Journal: International journal of pharmaceutics

Article Title: Lipidation of polyethylenimine-based polyplex increases serum stability of bioengineered RNAi agents and offers more consistent tumoral gene knockdown in vivo

doi: 10.1016/j.ijpharm.2018.06.026

Figure Lengend Snippet: Delivery of BERA/GFP-siRNA and its knockdown efficiency in human carcinoma cells in vitro. (A) GFP fluorescence imaging demonstrated the knockdown of GFP by BERA/GFP-siRNA in SK-Hep1-Luc-GFP cells, delivered by Lipofectamine 3000 (LF3000), in vivo-jet PEI (IVJ) or LPP nanocomplex. This was further confirmed by measuring GFP fluorescence intensity (B) and mRNA levels (C) in SK-Hep1-Luc-GFP cells, attributable to the successful release of GFP-siRNA (D) from precursor GFP-siRNA levels (E). Likewise, delivery of BERA/GFP-siRNA and subsequent knockdown of GFP were shown in A549-Luc-GFP cells (F, G, H and I). Cells were treated with 5 nM BERA/GFP-siRNA for 72 h. GFP fluorescence intensity was measured at Ex/Em = 488/509 nm and RNA levels were determined by qPCR with gene specific primers. Values are mean ± SD of triplicate treatments (N = 3 per group). *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: Human cell Lines The human HCC cell line SK-Hep1 and lung cancer cell line A549 were bought from American Type Culture Collection (Manassas, VA, USA).

Techniques: In Vitro, Fluorescence, Imaging, In Vivo

Journal: International journal of pharmaceutics

Article Title: Lipidation of polyethylenimine-based polyplex increases serum stability of bioengineered RNAi agents and offers more consistent tumoral gene knockdown in vivo

doi: 10.1016/j.ijpharm.2018.06.026

Figure Lengend Snippet: Delivery and effectiveness of BERA/GFP-siRNA in orthotopic HCC xenograft mouse models in vivo. (A) GFP-siRNA was successfully delivered to orthotopic HCC tumor tissues, leading to significantly lower levels of GFP mRNA. (B and C) Target siRNA molecules were also distributed to mouse lung and liver tissues. Orthotopic HCC xenograft mouse models were established after grafting SK-Hep1-Luc-GFP cells into the left liver lobe. Mice were treated i.v. with 1 mg/kg of BERA/GFP-siRNA-loaded LPP every other day for 4 times. IVJ-PEI formulated BERA/GFP-siRNA was used for comparison. GFP-siRNA and mRNA levels were determined by qPCR assays. Values are mean ± SD (N = 3 per group). *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: Human cell Lines The human HCC cell line SK-Hep1 and lung cancer cell line A549 were bought from American Type Culture Collection (Manassas, VA, USA).

Techniques: In Vivo

Journal: International Journal of Biological Sciences

Article Title: HPV E7 inhibits cell pyroptosis by promoting TRIM21-mediated degradation and ubiquitination of the IFI16 inflammasome

doi: 10.7150/ijbs.50074

Figure Lengend Snippet: HPV E7 interacts with IFI16 and the E3 ligaseTRIM21. (A) Mass spectrum data of TRIM21 among HPV 11E7-interacting proteins identified by mass spectrometry. (B) Immunoblot analysis of HaCaT cells stably expressing HPV 11E7 transfected with poly(dA:dT) for the indicated times, followed by immunoprecipitation with anti-FlagM2 beads. (C) Immunoblot analysis of HeLa cells transfected with poly(dA:dT) for the indicated times, followed by immunoprecipitation with IFI16, HPV 18E7, TRIM21 or immunoglobulin G (IgG)-conjugated magnetic beads. (D) Coimmunoprecipitation and immunoblot analysis of 293T cells cotransfected for 36 hr with Flag-IFI16 plus Myc-TRIM21 or Myc-TRIM21 mutant vectors, followed by immunoprecipitation with anti-Flag-M2 beads. (E) Coimmunoprecipitation and immunoblot analysis of 293T cells cotransfected for 36 hr with Flag-HPV 18E7 plus Myc-TRIM21 or Myc-TRIM21 mutant vectors, followed by immunoprecipitation with anti-FlagM2 beads. (F) Coimmunoprecipitation and immunoblot analysis of 293T cells cotransfected for 36 hr with Myc-TRIM21 and Flag-IFI16 or Flag-IFI16 mutant vectors, followed by immunoprecipitation with anti-FlagM2 beads.

Article Snippet: Antibodies specific for IFI16 (sc-8023, 1:1000), TRIM21 (sc25351, 1:1000), ubiquitin (sc-471120, 1:1000), and GFP (sc-390394) were from Santa Cruz, Inc. Antibodies against Flag (M20008M), Myc (M20002M), mouse IgG (B30010M), and rabbit IgG (B30011S) were obtained from Abmart.

