Journal: Current Research in Pharmacology and Drug Discovery

Article Title: Jagged-1 is induced by mTOR inhibitors in renal cancer cells through an Akt/ALK5/Smad4-dependent mechanism

doi: 10.1016/j.crphar.2022.100117

Figure Lengend Snippet: The impact of PI3K, Akt, and MEK on the induction of JAG1 expression by mTOR inhibitors in RCC cells. A) RCC4 cells were pre-treated with either DMSO vehicle, LY294002 (10 ​μM), MK2206 (1 ​μM), U0126 (10 ​μM), or ALK5i-II (200 ​nM) for 2 ​h before 24 ​h of treatment with Rap (20 ​nM) followed by Western blotting for JAG1, and P-Akt1 T308 , P-PRAS40 T246 , and β-Actin. B) 786-O cells were pre-treated with either DMSO vehicle, LY294002 (10 ​μM), MK2206 (1 ​μM), and U0126 (10 ​μM) for 2 ​h before 24 ​h of treatment with Rap (20 ​nM) followed by Western blotting for JAG1, P-Akt1 T308 , P-PRAS40 T246 , and β-Actin. C) RCC4 cells were pre-treated with either DMSO vehicle, LY294002 (20 ​μM and 40 ​μM), or ZSTK474 (0.1, 1, 10 ​μM) for 2 ​h before 24 ​h of treatment with Rap (20 ​nM) and Western blotted as above. D) RCC4 cells were pretreated with various doses of MK2206 or vehicle (DMSO) 2 ​h before 24 ​h of treatment with 20 ​nM Rap followed by Western blotting for JAG1, P-PRAS40 T246 , and β-Actin. E) RCC4 cells were treated either with DMSO vehicle or various doses of MK2206 (1–1000 ​nM) 2 ​h before 24 ​h treatment with 20 ​nM Rap followed by Western blotting for JAG1, P-PRAS40 T246 , and β-Actin. F) RCC4 cells were infected with empty control AdMax or Myr-Akt1 expressing AdMax adenovirus 24 ​h before a 24-h treatment with either 1 ​μM ZSTK474 or 20 ​nM Rap followed by Western blotting for JAG1, P-Akt1 T308 , and P-PRAS40 T246 . G, H) RCC4 cells (G) or 786-O cells (H) were infected with AdMax adenoviral expressing constitutive active Akt (Myr-Akt1), Myr-Akt T308A , Myr-Akt S473A , Myr-Akt T308A/S473A , or empty vector (AdMax control) for 24 ​h before a 24-h treatment with either 20 ​nM Rap or DMSO vehicle followed by Western blotting for JAG1, Akt, Myc-tag, P-Akt T308 , P-Akt S473 , and β-Actin, as shown in the respective blots.

Article Snippet: Replication-incompetent adenoviral constructs for the expression of constitutively active and phosphorylation mutants of Akt1 and ALK5 were developed and packaged using an AdMax kit (Microbix Biosystems, Inc.) as described in a previous report from our group ( ).

Techniques: Expressing, Western Blot, Infection, Control, Plasmid Preparation

Journal: Current Research in Pharmacology and Drug Discovery

Article Title: Jagged-1 is induced by mTOR inhibitors in renal cancer cells through an Akt/ALK5/Smad4-dependent mechanism

doi: 10.1016/j.crphar.2022.100117

Figure Lengend Snippet: Role of the TGF-β type I receptor (ALK5) and Smad4 in the induction of JAG1 expression by mTOR inhibitors in RCC cells. A) RCC4 cells were treated with 20 ​nM Rap, 1 ​μM KU-0063794, or 30 ​nM BEZ235 for 24 ​h, and then P-Smad2 S465/S467 and β-Actin were assessed by Western blot. B) RCC4 cells were treated with various doses of the TGF-β receptor type I inhibitor (ALK5i-II) or vehicle control 2 ​h before a 24 ​h treatment with DMSO vehicle or 20 ​nM Rap. Cell lysates were Western blotted for expression of JAG1 or β-Actin. C) RCC4 cells were infected for 24 ​h with empty Admax control, AdMax expressing kinase-dead (KD) ALK5 or constitutively active (CA) ALK5, followed by a 24 ​h treatment with vehicle control or 20 ​nM Rap, and cell lysates were assessed for expression of JAG1, P-Smad2 S465/S467 , P-Akt T308 , and β-Actin. D, E) RCC4 cells were stably silenced for the expression of Smad4 by lentiviral transduction of three different shRNAs targeting Smad4 versus scrambled shRNA as described in “Materials and Methods” and then treated with vehicle or Rap (D) or vehicle and KU-0063794 (E) , followed by Western blotting for JAG1, Smad4, and β-Actin. F) RCC4 cells were pre-treated with vehicle or 1 ​μM MK2206 for 2 ​h before a 24 ​h of treatment with vehicle or 20 ​nM Rap, and P-Smad S465/S467 was assessed by Western blot. All blots were reprobed for expression of β-Actin as a loading control.

Article Snippet: Replication-incompetent adenoviral constructs for the expression of constitutively active and phosphorylation mutants of Akt1 and ALK5 were developed and packaged using an AdMax kit (Microbix Biosystems, Inc.) as described in a previous report from our group ( ).

Techniques: Expressing, Western Blot, Control, Infection, Stable Transfection, Transduction, shRNA