Journal: Autophagy

Article Title: A new transcription factor ATG10S activates IFNL2 transcription by binding at an IRF1 site in HepG2 cells

doi: 10.1080/15548627.2020.1719681

Figure Lengend Snippet: Transcription factor IRF1 Binding Domain (BD) is crucial for ATG10S promotion of IFNL2 transcription. (A) Diagrams of LP1-1-GFP constructs with IRF1-BD deletion (ΔBD) and four pairs of oligonucleotide deletions in the BD. All of the mutants were cloned into pEGFP-ΔCMV-N1 (GFP as a reporter gene) and pGL3-basic (luciferase gene as a reporter gene) vectors. (B) GFP expression directed by LP1-1 and LP1-1-ΔBD in HepG2 cells via co-transfected with ATG10 or ATG10S mRNA were analyzed by RT-PCR (upper) and western blotting (bottom), Ctrl, non-transfection control. (C) The luciferase activity driven by LP1-1 and LP1-1-ΔBD under co-transfected with ATG10 or ATG10S mRNA were examined by luciferase assay. (D–F) ATG10/ATG10S-mediated activations of the oligonucleotide-deleted LP1-1 mutants (LP1-1-BDΔ1, LP1-1-BDΔ1-2, LP1-1-BDΔ2-3, and LP1-1-BDΔ3) compared with LP1-1 were detected using RT-PCR (D), western blotting (E), and luciferase assay (F). RLU, relative light unit. * indicates P < 0.05, *** P < 0.001. All of the statistics data are expressed as the mean ± SD (n = 3)

Article Snippet: The 3 units were deleted from the 5ʹ-terminal or 3ʹ-terminal sequentially and inserted into the pEGFP-ΔCMV-N1 vector and pGL3.0-basic vector (Promega, E1751), forming four truncated mutants: BDΔ1, BDΔ1-2, BDΔ2-3 , and BDΔ3 ( ).

Techniques: Binding Assay, Construct, Clone Assay, Luciferase, Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Activity Assay

Journal: Autophagy

Article Title: A new transcription factor ATG10S activates IFNL2 transcription by binding at an IRF1 site in HepG2 cells

doi: 10.1080/15548627.2020.1719681

Figure Lengend Snippet: Crucial nucleotides of the IFNL2 LP1-1 segment for ATG10S binding in the core motif (CM) of the IRF1-binding domain. (A) Diagram of CM deletion and six nucleotide-point mutations in the CM. These mutants were cloned into pEGFP-ΔCMV-N1 and pGL3-basic vectors. (B–D) Analysis of transcription activity driven by LP1-1-ΔCM or the point mutation constructs (LP1-1-CM1[C > T], LP1-1-CM2[A > G], LP1-1-CM3[A > C], LP1-1-CM4[G > T], LP1-1-CM5[A > G] or LP1-1-CM6[C > T] mutants) compared with LP1-1 under ATG10 or ATG10S overexpression were detected by RT-PCR (B), western blotting (C), and luciferase assay (D). RLU, relative light unit. *** indicates P < 0.001. All of the data are expressed as the mean ± SD (n = 3). (E) DNA IP analysis of the interactions between ATG10S or ATG10 protein and IFNL2 promoter LP1-1 or LP1-1 DNA mutation fragments (LP1-1-CM1[C > T], LP1-1-CM2[A > G], LP1-1-CM3[A > C], LP1-1-CM4[G > T], LP1-1-CM5[A > G] or LP1-1-CM6[C > T]). These IFNL2 promoter mutants were separately co-transfected with ATG10S or ATG10 into HepG2 cells. The cell lysates were immunoprecipitated with anti-FLAG antibody (FLAG-labeled with ATG10 and ATG10S) and followed PCR determination for these mutated LP1-1 DNAs

Article Snippet: The 3 units were deleted from the 5ʹ-terminal or 3ʹ-terminal sequentially and inserted into the pEGFP-ΔCMV-N1 vector and pGL3.0-basic vector (Promega, E1751), forming four truncated mutants: BDΔ1, BDΔ1-2, BDΔ2-3 , and BDΔ3 ( ).

Techniques: Binding Assay, Clone Assay, Activity Assay, Mutagenesis, Construct, Over Expression, Reverse Transcription Polymerase Chain Reaction, Western Blot, Luciferase, Transfection, Immunoprecipitation, Labeling