Journal: Nature Communications

Article Title: Anti-proteolytic regulation of KRAS by USP9X/NDRG3 in KRAS-driven cancer development

doi: 10.1038/s41467-024-54476-8

Figure Lengend Snippet: a Cell growth inhibition by shRNA-mediated depletion of NDRG3 expression in pancreatic and lung cancer cell lines expressing wild-type (KRAS-WT) or mutant (KRAS-Mutant) KRAS. b Quantification of the inhibition of anchorage-independent cell growth by shRNA-mediated depletion of NDRG3 expression in KRAS-WT or KRAS-Mutant pancreatic and lung cancer cell lines. (n = 4 biological replicates/cell line). c Differential inhibition of AKT and ERK1/2 activities by NDRG3 depletion in KRAS-WT and KRAS-Mutant cells. (n = 3 biological replicates/cell line). d Effects of NDRG3 depletion on the tumorous growth of KRAS-WT or KRAS-Mutant cells in nude mice (n = 5 mice/cell line). e A heatmap showing the genomic activity (Z-score) of upstream regulators for NDRG3-dependent DEGs in different pancreatic cancer cell lines. Ras-related signaling pathways are denoted in red, while the pathways not significantly enriched in IPA are shown in dark gray (left). A differential genomic activity profile of RAS-related signaling pathways between KRAS-WT and KRAS-Mutant cells is shown (right, n = 7 NDRG3-dependent RAS-related pathways). The genomic activity of an upstream pathway was estimated by IPA analysis of NDRG3-dependent DEGs (≥|1.5| fold change) for each pathway as identified from RNA-Seq. f Upstream regulatory pathways whose NDRG3-dependent genomic activity is highly correlated with cell growth inhibition caused by NDRG3 depletion. g Analysis of NDRG3-dependent changes in AKT and ERK1/2 activities induced by ectopic expression of KRAS G12D in BxPC-3 cells. h NDRG3 dependence of the tumorous growth of KRAS G12D -expressing BxPC-3 cells in nude mice (n = 8 mice/group). i Histogram of Ki-67-positive cells and p-ERK1/2-positive areas in KRAS G12D -expressing BxPC-3 tumor tissues (n = 12 areas/group). Data are presented as median (center), with the box plots extending from the 25th to 75th percentiles and the whiskers (the minimum and maximum values) ( a ), median and quartile ( b ), or mean ± SEM ( c , d , h ) or ±SD ( e , i ). Statistical analysis was done with two-sided Student’s t-Test ( a – d ), one-way ANOVA ( e , I ), or two-way ANOVA ( h ), followed by Dunnett’s multiple comparison test. Source data are provided as a Source Data file.

Article Snippet: For stable knock-down of NDRG3 by targeting the CDS ****or 3’-UTR region and of GFP, lentiviral particles containing specific shRNA expression vector (pLKO.1; Addgene) were produced by transfecting the shRNA expression plasmid and lentiviral packaging mix into HEK293T cells.

Techniques: Inhibition, shRNA, Expressing, Mutagenesis, Activity Assay, Protein-Protein interactions, RNA Sequencing, Comparison