Journal: Cell Death & Disease

Article Title: TXNL1-XRCC1 pathway regulates cisplatin-induced cell death and contributes to resistance in human gastric cancer

doi: 10.1038/cddis.2014.27

Figure Lengend Snippet: The XRCC1 levels are elevated in cisplatin-resistant gastric cancer cells and mediate resistance to cisplatin. ( a ) The XRCC1 expression in BGC823 and BGC823/DDP cells was analyzed by western blotting. ( b ) The viability of BGC823 cells transfected with RFP-XRCC1 for 48 h followed by exposure to 0, 0.4, 0.6, 0.8, or 1.0 μ g/ml of cisplatin for 48 h was determined by CCK-8 assay. ( c ) The viability of BGC823/DDP cells transfected with XRCC1 shRNA for 48 h followed by exposure to 0, 2.5, 5, 10, or 15 μ g/ml of cisplatin for 48 h was determined by CCK-8 assay. ( d ) BGC823 cells transfected with RFP-XRCC1 for 48 h were subjected to clonogenic survival assay 2 weeks after treatment with 0, 0.2, 0.4, or 0.8 of μ g/ml of cisplatin for 2 h. ( e ) Quantify numbers of colony in BGC823 cells transfected with RFP-XRCC1, each colony containing cells >50 were counted. ( f ) The BGC823/DDP cells transfected with XRCC1 shRNA for 48 h were subjected to clonogenic survival assay 3 weeks after treatment with 0, 5, 10, or 15 μ g/ml of cisplatin for 2 h. ( g ) Quantification of colonies in BGC823/DDP cells transfected with XRCC1 shRNA, each colony containing >50 cells were counted. Results are representative of at least three independent experiments and shown as the mean±S.D. * P <0.05, ** P <0.01, *** P <0.001

Article Snippet: To confirm the role of plasmid expressed siRNA, another nude small interfering RNA (siRNA) specific for XRCC1 (5′-GGUCCUUCUAUAUCUGUAAdTdT-3′) and nonspecific control siRNA was synthesized (Ribobio, Guangzhou, China).

Techniques: Expressing, Western Blot, Transfection, CCK-8 Assay, shRNA, Clonogenic Cell Survival Assay

Journal: Cell Death & Disease

Article Title: TXNL1-XRCC1 pathway regulates cisplatin-induced cell death and contributes to resistance in human gastric cancer

doi: 10.1038/cddis.2014.27

Figure Lengend Snippet: XRCC1 restrains cisplatin-induced cell death by regulation of DNA damage-induced apoptosis. ( a ) The BGC823 cells transfected with RFP-XRCC1 for 48 h followed by exposure to 0.8 μ g/ml of cisplatin for additional 24 h, and analyzed by γ H2AX foci staining. ( b ) Quantify γ H2AX foci-positive cells of BGC823 cells transfected with RFP-XRCC1. The cells with γ H2AX foci >5 were counted as positive cells and expressed as a percentage. ( c ) Western blotting to determine the expression of XRCC1 and γ H2AX in BGC823 cells transfected with RFP-XRCC1. ( d ) BGC823/DDP cells transfected with XRCC1 shRNA for 48 h followed by exposure to 5 μ g/ml of cisplatin for additional 24 h to give γ H2AX foci staining. ( e ) Quantify γ H2AX foci-positive cells of BGC823/DDP cells transfected with XRCC1 shRNA. The cells with γ H2AX foci >5 were counted as positive cells and expressed as a percentage. ( f ) Western blotting to determine the level of XRCC1 and γ H2AX in BGC823/DDP cells transfected with XRCC1 shRNA. ( g ) The BGC823 cells transfected with RFP-XRCC1 48 h followed by exposure to 0.8 μ g/ml of cisplatin for 48 h, and the cell death rate was determined by the TUNEL assay ( × 1000). ( h ) Quantification of TUNEL-positive BGC823 cells transfected with RFP-XRCC1. ( i ) Western blotting was applied to determine the level of cleaved forms of PARP1 in BGC823 cells transfected with RFP-XRCC1. ( j ) The BGC823/DDP cells transfected with XRCC1 shRNA for 48 h followed by exposure to 5 μ g/ml of cisplatin for 48 h, and using TUNEL assay to determine the cell death rate. ( k ) Quantify TUNEL-positive cells of BGC823/DDP cells transfected with shXRCC1. ( l ) Western blotting was used to determine the level of cleaved forms of PARP1. Results are representative of at least three independent experiments and shown as the mean±S.D. ** P <0.01, *** P <0.001

Article Snippet: To confirm the role of plasmid expressed siRNA, another nude small interfering RNA (siRNA) specific for XRCC1 (5′-GGUCCUUCUAUAUCUGUAAdTdT-3′) and nonspecific control siRNA was synthesized (Ribobio, Guangzhou, China).

