Journal: PLoS ONE

Article Title: Differential Processing of let-7 a Precursors Influences RRM2 Expression and Chemosensitivity in Pancreatic Cancer: Role of LIN-28 and SET Oncoprotein

doi: 10.1371/journal.pone.0053436

Figure Lengend Snippet: A, RRM2 mRNA expression in pancreatic cancer cell lines relative to expression identified in HPDE. Columns, mean of triplicate; bars, SD. n = 3. B, Western blotting analysis of RRM2 (∼45 kDa) and β-actin (45 kDa) in whole cell lysates of HPDE and pancreatic cancer cell lines. C, Expression of let- 7 family members in pancreatic cancer cell lines relative to expression in HPDE. Columns , mean of triplicate; bars , SD. n = 3; * P <0.05. D, RRM2 is a direct target of let-7 . 293TA and MIA PaCa-2 cells were virally infected for expression of precursors of let-7a-1 , let-7a-2 , let-7a-3 , let-7b , and miR-214 ( negative control ) and subsequently transfected with a RRM2 3′ UTR luciferase reporter construct. Luciferase activities measured 36 h after transfection (normalized relative to renilla activity) were plotted. Columns , mean of triplicate; bars , SD. n = 3. * p <0.05, ** p <0.01.

Article Snippet: We then tested the direct interaction of let-7 with RRM2 by transfecting a luciferase-expression construct fused to the 3′ UTR of RRM2 into 293Ta (ATCC-CRL-9078) and MIA PaCa-2 transiently overexpressing let-7 members.

Techniques: Expressing, Western Blot, Infection, Negative Control, Transfection, Luciferase, Construct, Activity Assay

Journal: PLoS ONE

Article Title: Differential Processing of let-7 a Precursors Influences RRM2 Expression and Chemosensitivity in Pancreatic Cancer: Role of LIN-28 and SET Oncoprotein

doi: 10.1371/journal.pone.0053436

Figure Lengend Snippet: A , Western blotting analysis of RRM2 (∼45 kDa) and β-actin (45 kDA) in whole cell lysates of MIA PaCa-2 overexpressing precursors of let-7 family members. Ratios of RRM2 to β-actin band intensities (normalized to control) from three experiments are indicated ( top ). Asterisks indicate significant reductions ( p <0.05) in RRM2 levels compared with control. B , Immunocytochemical detection of RRM2 in exponentially growing MIA PaCa-2 overexpressing pre- let-7 family members. Original magnification, x20. C , MIA PaCa-2 cells stably overexpressing pre- let-7 family members ( red ) or vector alone ( blue ) were treated with gemcitabine (0.1 nM to 100 µM), and percent inhibition of cellular proliferation was measured using an MTT assay. Points , mean of triplicate; bars , SE. n = 3. Gemcitabine IC 50 estimations indicated ( parentheses ).

Article Snippet: We then tested the direct interaction of let-7 with RRM2 by transfecting a luciferase-expression construct fused to the 3′ UTR of RRM2 into 293Ta (ATCC-CRL-9078) and MIA PaCa-2 transiently overexpressing let-7 members.

Techniques: Western Blot, Stable Transfection, Plasmid Preparation, Inhibition, MTT Assay

Journal: PLoS ONE

Article Title: Differential Processing of let-7 a Precursors Influences RRM2 Expression and Chemosensitivity in Pancreatic Cancer: Role of LIN-28 and SET Oncoprotein

doi: 10.1371/journal.pone.0053436

Figure Lengend Snippet: A, Western blotting analysis of hENT1 (∼50–55 kDa), hENT2 (50 kDa), hCNT1 (72 kDa), hCNT3 (77 kDa), CDA (55 kDa), dCK (30 kDa), RRM1 (94 kDa), RRM2 (45 kDa), and β-actin (45 kDA) levels in whole cell lysates of Capan-1 and Capan-1-GR cells. B , RRM2 protein increased in a gemcitabine dose-dependent fashion in Capan-1-GR cells. C, Western blotting analysis of RRM2 (∼45 kDa) and β-actin (45 kDA) levels in cells with acquired gemcitabine resistance. Ratios of RRM2 to β-actin band intensities (normalized to untreated cells) from three experiments are indicated ( top ). Asterisk indicates significantly higher RRM2 expression in gemcitabine-resistant cells ( p <0.05) compared with untreated cells. D and E, Relative expression of precursor and mature let-7a in Capan-1 ( D ) and L3.6pl ( E ) cells induced to acquire gemcitabine resistance. Columns, mean of triplicate; bars, SD. n = 3. F , Differential miRNA expression in Capan-1-GR compared with Capan-1. Putative RRM2-modulating miRNAs and their extent of reduction in Capan-1-GR cells are shown ( right ).

