Journal: Frontiers in Molecular Biosciences

Article Title: RNA-Binding Protein MSI2 Binds to miR-301a-3p and Facilitates Its Distribution in Mitochondria of Endothelial Cells

doi: 10.3389/fmolb.2020.609828

Figure Lengend Snippet: MSI2 binds to miR-301a-3p. (A) Schematic representation of biotinylated miRNA pull-down assay followed by label-free quantitative proteomic analysis. 5′biotinylated miR-301a-3p or random sequence (control group) was incubated with streptavidin beads and whole cell extracts of HUVECs. After washing and elution, the pull-down proteins were analyzed by MS/MS. (B) Relative abundances of MSI2 protein identified in three replicate experiments of label-free quantitative proteomic analysis for the product of biotinylated miR-301a-3p pull-down [(Btn) miR-301a-3p]. Biotinylated random small sequence [(Btn) random] was used as a negative control. (C) Representative MS/MS spectrum of a parent ion of MSI2 protein in the label-free quantitative proteomic analysis. The peptide sequence identified by this spectrum was shown in the upper right side. (D) Western blotting analysis of MSI2 protein in the product of the biotinylated miR-301a-3p pull-down assay. WCE: whole cell extracts; Beads+Extract: the mixture of streptavidin–agarose beads and whole cell extracts of HUVECs. (E) miR-301a-3p level in the immunoprecipitation products obtained by incubating anti-MSI2 antibody with the HUVECs lysates. The result was reported as percentage of the input sample (% input). The normal IgG was used as control (IP IgG). (F) The representative sensorgram of bio-layer interferometry (BLI) analysis for the binding kinetics of MSI2 and 5′ biotinylated miR-301a-3p. The blue curves represented the measured responses for each tested concentration of MSI2 protein. The overlapped red curves showed the global fitting results of the binding data. Wilcoxon rank-sum test was used for statistical analysis in (E) . Median with interquartile range was shown for (E) . *** P < 0.001.

Article Snippet: The pCMV3-MSI2 expression plasmid was purchased from Sino Biological (#HG13069-NF, Beijing, China).

Techniques: Pull Down Assay, Sequencing, Incubation, Tandem Mass Spectroscopy, Negative Control, Western Blot, Immunoprecipitation, Binding Assay, Concentration Assay

Journal: Frontiers in Molecular Biosciences

Article Title: RNA-Binding Protein MSI2 Binds to miR-301a-3p and Facilitates Its Distribution in Mitochondria of Endothelial Cells

doi: 10.3389/fmolb.2020.609828

Figure Lengend Snippet: MSI2 exists in mitochondria and facilitates the distribution of miR-301a-3p in mitochondria. (A) Western blotting analysis of mitochondrial fractions of HUVECs for MSI2 and Cytochrome C. Mito: mitochondria; WCE: whole cell extracts. (B) Fluorescence colocalization of MSI2 and mitochondria in different types of cells. MSI2 was labeled with its Alexa Fluor 488-conjugated antibody (green). Mitochondria and nuclei were stained, respectively, with MitoTracker (red) and DAPI (blue). Yellow areas in the merged images represented the colocalization of MSI2 and mitochondria. R value represented the Mander's overlap coefficient between MSI2 and mitochondria calculated by Image-Pro Plus 6.0 software. Bar=10 μm. (C,D) The relative levels of miR-301a-3p in mitochondria (C) and cytosol (D) of HUVECs transfected with MSI2 siRNA compared to those in the control group. The 12S rRNA (mitochondria) and GAPDH (cytosol) were used as internal standards. (E,F) The relative levels of miR-301a-3p in mitochondria (E) and cytosol (F) of HUVECs transfected with MSI2 expression vector compared to those in the control group. (G) Western blotting analysis of MSI2 levels in the whole, cytosolic and mitochondrial fractions of HUVECs treated with H 2 O 2 for 0, 12, and 18 h. (H) Western blotting analysis of MSI2 levels in the whole, cytosolic and mitochondrial fractions of HUVECs treated with GO for 24 h. (I,J) Immunoprecipitation analysis of the binding of Ago2 and MSI2 in HUVECs mitochondria. The immune complexes were formed by incubating mitochondrial lysates with anti-MSI2 (MSI2 IP) and then immunoblotted with anti-Ago2 antibody (I) , or by incubating mitochondrial lysates with anti-Ago2 (Ago2 IP) and then immunoblotted with anti-MSI2 antibody (J) . Mitochondrial lysates were used as input sample and normal IgG was used as the negative control (IgG). Two-tailed Student's t -test was used for statistical analysis in (C–F,H) . One-way analysis of variance (ANOVA) was used for (G) . Data are presented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The pCMV3-MSI2 expression plasmid was purchased from Sino Biological (#HG13069-NF, Beijing, China).

Techniques: Western Blot, Fluorescence, Labeling, Staining, Software, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Binding Assay, Negative Control, Two Tailed Test

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 contributes to the maintenance of CD44v6+ liver cancer stem cells via notch1 signaling pathway

doi: 10.1186/s13046-019-1508-1

Figure Lengend Snippet: MSI2 was closely related to CD44v6 and predicted poor prognosis. a Western blot analysis of CD44v6 and MSI2 protein levels in HCC tissues and adjacent non-tumor tissues selected randomly. β-actin was used as a normalized control. b Analysis of CD44v6 protein levels relative to β-actin in 28 pairs of HCC tissues and adjacent non-tumor tissues ( n = 28, * p = 0.0179, t test). c Analysis of MSI2 protein levels relative to β-actin in 28 pairs of HCC tissues and adjacent non-tumor tissues ( n = 28, ** p = 0.0012, t test). d and e Kaplan–Meier survival analysis of overall survival and disease-free survival were compared according to the expression levels of CD44v6 in HCC tissues. Patients with high CD44v6 expression had shorter overall survival ( d , median survival = 24 months Vs. 36 months, log-rank test, n = 82, * p = 0.0486) and disease-free survival ( e , median survival = 20 months Vs. 36 months, log-rank test, n = 82, ** p = 0.0426). f and g Kaplan–Meier survival analysis of overall survival and disease-free survival were compared according to the expression levels of MSI2 in HCC tissues. Patients with high MSI2 expression had shorter overall survival ( f , median survival = 18 months Vs. 40 months, log-rank test, n = 82, **** p <0.0001) and disease-free survival ( g , median survival = 12 months Vs. 38 months, log-rank test, n = 82, **** p <0.0001). h Representative images of IHC staining of MSI2 in tumor and adjacent non-tumor tissues. And analysis of MSI2 expression in tumor and adjacent non-tumor tissues by paired t test. Scale bars: 200 μm and 50 μm. i The expression of MSI2 and CD44v6 in tumor tissues from the same HCC patient were analyzed by IHC staining and found that MSI2 was positively correlated with CD44v6 ( n = 82, r = 0.6093, **** p <0.0001, Pearson’s correlation). Scale bars: 200 μm and 50 μm

