Journal: International journal of cancer

Article Title: Combined inhibition of RAD51 and CHK1 causes synergistic toxicity in cisplatin resistant cancer cells by triggering replication fork collapse.

doi: 10.1002/ijc.35164

Figure Lengend Snippet: FIGURE 2 Synergistic toxicity evoked by combined treatment with the CHK1 inhibitor PF477736 (PF) and the RAD51 inhibitor B02 in J82CisPt. (A) The viability of J82CisPt cells was analyzed after treatment with differently combined low and moderate toxic doses of CHK1i PF477736 and RAD51i B02 as indicated. Cell viability was measured after a 72 h treatment period using the AlamarBlue Assay. Based on the data obtained from three independent experiments each performed in quadruplicate, the combination indices (CIs) were calculated using CompuSyn software (CI < 0.9 indicating synergistic effects, CI ≈1 additive effects and CI > 1.2 antagonistic effects). (B) Protein expression and activation of different apoptosis-related factors were examined via Western Blot analyses using protein extracts of J82CisPt

Article Snippet: Cytostatics, DDR modifiers and other compounds were obtained from the following suppliers: Doxorubicin (Cellpharm [Bad Vilbel, Germany]), OH-Urea (Sigma [Steinheim, Germany]), 5-Fluorouracil (Medac [Wedel, Germany]), Carboplatin (TEVA [Ulm, Germany]), Oxaliplatin (Accord Healthcare [Munich, Germany]), hydrogen peroxide (H2O2) (Carl Roth GmbH [Karlsruhe, Germany]), checkpoint kinase 1 (CHK1) inhibitor PF477736 and LY2603618 (Sigma [Steinheim, Germany]), RAD51 inhibitor B02 and RI(dl)2 (Tocris Bioscience [Bristol, UK]), MRE11 inhibitor Mirin (Abcam [Cambridge, UK]), PARP inhibitor Niraparib (MedChemExpress [Monmouth Junction, NJ, USA]), HDAC inhibitors Entinostat (Selleckchem [Munich, Germany]) and Vorinostat (Sigma [Steinheim, Germany]).

Techniques: Alamar Blue Assay, Software, Expressing, Activation Assay, Western Blot

Journal: International journal of cancer

Article Title: Combined inhibition of RAD51 and CHK1 causes synergistic toxicity in cisplatin resistant cancer cells by triggering replication fork collapse.

doi: 10.1002/ijc.35164

Figure Lengend Snippet: FIGURE 3 S-phase arrest in J82CisPt cells following treatment with B02 and PF477736. (A, B) Inhibitors (10 μM B02 ± 1 μM PF477736) were added 24 h after seeding and cell cycle distribution was analyzed after a treatment period of 24 h (A) and 72 h (B) employing propidium iodide staining and flow cytometric analysis. Data are presented as mean + SD from n = 3 independent experiments. (C) The EdU incorporation of J82CisPt cells was analyzed after treatment with 10 μM B02 or/and 1 μM PF477736. EdU incorporation was analyzed after 24 h treatment period with an EdU pulse of 2 h. The graph shows the mean + SD of n = 3 independent experiments (1000–2000 nuclei analyzed per sample). The scale bars in the representative pictures correspond to 50 μm. ***p ≤.001; **p ≤.01; *p ≤.05; significant compared to control (*), to B02 mono- treatment (#) and to PF477736 mono-treatment (+). [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Cytostatics, DDR modifiers and other compounds were obtained from the following suppliers: Doxorubicin (Cellpharm [Bad Vilbel, Germany]), OH-Urea (Sigma [Steinheim, Germany]), 5-Fluorouracil (Medac [Wedel, Germany]), Carboplatin (TEVA [Ulm, Germany]), Oxaliplatin (Accord Healthcare [Munich, Germany]), hydrogen peroxide (H2O2) (Carl Roth GmbH [Karlsruhe, Germany]), checkpoint kinase 1 (CHK1) inhibitor PF477736 and LY2603618 (Sigma [Steinheim, Germany]), RAD51 inhibitor B02 and RI(dl)2 (Tocris Bioscience [Bristol, UK]), MRE11 inhibitor Mirin (Abcam [Cambridge, UK]), PARP inhibitor Niraparib (MedChemExpress [Monmouth Junction, NJ, USA]), HDAC inhibitors Entinostat (Selleckchem [Munich, Germany]) and Vorinostat (Sigma [Steinheim, Germany]).

Techniques: Staining, Control

Journal: International journal of cancer

Article Title: Combined inhibition of RAD51 and CHK1 causes synergistic toxicity in cisplatin resistant cancer cells by triggering replication fork collapse.

doi: 10.1002/ijc.35164

Figure Lengend Snippet: FIGURE 4 Hampered replication occurring after combined treatment of J82CisPt with PF477736 and B02. (A) 24 h after seeding, J82CisPt cells were treated with either 10 μM B02, 1 μM PF477736 or both substances for 6 h. After the treatment, cells were incubated for 20 min with CldU, followed by 20 min incubation with IdU. The BrdU analogs were labeled by immunofluorescence, staining was analyzed microscopically and fiber lengths were measured using ImageJ. Data presented are from two independent experiments, whereby 200 fibers were measured for each sample. Each dot represents one analyzed fiber and the black lines show the mean ± SEM. The mean value is also given above the graphs. Upper panel, nascent DNA elongation, graphically displayed as IdU track lengths of bi-colored DNA fibers. Middle panel, table summarizing the evaluation of proportions of origins and terminations in the total fiber population (ns, not significant). Lower panel, as measure of DNA replication fork stalling fork asymmetry was determined from three-colored replication origins as the ratio of the longer red IdU fiber track length versus the shorter red IdU fiber track length departing from the same green CldU track. (B) Formation of RPA foci in the nuclei of J82CisPt cells was analyzed after 6 and 24 h treatment with 10 μM B02 or/and 1 μM PF477736 via immunocytochemical staining. Data are shown with each dot representing one analyzed nucleus and the black lines showing the mean ± SEM from three independent experiments, where in each case 50 nuclei were counted. The scale bars in the representative pictures correspond to 10 μm. ***p ≤.001; **p ≤.01; *p ≤.05; significant compared to control (*), to B02 mono-treatment (#) and to PF477736 mono-treatment (+). [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Cytostatics, DDR modifiers and other compounds were obtained from the following suppliers: Doxorubicin (Cellpharm [Bad Vilbel, Germany]), OH-Urea (Sigma [Steinheim, Germany]), 5-Fluorouracil (Medac [Wedel, Germany]), Carboplatin (TEVA [Ulm, Germany]), Oxaliplatin (Accord Healthcare [Munich, Germany]), hydrogen peroxide (H2O2) (Carl Roth GmbH [Karlsruhe, Germany]), checkpoint kinase 1 (CHK1) inhibitor PF477736 and LY2603618 (Sigma [Steinheim, Germany]), RAD51 inhibitor B02 and RI(dl)2 (Tocris Bioscience [Bristol, UK]), MRE11 inhibitor Mirin (Abcam [Cambridge, UK]), PARP inhibitor Niraparib (MedChemExpress [Monmouth Junction, NJ, USA]), HDAC inhibitors Entinostat (Selleckchem [Munich, Germany]) and Vorinostat (Sigma [Steinheim, Germany]).

