Fig 2: HUWE1 induced by the dCas9 SAM system suppresses GBM progression in vivo and in vitro. (A) Schematic diagram of the dCas9 SAM systems. In brief, single‐guide RNA binding to the HUWE1 promoter regions results in recruitment of multiple activator domains of transcription factors VP64, HSF1 and P65 to transcriptionally activate HUWE1. (B) Western blotting and qRT‐PCR analyses of HUWE1 in the GBM2 cells infected with different dCas9 SAM vectors. (C) Growth curves of HUWE1‐overexpressing cells. (D) Representative images of Transwell migration and invasion assays of HUWE1‐overexpressing cells. (E) Kaplan‐Meier survival analysis of the mice bearing HUWE1‐overexpressing and NC cell‐derived xenograft tumors. (F) In vivo bioluminescent images and quantification of xenograft tumors in the mouse brain injected with HUWE1‐overexpressing and NC cells. (G) Representative H&E staining images of xenograft tumors in the mouse brains. Scale bar = 100 μm. (H) Representative IHC staining images of HUWE1 and Ki67 in xenografts. Scale bar = 50 μm. (I) Representative H&E staining images of the indicated xenografts and normal brain. Scale bar = 150 μm. Data are presented as mean ± SD. **, P < 0.01; ***, P < 0.001. Abbreviations: sgRNA, single‐guide RNA; OE, over expression; NC,negative control; IHC, immunohistochemistry; SD, Standard Deviation

Fig 3: HUWE1 is essential for the degradation of N‐Myc in GBM cells. (A) Venn diagram shows the intersection among significantly altered genes in the mass spectrometry (MS)‐detected genes with upregulated expression (n = 2051) in HUWE1‐KD cells, MS data of anti‐HUWE1 coimmunoprecipitation (n = 567), and the Ubibrowser platform‐predicted substrates of HUWE1 with a high or moderate confidence level (n = 700). (B) Western blotting analyses of N‐Myc and HUWE1 in GBM patient specimens and normal tissues. (C) Representative immunofluorescence staining of HUWE1 (in red), N‐Myc (in green) and Hoechst (in blue) in LN229 and GBM2 cells. Scale bar = 5 μm. (D) PLAs of HUWE1/N‐Myc complexes and HUWE1/P53 complexes in situ in LN229 and GBM2 cells. PLAs of P53/MDM2 was set as positive control. Scale bar = 5 μm. (E) Co‐immunoprecipitation analyses of the interaction between endogenic HUWE1 and N‐Myc in GBM cells. (F) Schematic diagram of the HUWE1 recognition motif on the HLH and MYC_N regions of N‐Myc. (G) Western blotting analyses of N‐Myc in GBM cells treated with CHX, MG132 and heclin alone or together at the indicated time. (H) In vivo ubiquitination assays of polyubiquitin chains of N‐Myc in HA‐Ub‐overexpressing GBM cells treated with or without heclin. (I) In vivo ubiquitination assays of polyubiquitin chains of N‐Myc in HA‐Ub‐overexpressing GBM cells infected with or without the shHUWE1 vector. (J) In vivo ubiquitination assays of polyubiquitin chains of N‐Myc in GBM cells transfected with mutant ubiquitin plasmids at the K6, K11, K48, and K63 sites. (K) In vivo ubiquitination assays of polyubiquitin chains of N‐Myc in GBM cells transfected with wild‐type or K6‐, K48‐ and K6‐K48‐mutant ubiquitin plasmids. Data are presented as mean ± SD. **, P < 0.01; ***, P < 0.001. Abbreviations: MS, mass spectrometry; IP, immunoprecipitation; IB, immunoblot; DMSO, Dimethyl sulfoxide; Ub, ubiquitin; CHX, cycloheximide; Ub, ubiquitin; KD, knockdown; NC, negative control; PLA, Proximity ligation assay; SD, Standard Deviation

Fig 4: N‐Myc binds to DLL1 promoter to activate NOTCH1 signaling pathway in GBM cells. (A) Left schematic diagram shows the top 20 genes positively correlated with MYCN in the TCGA GBM RNA‐seq database. The right schematic diagram shows positively correlated genes (adjusted P < 0.05 and r > 0) of MYCN that were enriched in KEGG pathway analysis using DAVID software. (B) Schematic diagram shows the promoter regions of DLL1 and DLL3 are divided into 9 parts for ChIP assays. (C) ChIP analyses of the N‐Myc‐bound promoter regions of DLL1 and DLL3. The corresponding isotype IgG was used as a negative control. (D) Dual‐luciferase reporter system analyses of luciferase activity in GBM cells co‐transfected with reporter plasmids containing the DLL1/DLL3 promoter sequences and MYCN expression plasmids and qRT‐PCR analyses of the expression of DLL1/DLL3 in GBM cells transfected with MYCN expression plasmids. (E, F) qRT‐PCR and Western blotting analyses of the expression of indicated proteins in HUWE1‐KD cells compared with NC cells. (G) Western blotting analyses of nuclear and cytoplasmic N1ICD in HUWE1‐KD cells. (H) Western blotting analyses of the expression of Hes family BHLH transcription factor 1 (HES1) and HEY1 in HUWE1‐KD cells. (I) Growth curves of HUWE1‐KD cells treated with DAPT (25 μmol/L) tested by CCK‐8 assays. (J, K) Representative images of Transwell migration and invasion assays of HUWE1‐KD cells treated with DAPT (25 μmol/L). Data are presented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Abbreviations: KD, knockdown; NC, negative control; TCGA, The Cancer Genome Atlas; ChIP, Chromatin immunoprecipitation; HEY1, Hes Related Family BHLH Transcription Factor With YRPW Motif 1; HES1, Hes Family BHLH Transcription Factor 1; SD, Standard Deviation

Fig 5: REDD1 stability is regulated by HUWE1 ubiquitin ligase.(A) HEK293 cells were transfected with 20 nM negative control siRNA or HUWE1−1 or −2 siRNAs in the indicated combinations for 3 days and MG-132 (20 µM) was added to cells for 4 hours followed by cell lysis. (B) HEK293 cells were transfected with 20 nM control or HUWE1 siRNAs for 3 days and rapamycin (40 nM) or PP242 (2 µM) were added to cells for 8 hours followed by cell lysis. (C,D) HEK293 cells were transfected with 20 nM negative control or HUWE1-1 (C) or −2 (D) siRNAs for 3 days followed by cycloheximide (40 µM) treatment and cell lysis at indicated time points.