Article Snippet: Then the protein A/G PLUS-Agarose was collected, washed and boiled, samples were immunoblotted with anti-MSI2 (ORIGENE, cat#TA506196S) or anti-LFNG (ABGENT, cat#AP9524c-400 1) antibodies.

Techniques: Western Blot, Expressing, Immunohistochemistry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 contributes to the maintenance of CD44v6+ liver cancer stem cells via notch1 signaling pathway

doi: 10.1186/s13046-019-1508-1

Figure Lengend Snippet: Knockdown of MSI2 significantly attenuated the stemness properties of CD44v6+ LCSC. a MSI2 expression levels were tested in CD44v6+ HCC cells and CD44v6- HCC cells by western blot in SNU-398 and MHCC-97 h cell lines. b Immunofluorescence images of CD44v6+ SNU-398 cells and CD44v6- SNU-398 cells for localization of MSI2 (red) and CD44v6 (green). Histogram analysis of the relative fluorescence intensity of CD44v6 and MSI2 in CD44v6+ cells and CD44v6- cells. Scale bar, 50 μm. c Representative images of spheres and histogram analysis in indicated cells. The inhibition of MSI2 decreased self-renewal property in vitro in CD44v6+ LCSCs, Scale bar, 200 μm. d and e Transwell migration and invasion assays showed that knockdown of MSI2 decreased the migration and invasion of CD44v6+ cells. Scale bar, 200 μm. f Colony formation assays showed that the ability of cell proliferation and colony formation of CD44v6+ cells was inhibited when MSI2 was down regulated. g 1 × 10 5 of MSI2 shRNA1 cells and the corresponding controls were injected into the left lobes of liver. Bioluminescence signals from MSI2 shRNA1 groups were weaker than that from corresponding control groups. Red arrows indicated the site of tumor formation. h Efficiency of tumor formation of MSI2 shRNA cells and corresponding control cells. Number of injected cells: 1 × 10 5 . n = 8. Black arrow means Mock group, blue arrow means NC group, red arrow means MSI2 shRNA1 group, and orange arrow means MSI2 shRNA2 group. i The expression of cancer stemness-related genes, including Nanog, Oct4 and Sox2 in MSI2 shRNA cells compared with corresponding control. β-actin was used as a normalized control. It showed the expression of stemness-related genes were decreased when MSI2 was knockdown in CD44v6+ LCSCs. For statistical analysis, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001, t test

Article Snippet: Then the protein A/G PLUS-Agarose was collected, washed and boiled, samples were immunoblotted with anti-MSI2 (ORIGENE, cat#TA506196S) or anti-LFNG (ABGENT, cat#AP9524c-400 1) antibodies.

Techniques: Expressing, Western Blot, Immunofluorescence, Fluorescence, Inhibition, In Vitro, Migration, Injection, shRNA

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 contributes to the maintenance of CD44v6+ liver cancer stem cells via notch1 signaling pathway

doi: 10.1186/s13046-019-1508-1

Figure Lengend Snippet: MSI2 maintained the stemness properties of CD44v6+ LCSCs via activating Notch1 signaling pathway. a External dataset from starBase v3.0 project with 374 HCC samples and 50 normal samples was used to analyze the correlation of MSI2 and Notch1. The results showed that MSI2 was positively related to Notch1 in clinical-pathology ( r = 0.458, p = 8.02e-21). b Immunofluorescence images of MSI2 (red) and Notch1 (green) in HCC tissues. MSI2 was co-localized with Notch1 in HCC tissues. Scale bar, 50 μm. c Immunofluorescence images of CD44v6+ SNU-398 cells and CD44v6- cells for localization of MSI2 (red) and Notch1 (green). Histogram analysis of the relative fluorescence intensity of MSI2 and Notch1 in CD44v6+ cells and CD44v6- cells. Scale bar, 50 μm. d. Silencing MSI2 decreased the expression of Notch1 receptor and Notch1 pathway target genes in CD44v6+ SNU-398 cells. β-actin was used as a normalized control. e Western blot showed that overexpression of MSI2 increased the expression of Notch1 receptor and Notch1 pathway target genes in CD44v6- SNU-398 cells. β-actin was used as a normalized control

Article Snippet: Then the protein A/G PLUS-Agarose was collected, washed and boiled, samples were immunoblotted with anti-MSI2 (ORIGENE, cat#TA506196S) or anti-LFNG (ABGENT, cat#AP9524c-400 1) antibodies.

Techniques: Immunofluorescence, Fluorescence, Expressing, Western Blot, Over Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 contributes to the maintenance of CD44v6+ liver cancer stem cells via notch1 signaling pathway

doi: 10.1186/s13046-019-1508-1

Figure Lengend Snippet: MSI2 activated Notch1 signaling through LFNG in CD44v6+ LCSCs. a A Notch RT 2 PCR Array was used to determine mRNA expression profiles between MSI2 shRNA and control CD44v6+ LCSCs. b Hypothesis diagram of MSI2 regulates Notch1 signaling pathway. c Relative mRNA of the most significantly regulated genes were detected by RT-PCR in MSI2 shRNA1 group and the corresponding control group. d Western blot showed that silencing MSI2 decreased the expression of LFNG in CD44v6+ cells while overexpression of MSI2 increased the expression of LFNG in CD44v6- cells. β-actin was used as a normalized control. e Efficiency of tumor formation of LFNG shRNA1 cells and the corresponding controls. Number of injected cells: 1 × 10 5 . n = 4. f Representative images of spheres and histogram analysis in indicated cells. The inhibition of LFNG decreased self-renewal property in vitro in CD44v6+ LCSCs, Scale bar, 200 μm. g The expression of cancer stemness-related genes, including Nanog, Oct4 and Sox2 in LFNG shRNA cells compared with corresponding controls. β-actin was used as a normalized control. The inhibition of LFNG decreased the expression of stemness-related genes in CD44v6+ LCSCs. h Silencing LFNG in CD44v6+ HCC cells decreased the expression of key components of Notch1 pathway (including Notch1, NICD, Hey1 and Hes1) but MSI2 had no significant change. β-actin was used as a normalized control. i Key components of Notch1 signaling reduction caused by MSI2 knockdown could be rescued by LFNG overexpression in CD44v6+ LCSCs. For statistical analysis, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001, t test

