Fig. 5

Verification of ARL4D as a subtype-specific tumor dependency of G3-MB. a Box plots showing ARL4D mRNA levels of four MB subgroups or NC in the indicated MB datasets. b Kaplan–Meier analysis of the overall survival of MB patients stratified by ARL4D expression level in Cavalli dataset. c Box plots showing the FPKM values of ARL4D from two RNA-Seq replicates of MB002, D425 lines versus UW228. d RT-qPCR analysis of ARL4D in MB002, D425 and UW228 lines. MB002 and D425 cells upon ARL4D knockdown were subjected to RT-qPCR analysis (e) and cell viability measurement at Day 0/2/4 (f). g UW228 cells were infected with shSCR or two separate clones of shARL4D lentiviruses individually at the same MOI as cells in e-f. Day 0-normalized cell viability was measured at Day 0/2/4. h-i FACS analyses of cell proliferation (h) and apoptosis (i) of MB002 and D425 cells upon ARL4D knockdown. j Kaplan-Meier survival curve of nude mice carrying orthotopic xenografts of MB002-GFP-luc cells stably expressing shARL4D-1 or shSCR. k-l Tumor growth of xenografted nude mice treated as described in (j) was monitored by IVIS weekly. The collected mice images with corresponding signal scale bars measured in p/s are shown in (k). The crossed white box indicates death of the treated mouse. Box plots showing the signals of total bioluminescence flux intensity for xenografted nude mice of each treatment condition and the data are presented as mean ± SEM in (l). All RT-qPCR and cell viability assays were performed in triplicate and the data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA (a), two-sided log-rank test (b and j), two-tailed t test (c) and two-way ANOVA (l)