Techniques: Transfection, Staining, Western Blot, Expressing, shRNA, TUNEL Assay

Journal: Cell Death & Disease

Article Title: TXNL1-XRCC1 pathway regulates cisplatin-induced cell death and contributes to resistance in human gastric cancer

doi: 10.1038/cddis.2014.27

Figure Lengend Snippet: The TXNL1 downregulates XRCC1 through the Ub-proteasome pathway. ( a ) The protein stability of XRCC1 in BGC823 or BGC823/DDP cells was assessed by western blotting after treatment with 50 μ g/ml of CHX for the indicated times. ( b ) Quantification of XRCC1 protein level of BGC823/DDP cells after treatment with 50 μ g/ml of CHX for the indicated times. ( c ) The expression of TXNL1 was determined by 2-DE-MS and western blotting. The arrows showed the location of TXNL1. ( d ) The viability of BGC823 and BGC823/DDP cells exposed to As 2 O 3 for 48 h was determined by CCK-8 assay. ( e ) The BGC823 cells and BGC823/DDP cells exposed to As 2 O 3 for 24 h were analyzed by western blotting to determine the level of TXNL1 and XRCC1. ( f ) BGC823 cells transfected with TXNL1 shRNA for 48 h followed by exposure to 0.8 μ g/ml of cisplatin for 24 h to give western blotting to determine the level of TXNL1, XRCC1, and γ H2AX. ( g ) The BGC823/DDP cells transfected with flag-TXNL1 for 48 h followed by exposure to 5 μ g/ml of cisplatin for 24 h for western blotting to determine the level of TXNL1, XRCC1, and γ H2AX. ( h ) The BGC823/DDP cells transfected with Flag-TXNL1 for 48 h followed by exposure to 5 μ g/ml of cisplatin for 24 h were analyzed by XRCC1 foci staining. ( i ) The BGC823/DDP cells exposed to 60 μ g/ml of irinotecan combined with 5 μ g/ml of cisplatin for 24 h were analyzed by western blotting to determine the level of XRCC1 and TXNL1. ( j ) The protein stability of XRCC1 in BGC823/DDP cells transfected with flag-TXNL1 for 48 h assessed by western blotting analysis after treatment with 50 μ g/ml of CHX at the indicated times. ( k ) Quantification of XRCC1 protein level of BGC823/DDP cells transfected with flag-TXNL1 after treatment with 50 μ g/ml of CHX at the indicated times. ( l ) The BGC823/DDP cells transfected with flag-TXNL1 for 48 h were treated with 10 μ M of MG132 or 50 μ M of PS-341 for 6 h. Western blotting was performed to confirm the levels of XRCC1 and immunoprecipitation was used to show the ubiquitination of XRCC1

Article Snippet: To confirm the role of plasmid expressed siRNA, another nude small interfering RNA (siRNA) specific for XRCC1 (5′-GGUCCUUCUAUAUCUGUAAdTdT-3′) and nonspecific control siRNA was synthesized (Ribobio, Guangzhou, China).

Techniques: Western Blot, Expressing, CCK-8 Assay, Transfection, shRNA, Staining, Immunoprecipitation, Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Carboxypeptidase M Is a Positive Allosteric Modulator of the Kinin B1 Receptor *

doi: 10.1074/jbc.M113.520791

Figure Lengend Snippet: CPM enhances B1R binding of its agonist at low concentration in primary human endothelial cells. HLMVEC were pretreated with 5 ng/ml IL-1β and 200 units/ml IFN-γ for 16 h (“cytokine-treated”) and then incubated with 50 μm CT peptide or 500 ng/ml CPM monoclonal antibody for 90 min. CPM expression (A) and activity (B) were determined. Inhibition by MGTA (dl-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid) was used as a positive control. C, cytokine-treated HLMVEC were then incubated with 50 μm CT peptide or 500 ng/ml CPM monoclonal antibody and 4 nm [3H]DAKD or 100 nm DAKD (4 nm [3H]DAKD+ 96 nm cold DAKD) for 90 min. Cells were washed, and specific binding was determined as described under “Experimental Procedures.” The data are expressed as % of control binding without CT-peptide or antibody. D and E, HLMVEC were transfected with specific CPM siRNA or nonspecific control (NC) siRNA using an Amaxa nucleofector. After 48 h cells were cytokine-treated as above, and CPM expression (D) and activity (E) were determined as well as the binding of B1R with its agonist, 4 nm [3H]DAKD or 100 nm DAKD (4 nm [3H]DAKD + 96 nm cold DAKD), for 90 min (F). The data in B, C, E, and F are expressed as the mean ± S.E. (n = 3). * = p < 0.05 versus control (Student's t test). The data in A and D are representative of three experiments.