Article Snippet: Then the protein A/G PLUS-Agarose was collected, washed and boiled, samples were immunoblotted with anti-MSI2 (ORIGENE, cat#TA506196S) or anti-LFNG (ABGENT, cat#AP9524c-400 1) antibodies.

Techniques: Expressing, shRNA, Reverse Transcription Polymerase Chain Reaction, Western Blot, Over Expression, Injection, Inhibition, In Vitro

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 contributes to the maintenance of CD44v6+ liver cancer stem cells via notch1 signaling pathway

doi: 10.1186/s13046-019-1508-1

Figure Lengend Snippet: MSI2 bound to LFNG mRNA and protein directly. a Schematic representation of MSI2 molecular interaction domains for interaction with RNA and with proteins. RRM, RNA recognition motif; PPD, protein-protein binding domain. b CD44v6+ cells and CD44v6- cells were isolated from SNU-398 cells. CD44v6+ cells were transfected with MSI2 shRNA1 or corresponding control virus, CD44v6- cells were transfected with Lv MSI2 or corresponding control virus. Lysates were precipitated with anti-MSI2 antibody and then immunoblotted (WB) for LFNG. Protein expression of MSI2 and LFNG was also analyzed. c CD44v6+ cells were transfected with MSI2 shRNA1 or corresponding control virus, CD44v6- cells were transfected with Lv MSI2 or corresponding control virus. RIP assays using anti-MSI2 antibody showed that MSI2 interacted with LFNG. The results of agarose electrophoresis of the PCR products were shown. d Schematic illustration the mechanism by which MSI2 activating Notch1 signaling pathway by binding to LFNG mRNA and protein directly

Article Snippet: Then the protein A/G PLUS-Agarose was collected, washed and boiled, samples were immunoblotted with anti-MSI2 (ORIGENE, cat#TA506196S) or anti-LFNG (ABGENT, cat#AP9524c-400 1) antibodies.

Techniques: Protein Binding, Isolation, Transfection, Expressing, Electrophoresis, Binding Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 contributes to the maintenance of CD44v6+ liver cancer stem cells via notch1 signaling pathway

doi: 10.1186/s13046-019-1508-1

Figure Lengend Snippet: Correlation of the expression of CD44v6 and MSI2 with clinical-pathological variables in HCC patients

Article Snippet: Then the protein A/G PLUS-Agarose was collected, washed and boiled, samples were immunoblotted with anti-MSI2 (ORIGENE, cat#TA506196S) or anti-LFNG (ABGENT, cat#AP9524c-400 1) antibodies.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Evasion of regulatory phosphorylation by an alternatively spliced isoform of Musashi2

doi: 10.1038/s41598-017-11917-3

Figure Lengend Snippet: Regulatory phosphorylation of Msi2 controls translational activation of target mRNAs and oocyte maturation. ( a ) Schematic alignment of Xenopus Msi1 regulatory phosphorylation motifs with similar motifs in Msi2 isoforms from Xenopus (Xe), Human (Hu) and mouse (Mm). ( b ) Immature stage VI oocytes were injected with antisense oligonucleotides to Msi1 and Msi2, incubated overnight and subsequently re-injected with water (No Rescue), or RNA encoding Xenopus Msi2 wild type (Msi2 WT) or mutant Msi2 S356A/S381A (Msi2 AA). Following re-injection, the oocytes were allowed to rest for 1 hour before being stimulated with progesterone to induce maturation. The extent of cell cycle rescue for each condition was assessed when 50% of Msi2 WT injected oocytes had completed GVBD. The combined data for three independent experiments are shown; error bars represent S.E.M. ( c ) Cell lysates from one of the experiments in panel (b) were analyzed for expression of the GST-tagged Msi2 WT or Msi2 AA proteins (arrowhead). When 50% of the Msi2 WT oocytes had reached GVBD (GVBD 50 ), they were segregated into those that had not (−) or had (+) completed GVBD. The filter was cropped to retain the 50–80 kD range, prior to western blotting. ( d ) Stage VI immature oocytes were injected with scrambled control antisense oligonucleotides (Con AS); Msi antisense oligonucleotides without (Msi AS) or with re-injection of RNA encoding Xenopus Msi2 or mutant Msi2 AA and subsequently stimulated with progesterone. Total RNA was prepared from immature stage VI oocytes (I), or Con AS when 50% of the oocytes had reached GVBD (GVBD 50 ), segregated into those that had not (−) or had (+) completed GVBD. Similarly, RNA was prepared from Msi AS at the time when Con AS reached GVBD 50 , or Msi AS oocytes expressing Msi2 when they reached GVBD 50 , segregated into those that had not (−) or had (+) completed GVBD. Total RNA was also prepared from Msi AS oocytes expressing Msi2 AA, which did not mature, at the time when the Msi AS + Msi2 reached GVBD 50 . Progesterone-dependent polyadenylation (indicated by a retarded mobility shift) of the endogenous Mos, cyclin B5 and Msi1 mRNAs was assessed by RNA ligation coupled PCR.

Article Snippet: Human Msi2 variant 2 (Accession NM_170721, Origene) was PCR amplified to include a 5′ Cla1 site, and a 3′ Xho1 site and cloned into Cla1/Xho1 digested pXen2. hMsi2 eGFP and hMsi2v2 eGFP. hMsi2 and hMsi2v2 were PCR amplified to include a 5′ HindIII site and a 3′ BamH1 immediately prior to the STOP codon and cloned into HindIII/BamH1 digested pEGFP-N1 (Clontech) to generate an in-frame C-terminal eGFP fusion protein.

