Journal: iScience

Article Title: Lipopolysaccharide induces placental mitochondrial dysfunction in murine and human systems by reducing MNRR1 levels via a TLR4-independent pathway

doi: 10.1016/j.isci.2022.105342

Figure Lengend Snippet: ROS generated by NOX2 activates ATM kinase in LPS-treated placental cells (A–C) Left , Equal amounts of HTR cells treated with control (water) or LPS were separated on an SDS-PAGE gel and probed for NOX2. Tubulin was probed as a loading control. Right , NOX2 levels relative to tubulin are shown (n = 4 biological replicates). (B) Left , Equal amounts of HTR cells were treated for 24 h with control (water) or LPS and, for second blot, 25 μM NOX2 inhibitor (using DMSO in control); lysates were separated on an SDS-PAGE gel and probed for MNRR1. Actin was probed as a loading control. Right , Relative MNRR1 levels are shown for each lane (n = 4 biological replicates). (C) HTR cells were treated with control (water) or LPS for the times shown, and ROS levels were measured as in Figure 1 C. Total ROS, black; mitochondrial ROS, red; total ROS with ATM inhibitor, gray (n = 2 biological replicates). (D) Equal amounts of HTR cells were treated with control (water) or H 2 O 2 for 16 h and lysates separated on an SDS-PAGE gel and probed for phospho-CHK2, total CHK2, and ATM kinase. Actin was probed as a loading control. (E) Left, Equal amounts of HTR cells treated with control (water) or LPS with either Vehicle (DMSO) or 100 μM N-acetyl cysteine for 24 h were separated on an SDS-PAGE gel and probed for YME1L1. Actin was probed as a loading control. Right , Relative YME1L1 levels are shown for each condition (n = 4 biological replicates). (F) TNFα and PTGS2 transcript levels relative to Actin were measured in HTR cells treated with Control (water), LPS, or LPS +20 μM MitoTempo (n = 4 biological replicates). (G) TNFα and PTGS2 relative transcript levels in HTR cells treated with Control (DMSO), LPS (LPS + DMSO) or LPS +25 μM NOX2 inhibitor (n = 4 biological replicates). (H) Equal amounts of YME1L1 −/− cells overexpressing WT or various mutants of YME1L1 were treated as control (water) or with LPS (1 μg/mL). Lysates were separated on an SDS-PAGE gel and probed for MNRR1, STARD7, and YME1L1. Actin was probed as a loading control.

Article Snippet: α-total CHK2 , Cell Signaling Technology , Cat # 6334; RRID: AB_11178526.

Techniques: Generated, Control, SDS Page

Journal: iScience

Article Title: Lipopolysaccharide induces placental mitochondrial dysfunction in murine and human systems by reducing MNRR1 levels via a TLR4-independent pathway

doi: 10.1016/j.isci.2022.105342

Figure Lengend Snippet:

Article Snippet: α-total CHK2 , Cell Signaling Technology , Cat # 6334; RRID: AB_11178526.

Techniques: Immunostaining, Recombinant, Luciferase, Reporter Assay, Plasmid Preparation, Purification, Fractionation, cDNA Synthesis, Mutagenesis, Isolation, Transfection, Generated, Software

Journal: Molecular cancer

Article Title: MutS homologue hMSH4: interaction with eIF3f and a role in NHEJ-mediated DSB repair.

doi: 10.1186/1476-4598-12-51

Figure Lengend Snippet: Figure 7 Immunoblotting analysis of IR-induced AKT (Ser473) activation. (A) The levels of AKT activation were measured by AKT Ser473 phosphorylation in 293T, 293T/eIF3f and 293T/eIF3f-hMSH4 cells treated with 10 Gy IR in comparison to untreated controls. Levels of p53 Ser15 phosphorylation and the total protein levels of AKT, hMRE11 and p53 were also analyzed. α-tubulin was used as a loading control. (B) The levels of Chk2 activation (Chk2 Thr68 phosphorylation) in 293T, 293T/eIF3f and 293T/eIF3f-hMSH4 cells in response to 10 Gy IR. Cell lysates were prepared at 1 hr post IR treatment. Untreated cells were analyzed as controls, while α-tubulin was used as a loading control.

