Journal: Cell Death & Disease
Article Title: I-BET726 suppresses human skin squamous cell carcinoma cell growth in vitro and in vivo
Figure Lengend Snippet: I-BET726 inhibits BRD4, SphK1, and Akt signalings in human skin SCC cells. A431 cells or the primary human skin SCC cell (“C1/C2”) were treated with I-BET726 (5–100 n m ), cells were further cultured for 24 h, expression of listed proteins in total cell lysates were tested by western blotting a , h ; relative SphK1 activities d and ceramide levels e were tested. Expression of BRD4 and Tubulin in the stable A431 cells with CRISPR/Cas9 BRD4-KO plasmid (“BRD4-KO-sL1/2”) or Cas9 control construct (“Cas9-C”) was shown b ; Relative SphK1 activities ( f , the left panel) and ceramide levels g , the left panel were tested. Cas9-C cells or the BRD4-KO-sL1/2 cells were treated with or without I-BET726 (50 n m ) for 72 h, cell viability was tested by MTT assay c . A431 cells were treated with I-BET726 (50 n m ), CPI203 (500 n m ), JQ1 (500 n m ) or AZD5153 (“AZD”, 100 n m ) for 24 h, the SphK1 activities ( f , the right panel) and ceramide levels ( g , the right panel) were tested. Stable A431 cells with the constitutively active S473D mutant Akt1 (“caAkt1”) construct or the empty vector (“Vector”) were treated with or without I-BET726 (50 n m ) for applied time periods, expression of listed proteins were shown i . Cell viability (MTT viability, j and apoptosis activation (Annexin V-FACS/TUNEL staining, k and l were tested, and results were quantified. Expression of listed proteins was quantified and normalized to the loading control ( a , h and i ). * p
Article Snippet: BRD4 knockout (KO) The CRISPR/Cas9 BRD4-KO plasmid (sc-400519-KO-2; Santa Cruz Biotechnology, Shanghai, China) was transfected to cultured A431 cells via the Lipofectamine 2000 protocol (Invitrogen, Shanghai, China).
Techniques: Cell Culture, Expressing, Western Blot, CRISPR, Plasmid Preparation, Construct, MTT Assay, Mutagenesis, Activation Assay, FACS, TUNEL Assay, Staining