Techniques: Activation Assay, Injection, Incubation, Mutagenesis, Expressing, Western Blot, Mobility Shift, Ligation

Journal: Scientific Reports

Article Title: Evasion of regulatory phosphorylation by an alternatively spliced isoform of Musashi2

doi: 10.1038/s41598-017-11917-3

Figure Lengend Snippet: Progesterone-dependent phosphorylation of Msi2 during oocyte maturation. ( a ) Immature Stage VI oocytes were injected with RNA encoding GST-tagged Xenopus Msi2 (XeMsi2 WT) or mutant Msi2 S356A/S381A (XeMsi2 AA), incubated overnight to express the protein and subsequently stimulated to mature with progesterone. Protein lysates were prepared at the indicated times and the phosphorylation of S381 or S356 assessed with phospho-specific antisera as indicated. The lower panel represents a GST western blot to show the relative levels of the expressed proteins. XeMsi2 WT expressing oocytes reached GVBD 50 after 4 hours and were segregated into those that had not (−) or had (+) completed GVBD. XeMsi2 AA oocyte samples were prepared 6 hours after progesterone treatment, but they had still not undergone GVBD. The filter was cropped to retain the 50–80 kD range, prior to western blotting. A representative experiment is shown. ( b ) Immature Stage VI oocytes were injected with RNA encoding GST-tagged Xenopus Msi2 (XeMsi2 WT) and incubated overnight. The oocytes were then stimulated to mature with progesterone. When the progesterone treated oocytes had completed GVBD, samples were prepared and either mock treated or incubated with λ phosphatase prior to analysis by GST western blotting. The filter was cropped to retain the 50–80 kD range, prior to western blotting. ( c ) 50–60 immature Stage VI oocytes were left untreated (UI) or injected with RNA encoding either GST-tagged XeMsi2 WT or XeMsi2 AA, incubated overnight and subsequently stimulated to mature with progesterone. The extent of GVBD in each cohort was assessed when 40–50% of the control (UI) oocytes reach GVBD. The results represent three independent experiments; error bars represent S.E.M.

Article Snippet: Human Msi2 variant 2 (Accession NM_170721, Origene) was PCR amplified to include a 5′ Cla1 site, and a 3′ Xho1 site and cloned into Cla1/Xho1 digested pXen2. hMsi2 eGFP and hMsi2v2 eGFP. hMsi2 and hMsi2v2 were PCR amplified to include a 5′ HindIII site and a 3′ BamH1 immediately prior to the STOP codon and cloned into HindIII/BamH1 digested pEGFP-N1 (Clontech) to generate an in-frame C-terminal eGFP fusion protein.

Techniques: Injection, Mutagenesis, Incubation, Western Blot, Expressing

Journal: Scientific Reports

Article Title: Evasion of regulatory phosphorylation by an alternatively spliced isoform of Musashi2

doi: 10.1038/s41598-017-11917-3

Figure Lengend Snippet: MAP kinase and Ringo signaling pathways mediate Msi2 S356 and S381 phosphorylation. ( a ) Immature oocytes (I) were injected with RNA encoding GST-tagged Xenopus Msi2 and incubated overnight to express the protein. The next morning the oocytes were split into two pools and either treated with DMSO (vehicle control) or U0126 for 1 hour prior to progesterone stimulation. When DMSO-treated oocytes reached GVBD 50 , oocytes were segregated into those that had not (−) or had (+) completed GVBD. UO126–treated oocyte lysates did not mature in response to progesterone and so time-matched (GVBD−) samples were prepared when DMSO treated oocytes reached GVBD50 (UO126 equiv). Lysates were probed with Msi2 S356 phospho-specific antisera, phospho-MAPK or GST antisera as indicated. The filter was cropped to retain the 50–80 kD range, prior to western blotting for the GST and pMsi2 S365 antibodies, and cropped to retain the 30–50 kD range for the phospho-MAPK western. ( b ) as ( a ), except probed with Msi2 S381 phospho-specific antisera. ( c ) Immature oocytes were co-injected with RNA encoding GST-tagged Xenopus Msi2 and either scrambled control or Ringo antisense oligonucleotides (C-AS or R-AS) and incubated overnight. Oocytes were then split into two pools and either left unstimulated (I) or stimulated with progesterone. Time matched protein lysates were prepared when progesterone stimulated C-AS oocytes reached GVBD 50 . C-AS oocytes were segregated into those that had not (−) or had (+) completed GVBD. Protein lysates were probed by western blotting with the indicated antibodies. The phospho-Msi2 images were over-exposed to demonstrate a low level of basal phosphorylation in immature oocytes. A lower exposure of these panels is shown in the Supplementary data. The filter was cropped prior to western blotting, as described for panel (a). In addition, the filter was cropped to retain the 30–50 kD range for the total MAPK western blot. ( d ) Progesterone-dependent polyadenylation of the endogenous Mos, cyclin B5 and Musashi1 mRNAs was assessed by RNA ligation coupled PCR in scrambled, control or Ringo antisense injected oocytes, essentially as described for Fig. .

Article Snippet: Human Msi2 variant 2 (Accession NM_170721, Origene) was PCR amplified to include a 5′ Cla1 site, and a 3′ Xho1 site and cloned into Cla1/Xho1 digested pXen2. hMsi2 eGFP and hMsi2v2 eGFP. hMsi2 and hMsi2v2 were PCR amplified to include a 5′ HindIII site and a 3′ BamH1 immediately prior to the STOP codon and cloned into HindIII/BamH1 digested pEGFP-N1 (Clontech) to generate an in-frame C-terminal eGFP fusion protein.

