. (F) Quantitative analysis of fluorescence intensity of EpCAM and CD31 in (G). Scale bar, 100 μm. In all images, data are shown as the mean ± SEM. Every experiment was independently repeated 2-3 times. *p<0.05; **p<0.01; ***p<0.001.

Fig 5: VEGF restores RhoJ silencing-induced CD31 reduction. (A) The expression of RhoJ, moesin and p-VEGFR2 in HUVEC cells stimulated by VEGF at different concentrations were detected by Western blotting. (B) The expression of moesin in HUVEC cells with RhoJ knockdown was detected by Western blotting assay. (C) The mRNA expression levels of RhoJ and moesin in HUVEC cells stimulated by VEGF (50 ng/mL) were detected by qPCR. (D) Representative images for IF analysis of EpCAM and CD31. Scale bar, 100 μm.