Journal: Oncogenesis

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

doi: 10.1038/s41389-021-00317-y

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

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

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

Journal: Oncogenesis

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

doi: 10.1038/s41389-021-00317-y

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

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

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

Journal: Oncogenesis

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

doi: 10.1038/s41389-021-00317-y

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

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

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

Journal: Oncogenesis

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

doi: 10.1038/s41389-021-00317-y

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

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

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

Journal: Oncogenesis

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

doi: 10.1038/s41389-021-00317-y

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

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

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

Journal: Oncogenesis

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

doi: 10.1038/s41389-021-00317-y

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

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

Techniques: Staining

Journal: Oncogene

Article Title: β-Catenin interacts with canonical RBPs including MSI2 to associate with a Wnt signalling mRNA network in myeloid leukaemia cells.

doi: 10.1038/s41388-025-03415-y

Figure Lengend Snippet: Fig. 4 β-Catenin interacts with MSI2 in myeloid cells. A Scatter plots showing MSI2 detection in β-catenin interactomes performed in cytosolic fractions of K562 and HEL cells. Vertical dashed red line indicates the threshold for 2-fold change in protein binding at log2 (=1) relative to IgG Co-IP. The horizontal red line represents threshold for p < 0.05 on log10 scale (=1.3). Highlighted red dots indicate enriched interactions (p < 0.05), green labels highlight position of MSI2, and blue labels highlight position of β-catenin bait. Representative immunoblots showing the level of β-catenin protein present in MSI2 Co-IPs derived from B K562 - RNase A, C K562 + 20 µg/mL RNaseA, D HEL - RNase A and E HEL + 20 µg/mL RNaseA whole cell lysates, ±5µM CHIR99021 overnight. ID immunodepleted lysate.

Article Snippet: Immunoblotting was performed as previously [8], using antibodies to β-catenin, MSI2, LEF-1, GAPDH (Proteintech, Manchester, UK), Lamin A/C (Merck Millipore, New Jersey, United States) and α-tubulin (Merck Millipore) already outlined in above sections.

Techniques: Protein Binding, Co-Immunoprecipitation Assay, Western Blot, Derivative Assay

Journal: Oncogene

Article Title: β-Catenin interacts with canonical RBPs including MSI2 to associate with a Wnt signalling mRNA network in myeloid leukaemia cells.

doi: 10.1038/s41388-025-03415-y

Figure Lengend Snippet: Fig. 5 MSI2 and β-catenin correlate and interact in primary AML patient samples. A Immunoblot of 18 myeloid leukaemia cell lines showing the relative level of β-catenin and MSI2 protein, with GAPDH used to assess protein loading. B Summary scatter plot showing the correlation (Spearman Rank R = 0.6, P < 0.03) between relative β-catenin and MSI2 protein expression across 18 myeloid cell lines as deduced from densitometry (normalised to GAPDH expression present within each cell line, AU = arbitrary units). C Immunoblot screen of 20 primary AML patient samples showing the relative level of β-catenin and MSI2 protein (light and dark exposures). * Denotes samples co-overexpressing both β-catenin and MSI2 relative to levels in CB MNC and CB CD34+ enriched fraction (pooled from five independent CB samples). X = Void sample containing no protein as deduced from negative GAPDH detection. D Summary scatter plot showing the correlation (Spearman Rank R = 0.79, P < 0.0001) between relative β-catenin and MSI2 protein expression across 20 primary AML patient samples as deduced from densitometry (normalised to GAPDH expression present in each sample, AU = arbitrary units). E Immunoblot showing the level of β-catenin and GAPDH protein present in an MSI2 Co-IP performed from primary AML patient sample #10 of sample screen.

Article Snippet: Immunoblotting was performed as previously [8], using antibodies to β-catenin, MSI2, LEF-1, GAPDH (Proteintech, Manchester, UK), Lamin A/C (Merck Millipore, New Jersey, United States) and α-tubulin (Merck Millipore) already outlined in above sections.

