Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 promotes EGF-induced EMT in pancreatic cancer via ZEB1-ERK/MAPK signaling

doi: 10.1186/s13046-020-1521-4

Figure Lengend Snippet: Nuclear and cytoplasmic location of MSI2 in PC cells by IF and the construction of MSI2 silencing stable PC cell lines via CRISPR/Cas9 system. a IF staining of MSI2 (TRITC, red) and nuclear (Hoechst, blue) in 4 PC cell lines (× 100 magnification). b and c MSI2 protein level in sg1-MSI2, sg2-MSI2 and scramble transfectedAsPC-1 ( b ) and BxPC-3 ( c ) cell lines detected by WB. The white bars: MSI2 protein expression in scramble groups. The black bars: MSI2 protein expression in sg1-MSI2 and sg2-MSI2 groups. **, P < 0.01 compared with the control

Article Snippet: Briefly, MSI2, ZEB1, c-Myc and pERK and IgG (Santa Cruz) antibodies were pre-incubated with magnetic beads (Bio-Rad, California, USA) at 4 °C.

Techniques: CRISPR, Staining, Expressing, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 promotes EGF-induced EMT in pancreatic cancer via ZEB1-ERK/MAPK signaling

doi: 10.1186/s13046-020-1521-4

Figure Lengend Snippet: Cell morphology in sg1-MSI2, sg2-MSI2 and scramble transfected AsPC-1 and BxPC-3 cells with or without EGF (50 ng/ml) treatment. a and b Under EGF treatment, the fibroblastoid-like phenotype in AsPC-1 ( a ) and BxPC-3 ( b ) cells in scramble, sg1-MSI2 and sg2-MSI2 groups

Article Snippet: Briefly, MSI2, ZEB1, c-Myc and pERK and IgG (Santa Cruz) antibodies were pre-incubated with magnetic beads (Bio-Rad, California, USA) at 4 °C.

Techniques: Transfection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 promotes EGF-induced EMT in pancreatic cancer via ZEB1-ERK/MAPK signaling

doi: 10.1186/s13046-020-1521-4

Figure Lengend Snippet: MSI2 silencing inhibited EGF-induced Cell invasion and migration in PC cells. a and b Cell invasion ( a ) and migration ( b ) in sg1-MSI2, sg2-MSI2 and scramble transfected AsPC-1 cells with or without EGF (50 ng/ml) treatment. c and d Cell invasion ( c ) and migration ( d ) in sg1-MSI2, sg2-MSI2 and scramble transfected BxPC-3 cells with or without EGF (50 ng/ml) treatment. Bars indicate ± S.E.*, P < 0.05; **, P < 0.01 compared with the control

Article Snippet: Briefly, MSI2, ZEB1, c-Myc and pERK and IgG (Santa Cruz) antibodies were pre-incubated with magnetic beads (Bio-Rad, California, USA) at 4 °C.

Techniques: Migration, Transfection, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 promotes EGF-induced EMT in pancreatic cancer via ZEB1-ERK/MAPK signaling

doi: 10.1186/s13046-020-1521-4

Figure Lengend Snippet: MSI2 silencing inhibited EGF-induced EMT and ZEB1-ERK/MAPK signaling. a and b. The protein expression involving EMT and ZEB1-ERK/MAPK signaling in sg1-MSI2, sg2-MSI2 and scramble transfected AsPC-1 ( a ) and BxPC-3 ( b ) cells with or without EGF (50 ng/ml) treatment. The white bars: target protein expression in scramble groups with or without EGF treatment. The black bars: target protein expression in sg1-MSI2 and sg2-MSI2 groups with or without EGF treatment. Bars indicate ± S.E.*, P < 0.05; **, P < 0.01 compared with the control

Article Snippet: Briefly, MSI2, ZEB1, c-Myc and pERK and IgG (Santa Cruz) antibodies were pre-incubated with magnetic beads (Bio-Rad, California, USA) at 4 °C.

Techniques: Expressing, Transfection, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 promotes EGF-induced EMT in pancreatic cancer via ZEB1-ERK/MAPK signaling

doi: 10.1186/s13046-020-1521-4

Figure Lengend Snippet: The effect of MSI2 silencing in EGF induced pEGFR expression. a and b The expression of pEGFR845, pEGFR992 and pEGFR1068 in sg1MSI2, sg2MSI2 and scramble transfected AsPC-1( a ) and BxPC-3 cells ( b ) with or without EGF treatment

Article Snippet: Briefly, MSI2, ZEB1, c-Myc and pERK and IgG (Santa Cruz) antibodies were pre-incubated with magnetic beads (Bio-Rad, California, USA) at 4 °C.

Techniques: Expressing, Transfection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 promotes EGF-induced EMT in pancreatic cancer via ZEB1-ERK/MAPK signaling

doi: 10.1186/s13046-020-1521-4

Figure Lengend Snippet: The co-staining of MSI2 with ZBE1, pERK and c-Myc in PC cells by IF. a and b MSI2 was co-stained with ZBE1, pERK and c-Myc in predominant nucleus in AsPC-1 ( a ) and BxPC-3 ( b ) cells (× 100 magnification)

Article Snippet: Briefly, MSI2, ZEB1, c-Myc and pERK and IgG (Santa Cruz) antibodies were pre-incubated with magnetic beads (Bio-Rad, California, USA) at 4 °C.

