Journal: BMC Biology

Article Title: TRIM25 promotes Capicua degradation independently of ERK in the absence of ATXN1L

doi: 10.1186/s12915-020-00895-0

Figure Lengend Snippet: Loss of ATXN1L results in CIC instability. a Representative Western blot of ATXN1L WT (HEK) and ATXN1L KO cell lines (A10, A30, B21) treated with MG132. DMSO was used as a negative control. Below: barplots showing quantification of CIC protein expression. b Representative Western blot of ATXN1L WT (HEK) and ATXN1L KO cell lines (A10, A30, B21), ectopically overexpressing FLAG-tagged ATXN1L. Below: barplots showing quantification of CIC protein expression. c Representative Western blot of siRNA knockdown of ATXN1 and ATXN1L in NHA. Scrambled siRNA was used as a negative control. Below: barplots showing quantification of CIC protein expression. d Representative Western blot of ATXN1L WT (HEK) and ATXN1L KO cell lines (A30) ectopically overexpressing FLAG-tagged wild-type ATXN1L or mutant ATXN1L-V485A. e Barplots showing quantification of ubiquitin Western blots normalized to total CIC Western blots. f Representative Western blot of CIC immunoprecipitation in ATXN1L WT (NHA) and ATXN1L KO (B82) cell lines treated with MG132. DMSO was used as a negative control. g Immunofluorescence images of proximity ligation assay showing CIC-ubiquitin interaction in ATXN1L WT (NHA) cell lines following siRNA knockdown of ATXN1L . Scrambled siRNA was used as a negative control. White bars denote 10 μm. h Tukey boxplots showing quantification of the number of CIC-ubiquitin foci/cell. Western blot quantifications were collected from 3 independent experiments and were normalized to vinculin unless specified otherwise. Error bars represent one standard deviation. PLA quantifications were collected from 65 individual cells. p values were calculated using the two-tailed independent Student’s t test. Statistically significant values are denoted (* p < 0.05, ** p < 0.01, *** p < 0.001). Individual data values can be found in Additional file : Table S10

Article Snippet: FLAG-tagged ATXN1L (#33242) [ ] and FLAG-tagged TRIM25 (#12449) [ ] constructs were purchased from Addgene.

Techniques: Western Blot, Negative Control, Expressing, Mutagenesis, Immunoprecipitation, Immunofluorescence, Proximity Ligation Assay, Standard Deviation, Two Tailed Test

Journal: BMC Biology

Article Title: TRIM25 promotes Capicua degradation independently of ERK in the absence of ATXN1L

doi: 10.1186/s12915-020-00895-0

Figure Lengend Snippet: ATXN1L-mediated CIC instability is independent of ERK activity. a Heatmap showing the top 20 upregulated gene sets in ATXN1L KO NHA and HEK cell lines. Terms related to the MAPK pathway are bolded. b ELISA quantification of phosphorylated ERK (pThr202/Tyr204) in ATXN1L WT and ATXN1L KO NHA and HEK cell lines. Quantifications were normalized to total ERK. c Representative Western blot of phosphorylated ERK (pThr202/Tyr204) in ATXN1L WT (NHA) and ATXN1L KO (B82, B16, B21) cell lines. d Relative mRNA expression of CIC target genes ETV1/4/5 , DUSP6 , and SPRY4 in ATXN1L WT (NHA) and ATXN1L KO (B82, B16, B21) cell lines. Gene expression was normalized to TBP, and the parental ATXN1L WT (NHA) cell line was used as a relative control. e Representative Western blot of ATXN1L WT (HEK) and ATXN1L KO (A10, A30, B21) cell lines treated with MEK/ERK inhibitors trametinib/LY3214996. DMSO was used as a negative control. Below: barplot quantifications of CIC expression. Quantifications were normalized to vinculin. f Representative Western blot of ATXN1L WT (HEK) cells treated with MEK/ERK inhibitor and/or ATXN1L siRNA. DMSO and scrambled siRNA were used as negative controls. Right: barplot quantification of CIC expression. Quantifications were normalized to vinculin. Western blot, ELISA, and RT-qPCR quantifications were collected from 3 independent experiments. Error bars represent one standard deviation. p values were calculated using the two-tailed independent Student’s t test. Statistically significant values are denoted (* p < 0.05, ** p < 0.01). Individual data values can be found in Additional file : Table S10

Article Snippet: FLAG-tagged ATXN1L (#33242) [ ] and FLAG-tagged TRIM25 (#12449) [ ] constructs were purchased from Addgene.

