Figures & data
Table S1 Blood biochemical test
Table S2 Blood routine test
Figure 1 Preparation of DMC.
Notes: Preparation of DMC and EZH2si complex: first, a novel gene carrier was prepared by a self-assembly method. MPEG-PCL and DOTAP were assembled into a new gene carrier, DOTAP/MPEG-PCL micelles (DMC). Then, EZH2 siRNA was carried into cancer cells by DMC.
Abbreviation: DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane.
![Figure 1 Preparation of DMC.Notes: Preparation of DMC and EZH2si complex: first, a novel gene carrier was prepared by a self-assembly method. MPEG-PCL and DOTAP were assembled into a new gene carrier, DOTAP/MPEG-PCL micelles (DMC). Then, EZH2 siRNA was carried into cancer cells by DMC.Abbreviation: DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane.](/cms/asset/e87e89fb-dfde-471e-aa14-07cc892c7252/dijn_a_12190698_f0001_c.jpg)
Figure 2 Physicochemical properties of DMC.
Notes: (A) Particle size of DMC; (B) zeta potential of DMC; (C) morphological characteristics of DMC by TEM observation; and (D) gel retardation assay of siRNA and complexes. Lane 1, DNA marker; lane 2, naked siRNA; lanes 3–6, different weight ratios of siRNA with DMC. siRNA was completely incorporated into DMC at a weight ratio of 1:50, and complexes were prepared without free siRNA.
Abbreviation: TEM, transmission electron microscope.
![Figure 2 Physicochemical properties of DMC.Notes: (A) Particle size of DMC; (B) zeta potential of DMC; (C) morphological characteristics of DMC by TEM observation; and (D) gel retardation assay of siRNA and complexes. Lane 1, DNA marker; lane 2, naked siRNA; lanes 3–6, different weight ratios of siRNA with DMC. siRNA was completely incorporated into DMC at a weight ratio of 1:50, and complexes were prepared without free siRNA.Abbreviation: TEM, transmission electron microscope.](/cms/asset/a7a1507a-7c32-4ba4-86fb-137f08a15875/dijn_a_12190698_f0002_c.jpg)
Figure 3 Transfection efficiency measurement of DMC.
Notes: DMC, containing 4 µg siRNA, was used to transfect U87 cells for 4 hours. The transfection efficiencies at both weight ratios (siRNA vs DMC as 1:100 and 1:150) were determined using (A) fluorescence microscope and (B) flow cytometry.
Abbreviation: FITC, fluorescein isothiocyanate.
![Figure 3 Transfection efficiency measurement of DMC.Notes: DMC, containing 4 µg siRNA, was used to transfect U87 cells for 4 hours. The transfection efficiencies at both weight ratios (siRNA vs DMC as 1:100 and 1:150) were determined using (A) fluorescence microscope and (B) flow cytometry.Abbreviation: FITC, fluorescein isothiocyanate.](/cms/asset/e7631c47-5081-445a-a45b-9a61f15df3c7/dijn_a_12190698_f0003_c.jpg)
Figure 4 RT-PCR, Western blot and MTT test of cell activity.
Note: When U87 cells were transfected with DMC, Consi-DMC or EZH2si-DMC for 72 hours, EZH2 expression was tested by RT-PCR (A) and Western blot (B), and cell activity was tested by MTT test (C). *P<0.01, EZH2si-DMC versus NS, DMC, Consi-DMC.
Abbreviations: RT-PCR, reverse transcription PCR; NS, normal saline.
![Figure 4 RT-PCR, Western blot and MTT test of cell activity.Note: When U87 cells were transfected with DMC, Consi-DMC or EZH2si-DMC for 72 hours, EZH2 expression was tested by RT-PCR (A) and Western blot (B), and cell activity was tested by MTT test (C). *P<0.01, EZH2si-DMC versus NS, DMC, Consi-DMC.Abbreviations: RT-PCR, reverse transcription PCR; NS, normal saline.](/cms/asset/25510bfb-3e4f-4f94-8fd1-48ac338f7429/dijn_a_12190698_f0004_b.jpg)
Figure 5 Cell apoptosis test.