Techniques: Incubation, Labeling, Immunofluorescence, Staining, Control

Journal: International journal of cancer

Article Title: Combined inhibition of RAD51 and CHK1 causes synergistic toxicity in cisplatin resistant cancer cells by triggering replication fork collapse.

doi: 10.1002/ijc.35164

Figure Lengend Snippet: FIGURE 5 S-phase-dependent formation of DNA damage and activation of DDR- and DNA repair-related mechanisms following treatment of J82CisPt with PF477736 and B02. (A) Protein expression and activation of different replication stress- and DDR-related factors was examined via Western Blot analyses with protein extracts of J82CisPt cells treated for 6 h or 24 h with 10 μM B02, 1 μM PF477736 or both substances. (B) Formation of DNA strand breaks was analyzed via alkaline Comet Assay after 24 h mono- and combination-treatment with 10 μM B02 and 1 μM PF477736. Tail intensity (% DNA in tail) is displayed as dot for every analyzed cell and the mean ± SEM calculated from n = 3; N = 50. ***p ≤.001; **p ≤.01; *p ≤.05; significant compared to control (*), to B02 mono-treatment (#) and to PF477736 mono-treatment (+). (C) To analyze in which cell cycle phase the damage predominantly occurs, a double staining with γH2AX antibody and propidium iodide was applied and examined by flow cytometry after a treatment period of 6 and 24 h in J82CisPt. Displayed representative images of the flow cytometrical analyses were generated using FlowJo software. (D) J82CisPt cells were co-treated with 10 μM B02 + 1 μM PF477736 for 24 h, afterwards immunocytochemical co-staining of γH2AX and RPA was performed to analyze the correlation of both markers. For γH2AX, the mean fluorescence intensity of the nuclei was measured and the number of RPA foci per nucleus were counted. Data are shown with each dot representing one analyzed nucleus and the black lines showing the mean ± SEM from two independent experiments, where in each case 50 nuclei were measured. The scale bar in the representative picture corresponds to 20 μm. ***p ≤.001; significant compared to nuclei with <10 RPA foci. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Cytostatics, DDR modifiers and other compounds were obtained from the following suppliers: Doxorubicin (Cellpharm [Bad Vilbel, Germany]), OH-Urea (Sigma [Steinheim, Germany]), 5-Fluorouracil (Medac [Wedel, Germany]), Carboplatin (TEVA [Ulm, Germany]), Oxaliplatin (Accord Healthcare [Munich, Germany]), hydrogen peroxide (H2O2) (Carl Roth GmbH [Karlsruhe, Germany]), checkpoint kinase 1 (CHK1) inhibitor PF477736 and LY2603618 (Sigma [Steinheim, Germany]), RAD51 inhibitor B02 and RI(dl)2 (Tocris Bioscience [Bristol, UK]), MRE11 inhibitor Mirin (Abcam [Cambridge, UK]), PARP inhibitor Niraparib (MedChemExpress [Monmouth Junction, NJ, USA]), HDAC inhibitors Entinostat (Selleckchem [Munich, Germany]) and Vorinostat (Sigma [Steinheim, Germany]).

Techniques: Activation Assay, Expressing, Western Blot, Alkaline Single Cell Gel Electrophoresis, Control, Double Staining, Flow Cytometry, Generated, Software, Staining, Fluorescence

Journal: International journal of cancer

Article Title: Combined inhibition of RAD51 and CHK1 causes synergistic toxicity in cisplatin resistant cancer cells by triggering replication fork collapse.

doi: 10.1002/ijc.35164

Figure Lengend Snippet: FIGURE 6 Combining B02 or PF477736 with other CHK1- or RAD51-inhibitors, respectively, likewise induces S-phase arrest, replication stress, and DNA damage in J82CisPt. J82CisPt cells were co-treated with 10 μM B02 + 1 μM LY2603618 (LY) (A) or 1 μM PF477736 (PF) + 30 μM RI(dl)2 (RI2) (B). Following 24 h treatment, propidium iodide-based cell cycle analysis was performed by flow cytometry with emphasis on the proportion of cells in S-phase. A total of 10,000 counts were measured for quantification. Induction of γH2AX and pRPA32 (S4, S8) was examined via Western Blot analyses with protein extracts of J82CisPt cells treated for 6 or 24 h with the corresponding combination or mono- treatments. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Cytostatics, DDR modifiers and other compounds were obtained from the following suppliers: Doxorubicin (Cellpharm [Bad Vilbel, Germany]), OH-Urea (Sigma [Steinheim, Germany]), 5-Fluorouracil (Medac [Wedel, Germany]), Carboplatin (TEVA [Ulm, Germany]), Oxaliplatin (Accord Healthcare [Munich, Germany]), hydrogen peroxide (H2O2) (Carl Roth GmbH [Karlsruhe, Germany]), checkpoint kinase 1 (CHK1) inhibitor PF477736 and LY2603618 (Sigma [Steinheim, Germany]), RAD51 inhibitor B02 and RI(dl)2 (Tocris Bioscience [Bristol, UK]), MRE11 inhibitor Mirin (Abcam [Cambridge, UK]), PARP inhibitor Niraparib (MedChemExpress [Monmouth Junction, NJ, USA]), HDAC inhibitors Entinostat (Selleckchem [Munich, Germany]) and Vorinostat (Sigma [Steinheim, Germany]).

Techniques: Cell Cycle Assay, Flow Cytometry, Western Blot

Journal: Cancer biology & therapy

Article Title: BRG1 promotes liver cancer cell proliferation and metastasis by enhancing mitochondrial function and ATP5A1 synthesis through TOMM40.

doi: 10.1080/15384047.2024.2375440

Figure Lengend Snippet: Figure 7. BRG1 induces the expession of TOMM40 in HCC cells. (a) HepG2 cells were stained with TOMM40 and BRG1 antibody after BRG1 knockdown or overexpression. (b and c) the percentage of positive cells was analyzed by ImageJ software (n = 10). **p < .01, ***p < .001 vs siRNA-NC, ##p < .01, ###p < .001 vs pcDNA3.1-NC. (d) HuH-7 cells were stained with TOMM40 and BRG1 antibody after BRG1 knockdown or overexpression. (e and f) the percentage of positive cells was analyzed by ImageJ software (n = 10). ***p < .001 vs siRNA-NC, ##p < .01, ###p < .001 vs pcDNA3.1-NC.

