Journal: Experimental & Molecular Medicine

Article Title: Extracellular signal-regulated kinase 5 increases radioresistance of lung cancer cells by enhancing the DNA damage response

doi: 10.1038/s12276-019-0209-3

Figure Lengend Snippet: Cells were plated at 500 cells per well in a 6-well plate and after 24 h treated with the indicated dose of IR and/or ERK5 siRNA. Cells were then maintained for another 12 days. The formed colonies were fixed and stained. Colonies containing >50 cells were counted, and the colony formation percentage was determined for each cell line with respect to the nontreated controls. Survival curves for advanced human lung adenocarcinoma A549 cells ( a ) and representative images of stained colonies formed by A549 cells ( b ), H1299 cells ( c ), HCT116 cells ( d ), and transformed nonneoplastic human bronchial epithelial BEAS-2B cells ( e ). * p < 0.01 compared with the respective control

Article Snippet: Short hairpin RNAs (shRNA) against mouse ERK5 were designed by using a web-based shRNA design program ( http://www.genscript.com ), and double-stranded oligonucleotides containing the specific sequences for open reading frames of the genes were cloned into a pRNAT-U6.1/Neo vector (GenScript, Piscataway, NJ, USA).

Techniques: Staining, Transformation Assay, Control

Journal: Experimental & Molecular Medicine

Article Title: Extracellular signal-regulated kinase 5 increases radioresistance of lung cancer cells by enhancing the DNA damage response

doi: 10.1038/s12276-019-0209-3

Figure Lengend Snippet: a A549 cells stably transfected with either empty vector or vector encoding ERK5 were treated with 5 Gy IR. After 16 h, all cells were harvested for flow cytometry analysis. Annexin V/PI-stained cells were analyzed, and the percentage of apoptotic cells was determined. The experiments were carried out independently in triplicate; representative data are shown. The data are presented as the mean ± SD. * p < 0.05. The Annexin V/PI double-staining profile of A549 cells is also included. b ERK5 was downregulated in H1299 and A549 cells through transfection with ERK5 siRNA. Cells were treated with IR, and cellular apoptosis was detected as in ( a ). c A549 cells stably transfected with either empty vector or vector encoding ERK5 were irradiated with IR at various doses and further cultured for 48 h. Whole-cell lysates were prepared and subjected to immunoblotting to detect cleaved caspase-8 (c-Casp8), cleaved caspase-3 (c-Casp3), cleaved caspase-9 (c-Casp9), and cleaved PARP (c-PARP) levels to reflect cellular apoptosis. ɑ-Tubulin levels were also detected as the loading control. d ERK5 was downregulated in H1299 and A549 cells after transfection with ERK5 siRNA. Cells were treated with IR, and cellular apoptosis was detected as in ( c ). The results shown are representative of three different experiments. Densitometric quantification of the immunoblot data in ( c , d ) is also shown. The data are presented as the mean ± SD; * p < 0.05, ** p < 0.01, and *** p < 0.001

Article Snippet: Short hairpin RNAs (shRNA) against mouse ERK5 were designed by using a web-based shRNA design program ( http://www.genscript.com ), and double-stranded oligonucleotides containing the specific sequences for open reading frames of the genes were cloned into a pRNAT-U6.1/Neo vector (GenScript, Piscataway, NJ, USA).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Flow Cytometry, Staining, Double Staining, Irradiation, Cell Culture, Western Blot, Control

Journal: Experimental & Molecular Medicine

Article Title: Extracellular signal-regulated kinase 5 increases radioresistance of lung cancer cells by enhancing the DNA damage response

doi: 10.1038/s12276-019-0209-3

Figure Lengend Snippet: A549 and H1299 cells were exposed to IR with or without ERK5 siRNA treatment. After the treatment time points, cells were processed for cell cycle analysis using PI staining. a Quantitative data showing the cell cycle distribution in A549 (left) and H1299 cells (right) after treatment with IR (5 Gy) and/or ERK5 siRNA. * p < 0.01 and ^ p < 0.05 compared with the respective control or indicated treatment. b Western blots for G2/M cell cycle-related proteins (Cyclin B1, Cdc2, and Cdc25C) at 6 and 24 h. Band intensity was quantified using ImageJ software. The results shown are representative of three different experiments. The data are presented as the mean ± SD. ^ p < 0.05, * p < 0.01, and ** p < 0.001

Article Snippet: Short hairpin RNAs (shRNA) against mouse ERK5 were designed by using a web-based shRNA design program ( http://www.genscript.com ), and double-stranded oligonucleotides containing the specific sequences for open reading frames of the genes were cloned into a pRNAT-U6.1/Neo vector (GenScript, Piscataway, NJ, USA).

