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. 2022 Jul 7;9(26):2202216. doi: 10.1002/advs.202202216

Figure 6.

Figure 6

The CD133–DNMT1 interaction maintains the quiescence of GSC. A,B) Immunofluorescence analysis of EdU‐labeled CD133+ cells expressing control shRNA, CD133 shRNA1, or CD133 shRNA2. A) Representative images of immunofluorescence are shown. B) The percentage of EdU‐positive cells is measured. Results are expressed as mean ± SD from six independent experiments; ***p < 0.001, Student's t‐test. Scale bar represents 10 µM. C) Analysis of the percentage of EdU‐positive cells in CD133+ cells and CD133‐ cells. Results are expressed as mean ± SD from six independent experiments; ***p < 0.001, Student's t‐test. D,E) In vivo CIdU and IdU incorporation assay is used to examine the proliferation of GSCs. D. CD133+ cells are intracranially implanted into immunocompromised mice brain. 7 days later, mice are intraperitoneally injected with CIdU. After 4 weeks, mice are intraperitoneally injected with IdU. 24 h later, mice are sacrificed and perfused. E) Glioblastoma orthotropic xenograft is assessed by immunofluorescence staining of CD133 (red), IdU (green), and CIdU (purple). White arrow indicates the CD133+ cells. Scale bar represents 10 µM. F) Immunofluorescence analysis the percentage of EdU‐positive cells in CD133+ cells expressing control shRNA, CD133 shRNA1, CD133 shRNA1+shR CD133, or CD133 shRNA1+shR CD133(1–862). Results are expressed as mean ± SD from six independent experiments; ***p < 0.001, Student's t‐test. shR, shRNA‐resistant. G) Analysis of the percentage of EdU‐positive cells in CD133+ cells expressing control shRNA or CD133 shRNA1 and control or p21 or p27. Results are expressed as mean ± SD from six independent experiments; **p < 0.01, *p < 0.05, Student's t‐test. H) Immunofluorescence analysis of γH2AX foci formation in CD133+ cells expressing control shRNA or CD133 shRNA1 and control or p21 or p27. The number of γH2AX foci‐positive cells is measured. Results are expressed as mean ± SD from three independent experiments; #p > 0.05, ***p < 0.001, Student's t‐test.