Journal: Molecular Therapy. Nucleic Acids

Article Title: Chimeric RNA-binding protein-based killing switch targeting hepatocellular carcinoma cells

doi: 10.1016/j.omtn.2021.08.012

Figure Lengend Snippet: Synthetic circuits composed of engineered chimeric RBPs and proteolysis-based sensing units (A) Schematic representation of synthetic circuit composed of ABA-responsive TEVp, tevD-MS2CP, and scMS2-dEGFP reporter. Results show high fluorescence intensity with the presence of both ABA and ABA-responsive TEVp, similar to the dEGFP positive control. (B) Schematic representation of synthetic circuit composed of nTEVp-CIBN-2A-CRY2-cTEVp, tevD-MS2CP, and scMS2-dEGFP reporter. Result shows high fluorescence intensity with the presence of both nTEVp-CIBN-2A-CRY2-cTEVp and blue-light pulses. (C) Schematic representation of synthetic circuit composed of Rapa-responsive TVMVp, CZ-nL7Ae-tvmvS-cL7Ae, and Kt-dEGFP reporter. Result shows high fluorescence intensity with the presence of both Rapa and Rapa-responsive TVMVp. (D) Schematic representation of synthetic circuit composed of nTEVp-YAP, 14-3-3-cTEVp, MS2CP-tevS-degron, and scMS2-dEGFP reporter. The depletion of YAP1, LATS1, or MOB1B by RNA interference (RNAi) successfully increased the dEGFP signal. The dEGFP intensity was decreased in the STK3 and LATS1 overexpression group, either in combination or alone. (E) Schematic representation of synthetic circuit composed of AFP-riboswitch-CZ-HCVp, degron-NZ-hcvS-MS2CP, and scMS2-dEGFP reporter. Results show that overexpressing AFP significantly increased dEGFP intensity in 293T cells loaded with the circuit, while in PLC/PRF/5 cells, AFP RNAi decreased dEGFP intensity. Welch’s t test was used to evaluate significant differences: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant.

Article Snippet: siRNAs used to knockdown AFP (sc-88864), YAP1 (sc-38637), LATS1 (sc-35797), and MOB1B (sc270319) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Fluorescence, Positive Control, Over Expression

Journal: Molecular Therapy. Nucleic Acids

Article Title: Chimeric RNA-binding protein-based killing switch targeting hepatocellular carcinoma cells

doi: 10.1016/j.omtn.2021.08.012

Figure Lengend Snippet: Apoptosis regulatory synthetic circuit specifically kills HCC cells (A) Schematic representation of the apoptosis regulatory circuit specifically targeting HCC cells. nTEVp-YAP, 14-3-3-cTEVp, and AFP-riboswitch-CZ-HCVp are introduced into the circuit to sense the state of the Hippo pathway and the existence of AFP. (B) Percentage of dead cells in each group. (C) Percentage of apoptotic cells in each group. (D) The synthetic circuit preferentially reduced cell numbers in the PLC/PRF/5 control strain. The knockdown of AFP and PPP2CA, either in combination or alone, decreased the reduction index. (E) Representative flow cytometry assay for cell death in mixed cell culture (IMR90 and PLC/PRF/5) and the percentage of dead cells in each group. PLC/PRF/5 stably expressing EBFP was used to distinguish from IMR90. 24 h after transfection, cells were stained with SYTOX AADvanced and subjected to flow cytometry.

Article Snippet: siRNAs used to knockdown AFP (sc-88864), YAP1 (sc-38637), LATS1 (sc-35797), and MOB1B (sc270319) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Flow Cytometry, Cell Culture, Stable Transfection, Expressing, Transfection, Staining

Journal: Molecular Therapy Oncolytics

Article Title: Programmable Synthetic Protein Circuits for the Identification and Suppression of Hepatocellular Carcinoma

doi: 10.1016/j.omto.2020.03.008

Figure Lengend Snippet: Design of Complete Circuits to Identify HCC (A) Schematic of the gene circuit sensing PPI between YAP and 14-3-3σ and output secNluc. (B) A more detailed schematic of the gene circuit. (C) The truth table of the circuit. (D) STK3 and LATS1, alone or together, were overexpressed in the 293T cell line loaded with the gene circuit. Nluc/Firefly was detected in each group. (E) YAP1, LATS1, and MOB1B were knocked down by siRNAs in the 293T cell line loaded with the gene circuit. Nluc/Firefly was detected in each group. (F) Schematic of the gene circuit sensing AFP and output secNluc. (G) A more detailed schematic of the gene circuit. (H) The truth table of the circuit. (I) AFP was overexpressed in the PLC/PRF/5 cell line loaded with the gene circuit. Nluc/Firefly was detected in each group. (J) AFP was knocked down by an siRNA in the PLC/PRF/5 cell line loaded with the gene circuit. Nluc/Firefly was detected in each group. Each experiment was independently performed three times in triplicate.

Article Snippet: A pool of three small interfering RNAs (siRNAs) was used to knockdown YAP1 (sc-38637, Santa Cruz Biotechnology, Dallas, TX, USA), LATS1 (sc-35797, Santa Cruz Biotechnology, Dallas, TX, USA), MOB1B (sc-88864, Santa Cruz Biotechnology, Dallas, Texas), and AFP (sc-270319, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: