Journal: bioRxiv

Article Title: IRES-mediated translation of Δ160p53 regulates p53 functions and fine-tunes cancer homeostasis

doi: 10.64898/2026.08.21.744132

Figure Lengend Snippet: (A) Western blot analysis of cell extracts from H1299 transfected with pcDNA and Δ133p53, and treated with 5-fluorouracil (5-FU; 20 µM) and thapsigargin (0.1 µM) for 16 h, probed with BiP antibody, p53 polyclonal antibody, and β-actin antibody. (B) Partial sequence of p53 transcript variants 5,6 and 7, which are known to produce Δ133p53, and the putative IRES sequence was marked. (C) Schematic representation of bicistronic plasmids; H1299 cells were transfected with the plasmids along with pRL-TK. At 48 h post-transfection, relative luciferase activity was calculated in the presence of different IRES sequences and the control sequence (null). (D) Schematic representation of the peGFPΔEMCVI3Δ160p53 construct; western blot analysis of cell extracts from H1299 cells transfected with pcDNA (negative control), Δ160p53 (positive control), and peGFPΔEMCVI3Δ160p53, probed with p53 polyclonal antibody, β-actin antibody, and green fluorescence protein (GFP) antibody. (E) Splicing assay: Semiquantitative polymerase chain reaction (PCR) analysis using two sets of primers (P1/P2 and P3/P4), RNA isolated from pRI3F-transfected (Lanes 2–7) and untransfected (Lane 1) H1299 cells. Lanes 2 and 5 show the reverse transcriptase negative control, and lanes 4 and 7 show the PCR product amplified from the pRI3F bicistronic plasmid (Positive control). (F) Cryptic promoter activity assay: pGL3-basic (Promoter-less luciferase vector as negative control), PG13 (p53 binding sites containing luciferase plasmid as positive control), and IRES-pGL3 plasmids were transfected into H1299 cells along with pRL-TK, and relative luciferase activity was measured. (G) Real-time PCR of Fluc mRNA in H1299 cells transfected with pGL3-basic, PG13, and IRES-pGL3 plasmids. Error bars indicate standard deviation (SD). All experiments were performed in three biological replicates (n = 3). The criterion for significance was determined using a two-tailed Student’s t-test (**P ≤ 0.01 or ***P ≤ 0.001).

Article Snippet: Rabbit anti-GFP-Tag antibody (Abclonal, Cat. No. AE011) and β-actin antibodies (Sigma) were horseradish peroxidase-conjugated, and no secondary antibodies were required for detection.

Techniques: Western Blot, Transfection, Sequencing, Luciferase, Activity Assay, Control, Construct, Negative Control, Positive Control, Fluorescence, Splicing Assay, Polymerase Chain Reaction, Isolation, Reverse Transcription, Amplification, Plasmid Preparation, Binding Assay, Real-time Polymerase Chain Reaction, Standard Deviation, Two Tailed Test