Fig 1: DIRAS family GTPase 2 (DIRAS2) affects cell-cycle progression and induces G0/G1 arrest. (A) The cell-cycle distribution in DIRAS2-oe-RKO and DIRAS2-oe-HT29 cells. (B) Volcano plot of differential genes in oe-DIRAS2 and its vector control cells. Red (upregulation), 604; green (downregulation); 562, Padj< 0.05, |log2 fold-change| > 0; n = 3 per group. (C, D) Kyoto Encyclopedia of Genes and Genomes pathway analysis between oe-DIRAS2 and its vector control cells. The top 10 most significantly changed signaling pathways are listed. (E) The cell cycle-related protein expression in oe-DIRAS2 RKO or HT29 cells and their controls.
Fig 2: The tumor-suppressing activity of DIRAS family GTPase 2 (DIRAS2) can be overridden by 26S proteasome non-ATPase regulatory subunit 2 (PSMD2). (A) DIRAS2 and PSMD2 were identified by western blot in RKO cells respectively transfected with control-vector, Flag-tagged DIRAS2, Ha-PSMD2, or both. (B) The effect of PSMD2 and DIRAS2 on cellular proliferation in RKO cells. (C) The impact of PSMD2 and DIRAS2 on cellular growth in RKO cells. (D) The correlation between DIRAS2 and PSMD2 expression. (E) A schematic diagram of DIRAS2 inhibiting cell growth through nuclear factor kappa light-chain enhancer of activated B-cells signaling pathways and being degraded by PSMD2 in a proteasome-mediated way (all *P<.05, ***P<.001).
Fig 3: DIRAS family GTPase 2 (DIRAS2) is stabilized by 26S proteasome non-ATPase regulatory subunit 2 (PSMD2) in a proteasome-mediated way. (A) RKO and HEK293 cells were transfected with FLAG-DIRAS2 and/or HA-PSMD2. Western blot and its quantification were used to determine the relationship between DIRAS2 and PSDM2. (B, C) The half-life of DIRAS2 was shortened by PSMD2 in RKO and HEK293 cells. (D-E) The half-life of DIRAS2 in RKO and HEK293 cells was lengthened after overexpressing PSMD2 under MG132 treatment.
Fig 4: DIRAS family GTPase 2 (DIRAS2) inhibits cell proliferation. (A) The identification of DIRAS2 in messenger RNA and protein levels after overexpression in RKO and HT29 cell lines. (B) The effect of DIRAS2 on cellular proliferation in RKO and HT29 cells. (C) The effect of DIRAS2 on cellular growth in RKO and HT29 cells. (D) Xenograft tumor assays using HT29-oe-NC and HT29-oe-DIRAS2 cells (n=6). (E) Tumor volumes of xenografts measured every four days after day 12. (F) Tumor weights of xenografts measured at the end of the assay.
Fig 5: DIRAS family GTPase 2 (DIRAS2) blocks nuclear factor kappa light-chain enhancer of activated B-cells signaling pathways in colorectal cancercells. (A) GSEA-enrichment analysis for the indicated pathways selected from the RNA sequencing gene set. (B) Western blot analysis for the expression of P65 in the cytoplasm and nucleus and its quantification in oe-DIRAS2 RKO or HT29 cells and their controls. (C) Immunofluorescence images of the expression of P65 in the cytoplasm and nucleus in oe-DIRAS2 RKO or HT29 cells and their controls (scale bar, 25 μm).
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