Data represent means SEM. depletion does not suppress the SG assembly under sorbitol-induced osmotic stress. ControlshRNA and = 3). n.s., not significant; *< 0.05, **< 0.01, as determined by Student test. All underlying numerical values are available in S1 Data. ATXN2, ataxin-2; EIF2, eukaryotic translation initiation factor 2 subunit ; LSM12, like-Sm protein 12; SEM, standard error of the mean; SG, stress granule.(TIFF) pbio.3001002.s002.tiff (3.6M) GUID:?0D6837B3-709F-4FB3-8AE3-059D47535177 S3 Fig: LSM12 depletion exacerbates the impairment of NCT caused by arsenite-induced oxidative stress. (A) LSM12 depletion facilitates the nuclear mislocalization of S-GFP under oxidative Mouse monoclonal antibody to MECT1 / Torc1 stress conditions. ControlshRNA and = 123C127 cells from 3 independent experiments). *< 0.05, **< 0.01, ***< 0.001 to controlshRNA cells at a given time point, as determined by Student test. (B) LSM12 depletion facilitates the cytoplasmic mislocalization of S-tdT under oxidative stress conditions. Data represent means SEM (= 103C118 cells from 3 independent experiments). n.s., not significant; *< 0.05, **< 0.01 to controlshRNA cells at a given time point, as determined by Student test. All underlying numerical values are available in S1 Data. GFP, green fluorescent protein; LSM12, like-Sm protein 12; NCT, nucleocytoplasmic transport; S-tdT, S-tdTomato; SEM, standard error of the mean.(TIFF) pbio.3001002.s003.tiff (5.9M) GUID:?D063EAE4-20CF-4C19-94AF-4512BD8F230D S4 Fig: deletion increases the toxicity of BM-131246 deletion exacerbates the poly(GR)-induced invaginations of the nuclear envelope. Control and = 15C17 confocal images obtained from 3 independent experiments; = 403C418 GFP-GR100Cpositive cells). ***< 0.001, as determined by Student test. (C) The abnormal morphology of the nuclear lamina was quantified as in Fig 3E. Data represent means SEM (= 18C19 confocal images obtained from 3 independent experiments; = 366C413 GFPCor GFP-GR100Cpositive cells). n.s., not significant; ***< 0.001, as determined by 2-way ANOVA with Tukey post hoc test. All underlying numerical values are available in S1 Data. ANOVA, analysis of variance; GFP, green fluorescent protein; LSM12, like-Sm protein 12; SEM, standard error of the mean.(TIFF) pbio.3001002.s004.tiff (2.7M) GUID:?1BDCC394-31D8-490D-93CC-9E960EE6FA2E S5 Fig: LSM12V135I overexpression does not alter the relative levels of endogenous LSM12 BM-131246 protein. (A) SH-SY5Y cells were transfected with an expression vector for FLAG, LSM12-FLAG, or LSM12V135I-FLAG. Total cell extracts were prepared 48 hours after transfection and immunoblotted with anti-FLAG, BM-131246 anti-LSM12, anti-ATXN2, anti-PABPC1, and anti-tubulin (loading control) antibodies. Overexpression of wild-type LSM12, but not LSM12V135I, increased the relative levels of endogenous ATXN2 protein, consistent with low levels of endogenous ATXN2 protein in LSM12-depleted cells (Fig 1C). (B) The abundance of each protein was quantified as in Fig 1C. Data represent means SEM (= 4). n.s., not significant; ***< 0.001, as determined by 1-way ANOVA with Dunnett post hoc test. All underlying numerical values are available in S1 Data. ANOVA, analysis of variance; ATXN2, ataxin-2; LSM12, like-Sm protein 12; SEM, standard error of the mean.(TIFF) pbio.3001002.s005.tiff (1.0M) GUID:?95705FA9-EBBE-47E2-B029-F8983A70DAF8 S6 Fig: Quantitative analyses of total mRNAs (RNA-seq) and translating ribosome-associated mRNAs (TRAP-seq) are reproducible between 2 biological replicates of controlshRNA and values are indicated in each plot. All underlying numerical values are available in S1 Data. LSM12, like-Sm protein 12; RNA-seq, RNA sequencing; TRAP-seq, translating ribosome affinity purification sequencing.(TIFF) pbio.3001002.s006.tiff (972K) GUID:?1C1E48AA-2EFA-451E-97FC-14D8C89026CF S7 Fig: deletion posttranscriptionally down-regulates expression. (A) = 3). **< 0.01, as determined by Student test. (B) A schematic representation of the locus and reporter constructs. Transcription of control and UTR reporters was driven by heterologous CMV promoter. A promoter region in the locus (from ?1,805 to +71 in relative to the transcription start site +1) was subcloned upstream of the NLUC-coding sequence to measure the promoter activity by the NLUC activity. (C) deletion posttranscriptionally decreases expression via the 5 UTR. Control and reporter and a FLUC expression vector (normalizing control). Luciferase reporter assays were performed as in Fig 5D. Data represent means SEM (= 3). n.s., not significant; *< 0.05, **< 0.01 as determined by Student test. All underlying numerical values are available in S1 Data. CMV, cytomegalovirus; EPAC1, exchange protein directly activated by cyclic AMP 1; FLUC, firefly luciferase; LSM12, like-Sm protein 12; NLUC, Nano-luciferase; SEM, standard error of the mean; UTR, untranslated region.(TIFF) pbio.3001002.s007.tiff (1.2M) GUID:?46414B86-0B22-41CB-B5D9-1DC335E45B54 S8 Fig: EPAC1 depletion is sufficient to phenocopy loss of function in poly(GR) toxicity. (A) SH-SY5Y cells were transfected with control or 2 independent siRNAs. Total cell extracts were prepared 72 hours after transfection and immunoblotted with anti-EPAC1 or anti-tubulin (loading control) antibodies. (B) deletion and EPAC1 depletion nonadditively disrupt the RAN gradient. Control and = 62C67 cells from 3 independent experiments). n.s., not significant; ***< 0.001, as determined by 2-way ANOVA with.