Article Snippet: D and E , HLMVEC were transfected with specific CPM siRNA or nonspecific control ( NC ) siRNA using an Amaxa nucleofector.

Techniques: Binding Assay, Concentration Assay, Incubation, Expressing, Activity Assay, Inhibition, Positive Control, Transfection

Journal: Cellular signalling

Article Title: Pharmacological inhibition of MALT1 protease activity suppresses endothelial activation via enhancing MCPIP1 expression

doi: 10.1016/j.cellsig.2018.05.009

Figure Lengend Snippet: (A) MALT1 protein levels in HUVEC, HAEC, HCAEC, HDMEC and HLMEC were measured by Western blot. (B) HUVECs were pretreated with DMSO or MI-2 (1 μM) for 30 minutes and then treated with or without inflammatory stimuli (10 ng/mL TNF α, 1 μ g/mL LPS or 10 ng/mL IL1 β ) for 8 hours. The protein levels of VCAM-1, ICAM-1 were detected by Western blot. The bands were quantified by Gel-Pro Analyzer software and presented as fold changes; n=3, *p<0.05 or **p<0.001 vs DMSO treatment. (C) HUVECs were pretreated with MI-2 (1 μM) for 30 minutes and then incubated with or without TNFα (10 ng/mL) for 4 hours. The relative mRNA levels of VCAM-1, ICAM-1, IL1β, TNFα MCP1 and CXCL1 were detected by QPCR using 2△△CT method. Data represent mean±SD, n=4. (D) HUVECs were transiently transfected with short interfering RNA targeting on MALT1 (si-MALT1) or nonspecific short interfering RNA (si-Control) by electroporation (Amaxa). Transfected cells were quiescent for 48 hours and then treated with or without TNFα (10 ng/mL) for 8 hours. Expression of MALT1 and VCAM-1 were detected using Western blot. Band intensity of VCAM-1 was quantified by Gel-Pro Analyzer software; n=3, **p<0.001 vs treated si-Control cells. (E&F) HUVECs were pretreated with DMSO or 1 μM of MLT-827 (E) or mepazine (F) for 30 minutes and then treated with or without inflammatory stimuli (10 ng/mL TNF) for 8 hours. The protein levels of VCAM-1, ICAM-1 were detected by Western blot. The bands were quantified by Gel-Pro Analyzer software and presented as fold changes; n=3, *p<0.05 or **p<0.001 vs DMSO treatment.

Article Snippet: Data represent mean±SD, n=4. (D) HUVECs were transiently transfected with short interfering RNA targeting on MALT1 (si-MALT1) or nonspecific short interfering RNA (si-Control) by electroporation (Amaxa).

Techniques: Western Blot, Software, Incubation, Transfection, Small Interfering RNA, Control, Electroporation, Expressing

Journal: Frontiers in Pharmacology

Article Title: Nonmuscle Myosin Heavy Chain ⅡA-Mediated Exosome Release via Regulation of the Rho-Associated Kinase 1/Myosin Light Chains/Actin Pathway

doi: 10.3389/fphar.2020.598592

Figure Lengend Snippet: Detection of NMMHC ⅡA, NMMHC ⅡB, and NMMHC ⅡC in exosomes extracted from microglial cells. For transfection, microglial cells were sub-cultured in 24-well plates at a density of 3 × 10 5 cells/ml. Cells were transfected with siRNA-MYH9, siRNA-MYH10, or siRNA-MYH14 at a final concentration of 100 nM for 48 h. The extracted exosomes were grouped based on whether they were extracted from control microglial cells, microglial cells transfected with siRNA, microglial cells transfected with siRNA negative control, microglial cells stimulated by 1 mg/ml LPS for 24 h, microglial cells transfected with siRNA followed by 24 h LPS stimulation, or microglial cells transfected with siRNA negative control followed by 24 h LPS stimulation. The NMMHC ⅡA, NMMHC ⅡB, and NMMHC ⅡC protein expression levels for each group of exosomes were examined by western blotting. Western blots were quantified. Nine independent experiments were analyzed. A histogram depicted the quantitative representations of the protein expression levels of CD9 and CD81 for each group. The data were averages with SD, n = 9. ** p < 0.01 vs. control microglial cells; ## p < 0.01 vs. microglial cells stimulated with LPS.

