IRE1/XBP1-dependent regulation of UBE2D3. (A) IRE1, XBP1s, and RIDD signatures were confronted to NFκB signaling gene signature using the TCGA GB dataset (IRE1 high/low n = 264/275; XBP1s high/low n = 261/210; and RIDD high/low n = 285/249). P values obtained with unpaired t-test comparing IRE1, XBP1s, and RIDD high vs low tumors. (B) Venn diagram of the intersection of XBP1s target genes identified by ChIPseq²¹ with RIDD targets.¹⁰ (C) Association of NFκB signature with NCSTN, UBE2D3, and UFM1 low/high GB from TCGA. NCSTN, UBE2D3, and UFM1 high/low groups were determined using the median of the mRNA expression as cut-off (high/low n = 263/263). P values obtained from unpaired t-test comparing NCSTN, UBE2D3, and UFM1 high vs low tumors. (D) UBE2D3 mRNA expression in TCGA GB categorized according to their IRE1, XBP1s, and RIDD signatures (IRE1 high/low n = 258/265; XBP1s high/low n = 261/210; and RIDD high/low n = 252/258). P values obtained with unpaired t-test comparing IRE1, XBP1s, and RIDD high vs low tumors. (E) UBE2D3 mRNA expression in XBP1s low/high TCGA GB (RNAseq dataset; high/low n = 80/86). P value obtained from unpaired t-test comparing XBP1s high vs low tumors. (F) Quantitation of UBE2D3 mRNA expression using RT-qPCR in U87 and RADH87 cells silenced for XBP1 (n = 7 and 4 for U87 and RADH87, respectively). ns: not significant according to unpaired t-test compared to control. (G) Western blot analysis of UBE2D3 in parental (NT), control (siCTR) and XBP1-silenced (siXBP1) U87 and RADH87 cells. Actin (ACT) was used as loading control. Data are representative of 3 biological replicates (see Supplementary Figure S4F). (H) Quantification of UBE2D3 mRNA expression by RT-qPCR in cells with active (parental U87 and RADH87 (par)) and inactive IRE1 signaling (U87 DN and RADH87 Q780*) (n = 4, mean ± SD). ^*P < .05 according to unpaired t-test compared to parental. (I) Quantitation of UBE2D3 expression with RT-qPCR in U87 DN and RADH87 Q* cells and transfected with XBP1s (n = 3). ^*P < .05, ^***P < .001 according to unpaired t-test comparing CTR to XBP1s conditions. (J) Putative XBP1s binding sites on human UBE2D3 promoter regions analyzed with MatInspector and TFBIND. (K) and (L) Gel shift assays performed on 4 putative XBP1s binding sites using U87 nuclear extracts after XBP1s overexpression (K). Validation of putative binding sites h1, h2, and h3 using gradual amounts of unlabeled probes used in competition assay (L).