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Negative Control, Quantitative RT-PCR, Standard Deviation, Two Tailed Test

Journal: BMC Biology

Article Title: TRIM25 promotes Capicua degradation independently of ERK in the absence of ATXN1L

doi: 10.1186/s12915-020-00895-0

Figure Lengend Snippet: CIC interacts with the E3-ligase TRIM25. a Volcano plot showing CIC-interacting proteins identified using CIC immunoprecipitation followed by mass spectrometry in ATXN1L KO NHA cells. Red data points are high confidence interactors. b Representative Western blot of CIC immunoprecipitation showing interaction with TRIM25 in ATXN1L WT (NHA) and ATXN1L KO (B82) cell lines. Right: barplot showing quantifications of TRIM25 Western blots. Quantifications were normalized to CIC Western blots. c Immunofluorescence images of proximity ligation assay showing FLAG-tagged CIC-S-TRIM25 interaction in ATXN1L WT cells treated with ATXN1L siRNA. Scrambled siRNA was used as a negative control. White bars denote 10 μm. Right: Tukey boxplots showing quantification of the number of FLAG-TRIM25 foci/cell. d Representative Western blot of ATXN1L WT (HEK) and ATXN1L KO (A10, A30, B21) cell lines treated with TRIM25 siRNA. Scrambled siRNA was used as a negative control. Below: barplot quantifications of CIC protein expression. Quantifications were normalized to vinculin. e Relative mRNA expression of CIC and CIC target genes ETV1/4/5 following treatment with TRIM25 siRNA for 48 h. Scrambled siRNA was used as a negative control. Gene expression was normalized to TBP. f Representative Western blot of ATXN1L WT (NHA) and ATXN1L KO (B82) cell lines ectopically overexpressing FLAG-tagged TRIM25. Empty FLAG vector was used as a negative control. Western blot and RT-qPCR quantifications were collected from 3 independent experiments. PLA quantifications were collected from 65 individual cells. Error bars represent one standard deviation. p values were calculated using the two-tailed independent Student’s t test. Statistically significant values are denoted (* p < 0.05, ** p < 0.01, *** p < 0.001). Individual data values can be found in Additional file : Table S10

Article Snippet: FLAG-tagged ATXN1L (#33242) [ ] and FLAG-tagged TRIM25 (#12449) [ ] constructs were purchased from Addgene.

Techniques: Immunoprecipitation, Mass Spectrometry, Western Blot, Immunofluorescence, Proximity Ligation Assay, Negative Control, Expressing, Plasmid Preparation, Quantitative RT-PCR, Standard Deviation, Two Tailed Test

Journal: BMC Biology

Article Title: TRIM25 promotes Capicua degradation independently of ERK in the absence of ATXN1L

doi: 10.1186/s12915-020-00895-0

Figure Lengend Snippet: TRIM25 and ATXN1L mediated CIC stability in glioma. a Representative Western blot of CIC, ATXN1L, and phosphorylated ERK (pThr202/Tyr204) expression in GBM cell lines. b Representative Western blot of CIC, ATXN1L, and phosphorylated ERK (pThr202/Tyr204) expression in BTIC cell lines. c Tukey boxplots showing H -scores of CIC immunohistochemistry staining on glioma samples. d Immunohistochemistry images of CIC staining on glioma samples. Black bars denote 200 μm. e Representative Western blot of BTIC cell lines in standard EGF/FGF culture conditions and following 16 h of MEK/ERK inhibition. f Representative Western blot of BTIC cell lines following siRNA knockdown of ATXN1L or TRIM25 . Fluorescent RNA was used as a negative control. g Relative mRNA expression of CIC and CIC target genes ( DUSP6 , SPRY4 , ETV1/4/5 ) following treatment with ATXN1L or TRIM25 siRNA for 48 h in LN18 and U251 cell lines. Scrambled siRNA was used as a negative control. Gene expression was normalized to TBP. h Representative Western blot of LN229 and U343 cell lines treated with MEK/ERK inhibitors trametinib/LY3214996 and/or ATXN1L siRNA. DMSO and scrambled siRNA were used as a negative control. Below: barplot quantifications of CIC protein expression. i Representative Western blot of U251 and U343 cell lines treated with ATXN1L and/or TRIM25 siRNA. Scrambled siRNA were used as a negative control. Below: barplot quantifications of CIC protein expression. Western blot and RT-qPCR quantifications were collected from three independent experiments. Error bars represent one standard deviation. p values were calculated using the two-tailed independent Student’s t test. Statistically significant values are denoted (* p < 0.05, ** p < 0.01). Individual data values can be found in Additional file : Table S10

Article Snippet: FLAG-tagged ATXN1L (#33242) [ ] and FLAG-tagged TRIM25 (#12449) [ ] constructs were purchased from Addgene.

Techniques: Western Blot, Expressing, Immunohistochemistry, Staining, Inhibition, Negative Control, Quantitative RT-PCR, Standard Deviation, Two Tailed Test

Journal: BMC Biology

Article Title: TRIM25 promotes Capicua degradation independently of ERK in the absence of ATXN1L

doi: 10.1186/s12915-020-00895-0

Figure Lengend Snippet: CIC, ATXN1L, and TRIM25 regulate cell cycle in vitro and in TCGA patient data. a Barplot displaying the frequency of TRIM25 alterations in the TCGA Pan-Cancer study. Bars represent a cancer subtype and split based on alteration type. b Scatter plot showing the Log2Fold change of differentially expressed genes shared between TRIM25 siRNA in BT549 and MDA-MB-231 breast cancer cell lines and CIC/ATXN1L knockout in NHA cell lines. Differentially expressed genes with directionally discordant change are colored (red/blue). c Scatter plot showing the Log2Fold change of differentially expressed genes shared between TCGA BRCA samples with TRIM25 amplification and TCGA type II LGG, PRAD, and STAD samples with CIC deletions. Differentially expressed genes with directionally concordant change are colored (red/blue). d Heatmap showing the top 20 enriched gene sets for directionally concordant upregulated differentially expressed genes shared between TCGA BRCA samples with TRIM25 amplification and TCGA type II LGG, PRAD, and STAD samples with CIC deletions. Highlighted terms are terms related to the cell cycle. e Kaplan-Meier curve showing the overall survival of TCGA BRCA patients with high (top 25%) and low (bottom 75%) TRIM25 expression. f Tukey barplots showing the expression of TRIM25 , ETV1 , ETV4 , and ETV5 in TCGA LIHC samples with high (top 25%) and low (bottom 75%) TRIM25 expression. g Kaplan-Meier curve showing the overall survival of TCGA LIHC patients with high (top 25%) and low (bottom 75%) TRIM25 expression. Statistically significant values are denoted ( *p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: FLAG-tagged ATXN1L (#33242) [ ] and FLAG-tagged TRIM25 (#12449) [ ] constructs were purchased from Addgene.

Techniques: In Vitro, Knock-Out, Amplification, Expressing

Journal: BMC Biology

Article Title: TRIM25 promotes Capicua degradation independently of ERK in the absence of ATXN1L

doi: 10.1186/s12915-020-00895-0

Figure Lengend Snippet: The proposed mechanistic model of the CIC-ATXN1L-TRIM25 interaction. a CIC-ATXN1L form a repressive complex which stabilizes CIC from degradation by TRIM25. b In the absence of ATXN1L, CIC is targeted by TRIM25 for degradation by ubiquitination and transported for degradation

Article Snippet: FLAG-tagged ATXN1L (#33242) [ ] and FLAG-tagged TRIM25 (#12449) [ ] constructs were purchased from Addgene.

Techniques:

Journal: Brain Pathology

Article Title: Abnormal scaffold attachment factor 1 expression and localization in spinocerebellar ataxias and Huntington’s chorea

doi: 10.1111/bpa.12872

Figure Lengend Snippet: Increased binding of SAFB1 to Ataxin 1 with an expanded glutamine tract . A. The top 10 most SAFB1 bound RNA transcripts with the total number of iCLIP SAFB1 cross‐link sites from three independent experiments. B. The red line depicts the location of the ATXN1 gene on chromosome 6. The majority of the SAFB1 binding sites occur in RNA encoded by exon 8 of ATXN1 and in the region containing CAG repeat sequence (blue hatched lines). A screengrab taken from the genome browser shows the sense (brown) and antisense (blue) SAFB1 iCLIP binding sites. The binding sites relative to the amino‐acids encoded by the SAFB1 bound sequences are shown in a 3′‐5′ orientation. Note the SAFB1 cross‐link site will correspond to the amino‐acid most 5′ to the sense sequence and 3′ to the antisense sequence. The numbers within the schematic of the sense binding sites show the number of SAFB1 cross‐link sites in each of these individual experiments. C i. Western blotting of protein lysates from RNA immunoprecipitation experiments, probed for SAFB1. SAFB1 is present in both ATXN1‐30Q transfected and ATXN1‐85Q transfected cell lysates immunoprecipitated using a SAFB1 antibody (IP). There is no SAFB1 protein detected in IgG control lysates (IgG). Expected molecular weight for SAFB1 is 102KDa (arrow). ii. Relative levels of ATXN1 RNA bound to SAFB1 protein. SAFB1 protein was immunoprecipitated from HeLa cells transfected with plasmids containing ATXN1‐30CAG (physiological) or ATXN1‐85CAG (pathological expansion). The relative expression level of ATXN1 RNA associated with SAFB1 protein is significantly increased in ATXN1‐85 CAG compared to ATXN1‐30 CAG (unpaired, two‐tailed t ‐test t (4) = 3.76, P = 0.01). Data are presented as mean ± SEM.

Article Snippet: After 24 h, HeLa cells were transfected with DNA plasmids containing FLAG‐tagged ataxin1 (ATXN1) with either a normal CAG repeat tract (pcDNA1 Flag ATXN1[30Q]) or a pathologically expanded CAG repeat tract (pcDNA1 Flag ATXN1[85Q]). pcDNA1 Flag ATXN1[30Q] and pcDNA1 Flag ATXN1[85Q] were kind gifts from Huda Zoghbi ( ) (Addgene plasmid #33236 and #33237, respectively).

Techniques: Binding Assay, Sequencing, Western Blot, RNA Immunoprecipitation, Transfection, Immunoprecipitation, Control, Molecular Weight, Expressing, Two Tailed Test