Techniques: Cell Cycle Assay, Staining, Control, Western Blot, Software

Journal: Experimental & Molecular Medicine

Article Title: Extracellular signal-regulated kinase 5 increases radioresistance of lung cancer cells by enhancing the DNA damage response

doi: 10.1038/s12276-019-0209-3

Figure Lengend Snippet: a A549 cells stably transfected with either empty vector or vector expressing ERK5 were treated with IR and further cultured for various time periods. Whole-cell lysates were prepared and subjected to immunoblotting to detect the levels of various checkpoint proteins. b ERK5 was downregulated in H1299 and A549 cells via transfection with ERK5 siRNA. Cells were treated with IR and further cultured for various time periods. Whole-cell lysates were prepared and subjected to immunoblotting to detect phosphorylated Chk1. ɑ-Tubulin was also detected as the loading control. c The expression levels of ERK5 in 226 lung adenocarcinoma tumor tissues and 20 normal tissues were obtained from TCGA. d A549 cells stably transfected with either empty vector or vector encoding ERK5 were harvested for RNA extraction and quantitative real-time PCR analysis using primers specific for human p53, p21, and GAPDH (internal control). The data represent the mean values of triplicate samples. * p < 0.05. e A549 cells were irradiated with IR at various doses, and the protein expression level of p53 was detected via western blotting. f A549 cells were transfected with control vector or ERK5 siRNA and treated with or without IR, and the protein expression level of p53 was detected via western blotting. g A549 cells were transfected with control vector or ERK5 siRNA. Transfected cells were then cotransfected with P53-Luc plasmid and pRL-TK Renilla vector. After 24 h, luciferase expression was measured. p53 luciferase activity was normalized to that of Renilla luciferase activity, and the results are expressed relative to the control values. The results are presented as the means ± SD from at least three independent experiments. * p < 0.05 versus control

Article Snippet: Short hairpin RNAs (shRNA) against mouse ERK5 were designed by using a web-based shRNA design program ( http://www.genscript.com ), and double-stranded oligonucleotides containing the specific sequences for open reading frames of the genes were cloned into a pRNAT-U6.1/Neo vector (GenScript, Piscataway, NJ, USA).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Expressing, Cell Culture, Western Blot, Control, RNA Extraction, Real-time Polymerase Chain Reaction, Irradiation, Luciferase, Activity Assay

Journal: Experimental & Molecular Medicine

Article Title: Extracellular signal-regulated kinase 5 increases radioresistance of lung cancer cells by enhancing the DNA damage response

doi: 10.1038/s12276-019-0209-3

Figure Lengend Snippet: a , b A549 cells stably transfected with either empty vector or vector expressing ERK5 were treated with 5 Gy IR and further cultured for 6 h. Whole-cell lysates were prepared and subjected to immunoblotting to detect ERK5 and phosphorylated H2AX (γH2AX) levels. ɑ-Tubulin levels were also detected as the loading control ( a ). Immunofluorescent staining for γH2AX was performed on control and ERK5 overexpression A549 cells fixed at 6 h following 5 Gy X-ray irradiation treatment ( b ). c , d H1299 and A549 cells were transfected with control vector or ERK5 siRNA, treated with or without IR, and further cultured for 6 h. Whole-cell lysates were prepared and subjected to immunoblotting to detect ERK5 and phosphorylated H2AX (γH2AX) levels. ɑ-Tubulin was also detected as the loading control ( c ). Quantitation of the number of pH2A.X foci in H1299 and A549 cells after 6 and 12 h of treatments are also shown ( d ). * p < 0.05 and ** p < 0.01 compared with the respective control or indicated treatment

Article Snippet: Short hairpin RNAs (shRNA) against mouse ERK5 were designed by using a web-based shRNA design program ( http://www.genscript.com ), and double-stranded oligonucleotides containing the specific sequences for open reading frames of the genes were cloned into a pRNAT-U6.1/Neo vector (GenScript, Piscataway, NJ, USA).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Expressing, Cell Culture, Western Blot, Control, Staining, Over Expression, Irradiation, Quantitation Assay

Journal: Experimental & Molecular Medicine

Article Title: Extracellular signal-regulated kinase 5 increases radioresistance of lung cancer cells by enhancing the DNA damage response

doi: 10.1038/s12276-019-0209-3

Figure Lengend Snippet: a – c Tumor growth was suppressed in LLC-bearing C57BL/6J mice treated with ERK5 knockdown combined with low-dose IR. When visible tumors were formed, local irradiation treatments (cumulative dose of 6 Gy) were fractionally administered on days 0, 2, and 4, and constructs expressing either Luc shRNA or ERK5 shRNA were injected into the tumor mass on days 1, 3, and 5. The tumor growth inhibitory effects of different treatments were compared ( a ). Tumor doubling time ( b ) and tumor delay time ( c ) are also shown. d – f Tumor growth was suppressed in LLC-bearing C57BL/6J mice treated with ERK5 knockdown combined with high-dose IR. When visible tumors were formed, local irradiation (total dose of 30 Gy) was fractionally administered on days 0, 2, 4, 6, 8, and 10, and a construct expressing either Luc shRNA or ERK5 shRNA was injected into the tumor mass on days 1, 3, and 5. The tumor growth inhibitory effects of different treatments were compared ( d ). Tumor doubling time ( e ) and tumor delay time ( f ) are also shown. g , h Blood vessel density within tumors was characterized by anti-CD31 immunostaining using an anti-mouse CD31 monoclonal antibody ( g ) and determined by the average number of vessels in 3 regions of the highest density at ×200 magnification in each section (the assay was repeated in 4 sections per mouse, and 3 mice were tested) ( h ). i Total protein was extracted from LLC tumors, and the intracellular VEGF level was detected via ELISA using 100 μg total protein per well. The data are presented as the mean ± SD; * p < 0.05 and ** p < 0.01 compared with the respective control or indicated treatment