Article Snippet: Antibodies used in this study include: α-Flag M2 (Sigma), α-α-tubulin (Sigma), α-GST (GE Healthcare Life Sciences, Piscataway, NJ), α-eIF3f (Rockland Immunochemical Inc.), α-Myc (Clontech), and α-hMSH4 [20], α-p-AKT (Ser473) (Cell Signaling Technology, Danvers, MA), α-pS15-p53 (Cell Signaling Technology), α-total p53 (Cell Signaling Technology), α-phospho-Chk2 Thr68 (Cell Signaling Technology). α-hMRE11 (Novus Biologicals, Littleton, CO), α-HDAC3 (Abcam), α-prefolding 3 (VBP1) (K-13, Santa Cruz), α-Rad51 (ab-1) (Novus).

Techniques: Western Blot, Activation Assay, Phospho-proteomics, Comparison, Control

Journal: Nanoscale

Article Title: 111 In-labelled polymeric nanoparticles incorporating a ruthenium-based radiosensitizer for EGFR-targeted combination therapy in oesophageal cancer cells † †Electronic supplementary information (ESI) available: Supplementary figures and tables. See DOI: 10.1039/c7nr09606b

doi: 10.1039/c7nr09606b

Figure Lengend Snippet: (a) CLSM images of OE21 cells treated with 111 In-hEGF-PLGA or 111 In-hEGF-PLGA- Ru1 (1 MBq mL –1 , 24 h) followed by immunofluorescence staining for γH2AX (green). DNA was stained with DAPI (blue). Equivalent non-radiolabelled hEGF-PLGA- Ru1 treatment was included for comparison. (b) Quantification of γH2AX foci/nucleus (in plane of view) for cells treated as in (a). Data average of two independent repeats ± S.D. A minimum of 100 nuclei per condition were counted. (c) Immunoblotting of OE21 whole-cell extracts after 24 h treatment with non-radiolabelled hEGF-PLGA- Ru1 , 111 In-hEGF-PLGA or 111 In-hEGF-PLGA- Ru1 (1 MBq mL –1 ) using anti-pChk2 (Thr68) or pChk1 (Ser345) antibodies, as indicated. Total Chk2 and Chk1 protein content is provided. β-Actin was used as a loading control. Cells treated with free 111 InCl 3 (specific activity equivalent) or free Ru1 (concentration equivalent) were included for comparison.

Article Snippet: Antibodies: p-Chk1 (Ser345) and p-Chk2 (Thr68) (Cell Signaling), γH2AX (Millipore), total Chk1 (Santa Cruz), total Chk2, α-tubulin (both Abcam) and β-actin (Sigma).

Techniques: Immunofluorescence, Staining, Western Blot, Activity Assay, Concentration Assay

Journal:

Article Title: DNA Damage during Reoxygenation Elicits a Chk2-Dependent Checkpoint Response

doi: 10.1128/MCB.26.5.1598-1609.2006

Figure Lengend Snippet: Reoxygenation-induced G2 arrest is dependent on the protein kinase Chk2. (A) Cells were untreated (−) or treated with 15 h of hypoxia (H), followed by indicated hours of reoxygenation (R). Cell types are HCT116 Chk2+/+, HCT116 Chk2+/+ stably expressing Chk2T68A, HCT116 Chk2−/−, HCT116 Chk2−/− stably expressing empty vector, and HCT116 Chk2−/− stably expressing Chk2+/+. Histogram profiles of DNA content were derived from FACS analysis from PI staining. The percentage of cells in the G2 phase of the cell cycle is shown for 9 h of reoxygenation next to each histogram. (B) Confirmation of stable integration of Chk2+/+-expressing vector in HCT116 Chk2−/− cells. Total protein (50 μg) was run and transferred to a polyvinylidene difluoride membrane and then probed with total Chk2 antibody and an antibody to GAPDH for loading control.