Article Snippet: NMMHC ⅡA siRNA, NMMHC ⅡB siRNA, NMMHC ⅡC siRNA, and control non-specific siRNA were synthesized by the Shanghai Biotechnology Corporation (China). siRNA transfection was performed using an ExFect Transfection Reagent (Shanghai Biotechnology Corporation, China) according to the manufacturer’s instructions.

Techniques: Transfection, Cell Culture, Concentration Assay, Negative Control, Expressing, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Nonmuscle Myosin Heavy Chain ⅡA-Mediated Exosome Release via Regulation of the Rho-Associated Kinase 1/Myosin Light Chains/Actin Pathway

doi: 10.3389/fphar.2020.598592

Figure Lengend Snippet: The NMMHC ⅡA-regulated ROCK1/MLC/actin pathway might be the potential mechanism for exosome release. The exosomes were divided on the basis of whether they were extracted from control microglial cells, microglial cells incubated with 1 mg/ml LPS for 24 h, and microglial cells transfected with siRNA-MYH9 at a final concentration of 100 nM for 48 h. (A) Microglial cells were immunofluorescently stained with anti-NMMHC ⅡA, anti-ROCK1, anti-MLC, or anti-actin, together with CD11b (green) and DAPI for nuclei (blue). Stained cells were examined by confocal microscopy. Scale bars = 10 μm. A histogram depicted the quantitative fluorescence intensity from five vision fields from nine independent experiments for each group of microglial cells. (B) The protein expression levels of anti-NMMHC ⅡA, anti-ROCK1, anti-MLC, and anti-actin were examined by western blotting. Western blots were quantified. A histogram depicted the quantitative representations of the protein expression levels of NMMHC ⅡA, ROCK1, MLC, and actin for each group. Nine independent experiments were analyzed. The data were averages with SD, n = 9. ** p < 0.01 vs. control microglial cells; ## p < 0.01 vs. microglial cells cultured for 24 h.

Article Snippet: NMMHC ⅡA siRNA, NMMHC ⅡB siRNA, NMMHC ⅡC siRNA, and control non-specific siRNA were synthesized by the Shanghai Biotechnology Corporation (China). siRNA transfection was performed using an ExFect Transfection Reagent (Shanghai Biotechnology Corporation, China) according to the manufacturer’s instructions.

Techniques: Incubation, Transfection, Concentration Assay, Staining, Confocal Microscopy, Fluorescence, Expressing, Western Blot, Cell Culture

Journal: BMC Molecular Biology

Article Title: DNA-PKcs plays a dominant role in the regulation of H2AX phosphorylation in response to DNA damage and cell cycle progression

doi: 10.1186/1471-2199-11-18

Figure Lengend Snippet: Depletion of DNA-PKcs inhibits DNA double-strand break (DSB) repair and sensitizes HeLa cells to ionizing radiation . A: Depletion of DNA-PKcs by specific siRNA in HeLa-H1 cells. B: Survival curves of DNA-PKcs-depleted cells (HeLa-H1) and control cells (HeLa-NC). C: Comet images of DNA DSB detected by neutral single cell gel electrophoresis 0-6 h post-irradiation. D: The repair kinetics of 4 Gy-induced DNA DSBs detected by comet assay. The tail moment was used as the endpoint of DNA DSB. Each bar represents the mean tail moment from three independent experiments. 100 individual comets were counted per time point for each experiment. # p < 0.01, as compared to the HeLa-NC cells at the same time point.

Article Snippet: The siRNA duplexes used in this study were synthesized by Genechem (Shanghai, China), including PDK-specific siRNA (sense strand: 5'-CAACAUAGAGCAGUACAUU-3'), GSK3β-specific siRNA (sense strand: 5'-GAGCAAAUCAGAGAAAUGAdtdt-3'), and non-specific control siRNA (sense strand: 5'-UUCUCCGAACGUGUCACGUdtdt-3').

