virus strains plv6 bmal luc addgene addgene 68833 plv ef1a ires neo addgene addgene 85139 plx313 renilla luciferase addgene addgene 118016 chemicals  (Addgene inc)

Journal: Molecular Cancer

Article Title: Double vulnerability of active-NRF2 lung squamous cell carcinoma to NRF2 and TRIM24

doi: 10.1186/s12943-025-02401-y

Figure Lengend Snippet: TRIM24 expression is required for PI3Kα stability and PI3K/AKT pathway activation in active-NRF2 cells. (A-B) Silencing of TRIM24 and/or MKRN1 expression was performed using control or specific siRNAs in the active-NRF2 HCC15 cell line. Two consecutive transfections were conducted (at day 1 and 4), and after 72 h cells were treated with RSL3 (4.5 µM) (24 h). Cells were collected for WB studies. (A) Images show the indicated WB. Graphs represent the signals corrected for β-Actin loading and normalized to the signal in CTR HCC15 cells (100%). (B) The images show PI3Kα and PTEN blots. The graphs show PI3Kα signal corrected for β-Actin loading and normalized to PI3Kα in HCC15 CTR cells (100%). (C) The graphs show PIK3CA mRNA levels in HCC15 cells or H226 cells depleted or not of TRIM24. RT-qPCRs were represented as RQ values compared to those of GAPDH , referred to untreated cells (considered 1). (D) Cells were treated as indicated; MG132 (10 µM) was added for the last 5 h prior to collection. Representative (indicated). The graphs show the PI3Kα signal corrected for β-Actin loading and normalized to the PI3Kα levels in mocked HCC15 cells (considered 1). An arrow points as at an upper band showing a change in mobility in calpain by MG132. Graphs as in (A) . (E) HCC15 cells were transfected twice with the indicated siRNA (see above), then cultured in medium without serum (90 min). Cells were then incubated for 10 min with 10% FBS in the presence or absence of MG132 added 5 h prior to collection. Extracts were tested by WB after PI3Kα IP. Graphs show ubiquitin and PI3Kα levels as well as Ab signal; graphs as in (A) . (F) HCC15 cells transfected or not with siTRIM24, and treated or not with MG132 (as in E ) were collected in association buffer; the supernatants were subjected to immunoprecipitation (IP) using PI3Kα or TRIM24 Ab. Upon PI3Kα IP, the presence of associated TRIM24 was tested in WB; likewise TRIM24 IP also associated PI3Kα (as tested by PI3Kα WB). Whole extracts are showed on the right. (G) HCC15 cells infected with lentiviral particles containing pLV-SFFV- PIK3CA (7–10 days) and were sorted with GFP marker. Later, double silencing of TRIM24 expression was performed using a control or a TRIM24 siRNA (at 24 and 96 h). Cells were treated with RSL3 (4.5 µM) (24 h). Cells were collected for cell viability analysis (propidium iodide). Student’s t -test used for statistics. (H) The organoids were transfected with siRNA for TRIM24 or control (1 week total). Images (bar: 200 μm) show the different organoids and the nº live cells (%) according to propidium iodide. Statistical analyses using unpaired t- test. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001

Article Snippet: For the experiments with oxidative stress, the ROS inducer RSL3 (4.5 μM) or Erastin (10 μM ) or their vehicle was added for the last 24 h. In the case of PIK3CA overexpression, infection was performed as described above, except that HEK-293T cells were transfected with pLV-SFFV- PIK3CA -WPRE-Ubc-Emerald (Addgene 203797).

Techniques: Expressing, Activation Assay, Control, Transfection, Cell Culture, Incubation, Ubiquitin Proteomics, Immunoprecipitation, Infection, Marker