Article Snippet: Total Chk2 (1:1,000) (Upstate Biotechnology), phospho-Thr 68 Chk2 (1:800) (Cell Signaling), Cdc25A (1:500) (Santa Cruz), total Cdc25C (1:1,000) (Santa Cruz), phospho-S216 Cdc25C (1:100) (Santa Cruz), α-tubulin (1:5,000) (Research Diagnostics Inc.), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:5,000) (Research Diagnostics Inc.) were all detected following separation on an 8% acrylamide gel.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Derivative Assay, Staining

Journal:

Article Title: DNA Damage during Reoxygenation Elicits a Chk2-Dependent Checkpoint Response

doi: 10.1128/MCB.26.5.1598-1609.2006

Figure Lengend Snippet: Reoxygenation-induced G2 arrest after Chk2 siRNA treatment is attenuated in the RKO colon carcinoma cell line. (A) Chk2 protein knockdown in RKO colon cancer cells using Chk2 siRNA. RKO cells treated with or without Chk2 siRNA were subjected to 15 h of hypoxia (H) followed by 3, 6, or 9 h of reoxygenation (R). Shown here are histograms generated by FACS analysis of PI-stained DNA. (B) Knockdown of Chk2 protein by siRNA. Immunoblots of protein extracts from cells treated with 15 h of hypoxia followed by reoxygenation were probed with antibody to total Chk2 (tChk2), total p53, and GAPDH. (C) G2 arrest induced by Chk2 siRNA treatment was specific. NT-siRNA oligonucleotide treatment had no effect on cell cycle response to hypoxia and reoxygenation. Histograms were generated by FACS analysis of PI-stained DNA. (D) Western blots on protein extracts from NT-siRNA-treated cells subjected to hypoxia and reoxygenation probed with antibodies to total Chk2 and α-tubulin. (E) Quantification of sub-G1 populations in RKO cells treated with or without Chk2 siRNA and exposed to hypoxia and reoxygenation.

Article Snippet: Total Chk2 (1:1,000) (Upstate Biotechnology), phospho-Thr 68 Chk2 (1:800) (Cell Signaling), Cdc25A (1:500) (Santa Cruz), total Cdc25C (1:1,000) (Santa Cruz), phospho-S216 Cdc25C (1:100) (Santa Cruz), α-tubulin (1:5,000) (Research Diagnostics Inc.), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:5,000) (Research Diagnostics Inc.) were all detected following separation on an 8% acrylamide gel.

Techniques: Generated, Staining, Western Blot

Journal:

Article Title: DNA Damage during Reoxygenation Elicits a Chk2-Dependent Checkpoint Response

doi: 10.1128/MCB.26.5.1598-1609.2006

Figure Lengend Snippet: Chk2 phosphorylation is ATM dependent during hypoxia and reoxygenation. (A) HCT116 Chk2+/+ and Chk2−/− cells were treated with 15 h hypoxia (H) and reoxygenation (R) for the indicated hours. Protein lysates were subjected to Western blotting. Phospho-T68 Chk2 is shown in the upper panel, total Chk2 protein is shown in the middle panel, and GAPDH loading control is shown in the bottom panel. Lysates from HCT116 Chk2−/− cells failed to react with either Chk2 antibody (right panels). (B) Chk2 phosphorylation in a lymphoblastoid cell line treated with hypoxia and reoxygenation. The lymphoblastoid cell lines GM0536 (ATM+/+) and GM1526 (ATM−/−) were subjected to 15 h of hypoxia and various hours of reoxygenation. Western blots of protein lysates were probed with antibodies to phospho-S1981 ATM (pATM), total ATM (tATM), phospho-T68 Chk2 (pChk2), and α-tubulin (Tubulin).

Article Snippet: Total Chk2 (1:1,000) (Upstate Biotechnology), phospho-Thr 68 Chk2 (1:800) (Cell Signaling), Cdc25A (1:500) (Santa Cruz), total Cdc25C (1:1,000) (Santa Cruz), phospho-S216 Cdc25C (1:100) (Santa Cruz), α-tubulin (1:5,000) (Research Diagnostics Inc.), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:5,000) (Research Diagnostics Inc.) were all detected following separation on an 8% acrylamide gel.

Techniques: Western Blot

Journal:

Article Title: DNA Damage during Reoxygenation Elicits a Chk2-Dependent Checkpoint Response

