Fig 1: MSI2 regulation of ERBB protein expression in EGFRmut cell lines.A Western blots of indicated cell lines, following MSI2 depletion by shRNA (sh1, sh2) or siRNA (h1, h2) or overexpression (MSI2) in three EGFRmut 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.
Fig 2: MSI2 supports proliferation and resistance to drugs inhibiting EGFR in EGFRmut NSCLC cell lines.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 EGFRmut (PC9, HCC827, and H1975) and KRASmut (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.
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.
Fig 4: MSI2 knockdown increases the sensitivity of EGFR mutant xenograft tumors to erlotinib treatment.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 ß-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-??Ct 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.
Fig 5: Expression of MSI2 and EGFR proteins in human NSCLC primary tumors.A H scores for MSI2 and EGFR in EGFRmut NSCLC tumor TMA samples (see Supp Table S4 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 cells46,47.
Supplier Page from Abcam for Recombinant Human MSI2 protein