Techniques: Western Blot, Expressing, Co-Immunoprecipitation Assay

Journal: Oncogene

Article Title: β-Catenin interacts with canonical RBPs including MSI2 to associate with a Wnt signalling mRNA network in myeloid leukaemia cells.

doi: 10.1038/s41388-025-03415-y

Figure Lengend Snippet: Fig. 6 MSI2 knockdown impairs Wnt signalling output through LEF-1 modulation. A Immunoblots showing MSI2, β-catenin and LEF1 level in K562 and KU812 cells harbouring MSI2 shRNA or non-targeting shRNA controls. GAPDH indicates protein loading. B Representative flow cytometric histograms showing intensity of the TCF-dependent expression of Venus Yellow Fluorescent Protein (YFP) from the β-catenin activated reporter (BAR) reporter, or negative control ‘found unresponsive’ BAR (fuBAR; containing mutated promoter binding sites) in K562 and KU812 cells ± MSI2 shRNA following treatment with 5 μM CHIR99021 overnight. The fuBAR (dashed), non-targeting control shRNA (grey filled), and two MSI2 shRNAs (blue or red) histograms are shown. Summary graphs showing the median fluorescence intensity (MFI) generated from the BAR/fuBAR in C K562 and D KU812 cells ±MSI2 shRNA with ±5 μM CHIR99021. E Immunoblots showing total β-catenin, LEF-1, and MSI2 subcellular localisation in K562 and KU812 cells lentivirally transduced with two different MSI2 shRNAs ±5 μM CHIR99021. Lamin A/C and α-tubulin indicate the purity/loading of the nuclear (N) and cytosol (C) fractions respectively. Densitometric quantitation of LEF-1 (relative to nuclear Lamin A/C, AU = arbitrary units) present in nuclear fractions of F K562 and G KU812 cells ± MSI2 shRNA with ±5 μM CHIR99021. Summary graph showing the fold change in LEF1 and TCF7L2 mRNA expression as assessed by RT-qPCR in (H) K562 and (I) KU812 cells expressing MSI2 shRNA relative to non-targeting control shRNA (represented by dotted line at y = 1). Fold change is relative to matched respective controls (black dashed line) and overall expression was normalised to the housekeeping gene β-actin (ACTB). Data represent mean ± 1 s.d (n = 3). Statistical analysis is denoted by *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 as deduced from a student’s t test or one-sample t-test for RT-qPCR data.

Article Snippet: Immunoblotting was performed as previously [8], using antibodies to β-catenin, MSI2, LEF-1, GAPDH (Proteintech, Manchester, UK), Lamin A/C (Merck Millipore, New Jersey, United States) and α-tubulin (Merck Millipore) already outlined in above sections.

Techniques: Knockdown, Western Blot, shRNA, Expressing, Negative Control, Binding Assay, Control, Generated, Transduction, Quantitation Assay, Quantitative RT-PCR

Journal: Oncogene

Article Title: β-Catenin interacts with canonical RBPs including MSI2 to associate with a Wnt signalling mRNA network in myeloid leukaemia cells.

doi: 10.1038/s41388-025-03415-y

Figure Lengend Snippet: Fig. 7 MSI2 regulates LEF-1 expression and binds LEF1 in a partly β-catenin-dependent fashion. A Immunoblot showing MSI2 level in MSI2 RIP performed from K562 cells. ID= immunodepleted lysate. Summary graphs showing the fold enrichment of TCF7L2, MYC, MYB, and LEF1 mRNA in MSI2 B RIP and C CLIP performed from K562 cells. Fold enrichment is relative to matched IgG RIPs (dashed black line). D Immunoblots showing MSI2 and β-catenin levels in K562 cell lines harbouring β-catenin shRNAs versus non-targeting shRNA control. GAPDH indicates protein loading. Summary graphs showing the fold change in LEF1 mRNA expression in input K562 cells harbouring β-catenin shRNA versus non-targeting shRNA control (dashed black line) used for E MSI2 RIP and F) MSI2 CLIP. Summary graphs showing the fold enrichment of LEF1 mRNA (versus IgG RIP) in MSI2 G) RIP and H CLIP generated from K562 cells ± β-catenin shRNAs. I Line graph summarising the fold change in LEF1 mRNA expression (relative to T = 0 h for each respective cell line) in K562 cells harbouring MSI2 shRNA (red) versus non-targeting shRNA control (black) treated with 2 µg/ml ActD for indicated times. J Immunoblot showing the levels of MSI2, β-catenin, and LEF-1 protein in K562 cells ±MSI2 shRNA with treatment of 2 µg/ml ActD for indicated timepoints. Data represents mean ± 1 s.d (n = 3). Statistical analysis is denoted by *p < 0.05 and **p < 0.01 as deduced from a student’s t-test.

Article Snippet: Immunoblotting was performed as previously [8], using antibodies to β-catenin, MSI2, LEF-1, GAPDH (Proteintech, Manchester, UK), Lamin A/C (Merck Millipore, New Jersey, United States) and α-tubulin (Merck Millipore) already outlined in above sections.

Techniques: Expressing, Western Blot, shRNA, Control, Generated