Article Snippet: After blocking with 5% nonfat milk for 1 h at room temperature, the membranes were incubated with indicated primary antibodies: anti-rabbit BRG1 antibody (1:1000, 21634–1-AP, Proteintech, China), antirabbit TOMM40 antibody (1:1000, 18409–1-AP, Proteintech, China), anti-rabbit ATP5A1 (1:1000, 14676–1-AP, Proteintech, China) and anti-mouse β-actin antibody (1:2000, 66009–1-Ig, Proteintech, China) overnight at 4°C.

Techniques: Staining, Knockdown, Over Expression, Software

Journal: Cancer biology & therapy

Article Title: BRG1 promotes liver cancer cell proliferation and metastasis by enhancing mitochondrial function and ATP5A1 synthesis through TOMM40.

doi: 10.1080/15384047.2024.2375440

Figure Lengend Snippet: Figure 8. BRG1 induces the expession of TOMM40 in HCC cells. (a) HepG2 cells were immunostained with TOMM40 and BRG1 antibody after BRG1 knockdown or overexpression. Scale bar, 20 μm. (b and c) the relative fluorescence intensity was analyzed by ImageJ software (n = 10). ***p < .001 vs siRNA-NC, ##p < .01, ###p < .001 vs pcDNA3.1-NC. (d) HuH-7 cells were immunostained with TOMM40 and BRG1 antibody after BRG1 knockdown or overexpression. Scale bar, 20 μm. (e and f) the relative fluorescence intensity was analyzed by Image J software (n = 10). ***p < .001 vs siRNA-NC, ###p < .001 vs pcDNA3.1-NC.

Article Snippet: After blocking with 5% nonfat milk for 1 h at room temperature, the membranes were incubated with indicated primary antibodies: anti-rabbit BRG1 antibody (1:1000, 21634–1-AP, Proteintech, China), antirabbit TOMM40 antibody (1:1000, 18409–1-AP, Proteintech, China), anti-rabbit ATP5A1 (1:1000, 14676–1-AP, Proteintech, China) and anti-mouse β-actin antibody (1:2000, 66009–1-Ig, Proteintech, China) overnight at 4°C.

Techniques: Knockdown, Over Expression, Fluorescence, Software

Journal: Cancer biology & therapy

Article Title: BRG1 promotes liver cancer cell proliferation and metastasis by enhancing mitochondrial function and ATP5A1 synthesis through TOMM40.

doi: 10.1080/15384047.2024.2375440

Figure Lengend Snippet: Figure 9. BRG1 activates TOMM40/ATP5A1 pathway in HCC cells. (a) The expressions of BRG1, TOMM40 and ATP5A1 were analyzed using Western blotting after BRG1 knockdown or overexpression in HepG2 cells. (b-d) the relative intensities of BRG1, TOMM40 and ATP5A1 were determined using ImageJ software (n = 3). **p < .01, ***p < .001 vs siRNA-NC, ##p < .01, ###p < .001 vs pcDNA3.1-NC. (e) The expressions of BRG1, TOMM40 and ATP5A1 were analyzed using Western blotting after BRG1 knockdown or overexpression in HuH-7 cells. (f-h) the relative intensity were determined using ImageJ software (n = 3). **p < .01, ***p < .001 vs siRNA-NC, ###p < .001 vs pcDNA3.1-NC. (i-l) the mRNA expressions level of BRG1, TOMM40 and ATP5A1 were analyzed using RT-qPCR.(n = 3). ##p < .01, ###p < .001 vs siRNA-NC, ***p < .001 vs pcDNA3.1-NC.

Article Snippet: After blocking with 5% nonfat milk for 1 h at room temperature, the membranes were incubated with indicated primary antibodies: anti-rabbit BRG1 antibody (1:1000, 21634–1-AP, Proteintech, China), antirabbit TOMM40 antibody (1:1000, 18409–1-AP, Proteintech, China), anti-rabbit ATP5A1 (1:1000, 14676–1-AP, Proteintech, China) and anti-mouse β-actin antibody (1:2000, 66009–1-Ig, Proteintech, China) overnight at 4°C.

Techniques: Western Blot, Knockdown, Over Expression, Software, Quantitative RT-PCR

Journal: Carcinogenesis

Article Title: miR-29c induces cell cycle arrest in esophageal squamous cell carcinoma by modulating cyclin E expression.

doi: 10.1093/carcin/bgr078

Figure Lengend Snippet: Fig. 1. miR-29c regulates the expression of cyclin E at the posttranscriptional level by targeting 3# UTR of cyclin E mRNA in ESCC cells. (A) The public miRNA database (microRNA Targets Version 5) predicted that cyclin E might be a target for miR-29c, and the 3# UTR of human cyclin E mRNA contains a highly conserved binding site from Position 470 to 492 for miR-29c. (B) The full-length 3# UTR of cyclin E complementary DNA containing miR-29c-binding site was cloned directly downstream of the firefly luciferase gene to create the pMIR-CCNE-Wt plasmid (Wt). The full-length 3# UTR of cyclin E complementary DNA (cDNA) deleted 22 nt miR-29c-binding sequence was cloned directly downstream of the firefly luciferase gene to create the pMIR-CCNE-Mut plasmid (Mut). (C) KYSE150 cells and EC9706 cells were transfected with 800 ng Wt or Mut reporter plasmid and the increasing doses of Pre-miR-29c or Pre-Scramble (0 nmol/l, 10 nmol/l, 20 nmol/l and 30 nmol/l). After transfected for 24 h, luciferase activity was measured by a dual-luciferase reporter assay. The result was expressed as relative luciferase activity (firefly LUC/renilla LUC). Columns, mean for three experiments; bars, SE. (D) EC9706 cells and KYSE150 cells were transfected with either 30 nmol/l Pre-miR-29c or Pre-Scramble for 48 h. Cyclin E protein levels were measured by western blotting and cyclin E mRNA levels were measured by reverse transcription–PCR.

Article Snippet: The primary antibodies used were as follows: cyclin E antibody (mouse monoclonal, 1:1000; Cell Signaling Technology, Beverly, MA), cyclin D1 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA), cyclin D2 Antibody (rabbit polyclonal, 1:800; Proteintech Group Inc., Chicago, IL), CDK2 Antibody (rabbit polyclonal, 1:1000; Santa Cruz Biotechnology Inc.), CDK6 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc.), b-actin (mouse monoclonal, 1:5000; Proteintech Group Inc.).

