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Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
STEM_1685_sm_supptable1.pdf120KSupplemental Table 1
STEM_1685_sm_supptable2.pdf47KSupplemental Table 2
STEM_1685_sm_supptable3.pdf10KSupplemental Table 3
STEM_1685_sm_supplFig1.tif714KFigure S1. Pathohistology of the glioma cases with clinical information. (A): Tumor from a 45- year old patient, male, astrocytoma (grade□) in the right frontal lobe; (B): Tumor from a 37- year old patient, female, astrocytoma (grade□) in the left lobus temporalis; (C): Tumor from a 48-year old patient, male, glioblastoma (grade □) in the right frontal lobe; (D): Tumor from a 36-year old patient, male, glioblastoma (grade □) in the left frontal lobe and lobus temporalis; (E): Tumor from a 57-year old patient, male, glioblastoma (grade □) in the right frontal lobe; (F): Tumor from a 48 years old patient, male, glioblastoma (grade □) in the left apical lobe and lobus temporalis.
STEM_1685_sm_supplFig2.tif776KFigure S2. Isolation and characterization of GSCs. (A): Morphology of the tumorspheres derived from glioma cell line U87 and six glioma specimens. Light microscopy ×200. (B): The expression of stem cell markers nestin, CD133 and CD44s (all in red) in the tumorspheres detected by immunofluorescence staining, with the nuclei counterstained by DAPI (blue), under LCM. Scale bar=20, 50, 30, 75, 50, 25, 50, 100, 25 μm (from left to right, up to low). (C): Morphology of the descendants derived from secondary spheres (denoted by yellow arrow) seeded into serum-containing medium (DMEM+10% FBS) for 5 days. Light microscopy ×200. (D): Different markers (GFAP, Oligo2, nestin and CD133 in red; MBP and β-tubulinlllin green) in tumorspheres seeded into serum-containing medium (DMEM+10% FBS) for 2 weeks. The tumorspheres were denoted by yellow arrows. The nuclei were counterstained by DAPI (blue). Scale bar=25, 50, 20, 10, 25, 500, 75, 50, 50 μm (from left to right, up to low). (E): Xenografts. U87 Sub, a tumor formed 8 weeks after subcutaneous implantation of 1×105 U87 tumorspheres; U87 Ort, a tumor formed 6 weeks after orthotopical implantation of 1×104 U87 tumorspheres; P3 Ort, a tumor formed 6 weeks after orthotopical implantation of 5×103 glioma tissue sample-3 tumorspheres. Light microscopy ×200.
STEM_1685_sm_supplFig3.tif495KFigure S3. GSCs expanded in adherent culture. (A): Morphology of adherent GSCs derived from glioma cell line U87 and human glioma specimen 2. Light microscopy ×200. (B): Immunostaining of adherent GSCs for stem cell markers as observed by LCM. Scale bar=50 μm.
STEM_1685_sm_supplFig4.tif384KFigure S4. Reconstitution of Cx43 in GSCs and knocking down of Cx43 in non-GSCs. (A): Morphology of tumorspheres 72 h after Ad-Cx43 transduction. (B): Morphology of adherent GSCs 48 h after Ad-Cx43 transduction. (C): Transduction efficacy estimated by flow cytometry; black, negative control; green, Ad-Cx43 transduced U87 tumorsphere cells. (D): The expression of GJA1 after the reconstitution of Cx43 in GSCs revealed by real time PCR. The data were analyzed by Student T test. (E): Immunofluorescent staining of Cx43 after the Ad-Cx43 transduction as detected by LCM. Nuclei were counterstained by DAPI (blue). Scale bar=100, 100, 50, 50 μm. (F): The expression of GJA1 after the knocking down of Cx43 in non-GSCs revealed by real time PCR. The data were analyzed by Student T test.
STEM_1685_sm_supplFig5.tif262KFigure S5. GJIC assessment after the reconstitution of Cx43 (A) and (B): Morphology of Mock transduced U87 (A) and Ad-Cx43 transduced U87 (B) tumorspheres after incubation with Alexa Fluo 568 hydrazide. (C): GJIC estimated by FRAP after reconstitution of Cx43. One cell of interest (denoted by green arrow) was exposed to a short pulse laser that causes bleaching of the fluorescence. The recovery of the fluorescence, resulting from diffusion of unbleached dye from adjacent cells, was measured and plotted as a function of time when recovery was achieved. The fluorescence values were normalized against a reference cell (denoted by white arrow). Data were analyzed by ANOVA test. Images were obtained by LCM. Nuclei were counterstained by DAPI (blue). Scale bar=50 μm (A and B), or =25, 50, 50 μm (C). Wt, wide type (non-transduced); Mock, mock-transduced.
STEM_1685_sm_supplFig6.tif52KFigure S6. The expression of GFAP (A) and CD133 (B) in U87 cell line after transduction of Cx43 as detected by real time RT-PCR. The data were analyzed by Student T test.
STEM_1685_sm_supplFig7.tif661KFigure S7. Immunostaining of human glioma specimen 1 (A) and 2 (B) for Cx43 (green), E- Cadherin (red). The nuclei were counterstained by DAPI (blue). Scale bar=5 μm.
STEM_1685_sm_supplFig8.tif63KFigure S8. The expression of GJAI and E-cadherin mRNA level in Ad-Cx43 transducted-U87 tumorspheres after knocking down of E-cadherin by siRNA. The data were analyzed by Student T test.
STEM_1685_sm_suppinformation.pdf129KSupplemental Information

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