Techniques: Injection, Incubation, Western Blot, Ligation

Journal: Scientific Reports

Article Title: Evasion of regulatory phosphorylation by an alternatively spliced isoform of Musashi2

doi: 10.1038/s41598-017-11917-3

Figure Lengend Snippet: Mammalian Msi2 can direct mRNA translational activation and Xenopus oocyte maturation. ( a ) Msi1/2 antisense rescue assay, essentially as described in the legend to Fig. , comparing cell cycle progression in GST-tagged Xenopus Msi2 and GST-tagged murine Msi2 expressing oocytes. Oocyte GVBD was scored when 100% of Xenopus Msi2 injected oocytes completed GVBD. The data represent the results of three independent experiments; error bars indicate S.E.M. ( b ) Equivalent cell lysates from one of the experiments in panel (a) were analyzed for expression of the GST-tagged Xenopus or murine Msi2 proteins (arrowheads). The filter was cropped to retain the 50–80 kD range, prior to western blotting. ( c ) Progesterone-dependent polyadenylation (retarded mobility shift) of the endogenous Mos mRNA was assessed by RNA ligation coupled PCR in Musashi antisense (Msi AS) injected oocytes, essentially as described for Fig. , with either Xenopus Msi2 (XeMsi2) or murine Msi2 (mMsi2) rescue as indicated. ( d ) RNA EMSA using either a biotinylated Mos probe (WT Mos) with an intact Msi binding element (MBE) or a mutant Mos probe with a disrupted MBE (Msi mut), incubated with unprogrammed rabbit reticulocyte lysate or lysate expressing GST, GST murine Msi1 (mMsi1) or GST murine Msi2 (mMsi2), as indicated. Specific Msi1 and Msi2 complexes (solid arrowheads) form with the WT Mos probe but not the Msi mut probe. A number of additional non-specific bands are observed with unprogrammed lysate alone (open arrowheads). ( e ) Western blot of unprogrammed, GST, GST mMsi1 or GST mMsi2 expressing reticulocyte lysates used in the gel shift assay ( d ).

Article Snippet: Human Msi2 variant 2 (Accession NM_170721, Origene) was PCR amplified to include a 5′ Cla1 site, and a 3′ Xho1 site and cloned into Cla1/Xho1 digested pXen2. hMsi2 eGFP and hMsi2v2 eGFP. hMsi2 and hMsi2v2 were PCR amplified to include a 5′ HindIII site and a 3′ BamH1 immediately prior to the STOP codon and cloned into HindIII/BamH1 digested pEGFP-N1 (Clontech) to generate an in-frame C-terminal eGFP fusion protein.

Techniques: Activation Assay, Rescue Assay, Expressing, Injection, Western Blot, Mobility Shift, Ligation, Binding Assay, Mutagenesis, Incubation, Electrophoretic Mobility Shift Assay

Journal: Scientific Reports

Article Title: Evasion of regulatory phosphorylation by an alternatively spliced isoform of Musashi2

doi: 10.1038/s41598-017-11917-3

Figure Lengend Snippet: Regulatory phosphorylation controls mammalian Msi2 activity. ( a , b ) Immature oocytes were injected with RNA encoding GST-tagged murine Msi2 and incubated overnight to express the protein. The oocytes were then split into two pools and either left untreated or stimulated with progesterone. When stimulated oocytes reached GVBD 50 , oocytes were segregated into those that had not (−) or had (+) completed GVBD. Equivalent cell lysates from each condition were probed with phospho-specific antisera that recognizes S278 of the mammalian Msi2 protein (equivalent to S356 of Xenopus Msi2), or S303 (the equivalent of Xenopus Msi2 S381) or GST antisera to detect the expressed protein, as indicated. Mobility shifted forms of Msi2 are indicated by an upper arrowhead on the lower panels. The filter was cropped to retain the 50–80 kD range, prior to western blotting. ( c ) A Msi1/2 antisense rescue assay, essentially as described in Fig. , comparing progression to GVBD in GST-tagged murine Msi1 (mMsi1 WT), S312A/S337A phosphorylation mutant mMsi1 (mMsi1 AA), human Msi2 (hMsi2 WT) or S278A/S303A phosphorylation mutant Msi2 (hMsi2 AA). Oocyte GVBD was scored when 50% of mMsi1 reached GVBD (for mMsi1 AA) or when 50% of hMsi2 reached GVBD (for hMsi2 AA). Two independent experiments are shown. ( d ) Lysate from unstimulated oocytes in panel (c) experiment 2 were analyzed by GST western blotting for expression of mMsi1 (upper panel) or hMsi2 (lower panel). The filter was cropped to retain the 50–80 kD range, prior to western blotting. ( e ) Western blot analysis of murine 32D cells prepared at different times during differentiation (30 mins and 1 hour, respectively) vs . basal proliferation conditions (Basal) probed with mammalian Msi2 S303 phospho-specific antisera (upper panel), Msi2 antisera, or tubulin. In 32D cells, the larger Msi2 isoform showed increased regulatory phosphorylation. The filter was cropped to retain the 30–50 kD range, prior to western blotting for Msi2 or cropped to retain the 40–60 kD range, prior to western blotting for tubulin. ( f ) Western blot analysis of human SHSY5Y cells undergoing proliferation (Prolif) or 1 hour after induction of differentiation (Diff) with retinoic acid. The Msi2 isoform showed increased regulatory S303 phosphorylation. A cross-reacting, non-specific band runs above the phosphorylated Msi2 isoform in the upper panel (asterisk). GAPDH serves as a loading control. The filter was cropped to retain the 30–50 kD range, prior to western blotting. The phospho-Msi2 image was over-exposed to more clearly demonstrate increased phosphorylation. A lower exposure of this panel is shown in the Supplementary data.

Article Snippet: Human Msi2 variant 2 (Accession NM_170721, Origene) was PCR amplified to include a 5′ Cla1 site, and a 3′ Xho1 site and cloned into Cla1/Xho1 digested pXen2. hMsi2 eGFP and hMsi2v2 eGFP. hMsi2 and hMsi2v2 were PCR amplified to include a 5′ HindIII site and a 3′ BamH1 immediately prior to the STOP codon and cloned into HindIII/BamH1 digested pEGFP-N1 (Clontech) to generate an in-frame C-terminal eGFP fusion protein.