Techniques: Expressing, Binding Assay, Clone Assay, Luciferase, Plasmid Preparation, Sequencing, Transfection, Activity Assay, Reporter Assay, Western Blot, Reverse Transcription

Journal: Carcinogenesis

Article Title: miR-29c induces cell cycle arrest in esophageal squamous cell carcinoma by modulating cyclin E expression.

doi: 10.1093/carcin/bgr078

Figure Lengend Snippet: Fig. 2. Inverse correlation between miR-29c expression and cyclin E protein in ESCC cell lines. (A) miR-29c expression in KYSE150, KYSE410, KYSE450, KYSE510 and EC9706 cells was analyzed by quantitative real-time PCR. The results were presented as relative miR-29c expression, RNU6B served as internal control. The relative value of miR-29c expression of KYSE450 is set at 1. (B and C) The cell lysates of KYSE150, KYSE410, KYSE450, KYSE510 and EC9706 cells were prepared and analyzed by western blotting. The density of each protein band was quantified using LANE 1D Analyzer V4.0 software (Beijing Sage Creation) and b-actin served as loading control. The relative value of cyclin E expression in KYSE450 is set at 1. (D) KYSE150 cells were transfected with the increasing doses of Pre-miR-29c (0 nmol/l, 10 nmol/l, 20 nmol/l and 30 nmol/l). Forty-eight hours after transfection, miR-29c level was detected by using quantitative real-time PCR. (E and F) The expression of cyclin E was measured by western blotting, after transfecting KYSE150 cells with the increasing doses of Pre-miR-29c (0 nmol/l, 10 nmol/l, 20 nmol/l and 30 nmol/l) for 48 h. The density of each protein band was quantified by LANE 1D Analyzer V4.0 software (Beijing Sage Creation) and b-actin served as loading control. Columns, mean for three experiments; bars, SE.

Article Snippet: The primary antibodies used were as follows: cyclin E antibody (mouse monoclonal, 1:1000; Cell Signaling Technology, Beverly, MA), cyclin D1 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA), cyclin D2 Antibody (rabbit polyclonal, 1:800; Proteintech Group Inc., Chicago, IL), CDK2 Antibody (rabbit polyclonal, 1:1000; Santa Cruz Biotechnology Inc.), CDK6 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc.), b-actin (mouse monoclonal, 1:5000; Proteintech Group Inc.).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot, Software, Transfection

Journal: Carcinogenesis

Article Title: miR-29c induces cell cycle arrest in esophageal squamous cell carcinoma by modulating cyclin E expression.

doi: 10.1093/carcin/bgr078

Figure Lengend Snippet: Fig. 3. miR-29c induced G1/S cell cycle arrest by suppression of cyclin E expression. (A) EC9706 and KYSE150 cells were transfected with 30 nmol/ l Pre-miR-29c, Pre-Scramble or only Lipofectmine 2000 (Mock). Forty-eight hours after transfection was treated with 100 ng/ml nocodazole for 20 h, cells were collected for cell cycle analysis by propidium iodide staining and flow cytometer analysis. The percentage value of G1 fraction between Pre-miR-29c transfected cells and Pre-Scramble or Mock transfected cells was analyzed. P , 0.01. (B) EC9706 cells were transfected with 30 nmol/l Pre-miR-29c along with the expression plasmid pEF-cyclin E, which contains cyclin E open reading frame without 3# UTR. Forty-eight hours after transfection, cells were treated with 100 ng/ml nocodazole for 20 h. The percentage of cells in G1/G0 was determined by flow cytometer. (C) EC9706 cells and KYSE150 cells were transfected with 30 nmol/l Pre-miR-29c, Pre-Scramble or Mock for 48 h. The cells were collected for western blotting using antibody against cyclin D1, cyclin D2, CDK2 and CDK6. b-Actin was used as loading control. Columns, mean for three experiments; bars, SE.

Article Snippet: The primary antibodies used were as follows: cyclin E antibody (mouse monoclonal, 1:1000; Cell Signaling Technology, Beverly, MA), cyclin D1 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA), cyclin D2 Antibody (rabbit polyclonal, 1:800; Proteintech Group Inc., Chicago, IL), CDK2 Antibody (rabbit polyclonal, 1:1000; Santa Cruz Biotechnology Inc.), CDK6 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc.), b-actin (mouse monoclonal, 1:5000; Proteintech Group Inc.).

Techniques: Expressing, Transfection, Cell Cycle Assay, Staining, Cytometry, Plasmid Preparation, Western Blot, Control

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 1. miR-206 targets cyclin D1. (A) Sequence alignment between miR-206 and the 3′UTRs of cyclin D1 from different species. In brackets the 3′UTR size. (B) Diagram of the luciferase reporter construct with the putative miR-206 binding site (WT 3′UTR) and mutations (3′UTR MUT). (C) Relative lucif- erase activity was measured in HeLa cells after transfection of reporter constructs along with pSP65-U1 (CTR) or pSP65–206 (miR-206). Relative Firefly luciferase values were determined by a ratio of Firefly to Renilla luciferase with the control set to 1.00. Values are the means ± SD of 3 separate experi- ments. **A Student t test performed between control and miR-206 transfected cells yielded P values < 0.01.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Sequencing, Luciferase, Construct, Binding Assay, Activity Assay, Transfection, Control

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 2. Expression kinetics of miR-206 and cyclin D1 in differentiating C2C12 cells. C2C12 myoblasts were seeded in GM at 1.5 × 104/cm2. Cells were shifted in DM 24 h after plating and left to differentiate for further 72 h. (A) Northern blot analysis of miR-206 expression in C2C12 cells after 24 h in GM (0) and at different time points upon shift to DM. (B) Western blot analysis of cyclin D1 and MyHC expression in C2C12 cells cultured as in (A). Equal RNA and protein loading was confirmed by detecting, snRNA U2 and β-tubulin, respectively. (C) MyHC immunofluorescence staining (green) of C2C12 cells after 24 h in GM (DM 0 h) and after 72 h in DM (DM 72 h). Nuclei were counterstained in blue (DAPI) and individual pictures of the same field, taken with a DC camera, were merged using a LEICA Microsystems Imaging Equipment. Bar = 20 μm.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Expressing, Northern Blot, Western Blot, Cell Culture, Immunofluorescence, Staining, Imaging

