Journal: bioRxiv

Article Title: Improved transfection methods of primary cultured astrocytes for observation of cytoskeletal structures

doi: 10.1101/2025.01.27.635031

Figure Lengend Snippet: (A) Lower magnification view of astrocytes cultured at high density (initial cell density of 0.6 × 10 5 cells/cm 2 ; cultured for 7 days) and low density (initial cell density of 0.3 × 10 5 cells/cm 2 ; cultured for 6 days). Phase contrast images show cells just before trypsin treatment (pre-transfection) and cultured for one day after electroporation. Scale bar = 200 μm. (B-D) Representative images of (B) EGFP-ezrin and mCherry-actin, (C) EGFP-actin and mCherry-lasp-2, and (D) EGFP-ezrin and mCherry-lasp-2 at 2, 3, and 2 days post-transfection, respectively. See supplementary movie 1 for (D); (D’) kymographs of magnified segment of stem process surface from supplemental movie 1 with a sequence interval of 20 sec. Arrows in (C) and (D) indicate examples of elliptical structures of fluorescent-tagged lasp-2. Asterisks in (D’) indicate examples of structures where lasp-2 and ezrin colocalized.

Article Snippet: The regions coding EGFP of the pLenti CMV EGFP Puro (658-5) vector (Addgene) were removed using BamHI and SalI and replaced with PCR products using NEBuilder HiFi DNA Assembly (New England BioLabs, MA, USA).

Techniques: Cell Culture, Transfection, Electroporation, Sequencing

Journal: bioRxiv

Article Title: Improved transfection methods of primary cultured astrocytes for observation of cytoskeletal structures

doi: 10.1101/2025.01.27.635031

Figure Lengend Snippet: (A) Lower magnification view of astrocytes before and after (1d, 2d and 5d) infection. Scale bar = 200 μm. (B-E) Representative images of (B) EGFP, (C) EGFP-lasp-2, (D) mCherry-lasp-2, and (E) mCherry-lasp-2 and added Sara Fluor 497 actin probe in astrocytes at 7, 4, 6, and 5 days post-infection with lentivirus vectors, respectively. (F) Time-lapse imaging of astrocytes infected with lentiviral coding with EGFP or EGFP-lasp-2 every 2 hours. Arrows in (C) to (E) indicate examples of elliptical structures of fluorescent-tagged lasp-2.

Article Snippet: The regions coding EGFP of the pLenti CMV EGFP Puro (658-5) vector (Addgene) were removed using BamHI and SalI and replaced with PCR products using NEBuilder HiFi DNA Assembly (New England BioLabs, MA, USA).

Techniques: Infection, Imaging

Journal: PLoS ONE

Article Title: Effects of Angiotensin II Type 2 Receptor Overexpression on the Growth of Hepatocellular Carcinoma Cells In Vitro and In Vivo

doi: 10.1371/journal.pone.0083754

Figure Lengend Snippet: A , the percentages of SMMC7721 cells transduced with 1–500 ifu/cell of either Ad-G-AT2R-EGFP or Ad-CMV-EGFP for 24 hrs in G1 phase, S phase and G2 phase. Columns, mean (n = 3); bars, SE. Data are derived from DNA histograms.* P <0.05 and ** P <0.01 versus Ad-CMV-EGFP–transduced cells. B , fluorescence intensity of SMMC7721 and LO2 cells transduced with 300 ifu/cell of either Ad-G-AT2R-EGFP or Ad-CMV-EGFP for 24 hrs was measured by flow cytometry. C , data showing percentages of SMMC7721 and LO2 cells in G1 phase (left) and S phase (right) under each treatment condition. Columns, mean (n = 3); bars, SE. Data are derived from DNA histograms. ** P <0.01 versus Ad-CMV-EGFP–transduced cells.

Article Snippet: On the following day, cells were transduced with Ad-G-AT2R-EGFP or the control vector Ad-CMV-EGFP and changes in cell morphology were observed using an Olympus IX71 fluorescence microscope (Olympus America Inc., PA, USA).

Techniques: Transduction, Derivative Assay, Fluorescence, Flow Cytometry

Journal: PLoS ONE

Article Title: Effects of Angiotensin II Type 2 Receptor Overexpression on the Growth of Hepatocellular Carcinoma Cells In Vitro and In Vivo

doi: 10.1371/journal.pone.0083754

Figure Lengend Snippet: A, growth curve of HCC SMMC7721 cells with 200/cell of Ad-G-AT2R-EGFP and Ad-CMV-EGFP–transduced. ** P <0.01. B , representative Western blots showing the CDK4 and cyclin D1 in SMMC7721 cells induced for 24 hrs with Ad-G-AT2R-EGFP and Ad-CMV-EGFP (100, 200 and 300 ifu/cell, respectively). Data are representative of three experiments.

Article Snippet: On the following day, cells were transduced with Ad-G-AT2R-EGFP or the control vector Ad-CMV-EGFP and changes in cell morphology were observed using an Olympus IX71 fluorescence microscope (Olympus America Inc., PA, USA).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Effects of Angiotensin II Type 2 Receptor Overexpression on the Growth of Hepatocellular Carcinoma Cells In Vitro and In Vivo

doi: 10.1371/journal.pone.0083754

Figure Lengend Snippet: A , SMMC7721 cells were transduced with either Ad-CMV-EGFP or Ad-G-AT2R-EGFP (500 ifu/cell) for 24 hrs as described in materials and methods and apoptotic cells were detected using the DeadEnd Colorimetric TUNEL. Two representative phase-contrast micrographs from each treatment condition. Representative fluorescence micrographs from Ad-CMV-EGFP–transduced and Ad-G-AT2R-EGFP–transduced cells, showing EGFP fluorescence, TUNEL-positive (apoptotic) cell (brown-colored nuclei), and merged (G+A) EGFP/AT2R in each treatment condition. Scale bars , 50 µm. B , quantification of the TUNEL-positive cells as a percent of the total number of cells in the dish. Columns, mean of three experiments; bars, SE. ** P <0.01 versus GFP group.