Techniques: Activity Assay, Injection, Incubation, Western Blot, Rescue Assay, Mutagenesis, Expressing

Journal: Scientific Reports

Article Title: Evasion of regulatory phosphorylation by an alternatively spliced isoform of Musashi2

doi: 10.1038/s41598-017-11917-3

Figure Lengend Snippet: The Msi2 variant 2 promotes transformation of NIH3T3 cells. ( a ) Schematic showing alternative exon usage between the canonical human Msi2 isoform (hMsi2) and the variant 2 isoform (hMsi2v2). Substitution of exons 1 and 2 for exon 1b in hMsi2v2 results in a novel 17 amino acid N-terminal domain. Exons 3–11 are shared between the two isoforms, but use of the alternate 11b exon results in a unique terminal 13 amino acid sequence in the truncated C-terminal domain. Lower panel show schematic alignment of the canonical human Msi2 protein (Accession NP_620412) with the alternatively spliced human Msi2 variant 2 protein (Accession NP_733839). RRM1 and RRM2, RNA recognition motifs 1 and 2; P, indicates position of the sites of regulatory phosphorylation in the canonical Msi2 isoform. ( b ) A Musashi antisense rescue assay comparing progression to oocyte GVBD in GST-tagged hMsi2 or hMsi2 variant 2 (Msi2 var2). Rescue was scored when 50% of hMsi2-injected oocytes reached GVBD. Data shown are from three independent experiments; error bars represent S.E.M. ( c ) Lysates from unstimulated oocytes in panel (b) were analyzed by GST western blotting. The filter was cropped to retain the 50–80 kD range, prior to western blotting. ( d ) RNA EMSA using a biotinylated murine Pou1f1 probe with consensus MBE (GUAGG), incubated with unprogrammed rabbit reticulocyte lysate or lysate expressing GST fusion proteins: GST human Msi2 (hMsi2), GST human Msi2 variant 2 (hMsi2 var2) or the GST moiety alone, as indicated. A specific Msi2 complex (solid arrowhead) is indicated. ( e ) A GST western blot of the reticulocyte lysates used in the gel shift assay ( d ). GST proteins are indicated by arrowheads. The hMsi2 var2 runs just above a non-specific band (asterisk). ( f ) 3000 NIH3T3 cells were plated per well of a 24 well ultra-low adherence tissue culture dish and transfected in quadruplicate with the indicated expression construct. Colony growth was scored at day 10 after transfection. The data are combined from three independent transfection experiments. *p < 0.05; **p < 0.01, ns, not significant; n=3. ( g ) qRT-PCR showing expression levels of Msi2 var2 RNA in human iPSCs and SH-SY5Y cells relative to level of GAPDH RNA. The combined data from three independent experiments are shown. *p < 0.05.

Article Snippet: Human Msi2 variant 2 (Accession NM_170721, Origene) was PCR amplified to include a 5′ Cla1 site, and a 3′ Xho1 site and cloned into Cla1/Xho1 digested pXen2. hMsi2 eGFP and hMsi2v2 eGFP. hMsi2 and hMsi2v2 were PCR amplified to include a 5′ HindIII site and a 3′ BamH1 immediately prior to the STOP codon and cloned into HindIII/BamH1 digested pEGFP-N1 (Clontech) to generate an in-frame C-terminal eGFP fusion protein.

Techniques: Variant Assay, Transformation Assay, Sequencing, Rescue Assay, Injection, Western Blot, Incubation, Expressing, Electrophoretic Mobility Shift Assay, Transfection, Construct, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Evasion of regulatory phosphorylation by an alternatively spliced isoform of Musashi2

doi: 10.1038/s41598-017-11917-3

Figure Lengend Snippet: Normalized expression of Msi1, Msi2 and Msi2 variant 2 transcripts in sixteen human tissues.

Article Snippet: Human Msi2 variant 2 (Accession NM_170721, Origene) was PCR amplified to include a 5′ Cla1 site, and a 3′ Xho1 site and cloned into Cla1/Xho1 digested pXen2. hMsi2 eGFP and hMsi2v2 eGFP. hMsi2 and hMsi2v2 were PCR amplified to include a 5′ HindIII site and a 3′ BamH1 immediately prior to the STOP codon and cloned into HindIII/BamH1 digested pEGFP-N1 (Clontech) to generate an in-frame C-terminal eGFP fusion protein.

Techniques: Expressing, Variant Assay

Journal: Scientific Reports

Article Title: Evasion of regulatory phosphorylation by an alternatively spliced isoform of Musashi2

doi: 10.1038/s41598-017-11917-3

Figure Lengend Snippet: Differential expression of Msi2 and Msi2 variant 2 transcripts in human cancer vs normal adjacent tissue.

Article Snippet: Human Msi2 variant 2 (Accession NM_170721, Origene) was PCR amplified to include a 5′ Cla1 site, and a 3′ Xho1 site and cloned into Cla1/Xho1 digested pXen2. hMsi2 eGFP and hMsi2v2 eGFP. hMsi2 and hMsi2v2 were PCR amplified to include a 5′ HindIII site and a 3′ BamH1 immediately prior to the STOP codon and cloned into HindIII/BamH1 digested pEGFP-N1 (Clontech) to generate an in-frame C-terminal eGFP fusion protein.

Techniques: Expressing, Variant Assay

Journal: Oncogenesis

Article Title: Musashi-2 (MSI2) regulates epidermal growth factor receptor (EGFR) expression and response to EGFR inhibitors in EGFR-mutated non-small cell lung cancer (NSCLC)

doi: 10.1038/s41389-021-00317-y

Figure Lengend Snippet: A Heatmap summarizes RPPA results for expression of EGFR, pEGFR(Y1068), pEGFR(Y1173), ERBB2, pERBB2(Y1248), ERBB3, and pERBB3 (Y1298) protein expression. Three independent isolates of cell lines were analyzed in each experiment. In stable derivatives of 344SQ, expressing high levels of endogenous MSI2, SCR, scrambled shRNA and NTC, and non-transfected cells are negative controls: M2-m1 and M2-m2 are two independent shRNAs depleting MSI2. In stable derivatives of 393p, expressing low levels of endogenous MSI2, GFP-3, and GFP-4 are negative controlsand M2a and M2b overexpress a MSI2 cDNA. B Western blots of indicated cell lines, following depletion (m1, m2, sh1, sh2) or overexpression (M2a, M2b, MSI2) of MSI2.NC, pLD and pLV are negative controls. MSI2 depletion was induced by the addition of 1 μg/ml of Doxycycline for 48 h. C , D Quantification of Western blot data fromat least three independent experiments by Image J software, with values normalized to β-actin. Error bars represented by SEM. Statistical analysis was performed using unpaired two tailed t -test. * p < 0.05, ** p < 0.01, *** p < 0.001 for all graphs.

