Fig. 1: UBIAD1 inhibits the Ras/ERK signaling pathway.

a UBIAD1 reduced cell viability in T24 bladder cancer cells. T24 cells were transiently transfected with pcDNA3.1-UBIAD1 plasmid. Twenty-four hours after transfection, cell viability was detected by the MTT assay. ***p < 0.001, Student’s t-test, n = 6 experiments. b UBIAD1 inhibited the proliferation of T24 cells. T24 cells were transiently transfected with pcDNA3.1-UBIAD1 plasmid. Twenty-four hours after transfection, cell proliferation was detected using the BrdU cell proliferation ELISA kit. **p < 0.01, Student’s t-test, n = 3 experiments. c UBIAD1 inhibited the growth of T24 cells. T24 cells were transiently transfected with pcDNA3.1-UBIAD1 plasmid. Twenty-four hours after transfection, cell growth was detected by cell counting. **p < 0.01, Student’s t-test, n = 3 experiments. d UBIAD1 inhibits ERK phosphorylation in T24 cells. T24 cells were transfected with increasing amounts of pcDNA3.1-UBIAD1. Twenty-four hours after transfection, total cell lysate was examined by western blotting (WB). The same experiment was repeated at least three times and representative data are shown. This protocol is the same for most immunoblotting analyses throughout the study. The right panel represents the ratio (mean ± SD) from densitometry analyses. **p < 0.01, ***p < 0.001, Student’s t-test. e The Ras/ERK signaling pathway is activated by UBIAD1 deficiency in HEK293T cells. HEK293T cells were transfected with sh-UBIAD1. Seventy-two hours after transfection, total cell lysate was exposed to antibodies and WB was performed, as indicated. The same experiment was repeated three times. f Upregulated p-ERK was abrogated by GFP-RBD. HEK293T cells were transfected with plasmids as indicated. After 72 h of transfection, the total cell lysate was exposed to antibodies and examined by WB. The same experiment was repeated three times. g UBIAD1 inhibited H-Ras-induced p-ERK. HEK293T cells were transfected with plasmids as indicated. Twenty-four hours, the total cell lysate was first exposed to antibodies and then examined by WB. The same experiment was repeated three times. The right panel represents the ratio (mean ± SD) from densitometer analyses. **p < 0.01, Student’s t-test. h The mutation of heix (heix^k11403/Df and heix^k11403/heix¹) produced melanotic masses in Drosophila larvae; w¹¹¹⁸ is the wild-type and heix^k11403/Df; tub P > heix is the rescue type. Melanotic masses was detected in long larvae following crosses performed for 2 weeks. N.D.: not detected, (n = 3 experiments, each with 50 larvae). i The mutation of heix increased p-ERK in Drosophila larvae. Total larvae lysate was exposed to antibodies and examined by WB as indicated in the material and methods. The same experiment was repeated three times. j Melanotic masses disappeared under U0126 treatment in the heix mutant larvae. Melanotic masses were detected in long larvae following 2 weeks crosses. ***p < 0.001, Student’s t-test, (n = 3 experiments, each with 50 larvae)