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1.
Figure 7

Figure 7. Current model for the involvement of Musashi-1 in glioma development.. From: RNA-Binding Protein Musashi1 Modulates Glioma Cell Growth through the Post-Transcriptional Regulation of Notch and PI3 Kinase/Akt Signaling Pathways.

The elevated level of the RNA-binding protein Musashi-1 leads to the down-regulation of Numb and PTEN (Imai et al., unpublished), resulting in increased activity of the Notch and PI3K/Akt-pathways, respectively. These events could lead to the enhanced self-renewal and survival of glioma cells and subsequent glioma development. In addition to these mechanisms, the downregulation of Cyclin B1 expression and prolongation of the cell cycle could be involved in Musashi1-mediated glioma development.

Jun Muto, et al. PLoS One. 2012;7(3):e33431.
2.
Figure 6

Figure 6. MSI1-KD changed the expression level of various genes.. From: RNA-Binding Protein Musashi1 Modulates Glioma Cell Growth through the Post-Transcriptional Regulation of Notch and PI3 Kinase/Akt Signaling Pathways.

(A) Immunoblots showing the expression of various cell-cycle markers and downstream genes of MSI1 in control and MSI1-KD in U251MG cell lines. MSI1-KD resulted in the up-regulation of Numb, Cyclin B1, and P16, the downregulation of cleaved Notch, and no change in CyclinD1. (B) Immunocytochemistry showed that Cyclin B1 was potently expressed in MSI1-KD cells compared with control cells. (Bar = 200 µm). (C) In PTEN-intact Daoy cells, the immunoblot analysis showed that MSI1-KD led to the up-regulation of PTEN, and down-regulation of cleaved Notch and p-Akt with no change in total Akt level. Error bars represent SEM (*P<0.01).

Jun Muto, et al. PLoS One. 2012;7(3):e33431.
3.
Figure 1

Figure 1. MSI1 expression in glioblastoma cell lines, medulloblastoma cell lines, and low-passage cells from glioblastoma patients.. From: RNA-Binding Protein Musashi1 Modulates Glioma Cell Growth through the Post-Transcriptional Regulation of Notch and PI3 Kinase/Akt Signaling Pathways.

(A) Immunoblots showing the MSI1 expression in human glioblastoma cell lines (U251MG, and KNS42), low-passage glioblastoma cells obtained from patients GM97, GM1600, GM1605, and a human medulloblastoma cell line (Daoy). (B and C) The selective depletion of MSI1 protein by treatment with shRNAs in U251MG cells (B) and Daoy cells (C). β-actin was used as a loading control in all the experiments.

Jun Muto, et al. PLoS One. 2012;7(3):e33431.
4.
Figure 4

Figure 4. Knockdown of MSI1 induces G2/M arrest.. From: RNA-Binding Protein Musashi1 Modulates Glioma Cell Growth through the Post-Transcriptional Regulation of Notch and PI3 Kinase/Akt Signaling Pathways.

MSI1-KD cells were analyzed by fluorescence-activated cell sorting. A representative cell-cycle profile for each treatment is indicated. 2N represents the G0–G1 phase and 4N represents the G2/M phase. An increased number of MSI1 KD-positive cells accumulated in the G2/M phase. (B) Time-lapse microscopy using a fluorescent ubiquitination-based cell-cycle indicator (Fucci) probe system showed that MSI1-KD cells retained the green fluorescence for a much longer time (4 days). Error bars represent SEM, *P<0.01.The spheres were dissociated, and the dissociated cells were fixed to coverslips and stained for PH3 in U251MG cells (C and D). Cell counts are plotted as bar graphs. An average of 25 high-power fields was counted. Error bars represent SEM, *P<0.01. (D) Representative images of control cells and MSI1-KD cells stained for PH3 and Hoechst. There were more PH3-positive MSI1-KD cells than control cells. (Bar = 500 µm).

Jun Muto, et al. PLoS One. 2012;7(3):e33431.
5.
Figure 5

Figure 5. MSI1-KD cells may undergo non-apoptotic cell death.. From: RNA-Binding Protein Musashi1 Modulates Glioma Cell Growth through the Post-Transcriptional Regulation of Notch and PI3 Kinase/Akt Signaling Pathways.

(A and B) SA-β-Gal staining was performed to assess the senescence in controls and MSI1-KD cells. (A) Representative images of control cells and MSI1-KD cells stained for SA-β-Gal (Bar = 200 µm). (C, D, E, and F) Colonies were dissociated, and the cells were fixed to coverslips and stained for TUNEL in U251 MG cells (C and D), and for cleaved Caspase-3 in U251MG cells (E and F). Cell counts are plotted as bar graphs. An average of 25 high power fields was counted. (G and H) The number of PI-positive cells (indicating dead cells) was greater in the MSI1-KD cell population than in the control cell population. Error bars represent SEM. *P<0.01.**P<0.01. (I) The number of annexinV-positive in the MSI1-KD cells (indicating apoptotic cells) was not different from that in the control cell population.

