Fig. 5
ABHD5 sustains the activity of RNASET2. a Representative images of immunofluorescent stainings of lysosome (stained by Lysotracker, red) and ABHD5 (green) in SW480 cells. Scale bar: 10 μm. b Western blots showing ABHD5 expression in the lysosome lysates of ABHD5 KD and control cells. LAMP1 was used as a reference. c Immunoprecipitation showing an interaction between ABHD5 and RNASET2. SUCLG2 was used as a positive control. d Yeast two-hybrid assay showing a negative result for the direct interaction between ABHD5 and RNASET2. e Western blots of RNASET2 expression in the lysosome lysates of ABHD5 KD and control cells. f A direct interaction between ABHD5 and PDIA5 was shown based on HuProtTM human protein chip. g Immunoprecipitation showing an interaction between ABHD5 and PDIA5. CALR was used as a positive control. h The interaction complex model between ABHD5, PDIA5, and RNASET2 predicted by protein-protein docking methods. i Competitive binding assays of ABHD5 and RNASET2 to PDIA5. j In vitro binding assay with wild type PDIA5 (W) or mutant PDIA5 (T) and His-tagged RNASET2 or ABHD5 as indicated. k ABHD5 knockout (ABHD5 KO) and control SW480 cells were transfected with wild type ABHD5 or mutant ABHD5 plasmid, and subjected to FU treatment (25 μM) for 6 h. The intracellular uracil was analyzed (n = 6, Student’s t-test). l ABHD5 KO and control SW480 cells were transfected with wild type ABHD5 or mutant ABHD5 plasmid, and subjected to FU treatment (25 μM) for 24 h. The apoptotic rates were analyzed (n = 5, Student’s t-test). m ABHD5 KO and control SW480 cells transfected with wild type ABHD5 or mutant ABHD5 plasmid were subcutaneously inoculated in nude mice, and intraperitoneal injection of PBS or FU (50 mg per kg) + calcium folinate (80 mg per kg) (i.p., once per week for 3 weeks). Tumor volume was measured every 3 days (n = 5, Two-way ANOVA). The quantitative data were presented as mean ± S.D (error bar) (N.S. no significance, **p < 0.01, ***p < 0.001)