Article Snippet: On the following day, cells were transduced with Ad-G-AT2R-EGFP or the control vector Ad-CMV-EGFP and changes in cell morphology were observed using an Olympus IX71 fluorescence microscope (Olympus America Inc., PA, USA).

Techniques: Transduction, TUNEL Assay, Fluorescence

Journal: PLoS ONE

Article Title: Effects of Angiotensin II Type 2 Receptor Overexpression on the Growth of Hepatocellular Carcinoma Cells In Vitro and In Vivo

doi: 10.1371/journal.pone.0083754

Figure Lengend Snippet: Representative Western blots show the p38 MAPK and pp38 MAPK in SMMC7721 cells transduced for 12-G-AT2R-EGFP and Ad-CMV-EGFP (500 ifu/cell) or mock-transduced and PP2A, JNK, pJNK, p42/44 MAPK(Erk1/2) and pp42/44 MAPK(pErk1/2) in SMMC7721 cells transduced for 24 hrs. Data are representative of three experiments.

Article Snippet: On the following day, cells were transduced with Ad-G-AT2R-EGFP or the control vector Ad-CMV-EGFP and changes in cell morphology were observed using an Olympus IX71 fluorescence microscope (Olympus America Inc., PA, USA).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Effects of Angiotensin II Type 2 Receptor Overexpression on the Growth of Hepatocellular Carcinoma Cells In Vitro and In Vivo

doi: 10.1371/journal.pone.0083754

Figure Lengend Snippet: A, Western blot analysis of AT2R-induced increase in cleaved caspase-3 and caspase-8 generation in SMMC7721 cells. Cells were infected after 24 hrs with either 300 or 500 ifu/cell of Ad-CMV-EGFP, Ad-G-AT2R-EGFP or mock-transduced. Data are representative of three experiments. B , AT2R-induced increase in caspase-3 activity in SMMC7721 cells. Columns, mean A 405 nm from three experiments; bars, SE. * P <0.05 and ** P <0.01 versus Ad-CMV-EGFP–transduced or mock-transduced cells.

Article Snippet: On the following day, cells were transduced with Ad-G-AT2R-EGFP or the control vector Ad-CMV-EGFP and changes in cell morphology were observed using an Olympus IX71 fluorescence microscope (Olympus America Inc., PA, USA).

Techniques: Western Blot, Infection, Activity Assay

Journal: PLoS ONE

Article Title: Effects of Angiotensin II Type 2 Receptor Overexpression on the Growth of Hepatocellular Carcinoma Cells In Vitro and In Vivo

doi: 10.1371/journal.pone.0083754

Figure Lengend Snippet: A , CCCD signals of cellular orthotopic injection animal models post transfection with Ad-CMV-EGFP, Ad-G-AT2R-EGFP tail vein injection and PBS as negative control. B , liver tumor tissues were separated from human hepatocellular carcinoma nude mice models. C , the effect of AT2R on tumor size. Data are expressed as mean ± SD. (* P <0.05, n = 8). D , RQ comparison of AT2R mRNA expression in animal models (M-PBS; M-GFP; M-AT2R) and SMMC7721 cells. M-AT2R, M-GFP and M-PBS, AT2R mRNA expression in liver tumor tissues of the nude models injected with Ad-G-AT2R-EGFP, Ad-CMV-EGFP, PBS. C-AT2R1 and C-AT2R10, AT2R mRNA expression in SMMC7721 cells following transduction with gradient doses of Ad-G-AT2R-EGFP (1ifu, 10 ifu/cell). Columns, mean (n = 3); * P <0.05 versus 1 and 10 ifu/cell of Ad-G-AT2R-EGFP–transduced cells.

Article Snippet: On the following day, cells were transduced with Ad-G-AT2R-EGFP or the control vector Ad-CMV-EGFP and changes in cell morphology were observed using an Olympus IX71 fluorescence microscope (Olympus America Inc., PA, USA).

Techniques: Injection, Transfection, Negative Control, Expressing, Transduction

Journal: PLoS ONE

Article Title: Effects of Angiotensin II Type 2 Receptor Overexpression on the Growth of Hepatocellular Carcinoma Cells In Vitro and In Vivo

doi: 10.1371/journal.pone.0083754

Figure Lengend Snippet: A , the Ki67 immunohistochemistry staining of liver tumor tissues from human hepatocellular carcinoma nude mouse models injected with Ad-CMV-EGFP, Ad-G-AT2R-EGFP or PBS via tail vein. AT2R-N, GFP-N and PBS-N, PBS instead of Anti-Ki67 primary antibody as negative control (×400). B , the immunohistochemistry staining scoring from different groups. Data are expressed as mean ± SD. (* P <0.05, n = 8).

Article Snippet: On the following day, cells were transduced with Ad-G-AT2R-EGFP or the control vector Ad-CMV-EGFP and changes in cell morphology were observed using an Olympus IX71 fluorescence microscope (Olympus America Inc., PA, USA).

Techniques: Immunohistochemistry, Staining, Injection, Negative Control