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 3. miR-206 controls cyclin D1 accumulation in C2C12 cells. C2C12 myoblasts were seeded in GM at 2.5 × 103/cm2. Cells were transfected 24 h after plaiting. (A) Northern blot analysis of miR-206 expression (upper) and western blot analysis of cyclin D1 expression (lower) in C2C12 cells 48 h after transfection with a control vector (CTR) or with a miR-206 expression vector (miR-206). Cells were kept in GM throughout the experiment. (B) The effect of miR-206 overexpression on C2C12 cell proliferation and differentiation was evaluated 48 h after transfection by 1 h BrdU incorporation and MyHC staining, respectively. Results are represented relative to the BrdU+ nuclei or nuclei in MyHC+ cells in CTR (set to 1.00), as individually assessed in each independent experiment. Values are the means ± SD of 3 separate experiments. *A Student t test performed between control and miR-206 transfected cells yielded P values < 0.05. (C) Immunofluorescence staining of cyclin D1 (pink) and MyHC (green) 48 h after transfection. Nuclei were counterstained in blue with DAPI. Individual pictures of the same field, taken with a DC camera, were merged using a LEICA Microsystems Imaging Equipment. To obtain cyclin D1 images, before merging, individual pictures were pseudocol- ored using a LEICA Microsystems Imaging software. Bar = 10 μm. (D) C2C12 myoblasts were seeded at low (LD) and high (HD) density in GM. Cells were shifted to DM the day after plating and analyzed after further 3 d. The panels show a northern blot analysis of miR-206 expression (left panel) and a western blot analysis of cyclin D1 and differentiation associ- ated marker expression (right panel) after 24 h in GM and 72 h after shift- ing to DM. Equal RNA and protein loading was confirmed by detecting, snRNA U2 and β-tubulin, respectively.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Transfection, Northern Blot, Expressing, Western Blot, Control, Plasmid Preparation, Over Expression, BrdU Incorporation Assay, Staining, Immunofluorescence, Imaging, Software, Marker

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 4. Inhibition of miR-206 rescues cyclin D1 in myotubes (A) Experimental scheme. C2C12 myoblasts were induced to differ- entiate in DM in the presence of AraC. After 3 d, AraC was washed out and cells left to recover in DM for further 24 h. Finally, pure myotubes were transfected with LNA against miR-206 and analyzed 48 h later. (B) Northern blot analysis of miR-206 and miR-1 expression (left panel) and western blot analysis of cyclin D1 expression (right panel) in pure myotubes transfected with a control LNA (LNA C) or anti-miR-206 LNA (LNA 206). Cyclin D1 expression in proliferating myoblasts is also shown (GM). Equal RNA and protein loading was confirmed by detecting, snRNA U2 and β-tubulin, respectively. (C) Double immunofluorescence staining of MyHC and cyclin D1 of pure myotubes transfected with a control LNA (LNA C) or anti-miR-206 LNA (LNA 206). Individual pictures of the same field, taken with a DC camera, were merged using a LEICA Microsystems Imaging Equipment. Bar = 10 μm.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Inhibition, Transfection, Northern Blot, Expressing, Western Blot, Control, Double Immunofluorescence Staining, Imaging

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 5. miR-206 inhibits cell proliferation in Ras-transformed fibro- blasts. (A) Expression levels of cyclin D1 in NIH3T3(Ras) cells as compared with NIH3T3(BN) cells. (B) Real-time PCR analysis of miR-206 expres- sion in NIH3T3(Ras) cells. Results are shown relative to untransformed NIH3T3(BN) cells set to value 1.00. Each sample was analyzed in tripli- cate, and values are the means ± SD of 3 independent experiments. **A Student t test performed between untransformed and transformed cells yielded P values < 0.01. (C) NIH3T3(Ras) cells were transfected with a control vector (CTR) or with a miR-206 expression vector (miR-206) and analyzed 24 h later. Upper, northern blot analysis of miR-206 expression; lower, western blot analysis of cyclin D1 expression. (D) Effect of miR-206 forced expression on cell proliferation as determined by 1 h BrdU incor- poration. Data are reported relative to BrdU+ nuclei in CTR (set to 1.00), as individually assessed in each independent experiment. Values are the means ± SD of 3 separate experiments. *A Student t test performed between control and miR-206 transfected cells yielded P values < 0.05. Equal RNA and protein loading was confirmed by detecting, snRNA U2, and β-tubulin, respectively.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Transformation Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Control, Plasmid Preparation, Northern Blot, Western Blot

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 6. Relationship between miR-206 downregulation and cyclin D1 expression in NSCLCs. (A) Northern blot analysis of miR-206 in different murine tissues. snRNA U2 levels were used as a loading control. (B) Real-time PCR analysis of miR-206 expression in human NSCLC tissues. Results are shown relative to the matched normal lung tissues set to value 1.00. Each sample was analyzed in triplicate, and values are the means ± SD of three independent experiments. **A Student t test performed between normal and tumor tissues yielded P values < 0.01. (C) Western blot analysis of cyclin D1 expression in normal and neoplastic lung tissues. Equal protein loading was confirmed by detecting actin. n, normal tissue; t = tumor tissue

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Expressing, Northern Blot, Control, Real-time Polymerase Chain Reaction, Western Blot

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 7. miR-206 inhibits cancer cell proliferation through repression of cyclin D1. (A) A549 and HeLa cells were transfected with a control vec- tor (CTR) or with a miR-206 expression vector (miR-206) and analyzed 72 h later. Top panel: northern blot analysis of miR-206 expression; lower panel, western blot analysis of cyclin D1 expression. Equal RNA and protein loading was confirmed by detecting, snRNA U2 and β-tubulin, respectively. (B) Effect of miR-206 forced expression on cell prolifera- tion as determined by 1 h BrdU incorporation and immunofluorescence staining. Data are reported relative to BrdU+ nuclei in CTR (set to 1.00), as individually assessed in each independent experiment. Values are the means ± SD of 3 separate experiments. *A Student t test performed between control and miR-206 transfected cells yielded P values <0.05.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Northern Blot, Western Blot, BrdU Incorporation Assay, Immunofluorescence, Staining

Journal: PloS one

Article Title: Kinetic analysis of mouse brain proteome alterations following Chikungunya virus infection before and after appearance of clinical symptoms.

doi: 10.1371/journal.pone.0091397

Figure Lengend Snippet: Figure 5. Western blot validations of differentially regulated proteins identified by 2D-DIGE and/or iTRAQ analyses. (A) Protein samples from each group used for proteomic analysis were minimally labeled with cyanine-3 dye. At the top, a representative protein profile of three biological replicates from brain lysates of mock (M), early (E), late paralytic (LP) and late tetanus-like (LT), separated by 10% SDS-PAGE is shown. WB with fluorescence-based methods was used to detect an overlaid fluorescent scan of the general protein patterns (Cy3 dye; green) and the specific immunoreactive proteins (FITC or Cy5 dye; red). To better visualize protein detection signals observed with each specific antibody used, corresponding cropped WB images are presented in grey levels. (B) The graphs correspond to the mean 6 S.D. of protein quantity measured by densitometry of the antigenic bands. Densitometry analyses were performed using TotalLab Quant v12.2 software (Nonlinear Dynamics), and data were normalized to levels of global protein pattern intensity. The values indicated under each graph correspond to fold changes from paired comparisons. The significance of the differential protein expression are indicated *, p,0.05; **, p,0.01; ***, p,0.001. A.U., arbitrary units. ANXA2: annexin A2; ARRB1: b-arrestin; GABRA1: c-aminobutyric acid receptor subunit alpha-1; GRASP1: GRIP-associated protein; ITGAV: integrin aV; MYPT1: myosin phosphatase target subunit 1; N-Ras: N-Ras; RABEP1: rabaptin-5; SYNGR3: synaptogyrin-3. doi:10.1371/journal.pone.0091397.g005