Techniques: Control, Single Cell Gel Electrophoresis, Irradiation

Journal: BMC Molecular Biology

Article Title: DNA-PKcs plays a dominant role in the regulation of H2AX phosphorylation in response to DNA damage and cell cycle progression

doi: 10.1186/1471-2199-11-18

Figure Lengend Snippet: Regulation of phosphoinositide-dependent kinase (PDK) on the phosphorylation of H2AX . A: RNAi mediated depletion of PDK protein. HeLa cells were transfected with 50 nM PDK specific siRNA molecules or non-specific (ns) control siRNA. Western blotting shows PDK expression. B: Phosphorylation of Akt at Ser473 and GSK3β at Ser9 was decreased in the DNA-PKcs depleted HeLa-H1 cells compared to control HeLa-NC cells. C: PDK regulates the phosphorylation of H2AX in response to DNA damage induced by 4 Gy of γ-irradiation. After 48 h incubation with 50 nM PDK-specific siRNA or non-specific (ns) control siRNA, cells were irradiated with 4 Gy γ rays, then harvested 0, 0.5, 1, 4, 10 h post-irradiation and analyzed by Western blotting. D: PDK regulates the phosphorylation of H2AX associated with cell cycle progression. After 24 h incubation with 50 nM PDK-specific siRNA or non-specific (ns) control siRNA, cells were synchronized in G1 phase by TdR double-blocking, then released and harvested after 5 h, for S-phase, and at 8, 9, and 10 h, for G2/M phase. The culture medium was supplemented with 50 nM siRNA molecules during the period of synchronization and cell cycle progression. Protein expression was assayed by Western blotting.

Article Snippet: The siRNA duplexes used in this study were synthesized by Genechem (Shanghai, China), including PDK-specific siRNA (sense strand: 5'-CAACAUAGAGCAGUACAUU-3'), GSK3β-specific siRNA (sense strand: 5'-GAGCAAAUCAGAGAAAUGAdtdt-3'), and non-specific control siRNA (sense strand: 5'-UUCUCCGAACGUGUCACGUdtdt-3').

Techniques: Phospho-proteomics, Transfection, Control, Western Blot, Expressing, Irradiation, Incubation, Blocking Assay

Journal: BMC Molecular Biology

Article Title: DNA-PKcs plays a dominant role in the regulation of H2AX phosphorylation in response to DNA damage and cell cycle progression

doi: 10.1186/1471-2199-11-18

Figure Lengend Snippet: Regulation of H2AX phosphorylation in response to DNA damage and cell cycle progression by GSK3β . A: The GSK3β inhibitor LiCl prolongs phosphorylated H2AX increase in response to 4 Gy irradiation. HeLa cells were pretreated with 40 μM LiCl for 2 h, irradiated with 4 Gy γ rays and harvested at 0, 0.5, 1, 4 and 10 h after irradiation. Protein expression was assayed by Western blotting. B: GSK3β inhibitor LiCl enhanced the phosphorylation of H2AX in G2/M phase cells. To release the cells from G1 block and inhibit GSK3β activity, synchronized HeLa cells were grown in DMEM medium supplemented with 40 μM LiCl. S-phase cells were harvested at 5 h and G2/M phase cells at 8, 9 and10 h after released. Protein expression was assayed by Western blotting. C: RNAi depletion of GSK3β. HeLa cells were transfected with 50 nM GSK3β siRNA or non-specific (ns) control siRNA molecules. GSK3β expression was determined by Western blotting. D: Effect of GSK3β depletion on the phosphorylation of H2AX induced by 4 Gy γ-irradiation. After 48 h incubation with 50 nM GSK3β-specific siRNA or control non-specific (ns), cells were irradiated with 4 Gy γ rays, and harvested at 0, 0.25, 1, 4 and 10 h post-irradiation. Protein expression was assayed by Western blotting. E: Effect of the PP2A inhibitor fostriecin on H2AX phosphorylation. HeLa cells were pretreated with 50 nM fostriecin for 2 h, and then irradiated with 4 Gy γ rays, and harvested at 0, 0.25, 1, 4 and 10 h post-irradiation. Protein expression was assayed by Western blotting.

Article Snippet: The siRNA duplexes used in this study were synthesized by Genechem (Shanghai, China), including PDK-specific siRNA (sense strand: 5'-CAACAUAGAGCAGUACAUU-3'), GSK3β-specific siRNA (sense strand: 5'-GAGCAAAUCAGAGAAAUGAdtdt-3'), and non-specific control siRNA (sense strand: 5'-UUCUCCGAACGUGUCACGUdtdt-3').

Techniques: Phospho-proteomics, Irradiation, Expressing, Western Blot, Blocking Assay, Activity Assay, Transfection, Control, Incubation