Article Snippet: To generate stable cell lines with inducible MSI2 knockdowns, self-complementary single-stranded DNA oligos (Supp Table ) were annealed and cloned into AgeI/EcoR1 sites of Tet-pLKO-puro vector (Addgene plasmid # 21915).

Techniques: Expressing, shRNA, Transfection, Western Blot, Over Expression, Software, Two Tailed Test

Journal: Oncogenesis

Article Title: Musashi-2 (MSI2) regulates epidermal growth factor receptor (EGFR) expression and response to EGFR inhibitors in EGFR-mutated non-small cell lung cancer (NSCLC)

doi: 10.1038/s41389-021-00317-y

Figure Lengend Snippet: A Western blots of indicated cell lines, following MSI2 depletion by shRNA (sh1, sh2) or siRNA (h1, h2) or overexpression (MSI2) in three EGFR mut NSCLC cell lines; H1650, HCC827, PC9, and H1975. Negative controls include GL2 and NC for depletion, and pLV for overexpression. MSI2 depletion was induced by the addition of 1 μg/ml of Doxycycline for 48 h. B , C Quantification of Western blot data from at least three independent experiments by Image J software. Error bars represented by SEM. Statistical analysis was performed using unpaired two tailed t -test. * p < 0.05, ** p < 0.01, *** p < 0.001 for all graphs.

Article Snippet: To generate stable cell lines with inducible MSI2 knockdowns, self-complementary single-stranded DNA oligos (Supp Table ) were annealed and cloned into AgeI/EcoR1 sites of Tet-pLKO-puro vector (Addgene plasmid # 21915).

Techniques: Western Blot, shRNA, Over Expression, Software, Two Tailed Test

Journal: Oncogenesis

Article Title: Musashi-2 (MSI2) regulates epidermal growth factor receptor (EGFR) expression and response to EGFR inhibitors in EGFR-mutated non-small cell lung cancer (NSCLC)

doi: 10.1038/s41389-021-00317-y

Figure Lengend Snippet: A Quantification of mRNA immunoprecipitation (RIP) results from assays performed in A549 and PC9 cell lysates using antibodies to MSI2, or IgG (negative control) antibodies, followed by quantitative RT-PCR. Data are normalized to positive control PTP4A1, TGFBR1 , and SMAD3 are additional positive controls; GAPDH is a negative control. Data shown reflect the average of three independent RIP experiments. Error bars indicate SEM. Statistical analysis was performed using unpaired two tailed t -test. p < 0.05, ** p < 0.01, *** p < 0.001 for all graphs. B Location of consensus binding sites for Musashi proteins in EGFR, as defined from studies by Bennett et al. and Wang et al. . Coding sequences are represented by thick lines; 3′ untranslated regions by thin line. 7- or 8-bp consensus sequences are indicated by arrows. Thick arrows indicate identical concensus sequences identified simultaneously by Wang and Bennett studies. Shorter consensus sequences are not indicated. Blue arrows indicate the positions of ssRNA oligos (MSI2-binding sites are underscored) used for REMSA. The localization of the fragments used to generate reporter vectors are depicted as Reporter 1 and Reporter 2. C Analysis of recombinant MSI2 protein binding with 3′UTR fragments of EGFR mRNA by RNA-EMSA. In all, 50 ng of recombinant MSI2 protein were incubated with 32 P-labeled ssRNA oligos, EGFR oligo 1, EGFR oligo 2, and Positive- and Negative control oligos alone, or in presence of 100-fold molar excess of unlabeled competitors, identical to the labeled probe. Competing ssRNA EGFR oligos 1 and 2 were identical to labeled probes and contained wild type (oligo wt) or mutant (oligo mut) MSI2-binding motifs.

Article Snippet: To generate stable cell lines with inducible MSI2 knockdowns, self-complementary single-stranded DNA oligos (Supp Table ) were annealed and cloned into AgeI/EcoR1 sites of Tet-pLKO-puro vector (Addgene plasmid # 21915).

Techniques: Immunoprecipitation, Negative Control, Quantitative RT-PCR, Positive Control, Two Tailed Test, Binding Assay, Recombinant, Protein Binding, Incubation, Labeling, Mutagenesis

Journal: Oncogenesis

Article Title: Musashi-2 (MSI2) regulates epidermal growth factor receptor (EGFR) expression and response to EGFR inhibitors in EGFR-mutated non-small cell lung cancer (NSCLC)

doi: 10.1038/s41389-021-00317-y

Figure Lengend Snippet: A Cell viability quantified by Cell Titer Blue (CTB) assay, in indicated cell lines with negative control (NC) for depletion, or depletion of MSI2 (sh1, sh2). B Quantification of viability by CTB assay, 96 h after doxycycline treatment, in cell lines expressing empty lentivirus (pLV) or the same vector overexpressing MSI2. C IC50 curves for viability of cell lines measured by CTB assay following 96 h treatment with erlotinib or afatinib. Representative data of one of three independent experiments are presented. D EGFR mut (PC9, HCC827, and H1975) and KRAS mut (A549) cell line derivatives expressing doxycycline-inducible anti-MSI2 shRNAs (sh1 and sh2) or negative control (NC) cells were incubated in complete medium in presence of 1 μg/ml of Doxycycline with indicated concentrations of erlotinib (Erl) or afatinib (Afa) for 96 h, then viability measured by CTB Assay. For A , B , and D , data presented represent the average of three independent experiments. Error bars represented by SEM. Statistical analysis was performed using unpaired two tailed t -test. * p < 0.05, ** p < 0.01, *** p < 0.001 for all graphs.

Article Snippet: To generate stable cell lines with inducible MSI2 knockdowns, self-complementary single-stranded DNA oligos (Supp Table ) were annealed and cloned into AgeI/EcoR1 sites of Tet-pLKO-puro vector (Addgene plasmid # 21915).

