Journal: Clinical and Translational Medicine

Article Title: LncRNA LINC00942 promotes chemoresistance in gastric cancer by suppressing MSI2 degradation to enhance c‐Myc mRNA stability

doi: 10.1002/ctm2.703

Figure Lengend Snippet: LNC942 promotes cisplatin (DDP) resistance by targeting MSI2 in vivo. (A) Xenografts in nude mice by inoculating SGC7901 cells that were stably overexpressing LNC942 or empty vector (NC) ( n = 5/group). The administration of drug dose and time was recorded; representative photographs of tumours excised from the mice on day 18 are shown. (B) The growth curve showed changes in the tumour volume in mice from different groups; growth was assessed during the treatment period (days 9–18). (C) Tumour weight of the tumours excised from the mice in each group. (D) Representative immunohistochemical images of cleaved‐caspase 3, MSI2, c‐Myc and CD44 in excised tumour tissues. Scale bars = 100 μm. (E) The expression of MSI2 and c‐Myc in GC tissue was analysed by immunohistochemistry staining. (F) The correlation between MSI2 and c‐Myc expression in gastric cancer (GC) tissue was analysed by chi‐square test ( p < .01). (G) Hypothetical model of LNC942 function in GC. High expression of LNC942 prevents the interaction between MSI2‐β‐Trcp and subsequently ubiquitin‐mediated degradation. Then, MSI2 stabilizes the c ‐ Myc mRNA stability in an m 6 A dependent manner. Increased expression of c‐Myc inhibits cell apoptosis and maintains stemness to promote DDP resistance. Data in B‐C are presented as the mean ± SEM; the p value was determined using a two‐tailed unpaired Student's t test. ns, p > .05; * p < .05; ** p < .01; *** p < .001

Article Snippet: Briefly, recombinant MSI2 protein (Proteintech) was covalently immobilized to a CM5 sensor chip through amine groups in a sodium acetate buffer (pH 4.5) at a final response unit (RU) of about 11545.

Techniques: In Vivo, Stable Transfection, Plasmid Preparation, Immunohistochemical staining, Expressing, Immunohistochemistry, Staining, Ubiquitin Proteomics, Two Tailed Test

Journal: Clinical and Translational Medicine

Article Title: LncRNA LINC00942 promotes chemoresistance in gastric cancer by suppressing MSI2 degradation to enhance c‐Myc mRNA stability

doi: 10.1002/ctm2.703

Figure Lengend Snippet: LNC942 interacts with MSI2 protein and prevents its ubiquitination. (A) Quantitative real‐time PCR (qRT‐PCR) detection of LNC942 expression in the cytoplasmic and nuclear fractions. GAPDH is the cytoplasmic marker, and Marat1 is the nuclear marker. (B) Identification of LNC942‐binding protein by human proteome microarray experiment. Upper: Schematic of the human proteome microarray experiment. Bottom: Protein spot subarray demonstrating strand‐specific binding of LNC942 sense strand to MSI2 protein. (C) RNA fluorescence in situ hybridization (FISH) and immunofluorescence (IF) assays showing the co‐localization of LNC942 (Cy3‐labelled, red) and MSI2 (Alexa488‐labelled, green) in SGC‐R cells. Blue: DAPI. All scale bars: 10 μm. (D) RNA immunoprecipitation (RIP) assays were performed with anti‐Flag antibody in SGC‐R cell transfected with vectors expressing Flag‐tagged full length (FL) or truncation mutants of MSI2. Left: Western blotting indicating the expression of Flag‐tagged FL or truncation mutants of MSI2. Middle: Schematic structures of MSI2 proteins and five truncated mutants of MSI2 variants. The binding score indicated the binding intensity between LNC942 and the domains of MSI2. Right: qRT‐PCR detection of LNC942 enrichment after RIP assays. (E) Deletion mapping of the MSI2‐binding region(s) in LINC00942. Left: Western blotting detecting the MSI2 enrichment after RNA pull‐down. AS: anti‐sense probe. Right: the biotin‐labelled LNC942 probes were quantified (300 ng) and detected by the dot blot assay. MB: methylene blue, used as internal reference. (F and G) qRT‐PCR (F) and western blotting (G) were performed for the detection of MSI2 expression in chemosensitive or resistant SGC7901 and BGC823 cells. (H) The protein levels of MSI2 in chemoresistant cells transfected with si‐NC or LNC942 siRNAs. (I) The MSI2 protein expression in SGC7901 and BGC823 cells stably expressing LNC942. (J) SGC‐R cells with or without LNC942 knock‐down were treated with cycloheximide (CHX) (100 μg/ml) for the indicated time points. MSI2 protein abundance was analysed through western blotting, followed by quantification using ImageJ. (K) Western blotting detection of MSI2 protein half‐life in LNC942 stably expressed SGC7901 cells with CHX (100 μg/ml) for the indicated time points. MSI2 protein abundance was quantified using ImageJ. (L) Western blotting of MSI2 in SGC‐R or BGC‐R cells transfected with or without si‐LNC942 following proteasome inhibition with MLN4924 (1 μM, 24 h). (M) SGC7901 cells stably expressing LNC942 were treated with 20 μM MG‐132 for 6 h and then subjected to IP assays to detect the MSI2 ubiquitination levels. Data in panels (A), (D), (F), (J) and (K) are represented as mean ± SD of the three independent experiments. The p value was determined using a two‐tailed unpaired Student's t test. ns, p > .05; * p < .05; ** p < .01; *** p < .001