Jun Muto, et al. PLoS One. 2012;7(3):e33431.
6.
Figure 2

Figure 2. Colony-forming cell assay and effects of MSI1-KD.. From: RNA-Binding Protein Musashi1 Modulates Glioma Cell Growth through the Post-Transcriptional Regulation of Notch and PI3 Kinase/Akt Signaling Pathways.

(A,B,C and D) MSI1 shRNA decreased the number of colony-forming cells. (A) Representative images of the secondary colonies formed by the control shRNA-treated and MSI1-KD U251MG cells. (Bar = 500 µm). The number of spheres was reduced in U251 cells exposed to 10 µM DAPT. (B) On Day 30, secondary spheres larger than 100 µm in diameter were counted. U251MG glioblastoma cells transduced with MSI1 shRNA and cells treated with 10 µM DAPT (γ-secretase inhibitor) generated significantly fewer spheroid cell colonies than cells transduced with control shRNA. Error bars represent SEM, *P<0.01(C) Representative images of the secondary colonies obtained in cells transduced with control shRNA and in MSI1-KD PTEN-intact Daoy cells exposed to 10 µM LY294002 (PI3 kinase inhibitor) and DAPT. (D) On Day 30, secondary spheres larger than 100 µm in diameter were counted. PTEN-intact Daoy cells transduced with MSI1 shRNA and cells exposed to LY294002 and DAPT generated significantly fewer spheroid colonies than cells transduced with control shRNA. Error bars represent SEM, *P<0.01(E and G). Representative images of the secondary colonies formed by cells expressing low endogenous MSI1. GM1600 () and KNS42 () transduced with control shRNA and MSI1 shRNA (F and H). On day 30, secondary spheres larger than 100 µm in diameter were counted. There was no significant difference between control shRNA and MSI1 shRNA in GM1600 () or KNS42 (). (I, J, and K) MSI1-KD inhibits colony formation. Proliferation was assessed in glioblastoma and medulloblastoma cell lines (cell density of 2×103 cells/well) were assessed for cell proliferation by cell counting. The total cell count in the MSI1-KD groups on day 15 was reduced by 83%, and 55% in the (I) U251MG and (J) Daoy cells, respectively, as compared to the control groups.

Jun Muto, et al. PLoS One. 2012;7(3):e33431.
7.
Figure 3

Figure 3. Xenografts expressing MSI1-KD showed reduced BLI and longer survival.. From: RNA-Binding Protein Musashi1 Modulates Glioma Cell Growth through the Post-Transcriptional Regulation of Notch and PI3 Kinase/Akt Signaling Pathways.

Briefly, 1×104 human glioblastoma cells were transplanted into the right striatum of NOD/SCID mice (n = 4), and tumor growth was monitored in live animals. The MSI1-KD group exhibited better survival than the control group (n = 4, *P<0.05). (A) Representative BLI images of mice receiving implants of MSI1 shRNA lentivirus-transfected U251MG cells on day 28. (B&D) Relative photon count of BLI compared with that day0 was shown. The tumor average photon count in MSI1-KD group was inferior to that in control groups in U251MG cells () and in Daoy cells () (C) Kaplan-Meier survival curves for the control and MSI1-KD mice in U251MG cells (*P<0.05). The survival time in the MSI1-KD group (49.3±6.1 days) was significantly longer than that in the shRNA control group in U251-MG cells (33.6±3.6 days; P<0.01). (E) Kaplan-Meier survival curves for the control and MSI1-KD mice are shown in Daoy cells (*P<0.05). The survival time of the MSI1-KD group (45.5±5.4 days) was significantly longer than that of the shRNA control group in Daoy cells (34.0±4.1 days; P<0.01). (F) Mouse brain transplanted with control cells (F-a) and MSI1-KD cells (F-b) was stained for hematoxylin and eosin (Bar = 1 mm). (G) Brain transplanted with control cells [G-(a, c)] and MSI1-KD cells [G-(b, d)] was immunostained for PH3 [(a, b) Bar = 800 µm (c, d) Bar = 200 µm]. In G-(a, b), surrounding arrows show the transplanted tumor area. G-(c, d) indicated the high magnification images of the squared region of G-(a, b), respectively.

Jun Muto, et al. PLoS One. 2012;7(3):e33431.

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