Fig 2: SERBP1 knockdown increased expression of genes linked to neurogenesis and nervous system development. a Enriched Gene Ontology terms related to genes upregulated upon SERBP1 knockdown in U251 cells. Gene set was analyzed using Panther [24] and GO terms were compiled using [25]. Most representative terms associated with nervous system development and function are listed. b Network analysis of genes implicated in neuronal differentiation affected by SERBP1 knockdown. The network was built using String [26] considering interaction (experimental evidence), text mining, and co-occurrence. Different colors were used to indicate clusters. c Heatmap shows that genes upregulated upon SERBP1 knockdown cells display increased expression during murine neurogenesis—0 day/stem vs. 4 days/differentiated cells. d qRT-PCR shows SERBP1 and ß-III Tubulin expression in neuronal stem cells (NSCs) and differentiated cells. e Genes upregulated upon SERBP1 knockdown showing decreased expression in GBM in reference to LGG are labeled in blue, genes that show reduced expression in GBM in reference to normal brain (cortex) are labeled in green and genes that are methylated (H3K27me3) in GBM cells [45] are labeled in orange. f Western blot showing that SERBP1 knockdown leads to a decrease in H3K27me3 in GBM cells

Fig 3: SERBP1 binding motif. a SERBP1 RNA binding motif obtained with RNACompete. b Fluorescence polarization assay shows SERBP1’s high affinity (KD ~ 47 nM) to a 7-mer RNA oligonucleotide (5'- GCGCGGG - 3'). c ~ 40% of transcripts determined via RIP-Seq as preferentially associated with SERBP1 display the identified GC-rich motif in their 3' UTR, a number much higher than expected by chance. d Results of luciferase assay showing that co-transfection of a SERBP1 expression vector increased the expression of reporter constructs containing the 3' UTR of genes displaying putative SERBP1 binding motifs. GAPDH was used as a negative control

Fig 4: SERBP1 regulates metabolism. a Enriched Gene Ontology (GO) terms related to genes downregulated upon SERBP1 in U251 cells. Gene set was analyzed using Panther [24] and GO terms were compiled using Revigo [25]; most representative terms associated with metabolism are listed. b Network analysis of genes implicated in metabolism affected by SERBP1 knockdown. Network was built using String [26] considering interaction (experimental evidence), text mining, and co-occurrence. Different colors were used to indicate clusters. c Schematic representation of one-carbon cycle, showing genes affected by SERBP1 knockdown. d, e qRT-PCR and Western blot analysis in U251 and U343 cells corroborated the impact of SERBP1 on the expression of critical genes implicated in metabolism. f Representative PHGDH immunostaining of tumors from the xenograft study for each group. Scale bar = 60 µm

Fig 5: SERBP1 affects cancer-related phenotypes and tumor growth. a Knockdown of SERBP1 in U251 cells decreased viability (MTS assay). b SERBP1 silencing diminished clonogenic potential, as measured by colony formation assays. c The Boyden chamber assay was used to evaluate SERBP1 impact on invasion; values of crystal violet absorbance showed that SERBP1 knockdown decreased invasion potential. d–f SERBP1 silencing increased apoptosis as indicated by PARP1 cleavage (d), annexin staining (e), and caspase (f). g GSC proliferation across time was followed with the Incucyte automated system. Decrease in SERBP1 levels impaired cell proliferation. i Knockdown of SERBP1 in GSC lines decreased viability (MTS assays). Data were analyzed with Student’s t test and presented as the mean ± standard deviation. Bonferroni correction was used for multiple comparisons. *p = 0.05; **p = 0.01; ***p = 0.001; ****p = 0.0001. i Intracranial xenografts were established using the shSERBP1 3565 GSC cell line (10 mice per group). Experimental group received Dox to induce expression of shSERBP1. Kaplan-Meier curves indicate that SERBP1 knockdown decreased tumor growth and expanded survival. j Representative Ki67 staining from the tumor boundary for each group. Scale bar = 100 µm