Article Snippet: Blots were saturated with 5% nonfat dried milk in PBS containing 0.05% (v/v) Tween 20 (PBS-T-milk) for 1 h. Western blot (WB) analyses were carried out with rabbit mono- or polyclonal antibodies directed against b-arrestin (1:5000, ARRB1, no. 4674, Cell Signaling Technology, Danvers, MA), GRIP-associated protein (1:500, GRASP1, no. sc-135681, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), annexin A2 (1:100, ANXA2, no. sc-9061, Santa Cruz), integrin aV (1:100, ITGAV, no. 10179, Santa Cruz), myosin phosphatase target subunit 1 (1:100, MYPT1, no. sc25618, Santa Cruz), rabaptin-5 (1:1000, RABEP1, no. sc-15351, Santa Cruz), N-Ras (1:500, N-Ras, no. sc-519, Santa Cruz), synaptogyrin-3 (1:1000, SYNGR3, no. sc-68936, Santa Cruz), or with a goat polyclonal antibody directed against c-aminobutyric acid receptor subunit alpha-1 (1:100, GABAARa1 or GABRA1, no. sc-31045, Santa Cruz), diluted in PBS-T-milk and incubated overnight at 4uC.

Techniques: Western Blot, Multiplex sample analysis, Labeling, SDS Page, Fluorescence, Software, Expressing

Journal: Biochimica et biophysica acta

Article Title: Nucleocytoplasmic shuttling of the Duchenne muscular dystrophy gene product dystrophin Dp71d is dependent on the importin α/β and CRM1 nuclear transporters and microtubule motor dynein.

doi: 10.1016/j.bbamcr.2014.01.027

Figure Lengend Snippet: Fig. 6. Dp71d interacts with IMPα2/IMPβ1. (A) Pull down assays were performed by incubating glutathione-sepharose beads preincubated with bacterially expressed GST (negative con- trol) or GST-Dp71d (full length) with nuclear extracts from C2C12 cells. Beads were recovered by centrifugation and eluted proteins were analysed by Western blot using specific anti- bodies against IMPα2 and IMPβ1 (upper panel). Inputs correspond to 5% of nuclear extract prior to pull down. Lower panel shows purified GST and GST-Dp71d proteins that were visualized by SDS-PAGE followed by Coomassie brilliant blue staining. (B) Complexation of the ZZ domain with IMPs in living cells. C2C12 cells transiently expressing TetraGFP or TetraGFP-ZZ proteins were immunoprecipitated using GFP-trap and immunoprecipitated proteins subjected to Western blot analysis using the indicated anti-IMP antibodies. Inputs correspond to 5% of nuclear extract prior to immunoprecipitation. Un, unbound fraction; B, bound fraction. (C) ALPHAScreen binding assays [43] were performed by incubating the His-tagged ZZ domain of Dp71d with increasing concentrations (0–30 nM) of bacterially expressed GST alone or GST-IMPβ1, -IMPα2, or predimerized GST-IMPα2/β1 (where GST- IMPβ1 was biotinylated and GST-IMPα2 unlabelled) — see Section 2. Triplicate data points from a single typical experiment are representative of three independent experiments fitted using SigmaPlot software to determine the apparent dissociation constants (Kd). ND, not able to be determined due to low binding.

Article Snippet: GFP fusion proteins were immunoprecipitated using the GFPTrap® bead system (Chromotek, Germany) in accordance with the manufacturer's instructions.

Techniques: Centrifugation, Western Blot, SDS Page, Staining, Expressing, Immunoprecipitation, Amplified Luminescent Proximity Homogenous Assay, Binding Assay, Software

Journal: Biochimica et biophysica acta

Article Title: Nucleocytoplasmic shuttling of the Duchenne muscular dystrophy gene product dystrophin Dp71d is dependent on the importin α/β and CRM1 nuclear transporters and microtubule motor dynein.

doi: 10.1016/j.bbamcr.2014.01.027

Figure Lengend Snippet: Fig. 7. Identification of a CRM1-recognised nuclear export signal (NES) in the carboxy-terminal domain of Dp71d. (A) C2C12 cells seeded on glass coverslips were incubated for 24 h with the CRM1-specific inhibitor Leptomycin B (LMB) diluted in methanol or with methanol alone (control). Cells were immunostained with primary anti-Dp71d antibody and a fluorescein- conjugated secondary antibody (green) and counterstained with DAPI (nuclei, blue). (B–D) C2C12 cells seeded on glass coverslips were transfected to express GFP-Dp71d, TetraGFP- Amino or TetraGFP-ZZCarboxyl (green) and 8 h post-transfection incubated for additional 12 h without (control) or with LMB. Schematics of the N- and C-terminal portions of Dp71d expressed in the TetraGFP-Amino and TetraGFP-ZZCarboxyl constructs respectively. The putative CRM1-recognised NES sequences located in each portion of Dp71d are shown. Cells were fixed, counterstained with DAPI (nuclei, blue), imaged by CLSM, and images such as those shown were analysed as per the legend to Fig. 1 for endogenous Dp71d (A) and GFP- based fusion proteins (B–D), as described in Section 2. Results represent the mean +/−SEM (n = 50), with significant differences in the absence or presence of LMB denoted by the p values. (E) Interaction between the C-terminal domain of Dp71d and CRM1 as determined by immunoprecipitation. Lysates from C2C12 cells transiently transfected to express TetraGFP, TetraGFP-Dp71d, TetraGFP-Amino or TetraGFP-ZZCarboxyl were immunoprecipitated using GFP-Trap and immunoprecipitated proteins subjected to Western analysis with antibodies against CRM1 and GFP. Inputs correspond to 5% of nuclear extract prior to immunoprecipitation; Un, unbound proteins; B, bound proteins. (F) C2C12 cells transiently transfected to express GFP fused to full-length Dp71d (GFP-Dp71d-NES WT; NES sequence within amino acids 505–515 is shown) or its mutant variant (GFP-Dp71d-NES mut; mutated residues within the NES are denoted in red) (green) were cultured on glass coverslips, fixed 24 h post-transfection, stained with DAPI (nuclei, blue) and subjected to CLSM analysis; typical single optical Z-sections are shown (scale bar, 10 μm). Fn/c values were determined as above, with significant differences between cells expressing WT or Mut NES denoted by the p values.

