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STEM_586_sm_suppinfofigure1.tif358KFigure S1. Immunocytochemical and FACS analyses of NPCs isolated from E14.5 rat embryo. (A): NPCs isolated from E14.5 rat embryo forebrain were induced to differentiation by being grown in N2 media without bFGF (10 ng/ml) for 2 or 4 days. Immunocytochemical analyses were performed to detect Nestin or CD133 (red). Nuclei were counter stained by DAPI (blue). In this condition of sensitivity, Nestin was stained at either the cell body or cytosol of neural progenitor cells (NPCs) depending on their cellular physiological status. (B): For FACS analysis of CD133 positive cells, the cells were incubated with PBS or PE-conjugated anti-human CD133/AC133 (Prominin1) antibody (eBioscience, San Diego, CA). Labeled cells were read using FACS Calibur (BD Biosciences, San Jose, CA) and obtained data was analyzed using Cell quest software (BD Biosciences). FL2 parameter on the FACS Calibur was set for detection of red fluorochromes. Measurement of auto-fluorescence was also routinely conducted for each condition tested. (C): Mouse embryonic stem cells (mESCs) were grown in Knockout DMEM on the 0.1% gelatin-coated dish for 2 days. NPCs were grown in N2 medium containing bFGF for 4 days. Quantitative RT-PCR analysis was performed with identical amount (5 μg) of total RNA to detect mRNA level of Oct-4, Sox2, Nestin, or HPRT.
STEM_586_sm_suppinfofigure2and3.tif245KFigure S2. Visualization of the transfection efficiencies of NPCs. NPCs were grown in N2 medium containing bFGF (10 ng/ml) and transfected with 2 μg pEGFP-C1 as described in “Materials and Methods.” The level of EGFP -positive cells was 32% when estimated 48 hours after transfection. The microscopic fields/ cell number (3/ 718-768 or 3/ 400-600) of EGFP-sh SC or EGFP-sh Sur8 expressing cell were estimated, respectively. The error bars indicate the standard deviation (SD) of three independent experiments. These data show the high transfection efficiency of NPCs in our system. Figure S3. Detection of exogenous and endogenous Sur8 proteins by newly generated anti-rabbit Sur8 antibody in HEK293 cells. Recombinant GST human Sur8 fusion protein (GST-Sur8) was expressed in Escherichia coli BL21 (DE3) and purified on a glutathione column [2]. A rabbit polyclonal anti- Sur8 antibody was generated by injecting two rabbits with the purified recombinant GST-Sur8. Both endogenous and exogenous Sur8 proteins were detected by immunoblot analysis of HEK293 cell lysates transfected with either pcDNA3.0 or pcDNA3.0-HA-hSur8. These results show the functionalities and specificity of the Sur8 antibody that we generated.
STEM_586_sm_suppinfofigure4.tif1501KFigure S4. Expression pattern of Sur8 in developing embryonic cerebral cortex. Experiments were similarly performed as described in Figure 1A. (A): Immunohistochemical analysis of sagital sections of the E14.5 rat embryo forebrain were performed with anti- Sur8, -Tuj1, -PCNA or anti-MAP2ab antibody. (B): Immunofluorescent images of Sur8 (green) and MAP2ab (red) were obtained using a Zeiss confocal microscope. The cerebral cortex is divided into three layers: CP, IMZ, SVZ (sub VZ), and VZ. The red arrow indicates the migration direction of the differentiating NPCs. These data are independent experimental results for Fig. 1A using different animals at the same stage of development. (C): The intensity of stained images of Sur8 or MAP2ab were quantified using Ti Nikon Fluorescence microscope software.
STEM_586_sm_suppinfofigure5.tif551KFigure S5. Multipotency of NPCs isolated from E14.5 rat embryo. (A): NPCs were grown in N2 medium containing bFGF (10 ng/ml). (B): Neurospheres were grown on coverslips coated with poly-D-lysine (20 μg/ml) in N2 medium containing bFGF (10 ng/ml) and EGF (20 ng/ml). (A-B): Differentiation was induced by growth in N2 medium depleted of both EGF and bFGF for 2 days. Cells were subjected to immunofluorescence analysis using anti-Nestin, Tuj1, -GFAP, -MBP (for NPCs), or -O4 (for neurospheres) antibody.