Article Snippet: Briefly, recombinant MSI2 protein (Proteintech) was covalently immobilized to a CM5 sensor chip through amine groups in a sodium acetate buffer (pH 4.5) at a final response unit (RU) of about 11545.

Techniques: Ubiquitin Proteomics, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Marker, Binding Assay, Microarray, Fluorescence, In Situ Hybridization, Immunofluorescence, RNA Immunoprecipitation, Transfection, Western Blot, Dot Blot, Stable Transfection, Knockdown, Quantitative Proteomics, Inhibition, Two Tailed Test

Journal: Clinical and Translational Medicine

Article Title: LncRNA LINC00942 promotes chemoresistance in gastric cancer by suppressing MSI2 degradation to enhance c‐Myc mRNA stability

doi: 10.1002/ctm2.703

Figure Lengend Snippet: LNC942 disrupts the interactions between MSI2 and the SCF β‐TRCP E3 ubiquitin ligase. (A) 293T cells were transfected with empty vector or Flag–Cullin family proteins and Myc‐MSI2 plasmids as indicated, followed by immunoprecipitation (IP) with anti‐Flag or anti‐Myc antibody and western blotting with the indicated antibodies. α‐Tubulin served as the loading control. After 48 h of transfection, the cells were treated with MG‐132 (20 μM) for 6 h before harvesting. (B) IP with anti‐Flag antibody was performed in SGC7901 cells transfected with empty vector or Flag–MSI2 plasmids. Western blotting was performed with the indicated antibodies. The cells were treated with MG‐132 (20 μM) for 6 h before harvesting. (C) Western blotting of input and anti‐Flag IP derived from SGC7901 cells transfected with Flag‐MSI2 plasmids. The cells were treated with MG‐132 (20 μM) for 6 h before harvesting. (D) Western blotting analysis of input and anti‐Flag IP derived from 293T cells transfected with Flag‐MSI2, HA‐β‐Trcp or HA‐FBXW7 plasmids. The cells were treated with MG‐132 (20 μM) for 6 h before harvesting. (E) Western blotting analysis of input and anti‐Flag IP derived from 293T cells transfected with HA‐β‐Trcp and the indicated Flag‐tagged MSI2 constructs. The cells were treated with MG‐132 (20 μM) for 6 h before harvesting. (F) IP was performed with anti‐Flag antibody in 293T cells transfected with Flag‐β‐Trcp and Myc‐MSI2 plasmids in the presence or absence of LNC942, followed by western blotting with the indicated antibodies. The cells were treated with MG‐132 (20 μM) for 6 h before harvesting. (G) The SGC7901 cells were co‐transfected with LNC942 and β‐Trcp plasmids, followed by western blotting of the MSI2 and β‐Trcp expression. (H) The SGC‐R cells were co‐transfected with LNC942 and β‐Trcp siRNAs, followed by western blotting of the MSI2 and β‐Trcp expression. (I) Western blotting detection of MSI2 protein half‐life in SGC‐R cells transfected with the indicated siRNAs and treated with cycloheximide (CHX) (100 μg/ml). The MSI2 protein abundance was quantified using ImageJ in the bottom panel. (J) 293T cells were co‐transfected with the indicated plasmids and treated with MG‐132 (20 μM) for 6 h before collection. MSI2 pull‐down experiments were conducted with nickel‐nitrilotriacetic acid (Ni‐NTA) beads, and the samples were analysed using western blotting with the indicated antibodies. Ub, ubiquitin. Data in (I) are shown as mean ± SD of three independent experiments

Article Snippet: Briefly, recombinant MSI2 protein (Proteintech) was covalently immobilized to a CM5 sensor chip through amine groups in a sodium acetate buffer (pH 4.5) at a final response unit (RU) of about 11545.

Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Control, Derivative Assay, Construct, Expressing, Quantitative Proteomics

Journal: Clinical and Translational Medicine

Article Title: LncRNA LINC00942 promotes chemoresistance in gastric cancer by suppressing MSI2 degradation to enhance c‐Myc mRNA stability

doi: 10.1002/ctm2.703

Figure Lengend Snippet: MSI2 promotes gastric cancer (GC) cell resistance to cisplatin in vitro. (A) The SGC‐R and BGC‐R cells were transfected with si‐NC or si‐MSI2 treated with cisplatin (DDP) at different concentrations for 24 h, and the cell viability was then determined using the MTS assay. (B) SGC‐R and BGC‐R cells were treated with MSI2 inhibitor, FK228 (.125 μg/ml) and different concentrations of DDP for 24 h, and the cell viability was then measured using the MTS assay. DMSO: solvent control of FK228. (C) PI/Annexin V staining and flow cytometry in chemoresistant cells following si‐NC or si‐MSI2 transfection and treatment with or without DDP (8 μg/ml) for 24 h. (D) Resistant cells were treated with DDP (8 μg/ml), FK228 (.125 μg/ml) or DDP plus FK228 for 24 h. The apoptosis was measured by flow cytometry, and the number of apoptosis cells was quantified. (E) Apoptotic cells among MSI2 knocked‐down SGC‐R and BGC‐R cells treated with DDP (8 μg/ml) for 24 h were measured using anti‐cleaved‐PARP1 and cleaved‐caspase 3 through western blotting. (F) The caspase 3 and PARP1‐cleaved activities of resistant cells treated with DDP (8 μg/ml), FK228 (.125 μg/ml) or their combination for 24 h were analysed through western blotting. (G) The SGC7901 and BGC823 cells with ectopic overexpression of MSI2 were treated with different concentrations of DDP for 24 h, and the resultant cell viability was determined using the MTS assay. (H) Apoptosis of sensitive cells with or without MSI2 overexpression in the presence or absence of DDP treatment (1 μg/ml, 24 h) was evaluated through flow cytometry and then quantified. (I) Western blotting was performed to detect cleaved‐caspase 3 and cleaved‐PARP1 in sensitive cells with MSI2 overexpression following DDP treatment (1 μg/ml, 24 h). (J) Stable SGC7901 cells overexpressing LNC942 were transfected with MSI2 siRNAs and treated with different concentrations of DDP for 24 h, following which the cell viability was measured using the MTS assay. (K) Cell viability of stable SGC7901 cells overexpressing LNC942 treated with FK228 (.125 μg/ml) and different concentrations of DDP for 24 h was measured using the MTS assay. (L and N) SGC7901 cells that were stably overexpressing LNC942 were transfected with MSI2 siRNAs for 48 h and then treated with DDP (1 μg/ml) for 24 h, after which the apoptotic cells were analysed through flow cytometry (L) and western blotting (N). (M and O) SGC7901 cells stably overexpressing LNC942 were co‐treated with FK228 (.125 μg/ml) and DDP (1 μg/ml) for 24 h. Apoptotic cells were analysed by flow cytometry (M) and western blotting (O). Data in (A‐D), (G), (H), (J‐M) are represented as mean ± SD of the three independent experiments; the p value was determined using a two‐tailed unpaired Student's t test. ns, p > .05; * p < .05; ** p < .01; *** p < .001

Article Snippet: Briefly, recombinant MSI2 protein (Proteintech) was covalently immobilized to a CM5 sensor chip through amine groups in a sodium acetate buffer (pH 4.5) at a final response unit (RU) of about 11545.

Techniques: In Vitro, Transfection, MTS Assay, Solvent, Control, Staining, Flow Cytometry, Western Blot, Over Expression, Stable Transfection, Two Tailed Test

Journal: Clinical and Translational Medicine

Article Title: LncRNA LINC00942 promotes chemoresistance in gastric cancer by suppressing MSI2 degradation to enhance c‐Myc mRNA stability

doi: 10.1002/ctm2.703

Figure Lengend Snippet: LNC942 increases MSI2 downstream c‐Myc expression. (A) Gene set enrichment analysis (GSEA) of MYC target gene sets in the expression profiles of SGC‐R cells expressing LNC942 siRNA or shMSI2 expression profiles from the GEO database (GSE70685). (B and C) The c ‐ Myc mRNA and c‐Myc protein levels in resistant and sensitive cells were determined through quantitative real‐time PCR (qRT‐PCR) (B) and western blotting (C), respectively. (D and E) qRT‐PCR (top panel) and western blotting (bottom panel) analyses of c ‐ Myc mRNA and c‐Myc protein expression in SGC‐R cells transfected with LNC942 (D) or MSI2 (E) siRNAs. (F) qRT‐PCR (top panel) and western blotting (bottom panel) analyses of c ‐ Myc mRNA and c‐Myc protein expressions in SGC7901 cells stably expressing LNC942. (G) qRT‐PCR (top panel) and western blotting (bottom panel) analyses of c ‐ Myc mRNA and c‐Myc protein expressions in SGC7901 cells transfected with MSI2 plasmid. (H) qRT‐PCR (top panel) and western blotting (bottom panel) analyses of c ‐ Myc mRNA and c‐Myc protein expression in SGC7901 cells stably expressing LNC942 and transfected with MSI2 siRNA. (I) qRT‐PCR analysis of the decay rate of c ‐ Myc mRNA upon LNC942 (left panel) or MSI2 (right panel) inhibition in SGC‐R cells. (J) LNC942 (left panel) or MSI2 (right panel) overexpression enhanced the half‐life of c ‐ Myc mRNA. (K) The enhancement of the c ‐ Myc mRNA half‐life in SGC7901 cells induced by LNC942 was blocked by silencing MSI2. Data in (B) and (D‐K) are represented as mean ± SD of the three independent experiments; the p value was determined using a two‐tailed unpaired Student's t test. ns, p > .05; * p < .05; ** p < .01; *** p < .001