Techniques: CtB Assay, Negative Control, Expressing, Plasmid Preparation, Incubation, Two Tailed Test

Journal: Oncogenesis

Article Title: Musashi-2 (MSI2) regulates epidermal growth factor receptor (EGFR) expression and response to EGFR inhibitors in EGFR-mutated non-small cell lung cancer (NSCLC)

doi: 10.1038/s41389-021-00317-y

Figure Lengend Snippet: A Growth curve of subcutaneous xenografts PC9 cells stably expressing lentiviral vector as negative control (NC) or shRNA to MSI2 (sh1), and treated with vehicle or erlotinib (ERL) for 24 days. N = 5/group. B Quantification of tumors at endpoint of experiment in A . C Western blot analysis of MSI2, protein levels from treated tumors. D Quantification of western blot data from C ; data normalized to β-actin. E Quantitative RT-PCR of mRNA collected from indicated xenograft tumors at the end of experiments. Negative controls are denoted NC. Data are normalized to 18S rRNA, as noted. Relative quantification (RQ) of gene expression was performed using 2 −ΔΔCt method. Data are presented as normalized average RQ means in each group ( n = 5) of animals. In all graphs, error bars represented by SEM. Statistical analysis was performed using unpaired two tailed t test. * p < 0.05, ** p < 0.01, *** p < 0.001 for all graphs.

Article Snippet: To generate stable cell lines with inducible MSI2 knockdowns, self-complementary single-stranded DNA oligos (Supp Table ) were annealed and cloned into AgeI/EcoR1 sites of Tet-pLKO-puro vector (Addgene plasmid # 21915).

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Negative Control, shRNA, Western Blot, Quantitative RT-PCR, Two Tailed Test

Journal: Oncogenesis

Article Title: Musashi-2 (MSI2) regulates epidermal growth factor receptor (EGFR) expression and response to EGFR inhibitors in EGFR-mutated non-small cell lung cancer (NSCLC)

doi: 10.1038/s41389-021-00317-y

Figure Lengend Snippet: A H scores for MSI2 and EGFR in EGFR mut NSCLC tumor TMA samples (see Supp Table for clinical characteristics). For MSI2 and EGFR IHC quantification, each spot was examined by board-certified pathologists (ED and NK) who assigned a score of 0 (no staining), 1+ (weak staining), 2+ (moderate staining), and 3+ (strong staining) within carcinomatous areas. The score for each of the two tumor spots was averaged for statistical analysis. The H-score, which ranges from 0 to 300, was calculated using the following formula: [1(% cells 1+) + 2 (% cells 2+) + 3 (% cells 3+)], which reflects staining intensity as well as percentage of positive cells , .

Article Snippet: To generate stable cell lines with inducible MSI2 knockdowns, self-complementary single-stranded DNA oligos (Supp Table ) were annealed and cloned into AgeI/EcoR1 sites of Tet-pLKO-puro vector (Addgene plasmid # 21915).

Techniques: Staining

Journal: bioRxiv

Article Title: The long non-coding RNA FAM30A regulates the Musashi2-RUNX1 axis and is required for LSC function in AML cells

doi: 10.1101/2024.10.13.618058

Figure Lengend Snippet: A. Plot depicting enriched proteins after RNA-pulldown followed by LC-MS/MS analysis using biotinylated FAM30A repeats (FAM) as bait vs bead control (BC) in KG-1a cells. B. Functional clustering analysis (GO) for significantly enriched proteins FAM/BC (P< 0.05). Highlighted in blue are biological processes associated with MSI2 protein. C. Schematic of the probes used for RNA pulldown: a single wild-type FAM30A repeat (FAM(1) WT) with the three MSI2-binding sites (highlighted in blue), and a FAM30A repeat (FAM (1) MMUT) with mutated MSI2-binding sites (in red). Beads only (BC) as well as a control RNA neighbouring the FAM30A repeats (CTRL) were used as specificity controls. A representative Western blot analysis for MSI2 association in RNA pulldowns is shown. D. Western blot analysis for validation of immunoprecipitated endogenous MSI2 and RT-qPCR analysis of MSI2-associated RNAs (lower) in KG-1a cells. E. Representative Western blot of stable KG-1a cell lines for MSI2 protein and its validated target SMAD3. ACTB was used as a loading control F. Quantitative analysis of Western blot (light grey) RT-qPCR analysis (dark grey) for MSI2, harvested in cells from E. Statistical significance was calculated using unpaired two-tailed student t-test. Data is displayed as mean values and error bars represent SEM.

Article Snippet: Then, magnetic Protein G Dynabeads (Life Technologies) were washed three times with Immunoprecipitation buffer and 5-10 μg/condition of anti-MSI2 (Bethyl) was added and incubated for 15 min.

Techniques: Liquid Chromatography with Mass Spectroscopy, Control, Functional Assay, Binding Assay, Western Blot, Immunoprecipitation, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: The long non-coding RNA FAM30A regulates the Musashi2-RUNX1 axis and is required for LSC function in AML cells

doi: 10.1101/2024.10.13.618058

Figure Lengend Snippet: A. GSEA analysis (C2, MSigDB) in KG-1a stable cell lines shows a significant enrichment for RUNX1 activation. B. Immunoprecipitation of MSI2 in KG-1a cells and subsequent RT-qPCR analysis of MSI2-associated RNAs, depicting MSI2-targets (blue), RUNX1 isoforms (green) and negative controls (black). C. Representative Western blot (left) for MSI2 and RUNX1 isoforms in stable KG-1a cell lines and quantitative analysis (right) of RUNX1C and total RUNX1 protein levels, normalised to control cells. ACTB was used as a loading control. D. Western blot analysis in stable K562 cell lines for RUNX1 and MSI2 protein. E. Quantitative analysis of relative luciferase activity in stable K562 cell lines transfected with constructs comprising RUNX1 binding sites to assess RUNX1 activation. Normalization was performed against control cells and the control reporter (CTRL Rep). Statistical significance was calculated using unpaired two-tailed student t-test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns – not significant). Data is displayed as mean values and error bars represent SEM.

Article Snippet: Then, magnetic Protein G Dynabeads (Life Technologies) were washed three times with Immunoprecipitation buffer and 5-10 μg/condition of anti-MSI2 (Bethyl) was added and incubated for 15 min.

Techniques: Stable Transfection, Activation Assay, Immunoprecipitation, Quantitative RT-PCR, Western Blot, Control, Luciferase, Activity Assay, Transfection, Construct, Binding Assay, Two Tailed Test