Journal: International Journal of Molecular Sciences
Article Title: TH301 Emerges as a Novel Anti-Oncogenic Agent for Human Pancreatic Cancer Cells: The Dispensable Roles of p53, CRY2 and BMAL1 in TH301-Induced CDKN1A /p21 CIP1/WAF1 Upregulation
doi: 10.3390/ijms26010178
Figure Lengend Snippet: TH301 induces cell cycle arrest at the G1-phase and causes major expression alterations in cell cycle control proteins. ( A ) Flow cytometry (FACS) analysis of PI-stained PDAC cells, after treatment with increasing concentrations (0, 10, 20, and 40 μM) of TH301 for 24 h (post-administration). Data from 3 replicates (N = 3) are presented as mean ± SD values. Statistical significance was defined with one-way ANOVA, and comparisons were made in between control (0.1% DMSO) and (TH301) treated cells (% of control). Asterisks indicate comparisons between control and treated cells, at significance levels of 0.05 and below (*: <0.05; **: <0.01; ***: <0.001). ( B ) Western blotting-mediated expression profiling of main G1-phase-specific cell cycle regulators (Cyclin D3, CDK6, CDK4, CDK2, p21, and p27), after treatment of PDAC cells with increasing concentrations (0, 10, 20, and 40 μM) of TH301 for 24 h (post-administration). β -Actin was used as loading control (reference) protein. ( C ) Quantification of protein expression, as it is normalized to β -Actin (protein of reference), of G1-specific, cell cycle-phase proteins (Cyclin D3, CDK6, CDK4, CDK2, p21, and p27), of human pancreatic cancer cells, as compared to control cells (control cell values were set to “1”).
Article Snippet: The membranes were probed with the following Primary Antibodies: p21 CIP1/WAF1 (Cell Signaling Technology, Danvers, MA, USA; #2947), p27 KIP1 (Cell Signaling Technology, #9932), CDK2 (Cell Signaling Technology, #2546), CDK4 (Cell Signaling Technology, #12790), CDK6 (Cell Signaling Technology, #3136), Cyclin D3 (Cell Signaling Technology, #2936), p53 (Cell Signaling Technology, #9282), Phospho-p53 (Ser 15 ) (Cell Signaling Technology, #9284), Caspase-3 (Cell Signaling Technology, #9662) Cleaved/Activated Caspase-3 (Cell Signaling Technology, #9664), Survivin (Cell Signaling Technology, #2808), LC3B(I/II) (Cell Signaling Technology, #43566), p62/SQSTM1 (Cell Signaling Technology, #5114), PARP1 (Cell Signaling Technology, #9532), BMAL1 (Cell Signaling Technology, #14020), REV-ERBα (Cell Signaling Technology, #13418), Phospho-Histone H2A.X (γH2AX) (Cell Signaling Technology, #9718), beta-Actin ( β -Actin) (Origene Technologies, Rockville, MD, USA; #TA310155), and CRY2 (Origene Technologies, #TA502905S).
Techniques: Expressing, Control, Flow Cytometry, Staining, Western Blot
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