STEM_586_sm_suppinfofigure6.tif589KFigure S6. Effects of Sur8 knock-down on neurogenesis. NPCs were infected with either EGFP-sh Sc or EGFP-sh Sur8, and were grown in uncoated 6-well plates in N2 medium containing bFGF (10 ng/ml) and EGF (20 ng/ml). (A): Seventy two hours after virus infection, the cells were photographed as black and white (B/W) (left panels) or fluorescent (right panels) images using a fluorescence microscope. Representative cells expressing EGFP-sh Sur8 show distinct neurite outgrowths, and are marked by red arrows. (B): Average length of neurites (left panel) and numbers of EGFP -positive cells (right panel) were quantitated. The data represent the mean ± SD of three separate experiments. *p < .05; **p < .005. (C): Immunofluorescent labeling for Tuj1 with detection of EGFP-sh Sc or EGFP-sh Sur8 by fluorescent or confocal microscopy. Nuclei were counterstained with DAPI. The white and yellow arrows indicate Tuj1-negative and -positive cells in the neurospheres, respectively.
STEM_586_sm_suppinfofigure7.tif201KFigure S7. Effects of Sur8 knock-down or overexpression on proliferation of NPCs. Cell were prepared as described in Fig. 5. (A): The proliferation marker Ki67 was monitored by immunofluorescent labeling using anti-Ki67 antibody. Right panel represents quantitative analysis data for relative numbers of Ki67 positive cells. The microscopic fields/ cell number (3 fields/ 200-250 or 3 fields/ 350-500) of EGFP-sh SC or EGFP-sh Sur8 expressing cell were measured/counted, respectively. The error bars indicate the SD of three independent experiments. The percentage of cells showing Ki67-positive cells was reduced from 69.8% to 27.6% in cells infected with EGFP -sh Sur8. *p<.05. (B): NPCs were transfected with pcDNA3.0 or pcDNA3.0-HA-hSur8 vector, and were further grown for 48 hours in N2 medium without bFGF. Sur8 and PCNA proteins were detected by immunocytochemical analysis by using anti-Sur8 or anti-PCNA antibody. Nuclei were counterstained with DAPI. Cells expressing EGFP-sh Sur8 (white arrows), not those expressing EGFP (yellow arrow), abolished the Ki67-positive signal.
STEM_586_sm_suppinfofigure8.tif106KFigure S8. Effects of Sur8 overexpression on the expression of CD133 in NPCs. NPCs were transfected with 0.5 μg pcDNA3.0 or pcDNA3.0-HA-hSur8, and were further grown for 48 hours in N2 medium with or without bFGF. (A): FACS analyses were performed to detect CD133-positive (+) cells. For each sample, 105 events positioned in the right side of the R1 gate cells were immediately collected using a FACS Calibur flow cytometer (right panels). (B): Quantitated data for CD133 signal was estimated by using the Cell quest software.
STEM_586_sm_suppinfofigure9.tif356KFigure S9. Effect of Sur8 knock-down on stemness of NPCs. (A-B): NPCs were infected with either EGFP-sh Sc or EGFP-sh Sur8 lentivirus and grown in N2 medium containing bFGF (10 ng/ml) and EGF (20 ng/ml) for 72 hours in uncoated 6-well plates for neurosphere formation. (A): Effects of Sur8 knock-down on neurosphere formation. Neurospheres were captured as B/W images (upper panels) or as fluorescent images (lower panels) (B): Seventy-two hours after virus infection, the cells were stained by immunocytochemistry using anti-Nestin antibody. Expression of EGFP -sh Sc or EGFP -sh Sur8 was confirmed by EGFP visualization under fluorescence microscopy. Nestin staining was significantly reduced in the neurosphere infected with EGFP -sh Sur8 compared with those infected with EGFP-sh Sc.