Article Snippet: Briefly, recombinant MSI2 protein (Proteintech) was covalently immobilized to a CM5 sensor chip through amine groups in a sodium acetate buffer (pH 4.5) at a final response unit (RU) of about 11545.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Transfection, Stable Transfection, Plasmid Preparation, Inhibition, Over Expression, Two Tailed Test

Journal: Clinical and Translational Medicine

Article Title: LncRNA LINC00942 promotes chemoresistance in gastric cancer by suppressing MSI2 degradation to enhance c‐Myc mRNA stability

doi: 10.1002/ctm2.703

Figure Lengend Snippet: MSI2 promotes the stability of c ‐ Myc in an m 6 A‐dependent manner. (A) Integrative genomics viewer tracks displaying Me‐RNA immunoprecipitation (RIP)‐seq and MSI2 CLIP‐seq (GEO: GSE69583) read distributions in c ‐ Myc mRNA. Significant peaks are indicated within a box. (B) The detection of m 6 A enrichment in c ‐ Myc mRNA in chemosensitive and chemoresistant cells by Me‐RIP and quantitative real‐time PCR (qRT‐PCR). (C) MSI2‐ RIP and qRT‐PCR analysis of the binding between MSI2 and c ‐ Myc mRNA with or without LNC942 knock‐down in SGC‐R and BGC‐R cells. (D) The qRT‐PCR detection of the mRNA level of c ‐ Myc in SGC‐R cell with or without METTL3‐METTL14‐WTAP complex knock‐down. (E) qRT‐PCR detection of the mRNA half‐life of c ‐ Myc after treatment with ActD (5 μg/ml) for the indicated time points. (F and G) LNC942 (F) or MSI2 (G) overexpressing SGC7901 cells were transfected with METTL3 or METTL3+METTL14+WTAP siRNAs, and the mRNA level of c ‐ Myc was measured through qRT‐PCR. (H) RIP and qRT‐PCR analysis of the m 6 A modifications of c ‐ Myc and the binding of MSI2 to c ‐ Myc in METTL3‐METTL14‐WTAP knock‐down or control SGC‐R cells. (I) Western blotting revealed that MSI2 and YTHDF2 (positive control) were pulled down with m 6 A‐containing RNA probes (ss‐m 6 A or ss‐3m 6 A) in SGC‐R cells. (J) Venn diagram illustrating the overlap of MSI2‐binding targets (GEO: GSE69583) and m 6 A‐contained genes (GEO: GSE98623). (K and L) Changes in the c ‐ Myc mRNA levels (K) or half‐life (L) in MSI2 or/and YTHDF2 knocked down SGC‐R cells. Data in (B‐H), (K) and (L) panels are represented as the mean ± SD of the three independent experiments; the p value was determined by a two‐tailed unpaired Student's t test. ns, p > .05; * p < .05; ** p < .01; *** p < .001

Article Snippet: Briefly, recombinant MSI2 protein (Proteintech) was covalently immobilized to a CM5 sensor chip through amine groups in a sodium acetate buffer (pH 4.5) at a final response unit (RU) of about 11545.

Techniques: RNA Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Binding Assay, Knockdown, Transfection, Control, Western Blot, Positive Control, Two Tailed Test

Journal: Clinical and Translational Medicine

Article Title: LncRNA LINC00942 promotes chemoresistance in gastric cancer by suppressing MSI2 degradation to enhance c‐Myc mRNA stability