Article Snippet: GFP fusion proteins were immunoprecipitated using the GFPTrap® bead system (Chromotek, Germany) in accordance with the manufacturer's instructions.

Techniques: Incubation, Control, Transfection, Construct, Immunoprecipitation, Western Blot, Sequencing, Mutagenesis, Variant Assay, Cell Culture, Staining, Expressing

Journal: Oncogene

Article Title: The p38-MK2-HuR pathway potentiates EGFRvIII-IL-1β-driven IL-6 secretion in glioblastoma cells.

doi: 10.1038/onc.2014.225

Figure Lengend Snippet: Figure 2. IL-1β activates the p38 MAPK–MK2 pathway in U87 and U87-EGFRvIII cells. (a–b) U87 and U87-EGFRvIII (4 × 105) cells were starved for 2 h, pre-treated with (a) p38 MAPK inhibitor SB203580 (SB, 10 μM) or (b) MK2 inhibitor sc-48 (10 μM) for 60 min and treated with IL-1β (10 ng/ml) for 15 min. Protein concentration of whole-cell lysates was determined and 25 μg cellular protein was subjected to western blot analysis as described (Yeung Y et al.8, see Supplementary Material) and analysed for p38 MAPK phosphorylation (p-p38), total p38 MAPK (p38), phosphorylated MK2 (p-MK2), total MK2 (MK2), phosphorylated Hsp27 (p-Hsp27), total Hsp27 (Hsp27), β-actin and β-tubulin (all Cell Signalling) as indicated. Relative levels of p-p38 and p-Hsp27 were normalised to total p38 and total Hsp27, respectively, expressed as fold increase compared with untreated (Ctr) cells. Representative blots and quantification of three (a) and five (b) independent experiments are shown. Data represent the mean ± s.e.m. (***Po0.001, one-way analysis of variance followed by Newman–Keuls post-test using Prism 5 GraphPad Software).

Article Snippet: Representative blots and data (mean± s.e.m., *Po0.05, **Po0.01, ***Po0.001, one-way analysis of variance followed by Newman–Keuls post-test) from three independent experiments is shown. (c) Whole-cell lysates from unstimulated U87± EGFRvIII (40 μg) were subjected to western blot analysis and analysed for EGFR and EGFRvIII, p38 MAPK (p38), total MK2 (MK2), phosphorylated Cdk1 (p-Cdk1), total Cdk1 (Cdk1), total HuR (HuR), phosphorylated HuR (pHuR (S202),30 CRM1 and β-tubulin (all Cell Signalling) as indicated.

Techniques: Protein Concentration, Western Blot, Phospho-proteomics, Software

Journal: The EMBO Journal

Article Title: The p57 CDKi integrates stress signals into cell-cycle progression to promote cell survival upon stress

doi: 10.1038/emboj.2012.122

Figure Lengend Snippet: The CDKi p57 is phosphorylated at T143 in vivo by stress-activated p38 SAPK. ( A ) HeLa cells were transfected with wild-type Flag-p57 and Flag–p57 T143A in the presence or absence of HA–p38 SAPK and myc-MKK6 DD for 48 h. Cell lysates were immunoprecipitated with anti-Flag agarose beads and analysed by western blot with anti-pp38, anti-p38, anti-myc, anti-phospoS/T and anti-Flag antibodies. ( B ) HeLa cells were transfected with Flag–p57 in the presence or absence of HA–p38 SAPK and myc-MKK6DD for 48 h. The p38 SAPK inhibitor SB203580 was added to a final concentration of 10 μM 24 h prior harvesting the cells. Cell lysates were analysed as in ( A ). ( C ) HeLa cells were transfected with Flag–p57. Forty-eight hours post transfection, cells were treated with 100 mM NaCl for the indicated times. Cell lysates were analysed as in ( A ). Representative western blots are shown. ( D ) Wild-type and p38 −/− MEF cells were grown on glass covers and treated with 100 mM NaCl, 600 μM H 2 O 2 or 7.5 mM ionomycin for 60 min prior to fixation. p57 phosphorylation at T143 was detected by indirect immunofluorescence. Nuclear DNA was stained with Hoeschst 33342. Pictures were taken using an inverted Olympus CKX 41 microscope and the Olympus Cell̂D imaging software. Representative pictures are shown.

Article Snippet: Commercially available antibodies used were as follows: mouse monoclonal anti-α-Tubulin (Sigma, S9026), mouse monoclonal anti-Flag (Sigma, S2220), rabbit polyclonal anti-Cdk2 (Santa Cruz, sc-163), rabbit polyclonal anti-cyclinD (Santa Cruz, sc-717), rabbit polyclonal anti-p57(Santa Cruz, sc-8298), mouse monoclonal anti-p57 (Santa Cruz, sc-56341), rabbit polyclonal anti-p21 (Abcam, ab7960), rabbit polyclonal anti-p27 (Santa Cruz, sc-528), rabbit polyclonal anti-p38α SAPK (Santa Cruz, sc-535), rabbit monoclonal anti-pp38 SAPK (Cell Signaling, clone 3D7), rabbit polyclonal anti IκBα (Santa Cruz, sc-371), rabbit polyclonal anti-Histone 3 (Abcam, ab-1791), rabbit polyclonal anti-Cdt1 (Santa Cruz, sc-28262), rabbit polyclonal anti-HSP27 (Stressgen, #SPA-523) and Mouse anti-phospho-serine/threonine (BD Transduction Laboratories).

Techniques: In Vivo, Transfection, Immunoprecipitation, Western Blot, Concentration Assay, Phospho-proteomics, Immunofluorescence, Staining, Microscopy, Imaging, Software

