Journal: bioRxiv

Article Title: H19 is a PERK-regulated long non-coding RNA that fine-tunes UPR signalling and inhibits endoplasmic reticulum stress-induced cell death

doi: 10.1101/2025.10.17.683008

Figure Lengend Snippet: (A) RT-qPCR analysis of H19 expression in HEK293T cells transfected with either H19-overexpressing plasmid ( H19 ) or control vector (plenti4/V5) for 48 hours. (B) Schematic representation of the ATF6-luciferase reporter plasmid. (C) MCF7 cells were co-transfected with the H19 -overexpressing plasmid or control vector, ATF6-luciferase reporter, and β-galactosidase plasmid. After 16 hours, cells were treated with thapsigargin (TG, 1 µM) for 24 hours. ATF6 transcriptional activity was assessed via firefly luciferase activity normalized to β-gal expression. (D–I) HEK293T cells were co-transfected with H19 -overexpressing plasmid or control vector, and either the HA-tagged XBP1 reporter plasmid (D–F) or ATF4 reporter plasmid (G–I). (D, G) Diagrams of reporter plasmid structures. (E, H) GFP (XBP1) and RFP (ATF4) fluorescence signals recorded at 8 and 24 hours post-treatment with TG (1 µM). (F, I) Western blot analysis of HA-tagged XBP1 and ATF4 proteins, respectively, normalized to H3(K4).

Article Snippet: The membrane was incubated overnight at 4°C with primary antibodies against ATF6 (Abcam, Cat #ab122897), spliced XBP1 (BioLegend, Cat #619502), PERK (Cell Signaling, Cat #C33E10), phospho-eIF2α (Cell Signaling, Cat #9721S), β-Actin (Sigma, Cat #A- 5060), HA-Tag (Cell Signaling, Cat #MA1-12429), and/or Tri-methyl-histone H3 (K4) (Cell Signaling, Cat #9727S).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Plasmid Preparation, Control, Luciferase, Activity Assay, Fluorescence, Western Blot