doi: 10.1002/ctm2.703

Figure Lengend Snippet: c‐Myc promotes gastric cancer cell resistance to cisplatin in vitro. (A) The assessment of the viability of SGC‐R cells transfected with control or c‐Myc siRNAs with or without cisplatin (DDP) treatment for 24 h by using the MTS assay. (B) The effect of c‐Myc inhibition using the chemical c‐Myc inhibitor 10058‐F4 (6.25 μg/ml) on the viability of resistant cells with or without DDP treatment for 48 h, as detected by the MTS assay. (C and D) SGC‐R cells transfected with control siRNA (si‐NC) or c‐Myc siRNAs were treated with or without DDP (8 μg/ml) for 24 h, and the apoptosis level was measured by flow cytometry (C) as well as western blotting (D). (E and F) SGC‐R cells were pre‐treated with 10058‐F4 (6.25 μg/ml) for 24 h, followed by treatment with or without DDP (8 μg/ml) for 24 h, and the apoptosis level was measured through flow cytometry (E) and western blotting (F). DMSO: solvent control of 10058‐F4. (G) SGC7901 cells stably overexpressing LNC942 were transfected with c‐Myc siRNAs and treated with different concentrations of DDP for 24 h, after which the cell viability was measured using the MTS assay. (H) SGC7901 cells stably overexpressing LNC942 were pre‐treated with 10058‐F4 (6.25 μg/ml) for 24 h and co‐treated with different concentrations of DDP for 24 h, after which the cell viability was measured using the MTS assay. (I and J) SGC7901 cells stably overexpressing LNC942 were transfected with c‐Myc siRNAs for 48 h and treated with DDP (1 μg/ml) for 24 h, after which the apoptotic cells were analysed through flow cytometry (I) and western blotting (J). (K and L) SGC7901 cells stably overexpressing LNC942 were pre‐treated with 10058‐F4 (6.25 μg/ml) for 24 h and co‐treated with DDP (1 μg/ml) for 24 h. Apoptotic cells were analysed through flow cytometry (K) and western blotting (L). (M) The size and number of 3D‐cultured spheroids of LNC942 overexpressed or control SGC7901 with MSI2 or c‐Myc inhibition were measured by a 3D culture system. Spheroid size >40 cells per case was counted by ImageJ as one spheroid. (N and O) Pluripotent transcription factors were analysed in LNC942 (N) or MSI2 (O)‐depleted cells by quantitative real‐time PCR (qRT‐PCR). Data in (A‐C), (E), (G‐I), (K), (M‐O) panels are represented as mean ± SD of the three independent experiments; the p value was determined using a two‐tailed unpaired Student's t test. ns, p > .05; * p < .05; ** p < .01; *** p < .001

Article Snippet: Briefly, recombinant MSI2 protein (Proteintech) was covalently immobilized to a CM5 sensor chip through amine groups in a sodium acetate buffer (pH 4.5) at a final response unit (RU) of about 11545.

Techniques: In Vitro, Transfection, Control, MTS Assay, Inhibition, Flow Cytometry, Western Blot, Solvent, Stable Transfection, Cell Culture, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Two Tailed Test

Journal: Nature Communications

Article Title: Mapping genomic and transcriptomic alterations spatially in epithelial cells adjacent to human breast carcinoma

doi: 10.1038/s41467-017-01357-y

Figure Lengend Snippet: MSI overexpression increases migration and invasion of MCF7 and MDA-MB-231 MCF7-expressing GFP and MSI2-GFP were counted by Vi-cell-XR, and equally plated on transwells with and without Matrigel. Migration a and invasion b qualities were assessed by counting cells 48 h after initial plating. MSI2 overexpression causes an increase in migration and invasion (Welch’s t test; p < 0.05). Box plots of all replicates within each experiment ( n = 3) are depicted. MDA-MB-231-expressing GFP and MSI2-GFP were plated on transwells with and without Matrigel. 48 h after initial plating, the transwells were counted for migration c and invasion d . MSI2 increased both the migrative capabilities and invasive tendencies of MDA-MB-231 (Welch’s t test; p < 0.05). As with the MCF7, box plots of all replicates within each experiment ( n = 4) are depicted. Migration e and invasion f were also assayed in MCF7 shRNA control and shRNA MSI2, respectively. The knockdown clones presented the opposite effect, with a significant decrease in the migration and invasion ability of MCF7 (Welch’s t test; p < 0.05). Proliferation assay results are summarised in Supplementary Fig.

Article Snippet: A total of 5 μg MSI2-GFP and GFP vectors (pCMV6-MSI2-AC-GFP and pCMV6-AC-GFP from Origene, Rockville, MD, USA) were transfected into MCF7 and MDA-MB-231 cells (source: ATCC) using endofectin transfection reagent (Genocopoeia, Rockville, MD, USA) according to the manufacturer’s instructions.

Techniques: Over Expression, Migration, Expressing, shRNA, Control, Knockdown, Clone Assay, Proliferation Assay

Journal: International Journal of Biological Sciences

Article Title: Prolonged DEHP exposure enhances the stemness and metastatic potential of TNBC cells in an MSI2-dependent manner

doi: 10.7150/ijbs.101598

Figure Lengend Snippet: Clone ID and target sequences of the scrambled and MSI2-targeting shRNAs

Article Snippet: The human MSI2 (NM-170721) 3′ UTR luciferase reporter clone was purchased from Origene Technologies, Inc., Rockville, USA.