Notes: When U87 cells were transfected with DMC (A), Consi-DMC (B) or EZH2si-DMC (C) for 72 hours, U87 cells with different treatments were stained with Annexin V-FITC/PI and tested by flow cytometry. (D) Statistics of tumor cell apoptosis. *P<0.05.
Abbreviations: FITC, fluorescein isothiocyanate; PI, propidium iodide.
![Figure 5 Cell apoptosis test.Notes: When U87 cells were transfected with DMC (A), Consi-DMC (B) or EZH2si-DMC (C) for 72 hours, U87 cells with different treatments were stained with Annexin V-FITC/PI and tested by flow cytometry. (D) Statistics of tumor cell apoptosis. *P<0.05.Abbreviations: FITC, fluorescein isothiocyanate; PI, propidium iodide.](/cms/asset/b6d971fb-0495-4f7b-9d38-ea1c5b7e9678/dijn_a_12190698_f0005_b.jpg)
Figure 6 Molecular mechanism of siEZH2-DMC antiglioma.
Notes: (A) The effect of siEZH2-DMC on apoptosis-related proteins by Western blotting analysis. (B) The phosphorylation of Akt and p44/42 MAPK in U87 was inhibited by siEZH2-DMC.
![Figure 6 Molecular mechanism of siEZH2-DMC antiglioma.Notes: (A) The effect of siEZH2-DMC on apoptosis-related proteins by Western blotting analysis. (B) The phosphorylation of Akt and p44/42 MAPK in U87 was inhibited by siEZH2-DMC.](/cms/asset/c27f4de6-ed1c-4e49-81b3-f8ce02fcea07/dijn_a_12190698_f0006_b.jpg)
Figure 7 Antiglioma effect of EZH2si-DMC in vivo.
Notes: (A) Tumor growth curves; (B) tumor weight; (C) tumor photos of NS, DMC, DMC-Consi and DMC-EZH2si treatment groups; and (D) body weight of different groups. (Mean ± SEM, n=5) (*P<0.01, DMC-EZH2si vs NS, DMC, DMC-Consi (A); *P<0.01, DMC-EZH2 vs NS, DMC, and DMC-Consi (B).
![Figure 7 Antiglioma effect of EZH2si-DMC in vivo.Notes: (A) Tumor growth curves; (B) tumor weight; (C) tumor photos of NS, DMC, DMC-Consi and DMC-EZH2si treatment groups; and (D) body weight of different groups. (Mean ± SEM, n=5) (*P<0.01, DMC-EZH2si vs NS, DMC, DMC-Consi (A); *P<0.01, DMC-EZH2 vs NS, DMC, and DMC-Consi (B).](/cms/asset/172cd13b-6d60-4539-9277-3edc6918484e/dijn_a_12190698_f0007_c.jpg)
Figure 8 Bioimaging analysis in the GL261 orthotopic mice model.
Notes: (A) Intracerebral glioma was seen by using fluorescence markers. Tumor was inhibited more effectively by α-MM, compared with other groups. (B) Life survival time assessment. Both α-M and α-MM extended life spans of mice, while mice in the α-MM group reached a longer survival time.
![Figure 8 Bioimaging analysis in the GL261 orthotopic mice model.Notes: (A) Intracerebral glioma was seen by using fluorescence markers. Tumor was inhibited more effectively by α-MM, compared with other groups. (B) Life survival time assessment. Both α-M and α-MM extended life spans of mice, while mice in the α-MM group reached a longer survival time.](/cms/asset/f444763e-d6c8-4160-beeb-8a101ed0f878/dijn_a_12190698_f0008_c.jpg)
Figure 9 EZH2 expression detection in vivo.
Notes: (A) Intracerebral glioma was seen by using fluorescence markers. Tumor was inhibited more effectively by DMC-EZH2si complex, compared with other groups. (B) Life survival time assessment. DMC-EZH2si complex extended life spans of mice. The EZH2 expression of NS (A), DMC (B) and DMC-Consi (C) were higher than DMC-EZH2si (D). Results indicated that the EZH2 was successfully knocked down by the DMC-EZH2si complex.
