Fig 2: UBE1a knockdown by shRNA increased ICP5 expression in HSV-1 infected cells. (a) UBE1-knockdown by Dox-induced shRNA (shUBE1) against UBE1. Western blotting data showed the expression levels of UBE1a and UBE1b. Vero cells were transduced with or without lentivirus encoding Dox-inducible shUBE1, which downregulates both UBE1a and UBE1b. Transduced cells were treated with 0–8 µg/mL Dox for 24 h, and cell lysates were analyzed by Western blotting, using a UBE1 antibody that recognized both UBE1a and UBE1b. (b) The cytotoxic effect of Dox on Dox-inducible shUBE1-Vero cells. Cells were treated with 0–8 µg/mL Dox and cultured for 24 h. The number of viable cells was evaluated by a cell viability assay. The values obtained from Dox-untreated cells were set as 100%. (c) UBE1 knockdown increased ICP5 expression in HSV-1 infected cells. Vero cells infected with or without HSV-1 were cultured with 6 µg/mL Dox. At 8 hpi, cells were fixed and stained with anti-ICP5 (green), UBE1a (red), and ICP27 (cyan) antibodies. (d) The correlation of fluorescence intensity between UBE1a and ICP5 in infected cells after UBE1-knockdown. The means ± standard deviations are shown (n = 47, 44 in each experiment). The y-axis is ICP5 intensity/UBE1a intensity calculated by using ImageJ.

Fig 3: Requirement of UBE1 and XIAP for the SNIPER(TACC3)-induced cytoplasmic vacuolization. (a) UBE1 silencing represses cytoplasmic vacuolization. U2OS cells were transfected with the indicated siRNA for 24 h and treated with 30 µM SNIPER(TACC3)-2 for 5 h. Phase-contrast images were obtained. (left). Scale Bars: 20 µm. Whole cell lysates were analyzed by western blotting with the indicated antibodies (right). (b) XIAP silencing represses cytoplasmic vacuolization. U2OS cells were transfected with the indicated siRNA for 24 h and treated with 15 µM SNIPER(TACC3)-2 for 18 h. Phase-contrast images were obtained (left). Whole cell lysates were analyzed by western blotting with the indicated antibodies (right).

Fig 4: Dysregulation of UBA1 and GARS in dorsal root ganglia sensory neurons from SMA mice. (A and C) Spinal column sections from lumbar segments 1 and 2 of late-symptomatic SMA and control mice labelled with UBA1a (A) or GARS (C) (green), SMI32 (magenta) and DAPI. DRG are outlined in overview panels, boxes indicate the area in lower panels. Arrows in bottom panel indicate sensory neurons with altered UBA1 (A) or GARS (C) expression. N = NF200-positive neurons; P = peripherin-positive neurons. Scale bars = 100 µm (overview), 10 µm (lower panels). (B and D) Late-symptomatic SMA DRG sensory neurons show a reduction in the nuclear to cytoplasmic ratio of UBA1 (B) and an increase in the nuclear to cytoplasmic ratio of GARS (D) compared to control mice. n = 3 mice per condition, n = 4 DRGs per mouse (14 sensory neurons were analysed per DRG); *P < 0.05, ***P = 0.001.

Fig 5: SNIPER(TACC3) induces accumulation of ubiquitylated protein aggregates in cancer cells. (a,b) SNIPER(TACC3) induces accumulation of ubiquitylated proteins in cancer cells. Cells were treated with 30 µM SNIPER(TACC3)-2 for 4 h, and fixed and stained with the indicated anti-ubiquitin specific antibodies and Hoechst 33342. Images were obtained by fluorescent microscopy. Scale Bars: 20 µm. (c) UBE1 silencing represses the SNIPER(TACC3)-induced accumulation of ubiquitylated proteins. U2OS cells were transfected with the indicated siRNA for 24 h and treated with 30 µM of SNIPER(TACC3)-2 for 3 h. Cells were fixed and stained with indicated anti-multi-ubiquitin antibody and Hoechst 33342. Scale Bars: 20 µm. (d) XIAP silencing represses the SNIPER(TACC3)-induced accumulation of ubiquitylated proteins. U2OS cells were transfected with the indicated siRNA for 24 h and treated with 15 µM of SNIPER(TACC3)-2 for 18 h. Cells were fixed and stained with indicated anti-multi-ubiquitin antibody. Scale Bars: 20 µm.