Techniques: Sequencing

Journal: International Journal of Biological Sciences

Article Title: Prolonged DEHP exposure enhances the stemness and metastatic potential of TNBC cells in an MSI2-dependent manner

doi: 10.7150/ijbs.101598

Figure Lengend Snippet: Involvement of MSI2 in DEHP-induced migration and invasion as predicted by NGS analysis. (A) Heatmap of DEGs in control and DEHP-exposed cells. (B) Venn diagram of total DEGs overlapping among different samples; volcano plot of the DEGs; pie chart of common differentially expressed GO terms in control and DEHP-exposed clones #1 and #2. (C) Bar chart of significantly enriched GO terms and the number of DEGs enriched in biological processes (green) and cellular components (orange); (* significant enrichment). (D) IPA-derived heatmap analysis of cellular movement under various conditions and the functions of the DEGs involved. (E) Downstream analysis of genes involved in the migration and invasion of tumor cell lines, highlighting increased MSI2 expression.

Article Snippet: The human MSI2 (NM-170721) 3′ UTR luciferase reporter clone was purchased from Origene Technologies, Inc., Rockville, USA.

Techniques: Migration, Control, Clone Assay, Derivative Assay, Expressing

Journal: International Journal of Biological Sciences

Article Title: Prolonged DEHP exposure enhances the stemness and metastatic potential of TNBC cells in an MSI2-dependent manner

doi: 10.7150/ijbs.101598

Figure Lengend Snippet: Prolonged DEHP exposure induces EMT and stemness in MDA-MB-231 cells in an MSI2-dependent manner. (A) Changes in the expression of the EMT markers α-SMA, β-catenin, SNAI1, and vimentin, as evaluated by western blotting. (B) Evaluation of the effect of MSI2 knockdown on the expression of EMT markers by western blotting. (C) Anchorage-independent growth/spheroid formation as evaluated by the soft agar colony formation assay. Scale bar = 50 µm. (D-E) Quantitative analysis of the number and size of colonies/spheroids originating from untreated and DEHP-exposed MDA-MB-231 cells. (F) Assessment of anchorage-independent growth/spheroid formation by soft agar colony formation assays in Scr-treated and MSI2-depleted cells. Scale bar = 50 µm. (G-H) Quantitative analysis of colony number and colony size in the soft agar colony formation assay (mean ± SD). (I) Changes in the expression of the stemness-related markers CD133, cMyc, and SOX-2 in untreated and DEHP-exposed clones evaluated by western blotting. (J) Evaluation of changes in the expression of stemness-related markers in Scr-treated and MSI2-depleted MDA-MB-231 cells and in DEHP-exposed clones; **P < 0.001.

Article Snippet: The human MSI2 (NM-170721) 3′ UTR luciferase reporter clone was purchased from Origene Technologies, Inc., Rockville, USA.

Techniques: Expressing, Western Blot, Knockdown, Soft Agar Assay, Clone Assay

Journal: International Journal of Biological Sciences

Article Title: Prolonged DEHP exposure enhances the stemness and metastatic potential of TNBC cells in an MSI2-dependent manner

doi: 10.7150/ijbs.101598

Figure Lengend Snippet: MSI2 knockdown reversed DEHP-induced migration and invasion in vitro and in vivo . (A) Cell morphology of Scr-treated and MSI2-silenced MDA-MB-231 and DEHP-exposed clones as observed by light microscopy. Scale bar = 150 µm. (B) Quantitative analysis of cell length (means ±SDs). (C) A wound healing assay was performed to validate the role of MSI2 in DEHP-induced migration in Scr- and MSI2-silenced MDA-MB-231 cells and in DEHP-exposed clones. (D) Quantitative analysis of cell migration at 20 h after wound formation (mean ± SD). (E-F) Matrigel-coated Transwell assay to evaluate the effect of MSI2 knockdown on cell invasion and quantitative analysis of cell invasion for (E) 12h and (F) 24h. (G) Zebrafish xenograft assay to evaluate the cell migration of Scr-treated and MSI2-silenced MDA-MB-231- and DEHP-exposed clones in 48 hpf Tg ( fli1:EGFP ) zebrafish embryos (fluorescence image captured at 24 hpi). (H-I) Quantification of embryos showing metastasis to SIV (mean ±SD, n=50) and fluorescence intensity analysis of cells that migrated to SIV in zebrafish embryos (fluorescence intensity reflects the cell number, fold change vs. control); ** P < 0.001.

Article Snippet: The human MSI2 (NM-170721) 3′ UTR luciferase reporter clone was purchased from Origene Technologies, Inc., Rockville, USA.