Techniques: Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 promotes EGF-induced EMT in pancreatic cancer via ZEB1-ERK/MAPK signaling

doi: 10.1186/s13046-020-1521-4

Figure Lengend Snippet: The interaction of MSI2 with EGF induced EMT and ZEB1-ERK/MAPK signaling was further verified by IP and rescue experiments. a AsPC-1 and BxPC-3 lysates were immunoprecipitated and WB. Input and IgG bands were used as positive and negative control, respectively. b MSI2 overexpression promoted EGF induced EMT and ZEB1-ERK/MAPK signaling which was inhibited by MSI2siRNA. Bars indicate ± S.E.*, P < 0.05; **, P < 0.01 compared with the control

Article Snippet: Briefly, MSI2, ZEB1, c-Myc and pERK and IgG (Santa Cruz) antibodies were pre-incubated with magnetic beads (Bio-Rad, California, USA) at 4 °C.

Techniques: Immunoprecipitation, Negative Control, Over Expression, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 promotes EGF-induced EMT in pancreatic cancer via ZEB1-ERK/MAPK signaling

doi: 10.1186/s13046-020-1521-4

Figure Lengend Snippet: MSI2 silencing inhibited the growth of pancreatic tumors in situ. a Pancreatic tumors and corresponding HE staining (× 100 magnification) in scramble groups. b Pancreatic tumors and corresponding HE staining (× 100 magnification) in sg1-MSI2 groups. c The statistic analysis of tumor volume between scramble and sg1-MSI2 groups. d and e The statistic data ( d ) and representative images ( e ) in IHC assays of target proteins expression in vivo in sg1-MSI2 and scramble groups. Bars indicate ± S.E.*, P < 0.05; **, P < 0.01 compared with the control

Article Snippet: Briefly, MSI2, ZEB1, c-Myc and pERK and IgG (Santa Cruz) antibodies were pre-incubated with magnetic beads (Bio-Rad, California, USA) at 4 °C.

Techniques: In Situ, Staining, Expressing, In Vivo, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 promotes EGF-induced EMT in pancreatic cancer via ZEB1-ERK/MAPK signaling

doi: 10.1186/s13046-020-1521-4

Figure Lengend Snippet: MSI2 silencing inhibited liver metastases in vivo. a Liver metastases and corresponding HE staining (× 100 magnification) in scramble groups. b Liver metastases and corresponding HE staining (× 100 magnification) in sg1-MSI2 groups. c The statistic analysis of liver metastasis number between scramble and sg1-MSI2 groups. d and e The statistic data ( d ) and representative images ( e ) in IHC assays of target proteins expression in vivo in sg1-MSI2 and scramble groups. Bars indicate ± S.E.*, P < 0.05; **, P < 0.01 compared with the control

Article Snippet: Briefly, MSI2, ZEB1, c-Myc and pERK and IgG (Santa Cruz) antibodies were pre-incubated with magnetic beads (Bio-Rad, California, USA) at 4 °C.

Techniques: In Vivo, Staining, Expressing, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 promotes EGF-induced EMT in pancreatic cancer via ZEB1-ERK/MAPK signaling

doi: 10.1186/s13046-020-1521-4

Figure Lengend Snippet: The relationship of MSI2 with ZEB1, E-cad, Vimentin and c-Myc expression in clinical samples

Article Snippet: Briefly, MSI2, ZEB1, c-Myc and pERK and IgG (Santa Cruz) antibodies were pre-incubated with magnetic beads (Bio-Rad, California, USA) at 4 °C.

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 promotes EGF-induced EMT in pancreatic cancer via ZEB1-ERK/MAPK signaling

doi: 10.1186/s13046-020-1521-4

Figure Lengend Snippet: MSI2, ZEB1, c-Myc, pERK, E-cad and Vimentin expression in human PC samples. a High expression of MSI2, ZEB1,c-Myc and Vimentin and abnormal expression of E-cad in one case of PC tissues. b Weak expression of MSI2, ZEB1, c-Myc and Vimentin and normal expression of E-cad in another one case of PC tissues

Article Snippet: Briefly, MSI2, ZEB1, c-Myc and pERK and IgG (Santa Cruz) antibodies were pre-incubated with magnetic beads (Bio-Rad, California, USA) at 4 °C.

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Musashi2 promotes EGF-induced EMT in pancreatic cancer via ZEB1-ERK/MAPK signaling

doi: 10.1186/s13046-020-1521-4

Figure Lengend Snippet: The relationship of MSI2, ZEB1, c-Myc, pERK and E-cad with the survival of 74 postoperative PC patients in Kaplan-Meier analysis. a High and low expression of MSI2 was plotted against overall survival time. b High and low expression of ZEB1 was plotted against overall survival time. c Normal and abnormal expression of E-cad was plotted against overall survival time. d High and low expression of c-Myc was plotted against overall survival time. e , f and g Co-expression of MSI2/ZEB1 ( e ), MSI2/E-cad ( f ) and MSI2/c-Myc ( g ) was plotted against overall survival time

Article Snippet: Briefly, MSI2, ZEB1, c-Myc and pERK and IgG (Santa Cruz) antibodies were pre-incubated with magnetic beads (Bio-Rad, California, USA) at 4 °C.

Techniques: Expressing

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