RAP2A is highly downregulated in GBM patients.

(A, B) Analysis of GBM patient versus control samples focused on the level of expression of human homologs of Drosophila ACD regulators by Hierarchical clustering (A) and the Gene Distance Matrix (B). Color-coded scale bar indicates fold level of expression. (C) Kaplan-Meier survival curves corresponding to patients in the TCGA (IDHwt) and Gravendeel (IDHwt) cohort in GBM datasets. Low RAP2A expression levels in human GBM are associated with a poor prognosis.

Drosophila RAP2A homolog Rap2l regulates ACD.

(A) Drosophila NBII lineage; the only NB per lineage expresses the transcription factor Dpn, while mature INPs express both Dpn and Ase; iINP, immature INP; mINP, mature INP; GMC, Ganglion Mother Cell. (B) Confocal micrographs showing a control larval brain NBII lineage with one NB (Dpn+ Ase-) and an NBII lineage in which Rap2l has been downregulated displaying an ectopic NB (eNB). (C) Rap2l downregulation in NBII lineages by the wor-Gal4 ase-Gal80 (an NBII-specific driver) causes Cno and Numb localization failures (green arrows) in dividing progenitors (NB or INPs), while aPKC localization is not significantly altered (red arrows). Data shown in the scaled bar graphs was analyzed with a U Mann-Whitney test (B) or a Chi-square test (C), *p<0.05, **p<0.01, ns, not significant, n=number of NB lineages analyzed (in B) or number of dividing cells analyzed (in C); scale bar: 10 μm.

RAP2A expression in GBM neurosphere cultures reduces the stem cell population.

(A) Different GBM cell lines show similar RAP2A mRNA levels and significantly lower levels than in control Astros. (B) RAP2A mRNA levels are significantly higher in the GB5 line after infecting this line with RAP2A (GB5-RAP2A). Data shown in the scaled bar graphs in A and B was analyzed with an ANOVA and a T-test, respectively; error bars show the SD; n= 2 (in A) and 3 (in B) different experiments. (C) Immunofluorescences of the GSC stem cell markers CD133, SOX2 and Nestin reveal a significant reduction in the protein intensity in the GB5 RAP2A-expressing neurospheres (GB5-RAP2A) compared to the control neurospheres (GB5). Data shown in the box plots was analyzed with a U Mann-Whitney for CD133 and Nestin and with a T-test for Sox; the central lines represent the median and the box limits the lower and upper quartiles, as determined by R software; crosses represent sample means; error bars indicate the SEM; n= number of sample points; p values: *p<0.05, **p<0.01, ***p<0.001, ns, not significant; scale bar: 10 μm.

RAP2A expression in GBM neurosphere cultures reduces the stem cell population.

RT-PCRs and Westerns blots show a significant decrease in the mRNA and protein expression levels, respectively, of the GSC markers CD133, SOX2 and Nestin in the GB5 RAP2A-expressing neurospheres (GB5-RAP2A) compared to the control neurospheres (GB5). Data shown in the scaled bar graphs was analyzed with an unpaired two-tailed Student’s t test; error bars show the SD; n= 3 different experiments; p values: *p<0.05, **p<0.01.

RAP2A expression in GBM neurosphere cultures decreases cell proliferation and sphere size.

(A) GB5 neurospheres expressing RAP2A (GB5-RAP2A) show a significantly lower number of Ki67 expressing cells per neurosphere than control GB5 neurospheres. Data shown in the box plots was analyzed with a T-test; the central lines represent the median and the box limits the lower and upper quartiles, as determined by R software; crosses represent sample means; error bars indicate the SEM; n= number of sample points. (B) GB5 neurospheres expressing RAP2A (GB5-RAP2A) show a significant decrease in their size compared to control GB5 neurospheres of the same stage. Data shown in the scaled bar graphs was analyzed with an unpaired two-tailed Student’s T-test; error bars show the SD; n= total number of neurospheres of 3 different experiments; p values: *p<0.05; scale bars: 10 μm (in A) and 100μm (in B).

RAP2A expression in GBM neurosphere cultures fosters ACD in GSCs.

(A) Early stage GB5 neurospheres expressing RAP2A (GB5-RAP2A) show an odd number of cells (3-5-7) significantly more frequently than control GB5 neurospheres, which show more frequently an even number of cells (4-6-8). (B) GB5-RAP2A dividing cells show a significant increase in the number of asymmetric NUMB localization in the progeny, compared to control GB5 dividing cells. Data shown in the bar graphs was analyzed with a Chi-Square test with Yates correction; n= number of neurosphere cell clusters analyzed. p values: **p<0.01; ***p<0.001scale bar: 20 μm.

Statistical analysis of asymmetric cell division expression levels in GBM patient samples.

A hierarchical clustering of genes showing significant or non-significant changes in their expression levels is shown. A t-test was applied to the microarray values of the asymmetric cell division regulator genes shown in Fig.1. The statistical values were presented in separate tables corresponding to the significant (A) and non-significant (B) changes in the indicated genes. The overall threshold p-value was 0.01. p-values were based on t distribution. Significance was determined using just Alpha.

(A) RAP2A expression levels analysis according to IDHwt GBM subtypes in the TCGA cohort. (B-D) Kaplan-Meier survival curves for each GBM subtype, Proneural (B), Mesenchymal (C), and Classical (D), based on high and low RAP2A expression levels.

Drosophila RAP2A homolog Rap2l regulates ACD.

Confocal micrographs showing a control larval brain NBII lineage with one NB (Dpn+ Ase-) and NBII lineages in which Rap2l has been downregulated displaying an ectopic NB (eNB). The phenotype of three different, independent Rap2lRNAi lines (KK, GD and BDSC) are shown. Data represented in the bar graph was analyzed with a U Mann-Whitney test; ****p<0.0001; n=number of NB lineages analyzed; the number of different brains analyzed per genotype is also indicated in brackets.