Techniques: Knockdown, Migration, In Vitro, In Vivo, Clone Assay, Light Microscopy, Wound Healing Assay, Transwell Assay, Xenograft Assay, Fluorescence, Control

Journal: International Journal of Biological Sciences

Article Title: Prolonged DEHP exposure enhances the stemness and metastatic potential of TNBC cells in an MSI2-dependent manner

doi: 10.7150/ijbs.101598

Figure Lengend Snippet: MSI2 regulates the PI3K/Akt/NFκB signaling axis. (A) Heatmap of DEGs from the co-IP protein complexes in control and DEHP-exposed cells. (B) Volcano plot of total DEGs in control and DEHP-exposed cells (red: upregulated; green: downregulated). (C) KEGG pathway analysis identified the 5 most significantly enriched pathways with DEG annotations, p values, and q values. (D-E) GSEA of TNFα and Akt signaling revealed a positive correlation in untreated MDA-MB-231 cells and DEHP-exposed clone #1 cells (enrichment score). (F) Evaluation of the expression of the PI3K/Akt/NFκB signaling markers PI3K p85, p-PI3K, Akt, p-Akt, Ikkα, Ikkβ, Ikkε, p-Ikkα/β, and NFκB in untreated MDA-MB-231 cells and DEHP-exposed clones.

Article Snippet: The human MSI2 (NM-170721) 3′ UTR luciferase reporter clone was purchased from Origene Technologies, Inc., Rockville, USA.

Techniques: Co-Immunoprecipitation Assay, Control, Expressing, Clone Assay

Journal: International Journal of Biological Sciences

Article Title: Prolonged DEHP exposure enhances the stemness and metastatic potential of TNBC cells in an MSI2-dependent manner

doi: 10.7150/ijbs.101598

Figure Lengend Snippet: NFκB controls DEHP-induced cell migration via MMP-9 regulation. (A) Total and nuclear NFκB p65 expression levels in Scr-treated and MSI2-depleted MDA-MB-231 and DEHP-exposed clones were evaluated by western blotting. (B) Gelatin zymography analysis of MMP-9 expression in Scr-treated and MSI2-depleted MDA-MB-231 cells and DEHP-exposed clones. (C) IF analysis of intracellular NFκB p65 (red) localization and expression. Nuclear staining (blue). Scale bar = 100 µm. (D-E) Evaluation of total and nuclear NFκB p65 expression levels following BAY 11--7082 treatment (10 µM, 24 h) by western blotting in MDA-MB-231 and DEHP-exposed clones. (F) MMP-9 expression/activity analysis by gelatin zymography in BAY 11-7082 (10 µM, 24 h)-treated MDA-MB-231 cells and DEHP-exposed clones. (G) IF analysis of intracellular NFκB p65 (red) localization and expression following BAY 11--7082 (10 µM, 24 h) treatment. Nuclear staining (blue). Scale bar = 100 µm. (H) Effect of BAY 11-7082 (10 µM, 24 h) treatment on cell migration as evaluated by a wound healing assay for 24 h. (I) Quantitative analysis of cell migration at 24 h after wound formation (mean ± SD); **P < 0.001.

Article Snippet: The human MSI2 (NM-170721) 3′ UTR luciferase reporter clone was purchased from Origene Technologies, Inc., Rockville, USA.

Techniques: Migration, Expressing, Clone Assay, Western Blot, Zymography, Staining, Activity Assay, Wound Healing Assay

Journal: International Journal of Biological Sciences

Article Title: Prolonged DEHP exposure enhances the stemness and metastatic potential of TNBC cells in an MSI2-dependent manner

doi: 10.7150/ijbs.101598

Figure Lengend Snippet: MSI2 interacts with vimentin and regulates its expression and subcellular distribution. (A) SDS-PAGE analysis of total cell lysates, Flag-MSI2 co-IP eluates, and antibodies (IgG). (B) LC/MS/MS analysis and protein identification evaluation (unique and shared proteins) of Flag-MSI2 co-IP protein complexes in untreated MDA-MB-231 and DEHP-exposed clone #1 cells performed by Proteome Discoverer software. (C) Unique peptide identification of LC/MS/MS vimentin (FANYIDK) and (D) MSI2 (IFVGGLSANTVVEDVKQYFEQFGK). x-axis: mass/charge ratio (m/z), y-axis: intensity of peak [count]. (E-F) Evaluation of MSI2 and vimentin coexpression in cell lysates and co-IP products by western blotting. (G) IF analysis of the intracellular localization and expression of MSI2 (red) and vimentin (green). Nuclear staining (blue). Scale bar = 50 µm. (H-I) Quantitative analysis of MSI2 (H) and vimentin (I) expression in Scr-treated and MSI2-depleted MDA-MB-231 cells and DEHP-exposed clone #1 cells (means ± SDs); **P < 0.001.

Article Snippet: The human MSI2 (NM-170721) 3′ UTR luciferase reporter clone was purchased from Origene Technologies, Inc., Rockville, USA.

