Journal: Journal of Biochemistry
Article Title: Characterization of UGT8 as a monogalactosyl diacylglycerol synthase in mammals
doi: 10.1093/jb/mvae084
Figure Lengend Snippet: Membrane lipid saturation-induced UPR was suppressed in UGT8 KO HeLa cells. (A) Immunofluorescence of HeLa cells transiently expressing C-terminal FLAG-tagged mUgt8. Cells were immunostained with indicated antibodies. Scale bar, 10 μm. (B, C) The mRNA level of CHOP in WT or UGT8 KO (#1) HeLa cells treated with SCD1 inhibitor (100 nM) for 24 h (B) or tunicamycin (2.5 μg/ml) for 6 h (C) was quantified using qRT-PCR ( n = 3). (D–G) PERK phosphorylation of WT or UGT8 KO (#1) HeLa cells treated with SCD1 inhibitor (100 nM) for 24 h (D, E) or tunicamycin (2.5 μg/ml) for 6 h (F, G) were evaluated using immunoblotting (representative data of n = 2 out of n = 3 independent experiments). The ratio of phosphorylated PERK (pPERK) to total PERK was quantified (E, G) ( n = 3). (H–J) WT, UGT8 KO (#1) and UGT8 KO (#1) stably expressing mUgt8(WT)-FLAG or mUgt8(H358A)-FLAG HeLa cells were treated with SCD1 inhibitor (100 nM) for 24 h. PERK phosphorylation was evaluated using immunoblotting (F) (representative data of n = 2 out of n = 3 independent experiments). The ratio of phosphorylated PERK (pPERK) to total PERK was quantified (I) ( n = 3). The mRNA level of CHOP was quantified using qRT-PCR (J) ( n = 3). Mean ± SD. * P < 0.05, *** P < 0.001, **** P < 0.0001, ns; not significant, two-tailed unpaired t -test (B, C, E, G), one-way ANOVA with Dunnett’s multiple comparisons test (I, J).
Article Snippet: The UGT8 inhibitor (compound 19) and SCD1 inhibitor (A939572) were purchased from Cayman Chemicals.
Techniques: Membrane, Immunofluorescence, Expressing, Quantitative RT-PCR, Phospho-proteomics, Western Blot, Stable Transfection, Two Tailed Test
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