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

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MCPIP1 modulates the miRNA‒mRNA landscape in keratinocyte carcinomas

doi: 10.1186/s13046-024-03211-8

Figure Lengend Snippet: Expression of miR-223-3p, miR-376c-3p, and miR-139-5p in human keratinocyte carcinomas and regulation of their biogenesis by MCPIP1. ( A ) RT‒qPCR analysis of hsa-miR-223-3p, hsa-miR-376c-3p and hsa-miR-139-5p expression in A431 cells expressing the control (NC, negative control) or MCPIP1-specific shRNA ( n = 5). ( B ) RT‒qPCR analysis of miRNA expression in HaCaT and SCC-25 keratinocytes ( n = 4). ( C ) RT‒qPCR analysis of miRNA expression in skin sections from BCC patients ( n = 12) and healthy individuals ( n = 7). ( D ) HEK293 cells were co-transfected with pcDNA3.0 (control), pcDNA3.0 MCPIP1 or pcDNA3.0 MCPIP1-D141N and pcDNA3.0 expressing pre-miR-376c. Left panel : Scheme indicating experimental design. Right panel : RT-qPCR analysis of miRNA expression ( n = 3). ( E ) Degradation assays were performed using 3xFLAG MCPIP1 or 3xFLAG MCPIP1-D141N immunoprecipitated from HaCaT cells with in vitro transcribed pre-miR-376c as a substrate. Left panel : Scheme indicating experimental design. Right panel : Image of RNA electrophoresis in 8% PAA gel. ( F ) Predicted secondary structure of hsa-pre-miR-376c. MiR-103a-3p was used as an internal control. * – P < 0.05; ** – P < 0.01, *** – P < 0.001, **** – P < 0.0001 by Student’s t test ( B , C ) or one-way ANOVA ( A , D ). Hsa, Homo sapiens

Article Snippet: The cell Cycle Regulation Antibody Sampler Kit (#9932) and Cell Cycle Regulation Antibody Sampler Kit II (#9870) were obtained from Cell Signaling Technology (Beverly, MA, USA), and anti-FLAG M2 (Sigma-Aldrich).

Techniques: Expressing, Control, Negative Control, shRNA, Transfection, Quantitative RT-PCR, Immunoprecipitation, In Vitro, Electrophoresis