Article Snippet: We then tested the direct interaction of let-7 with RRM2 by transfecting a luciferase-expression construct fused to the 3′ UTR of RRM2 into 293Ta (ATCC-CRL-9078) and MIA PaCa-2 transiently overexpressing let-7 members.

Techniques: Western Blot, Expressing

Journal: PLoS ONE

Article Title: Differential Processing of let-7 a Precursors Influences RRM2 Expression and Chemosensitivity in Pancreatic Cancer: Role of LIN-28 and SET Oncoprotein

doi: 10.1371/journal.pone.0053436

Figure Lengend Snippet: A and B, Relative expression of primary let-7a transcripts ( A ) and mature let-7a ( B ) in 2 normal pancreatic tissues and 10 PDAC samples representing various tumor stages. C , Ratios of mature to precursor let-7a transcripts calculated from data points in A and B . D, Western blotting analysis of RRM2 (45 kDa) in total lysates (50 µg) of 6 matched normal-PDAC pairs. Ratios of RRM2 band intensities in PDACs compared to matched normal tissues indicated ( top ). Asterisk indicates significantly higher RRM2 expression in PDAC tissues ( p <0.05) compared with matched normal tissues. E and F, Relative expression of primary let-7a transcripts ( E ) and mature let-7a ( F ) in matched normal-PDAC pairs. G , Ratios of mature to precursor let-7a transcripts calculated from data points in E and F . Asterisk indicates significantly lower mature to precursor let-7a ratio in PDAC tissues ( p <0.05) compared with matched normal tissues.

Article Snippet: We then tested the direct interaction of let-7 with RRM2 by transfecting a luciferase-expression construct fused to the 3′ UTR of RRM2 into 293Ta (ATCC-CRL-9078) and MIA PaCa-2 transiently overexpressing let-7 members.

Techniques: Expressing, Western Blot

Journal: OncoTargets and therapy

Article Title: Monitoring the effects of doxorubicin on 3D-spheroid tumor cells in real-time

doi: 10.2147/OTT.S112566

Figure Lengend Snippet: Formation of 3D-spheroids after 3 days of culturing 12 cell lines: prostate (DU145), testis (F9), embryo (NIH3T3), muscle (C2C12), embryonic kidney (293T), neuroblastoma (SH-SY5Y), adenocarcinomic alveolar basal epithelial (A549), cervical cancer (HeLa), HeLa contaminant (HEp2), pituitary epithelial-like (GH3), embryo (PA317) and osteosarcoma (U2OS) cells. *Indicates eight cell lines selected for further DXR cytotoxicity study. Abbreviation: DXR, doxorubicin.

Article Snippet: Cell lines from the American Type Culture Collection, Manassas, VA, USA in the current study were osteosarcoma (U2OS), cervical cancer (HeLa), HeLa contaminant (HEp2), neuroblastoma (SH-SY5Y), muscle (C2C12), pituitary epithelial-like (GH3), embryo (NIH3T3), prostate (DU145), testis (F9), adenocarcinomic alveolar basal epithelial (A549), embryonic kidney (293T), and embryo (PA317) cells.

Techniques:

Journal: OncoTargets and therapy

Article Title: Monitoring the effects of doxorubicin on 3D-spheroid tumor cells in real-time

doi: 10.2147/OTT.S112566

Figure Lengend Snippet: Compiled still shot photos of eight cell lines, NIH3T3, C2C12, 293T, SH-SY5Y, A549, HeLa, PA317 and U2OS in a 96 well plate, 5 days after doxorubicin treatment. One well of 293T cells was contaminated and revealed dark coloration. Other dark colorations at DXR 320 µM were due to its high concentration. Abbreviation: DXR, doxorubicin.

Article Snippet: Cell lines from the American Type Culture Collection, Manassas, VA, USA in the current study were osteosarcoma (U2OS), cervical cancer (HeLa), HeLa contaminant (HEp2), neuroblastoma (SH-SY5Y), muscle (C2C12), pituitary epithelial-like (GH3), embryo (NIH3T3), prostate (DU145), testis (F9), adenocarcinomic alveolar basal epithelial (A549), embryonic kidney (293T), and embryo (PA317) cells.

Techniques: Concentration Assay