Article Snippet: Short hairpin RNAs (shRNA) against mouse ERK5 were designed by using a web-based shRNA design program ( http://www.genscript.com ), and double-stranded oligonucleotides containing the specific sequences for open reading frames of the genes were cloned into a pRNAT-U6.1/Neo vector (GenScript, Piscataway, NJ, USA).

Techniques: Knockdown, Irradiation, Construct, Expressing, shRNA, Injection, Immunostaining, Enzyme-linked Immunosorbent Assay, Control

Journal: Experimental & Molecular Medicine

Article Title: Extracellular signal-regulated kinase 5 increases radioresistance of lung cancer cells by enhancing the DNA damage response

doi: 10.1038/s12276-019-0209-3

Figure Lengend Snippet: Mice were given a s.c. injection of A549 cells (2 × 10 6 ) and monitored for tumor growth until the tumor size reached approximately 50 mm 3 . Then, local irradiation with 6 Gy was fractionally administered on days 0, 2, and 4, and a construct encoding either siCtrl or siERK5 was injected into the tumor mass on days 1, 3, and 5. a The tumor growth inhibitory effects of different treatments were compared. b Tumor weight/mouse at the end of the study. Tumor doubling time ( c ) and mean body weight per mouse ( d ) are also shown. e Determination of tumor necrosis after combined treatment with ERK5 siRNA and IR. Tumor necrosis areas are shown by H&E staining and were observed under a light microscope (×100). The viable tumor cells are indicated by a blue arrow. Tumor necrosis was determined with ImageJ software. Two sections/mouse from three mice were prepared. f Determination of tumor apoptosis after combined treatment with ERK5 siRNA and IR. TUNEL assays were used to detect apoptotic cells (original magnification, ×200). Cells positive for TUNEL staining are indicated by a white arrow. The ratio of apoptotic cells to total cells: TUNEL-positive cells were counted in three fields with the highest density of positively stained cells in each section to determine the percentage of apoptotic cells. g , h Mice were given a s.c. injection of A549 cells (2 × 10 6 ) and monitored for tumor growth until the tumor size reached approximately 50 mm 3 . Then, mice were treated with local irradiation (6 Gy, fractionally administered on days 0, 2, and 4), XMD8-92 (25 mg/kg) or a combination of the treatments. The tumor growth inhibitory effects of different treatments were compared ( g ). Tumor weight/mouse at the end of the study is shown ( h ). The data are presented as the mean ± SD, * p < 0.05 and ** p < 0.01 compared with the respective control or indicated treatment

Article Snippet: Short hairpin RNAs (shRNA) against mouse ERK5 were designed by using a web-based shRNA design program ( http://www.genscript.com ), and double-stranded oligonucleotides containing the specific sequences for open reading frames of the genes were cloned into a pRNAT-U6.1/Neo vector (GenScript, Piscataway, NJ, USA).

Techniques: Injection, Irradiation, Construct, Staining, Light Microscopy, Software, TUNEL Assay, Control

Journal: Frontiers in Molecular Neuroscience

Article Title: MicroRNA-133b Negatively Regulates Zebrafish Single Mauthner-Cell Axon Regeneration through Targeting tppp3 in Vivo

doi: 10.3389/fnmol.2017.00375

Figure Lengend Snippet: miR-shRNA based gene silence of tppp3 diminishes regenerative length of M-cell (A) Diagram of miR-shRNA system. Sequence of ShRNA-5 was presented here, with the guide strand (bottom) highlighted in dark blue. (B) Quantitative RT-PCR analysis exhibited a deduction of tppp3 mRNA in shRNA-5 expressing embryos. (C) Confocal imaging of M-cell at 2 dpa. White asterisk: ablation point. Scale bar: 50 μm. (D) Regeneration length at 2 dpa. Student's two-tailed t -test, control vs. shRNA-1, P = 0.5807; control vs. shRNA-5, P = 0.0025. ** P < 0.01. Error bars represent S.E.M.

Article Snippet: Based on the Web-based shRNA design tool ( https://www.genscript.com ), five shRNAs (shRNA1-shRNA5) targeting the tppp3 gene were selected (Figure ). mCherry was used as a fluorescent reporter to mark the zebrafish embryos that expressed the miR-shRNA (Figure ).

Techniques: shRNA, Sequencing, Quantitative RT-PCR, Expressing, Imaging, Two Tailed Test