STEM_586_sm_suppinfofigure10.tif156KFigure S10. Effects of the MEK inhibitors on differentiation and proliferation of NPCs (A and B): The experiments are similar with those described in Fig. 6D, except that the applied drug was U0126 was used instead of PD98059 (A), or cells were co-transfected with HA-Sur8 and dn-MEK instead of the drug treatment (B). (C-D): A FACS analysis was performed as described in Fig. 2 and 3 to measure the number of Tuj1 and PCNA positive cells. For each sample, 105 events positioned in the right side of the R1 gate cells were immediately collected using a FACS Calibur flow cytometer, and result for Tuj1 (C) and PCNA (D) signals were quantitated using Cell quest software. The error bars indicate the SD of three independent experiments. *p< .05
STEM_586_sm_suppinfofigure11.tif688KFigure S11. Effects of Sur8 knockdown on differentiation of NPCs in the developing Rat embryos. (A-C): The ex vivo experiments are basically same experiments as Fig. 7. The lower magnification images for Tuj1 (A), GFAP (B) and MBP (C) are provided here to show many more cells. The right panels are high magnified images of Tuj1 staining (upper-right) and quantified data for Tuj1 positive cells (lower-right). The error bars indicate the SD of three independent tissue staining images. Statistical differences among the groups were analyzed by Student's t test and *** p < .0005 was considered statistically significant. The signals for both GFP-positive and Tuj1-positive cells are marked by yellow arrows.
STEM_586_sm_suppinfofigure12.tif468KFigure S12. Expression of Sur8 during the endothelial differentiation of mESCs. (A-C): For maintenance of undifferentiated condition (0 D), mESCs were grown on 0.1% gelatin-coated dishes. To induce endothelial differentiation, mESCs were grown on collagen IV-coated dishes without LIF for 1, 2 or 4 days. (A): The morphologies of mESCs were captured as B/W images during the differentiation of 1, 2, 4 days. (B): Cell lysates were subjected to immunoblotting analyses using anti-Oct-4, -Sur8, -p-ERK, -pAKT, -PCNA, or -β-actin antibody. (C): The mESCs of undifferentiated (0 D) and differentiated for 4 days (4 D) were subjected to immunocytochemical analyses to detect Sur8, Oct-4, Flk-1 or BrdU . Nuclei were counterstained with DAPI.
STEM_586_sm_suppinfofigure13.tif171KFigure S13. Effects of Sur8 knock-down on differentiation of pluri-potent mESCs. (A-C): The mESCs were infected with either EGFP -sh Sc or EGFP -sh Sur8, and were further grown for 7 days in Knockout DMEM containing LIF. (A): Cells were subjected to immunocytochemical analyses to detect Sur8, and expression of EGFP -sh Sc or GFP-sh Sur8 was confirmed by EGFP visualization under fluorescence microscopy. Nuclei were counterstained with DAPI. (B): To check knock-down of Sur8 by EGFP -sh Sur8 lentivirus, cell lysates were subjected to immunoblotting analysis using anti-Sur8 or -β-actin antibody. (C): For alkaline phosphatase assay, mESCs were stained as described in Materials and Methods. Colonies expressing ALP-positive cells were divided into the status of Undifferentiated (95∼100 %), Mixed (30-50 %) ,and Differentiated (30 %) and were quantified by counting the number of colony-forming units (CFUs).
STEM_586_sm_suppinfofigure14.tif88KFigure S14. Effects of the proteasomal inhibitor MG132 and lysosomal inhibitor Luepeptin on Sur8 protein stability in NIH3T3 cell line. (A): NIH3T3 cells were grown in DMEM (10% FBS) for 12 hours) with MG132 (0, 2.5, 5, 10, or 20 μM). (B): NIH3T3 cells were grown in DMEM (10% FBS) for the 12 hours with MG132 (20 μM), or leupeptin (5 μg/ml). WCLs were subjected to IB analyses using the indicated antibodies.
STEM_586_sm_suppinfofigure15a-15b.tif117KFigure S15. Controls of the FACS analyses. The right side of the R1 gate cells were immediately collected using a FACSCalibur flow cytometer, and results were analyzed using cell quest software (BD Biosciences). (A-D): The results are dot-plot cytogram of Figure2, 3, 6, and Fig. S10C, S10D.
STEM_586_sm_suppinfofigure15c-15d.tif120KFigure S15. Controls of the FACS analyses. The right side of the R1 gate cells were immediately collected using a FACSCalibur flow cytometer, and results were analyzed using cell quest software (BD Biosciences). (A-D): The results are dot-plot cytogram of Figure2, 3, 6, and Fig. S10C, S10D.
STEM_586_sm_suppinfotableS1.tif18KTable S1. RT-PCR primer pairs used in this study.
STEM_586_sm_suppinfo.doc46KSupporting Information

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