Fig 1: The SHP-2 inhibitor PHPS1 reversed the effects of UBE2D3 knockdown. U87 and T98G cells transduced with shUBE2D3-1 or shNC and then treated with 10 µM PHPS1. (A) Expression of STAT3, and p-STAT3. (B) Cell proliferation was assessed. (C–E) Extracellular acidification rate (C), glycolytic capacity, glycolytic reserve (D) and oxygen consumption rate (E). *p < 0.05; **p < 0.01.
Fig 2: Knockdown of UBE2D3 suppressed the proliferation, glycolysis and STAT3 phosphorylation, but induced apoptosis in glioma cells. (A) The protein level of UBE2D3 in U87 and T98G cells transduced with shUBE2D3-1 and shUBE2D3-2 lentivirus. (B, C) Cell proliferation (B) and apoptosis (C) in U87 and T98G cells after the knockdown of UBE2D3. (D, E) Lactate production (D) and glucose uptake (E). (F–H) Extracellular acidification rate (F), glycolytic capacity, glycolytic reserve (G) and oxygen consumption rate (H) in U87 and T98G cells after the knockdown of UBE2D3. (I) Expression of HK-2 and PFKL in GBM cells after the knockdown of UBE2D3. (J) Expression of STAT3, and p-STAT3 in GBM cells after the knockdown of UBE2D3. GBM, glioblastoma multiforme; HK-2, hexokinase-2; PFKL, 6-phosphofructokinase; STAT3, Signal transducer and activator of transcription 3. *p < 0.05; **p < 0.01; ***p < 0.001.
Fig 3: UBE2D3 was highly expressed in GBM and enriched in different pathways. (A) The expression of UBE2D3 in GBM and normal brain tissue. Data were extracted from TCGA-GBM dataset. (B–D) GSEA analysis of cell glycolysis (B), STAT3 pathway (C), and cell apoptosis (D) between UBE2D3 high and low group. GBM, glioblastoma multiforme; GSEA, Gene Set Enrichment Analysis.
Fig 4: Ube2N is required for RIG-I and MAVS activation in MEFs.(a–c) Small hairpin RNAs (sh-Ube2D1/2/3/4 & N) as indicated was transduced into MEFs with lentivirus. Eighty four hours after transduction, the cells were infected with VSV. Sixteen hours post infection, the cells were collected and IFN ß (a) and IFN a (b) production were measured by qPCR. Immunoblotting were also performed to examine knockdown efficiency (c). *P<0.05 and ***P<0.001. NS indicates no statistically significant difference. (d) MEFs were transduced with or without sh-Ube2N. Eighty four hours after transduction, Flag-MAVS was further transduced into MEFs by retrovirus for forty eight hours. The cells were then infected with VSV. Sixteen hours post infection, the cells were collected to measure cytokine production by qPCR. See also Supplementary Fig. 4d. (e) MEFs were treated as described in a except that the cells were collected to isolate P5 fractions. P5 fractions were subjected to SDD-AGE to examine MAVS aggregation and IRF3 dimerization assay in vitro. The original full blot can be found in Supplementary Fig. 9a. (f) MEFs were treated as described in d except that Flag-Ube2D3 or Flag-Ube2N but not Flag-MAVS was transduced into the cells by retrovirus as indicated. See also Supplementary Fig. 4e.
Fig 5: Modification sites of RIG-I by polyubiquitin chains.(a) The ubiquitination reaction was performed using purified components (E1, Ube2D3, Riplet, ubiquitin (WT or K63), His-flag-RIG-I). His-flag-RIG-I was then immunoprecipitated by M2 beads and eluted with flag peptide, followed by SDS-PAGE and silver-staining. (b) Ubiquitination sites of RIG-I identified by mass spectrometric analysis. (c) pcDNA3-flag-RIG-I (wild type and various mutant forms) were transfected into HEK293T (Rig-i-/-) cells. Thirty six hours after transfection, the cells were infected with or without VSV for twelve hours, which then were collected to measure IFN induction by qPCR. *P<0.05 and ***P<0.001. (d) pcDNA3-HA-ubiquitin and pcDNA3-flag-sumo or pcDNA3-flag-RIG-I (wild type and various mutant forms) were transfected into HEK293T (Rig-i-/-) cells. Thirty six hours after transfection, the cells were infected with or without VSV. Twelve hours post infection, the cells were collected for IP with M2 beads and IP products were subjected to immunoblotting. The original full blot can be found in Supplementary Fig. 9d. (e) Wild type and various mutant forms of RIG-I were transfected into HEK293T (Rig-i-/-), HEK293T (Rig-i-/- & Ube2D3-/-) or HEK293T (Rig-i-/- & Ube2N-/-) cells. Thirty six hours after transfection, the cells were infected with VSV for twelve hours, which then were collected to measure IFN induction by qPCR. *P<0.05 and ***P<0.001.
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