Techniques: Expressing, SDS Page, Co-Immunoprecipitation Assay, Liquid Chromatography with Mass Spectroscopy, Software, Western Blot, Staining

Journal: International Journal of Biological Sciences

Article Title: Prolonged DEHP exposure enhances the stemness and metastatic potential of TNBC cells in an MSI2-dependent manner

doi: 10.7150/ijbs.101598

Figure Lengend Snippet: miR-155-5p negatively regulates MSI2 expression and MSI2-induced migration. (A) Evaluation of prediction-based MSI2-targeting miRNAs via the TargetScanHuman (Release 7.2) and miRBD miRNA target prediction databases. Conserved sites, site length and predicted position of the miR-155-5p binding site in the MSI2 3′UTR. (B) Evaluation of miR-155-5p levels by qPCR in untreated MDA-MB-231 cells and DEHP-exposed clones. (C) Effects of miR-155-5p mimic treatment (10, 25, or 50 nM) on cell migration, as evaluated by a wound healing assay 20 h after scratching. (D) Quantitative analysis of cell migration at 20 h after wound formation (means ± SDsSDs). (E) Evaluation of changes in the expression of MSI2 and vimentin in miR-155-5p mimic-treated (10, 25, 50 nMol) DEHP-exposed clones. (F-G) Evaluation of the effect of the miR-155-5p mimic on MSI2 (F) and vimentin (G) mRNA levels in DEHP-exposed clone #1 cells (means ± SDsSD). (H) Evaluation of miR-155-5p specificity with respect to the MSI2 3′UTR via a luciferase reporter assay via MSI2 3′UTR luciferase expression clone transfection in 293T cells (mean ±SD); **P < 0.001.

Article Snippet: The human MSI2 (NM-170721) 3′ UTR luciferase reporter clone was purchased from Origene Technologies, Inc., Rockville, USA.

Techniques: Expressing, Migration, Binding Assay, Clone Assay, Wound Healing Assay, Luciferase, Reporter Assay, Transfection

Journal: International Journal of Biological Sciences

Article Title: Prolonged DEHP exposure enhances the stemness and metastatic potential of TNBC cells in an MSI2-dependent manner

doi: 10.7150/ijbs.101598

Figure Lengend Snippet: MSI2 knockdown reduces DEHP-induced breast cancer metastasis in vivo . (A) The effect of prolonged DEHP treatment on TNBC cell metastasis was evaluated in a mouse metastasis model. Untreated and DEHP-exposed MDA-MB-231 cells (Scr and MSI2 knockdown) were implanted into the mammary fat pads of 8-week-old female BALB/c nude mice. The average tumor size was recorded. The mice were sacrificed and processed for evaluation of metastasis. (B) Evaluation of average tumor sizes in different groups (mean ±SD). (C-D) Evaluation of tumor growth and size measurements of excised tumors (means ± SDs). (E) Evaluation of lung metastasis by morphological changes and changes in the size of the excised lungs. (F) Lung metastasis evaluation via IHC analysis of the metastasis-associated markers vimentin, N-cadherin, E-cadherin, and HLA-ABC. Scale bar = 30 µm. *P < 0.05, **P < 0.001.

Article Snippet: The human MSI2 (NM-170721) 3′ UTR luciferase reporter clone was purchased from Origene Technologies, Inc., Rockville, USA.

Techniques: Knockdown, In Vivo

Journal: International Journal of Biological Sciences

Article Title: Prolonged DEHP exposure enhances the stemness and metastatic potential of TNBC cells in an MSI2-dependent manner

doi: 10.7150/ijbs.101598

Figure Lengend Snippet: Comparisons of MSI2 expression between tumor and adjacent normal tissues from breast cancer patients

Article Snippet: The human MSI2 (NM-170721) 3′ UTR luciferase reporter clone was purchased from Origene Technologies, Inc., Rockville, USA.

Techniques: Expressing

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

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: Figure 1. 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 (GVBD50), 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

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: Phospho-proteomics, Activation Assay, Injection, Incubation, Mutagenesis, Expressing, Western Blot, Control

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: Figure 2. 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 GVBD50 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: Phospho-proteomics, Injection, Mutagenesis, Incubation, Western Blot, Expressing, Control

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: Figure 3. 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 GVBD50, 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 GVBD50. 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. 1d.

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: Protein-Protein interactions, Phospho-proteomics, Injection, Incubation, Control, 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: Figure 4. 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. 1b, 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. 1d, 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, Gel Shift

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: Figure 5. 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 GVBD50, 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. 1b, 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

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: Phospho-proteomics, 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: Figure 6. 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

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, Phospho-proteomics, Rescue Assay, Injection, Western Blot

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: Fig. 3 MSI2 directly binds to EGFR and ERBB3 mRNA. 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.18 and Wang et al.19. 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 32P-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: MSI2 ORF (NM_138962.2) was amplified by PCR with specific primers and high-fidelity Ex Taq DNA polymerase (Takara Bio USA, Inc., Mountain View, CA) using a cDNA containing human MSI2 obtained from OriGene (Rockville, MD) as a template, and cloned into XbaI/XhoI sites of pLV-CMV-puro vector (a kind gift from Dr. A. Ivanov).

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