![Figure 9 EZH2 expression detection in vivo.Notes: (A) Intracerebral glioma was seen by using fluorescence markers. Tumor was inhibited more effectively by DMC-EZH2si complex, compared with other groups. (B) Life survival time assessment. DMC-EZH2si complex extended life spans of mice. The EZH2 expression of NS (A), DMC (B) and DMC-Consi (C) were higher than DMC-EZH2si (D). Results indicated that the EZH2 was successfully knocked down by the DMC-EZH2si complex.](/cms/asset/d8436335-2d50-49e5-b386-83c70b6dfcd3/dijn_a_12190698_f0009_c.jpg)
Figure 10 Cell proliferation detection.
Notes: (A) Cell proliferation was assessed by counting the number of Ki67-positive cells in the field (five high power fields per slide). (B) Mean cell proliferation every five fields was added. EZH2si-DMC was superior to other controls in inhibiting cell proliferation. EZH2si-DMC significantly inhibited cell proliferation. *P<0.01, EZH2si-DMC versus NS, DMC, Consi-DMC.
![Figure 10 Cell proliferation detection.Notes: (A) Cell proliferation was assessed by counting the number of Ki67-positive cells in the field (five high power fields per slide). (B) Mean cell proliferation every five fields was added. EZH2si-DMC was superior to other controls in inhibiting cell proliferation. EZH2si-DMC significantly inhibited cell proliferation. *P<0.01, EZH2si-DMC versus NS, DMC, Consi-DMC.](/cms/asset/7fe01e7c-c9ad-46ff-8d76-d9bf82fd3aa2/dijn_a_12190698_f0010_c.jpg)
Figure 11 Cell apoptosis detection.
Notes: (A) Cell apoptosis was assessed by counting the number of TUNEL-positive cells in the field (five high power fields per slide), and EZH2si-DMC was superior to other controls in increasing cell apoptosis. (B) Mean apoptosis rate every five fields. EZH2si-DMC significantly increased apoptosis (*P<0.01, EZH2si-DMC vs NS, DMC and Consi-DMC).
![Figure 11 Cell apoptosis detection.Notes: (A) Cell apoptosis was assessed by counting the number of TUNEL-positive cells in the field (five high power fields per slide), and EZH2si-DMC was superior to other controls in increasing cell apoptosis. (B) Mean apoptosis rate every five fields. EZH2si-DMC significantly increased apoptosis (*P<0.01, EZH2si-DMC vs NS, DMC and Consi-DMC).](/cms/asset/5876de9a-7bab-48b1-a050-772fd73350bb/dijn_a_12190698_f0011_c.jpg)
Figure S1 RT-PCR and MTT test of cell activity.
Notes: When GL261 cells were transfected with DMC, Consi-DMC or EZH2si-DMC for 72 hours, EZH2 expression was tested by RT-PCR (A) and cell activity was tested by MTT test (B). (siEZH2-1:GGATACAGCCTGTGCACAT; siEZH2-2:GCTTTGGACAACAAGCCTT; siEZH2-3:GCAAATTCTCGGTGTCAAA).
Abbreviation: RT-PCR, reverse transcription PCR.
![Figure S1 RT-PCR and MTT test of cell activity.Notes: When GL261 cells were transfected with DMC, Consi-DMC or EZH2si-DMC for 72 hours, EZH2 expression was tested by RT-PCR (A) and cell activity was tested by MTT test (B). (siEZH2-1:GGATACAGCCTGTGCACAT; siEZH2-2:GCTTTGGACAACAAGCCTT; siEZH2-3:GCAAATTCTCGGTGTCAAA).Abbreviation: RT-PCR, reverse transcription PCR.](/cms/asset/200569f3-2427-4e5f-bb27-9b497874dbd9/dijn_a_12190698_sf0001_b.jpg)