Techniques: Mass Spectrometry, Western Blot, Stable Transfection, Expressing, Transfection, Immunoprecipitation, Magnetic Beads, Mutagenesis

Journal: International Journal of Biological Sciences

Article Title: HPV E7 inhibits cell pyroptosis by promoting TRIM21-mediated degradation and ubiquitination of the IFI16 inflammasome

doi: 10.7150/ijbs.50074

Figure Lengend Snippet: HPV E7 promotes the K33-linked ubiquitination and degradation of IFI16 mediated by TRIM21. (A) Immunoblot analysis of lysates in control HeLa cells or TRIM21 knockout stable HeLa cells transfected with poly(dA:dT) for the indicated times. (B) Immunoblot analysis of IFI16 in the lysates of TRIM21 knockout stable HeLa cells treated with CHX (40 μg/ml) for the indicated number of hours after transfection with poly(dA:dT) for 1 hr. (C) Immunoblot analysis of 293T cells cotransfected with the Myc-TRIM21 and Flag-IFI16 vectors for 36 hr. (D) Immunoblot analysis of 293T cells cotransfected with the Myc-TRIM21 and Flag-IFI16 vectors with or without MG132 treatment. (E) Immunoblot analysis of 293T cells cotransfected with the Myc-TRIM21 and Flag-IFI16 vectors treated with CHX (40 μg/ml) for the indicated number of hours. (F) Immunoblot analysis of 293T cells cotransfected with Myc-TRIM21and the Flag-IFI16 or GFP-HPV 18E7 vector for 36 hr. (G) Immunoblot analysis of ubiquitinated IFI16 in control HeLa cells or TRIM21 knockout stable HeLa cells transfected with poly(dA:dT) for the indicated times and treated with MG132 for 6 hr before cell harvest. (H) Immunoblot analysis of ubiquitinated IFI16 in 293T cells cotransfected with Myc-TRIM21and the Flag-IFI16 or HA-ub vector for 36 hr with or without MG132 treatment for 6 hr before cell harvest. (I) Immunoblot analysis of ubiquitinated IFI16 in 293T cells transfected with combinations of the Myc-TRIM21, Flag-IFI16, HA-ub, and GFP-HPV 18E7 vectors for 36 hr and treated with MG132 for 6 hr before cell harvest. (J) Immunoblot analysis of ubiquitinated IFI16 in 293T cells transfected with combinations of the Myc-TRIM21, Flag-IFI16, HA-ub, and HA-ub mutant (each of the Lys residues were replaced by an Arg residue) vectors for 36 hr and treated with MG132 for 6 hr before cell harvest. (K) Immunoblot analysis of ubiquitinated IFI16 in 293T cells transfected with combinations of the Myc-TRIM21, Flag-IFI16, HA-ub, HA-ub mutant (all Lys residues but one was replaced with Arg residues) vectors for 36 hr and treated with MG132 for 6 hr before cell harvest. Data are representative of at least three independent experiments.

Article Snippet: Antibodies specific for IFI16 (sc-8023, 1:1000), TRIM21 (sc25351, 1:1000), ubiquitin (sc-471120, 1:1000), and GFP (sc-390394) were from Santa Cruz, Inc. Antibodies against Flag (M20008M), Myc (M20002M), mouse IgG (B30010M), and rabbit IgG (B30011S) were obtained from Abmart.

Techniques: Ubiquitin Proteomics, Western Blot, Control, Knock-Out, Transfection, Plasmid Preparation, Mutagenesis, Residue

Journal: International Journal of Biological Sciences

Article Title: HPV E7 inhibits cell pyroptosis by promoting TRIM21-mediated degradation and ubiquitination of the IFI16 inflammasome

doi: 10.7150/ijbs.50074

Figure Lengend Snippet: TRIM21 downregulates cell pyroptosis induced by poly(dA:dT). (A) Microscopy imaging of cell death in control HeLa cells or TRIM21 knockout stable HeLa cells transfected with poly(dA:dT) for 18 hr. (B) Flow cytometry analysis of propidium iodide-positive control HeLa cells or TRIM21 knockout stable HeLa cells transfected with poly(dA:dT) for 16 hr. (C) Cell viability assay in control HeLa cells or TRIM21 knockout stable HeLa cells transfected with poly(dA:dT) for the indicated times. (D) LDH assay in control HeLa cells or TRIM21 knockout stable HeLa cells transfected with poly(dA:dT) for the indicated times. (E) Immunoblot analysis of GSDMD and caspase-1 in the lysates of control HeLa cells or TRIM21 knockout stable HeLa cells transfected with poly(dA:dT) for 24 hr. (F) ELISA analysis of IL-18 and IL-1β in control HeLa cells or knockout stable HeLa cells transfected with poly(dA:dT) for 24 hr. Data are presented as mean ± SD of duplicate samples and are representative of at least three independent experiments. P values are determined by two-tailed Student's t test. ** p < 0.01, *** p < 0.001.

Article Snippet: Antibodies specific for IFI16 (sc-8023, 1:1000), TRIM21 (sc25351, 1:1000), ubiquitin (sc-471120, 1:1000), and GFP (sc-390394) were from Santa Cruz, Inc. Antibodies against Flag (M20008M), Myc (M20002M), mouse IgG (B30010M), and rabbit IgG (B30011S) were obtained from Abmart.

Techniques: Microscopy, Imaging, Control, Knock-Out, Transfection, Flow Cytometry, Positive Control, Viability Assay, Lactate Dehydrogenase Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: HPV E7 inhibits cell pyroptosis by promoting TRIM21-mediated degradation and ubiquitination of the IFI16 inflammasome

doi: 10.7150/ijbs.50074

Figure Lengend Snippet: Schematic diagram showing that HPV E7 interacted with IFI16 and promoted the ubiquitin-mediated degradation of IFI16 by recruiting the E3 ligase TRIM21, resulting in the inhibition of cell pyroptosis during HPV infection.

Article Snippet: Antibodies specific for IFI16 (sc-8023, 1:1000), TRIM21 (sc25351, 1:1000), ubiquitin (sc-471120, 1:1000), and GFP (sc-390394) were from Santa Cruz, Inc. Antibodies against Flag (M20008M), Myc (M20002M), mouse IgG (B30010M), and rabbit IgG (B30011S) were obtained from Abmart.

Techniques: Ubiquitin Proteomics, Inhibition, Infection