Journal: The EMBO Journal

Article Title: The p57 CDKi integrates stress signals into cell-cycle progression to promote cell survival upon stress

doi: 10.1038/emboj.2012.122

Figure Lengend Snippet: p38 SAPK and p57 promote cell survival upon stress. ( A ) Cell viability was assessed by FACS in wild-type, p38 −/− and p57 −/− MEFs 24 h after the addition of the indicated amounts of NaCl. Statistical significance was determined by one-way ANOVA followed by a Dunnett's multiple comparison test. A value of P <0.05 was considered statistically significant and represented by (*) in the bar graph. ( B ) Cell viability was assessed on wild-type, p38 −/− and p57 −/− MEF cells 24 and 48 h after the addition of 200 mM NaCl. ( C ) Cell viability upon 200 mM NaCl treatment is compromised when p38 SAPK is inhibited by SB203580 in wild-type MEFs. Reintroduction of wild-type p57 but not p57 T143A into p57 −/− rescued cell viability 24 h after the addition of NaCl. ( D ) HeLa cells were transfected with Flag–p57. Forty-eight hours post transfection, cells were incubated in the presence or the absence of the specific p38 SAPK inhibitor Birb 0796. Birb 0796 was added to a final concentration of 0.5 μM 2 h prior to the treatment with 600 μM H 2 O 2 and 7.5 mM ionomycin for 60 min. Cell lysates were analysed by western blot with anti-pp38, anti-p38, anti-pHSP27, anti-pp57 and anti-Flag antibodies. ( E , F ) Cell viability is compromised in p38 −/− and p57 −/− MEFs after oxidative and ionomycin stress. Reintroduction of wild-type p57 but not p57 T143A into p57 −/− MEF cell rescued cell viability 24 h after oxidative and ionomycin stress. ( G ) HeLa cells were transfected with Flag–p57. Forty-eight hours post transfection, cells were incubated in the presence or the absence of the specific p38 SAPK inhibitor Birb 0796. Birb 0796 was added to a final concentration of 0.5 μM 2 h prior to the treatment with 5 mJ of UV for 60 min. Cell lysates were analysed as in ( D ). ( H ) Cell viability is compromised in p38 −/− MEFs but not in wild type and p57 −/− 24 h after irradiating the cells with 5 mJ of UV. Data are represented as mean±s.e.m.

Article Snippet: Commercially available antibodies used were as follows: mouse monoclonal anti-α-Tubulin (Sigma, S9026), mouse monoclonal anti-Flag (Sigma, S2220), rabbit polyclonal anti-Cdk2 (Santa Cruz, sc-163), rabbit polyclonal anti-cyclinD (Santa Cruz, sc-717), rabbit polyclonal anti-p57(Santa Cruz, sc-8298), mouse monoclonal anti-p57 (Santa Cruz, sc-56341), rabbit polyclonal anti-p21 (Abcam, ab7960), rabbit polyclonal anti-p27 (Santa Cruz, sc-528), rabbit polyclonal anti-p38α SAPK (Santa Cruz, sc-535), rabbit monoclonal anti-pp38 SAPK (Cell Signaling, clone 3D7), rabbit polyclonal anti IκBα (Santa Cruz, sc-371), rabbit polyclonal anti-Histone 3 (Abcam, ab-1791), rabbit polyclonal anti-Cdt1 (Santa Cruz, sc-28262), rabbit polyclonal anti-HSP27 (Stressgen, #SPA-523) and Mouse anti-phospho-serine/threonine (BD Transduction Laboratories).

Techniques: Comparison, Transfection, Incubation, Concentration Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Transgenic Expression of MicroRNA-185 Causes a Developmental Arrest of T Cells by Targeting Multiple Genes Including Mzb1 *

doi: 10.1074/jbc.M113.503532

Figure Lengend Snippet: miR-185 targets a number of genes in developing thymocytes. A, relative mRNA levels of Mzb1, Nfatc3, and Camk4 in DN3 thymocytes, normalized to the endogenous Gapdh levels, were determined by real-time quantitative PCR. WT values were set to 1. Data shown are of the mean ± S.E. of at least three independent experiments performed in triplicates. Bars are representative of WT (white), Tg-25 (light gray), Tg-35 (dark gray), and Tg-6 (black) mice. (n.s. = nonsignificant, *, p < 0.05, **, p < 0.01, ***, p < 0.001; versus the threshold set as 1; one sample Student's t test) B, immunoblot analysis of Mzb1 and NFATc3 expression in miR-185 Tg-6 DN3 thymocytes when compared with the wild type control. β-Actin was used as the endogenous control. C, Mzb1 protein expression levels in human thymocytes obtained from five normal individuals (C1–C5) and four patients with 22q11.2 deletion syndrome (P1–P4). β-Actin was used as the endogenous control. Band intensities of Mzb1 and β-actin were measured using the ImageJ software. The Mzb1/β-actin ratio was calculated by dividing the band intensity of Mzb1 to that of the β-actin for each sample. Relative Mzb1 levels were then determined for each experiment (Exp #1–Exp. #3.) indicated as a group. This was done by normalizing the Mzb1/β-actin ratio for each sample relative to the first control sample. The first control sample was set as 1 in each of three independent experiments. D, the Mzb1 CDS and 3′-UTR each contain one putative miR-185 binding site. The diagram shows conserved miR-185 base pairing with human and murine Mzb1 mRNA. Mutated Mzb1 sequences are underlined. E, miR-185 directly targets Mzb1 CDS. A representative blot was shown from HEK293T cells transfected with the plasmid (pEF1) containing either wild type Mzb1 CDS-Myc or mutant Mzb1 CDS-Myc fusion, along with the empty vector (white) or pCDNA3.1/miR-185 (black). A GFP-expressing plasmid (pEGFP) was used as the transfection control. Band intensities of Myc and GFP were calculated for each lane using the ImageJ software. Relative Myc levels in the wild type Mzb1 or mutant Mzb1 transfectants were determined by normalizing the Myc/GFP ratio of pCDNA3.1/miR-185 to that of the pCDNA3.1 control, which was set as 1. F, graph shows the mean ± S.E. of relative Myc/GFP levels from four independent experiments performed in at least duplicates (n.s. = nonsignificant, *, p < 0.05, **, p < 0.01, ***, p < 0.001; two-tailed unpaired Student's t test). G, the Mzb1 3′-UTR is a direct target of miR-185. H, validation of additional miR-185 targets. G–H, luciferase activity was normalized to the β-galactosidase of COS-1 cells transfected with the luciferase plasmids containing the indicated 3′-UTR, along with either the empty vector or pCDNA3.1-miR-185. Normalized luciferase activity of the pCDNA3.1/miR-185 (black) transfectant was determined relative to that of the empty pCDNA3.1 vector (white), which was set as 1. Btk 3′-UTR, a previously validated target of miR-185, was used as a positive control. Data shown are of the mean ± S.E. from four independent experiments performed in at least triplicates (*, p < 0.05, **, p < 0.01, ***, p < 0.001; two-tailed unpaired Student's t test).

Article Snippet: Immunoblotting was performed as described previously with the following antibodies: Mzb1 (11454-1-AP, Proteintech), NFATc3 (SC-8321, Santa Cruz Biotechnology), β-actin (4967, Cell Signaling), GFP (632380, Clontech), Myc epitope (2272, Cell Signaling), anti-rabbit HRP-conjugated secondary antibody, and anti-mouse IgG HRP-conjugated secondary antibody ( 19 ).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Software, Binding Assay, Transfection, Plasmid Preparation, Mutagenesis, Two Tailed Test, Luciferase, Activity Assay, Positive Control