doi: 10.1128/MCB.26.5.1598-1609.2006

Figure Lengend Snippet: Chk2 targets are phosphorylated following hypoxia and reoxygenation. (A) Cdc25C is phosphorylated during reoxygenation and is dependent on Chk2. HCT116 Chk2+/+ and Chk2−/− cells were treated with 15 h of hypoxia (H) and various times of reoxygenation (R). The top panel shows phosphorylated (p) forms migrating more slowly than total Cdc25C (t). An α-tubulin loading control is shown in the bottom panel. (B) Lambda protein phosphatase (λptse) treatment of protein extracts from HCT116 Chk2+/+ and Chk2−/− cells harvested after hypoxia and reoxygenation. (C) Cdc25A is not phosphorylated or degraded during hypoxia or reoxygenation treatment. HCT116 Chk2+/+ and Chk2−/− protein lysates from cells treated with 15 h of hypoxia and indicated times of reoxygenation were subjected to Western blotting and detection with total Cdc25A antibody, as shown in the top panel. GAPDH loading control is shown in the bottom panel. (D) Cdc25A is phosphorylated and degraded in response to UV stress. HCT116 Chk2+/+ and Chk2−/− cells were subjected to 10 J/m2 UV light. Cells were lysed after 1 h. Cdc25A was detected with total Cdc25A antibody after Western blotting, as shown in the top panel. α-Tubulin loading control is shown in the bottom panel. (E and F) Cdc2 phosphorylation during reoxygenation is Chk2 dependent. Protein lysates from HCT116 Chk2+/+ and Chk2−/− cells or from RKO cells treated with Chk2 siRNA were treated with 15 h hypoxia and indicated times of reoxygenation and were probed with phospho-specific Tyr15 Cdc2 (pCdc2) antibody after Western blotting, as shown in the upper panels. GAPDH loading control is shown in the lower panels.

Article Snippet: Total Chk2 (1:1,000) (Upstate Biotechnology), phospho-Thr 68 Chk2 (1:800) (Cell Signaling), Cdc25A (1:500) (Santa Cruz), total Cdc25C (1:1,000) (Santa Cruz), phospho-S216 Cdc25C (1:100) (Santa Cruz), α-tubulin (1:5,000) (Research Diagnostics Inc.), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:5,000) (Research Diagnostics Inc.) were all detected following separation on an 8% acrylamide gel.

Techniques: Western Blot

Journal:

Article Title: DNA Damage during Reoxygenation Elicits a Chk2-Dependent Checkpoint Response

doi: 10.1128/MCB.26.5.1598-1609.2006

Figure Lengend Snippet: Chk2−/− cells are sensitive to hypoxia and reoxygenation. (A) Colony-forming efficiency. Decreased survival of HCT116 Chk2−/− cells during reoxygenation is proportional to length of exposure to hypoxia. HCT116 Chk2+/+ and Chk2−/− cells were untreated or treated with hypoxia for 4, 8, 12, or 24 h and then incubated undisturbed for 2 weeks. Colonies were stained with crystal violet and counted. Results are expressed in log values. (B) Chk2+/+ and Chk2−/− cells are equally as sensitive to ionizing radiation. HCT116 Chk2+/+ and Chk2−/− cells were subjected to 0, 2, 4, 6, and 8 Gy gamma irradiation (γIR) and then allowed to grow undisturbed for 2 weeks. Colonies were stained with crystal violet and counted. Colony numbers are expressed in log values. (C) Increased apoptosis in Chk2−/− cells during reoxygenation. HCT116 Chk2+/+ and Chk2−/− cells were either untreated or placed in hypoxia for 15 h. Upon reoxygenation, cells were stained with Hoechst 33342 and PI. Nuclear morphology was assessed for each condition. Graphs are presented as the percentages of apoptotic cells in the total population. (D) Cells deficient in ATM are sensitive to reoxygenation. ATM+/+ (YZ3) or ATM−/− (pEBS) cells were treated with 15 h of hypoxia and then stained with Hoechst 33342 and PI at the indicated times. Results are reported as the percentages of apoptotic cells in the total population.

Article Snippet: Total Chk2 (1:1,000) (Upstate Biotechnology), phospho-Thr 68 Chk2 (1:800) (Cell Signaling), Cdc25A (1:500) (Santa Cruz), total Cdc25C (1:1,000) (Santa Cruz), phospho-S216 Cdc25C (1:100) (Santa Cruz), α-tubulin (1:5,000) (Research Diagnostics Inc.), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:5,000) (Research Diagnostics Inc.) were all detected following separation on an 8% acrylamide gel.

Techniques: Incubation, Staining, Irradiation

Journal:

Article Title: DNA Damage during Reoxygenation Elicits a Chk2-Dependent Checkpoint Response

doi: 10.1128/MCB.26.5.1598-1609.2006

Figure Lengend Snippet: Chk2+/+ cells have a survival advantage during reoxygenation. HCT116 Chk2+/+ and Chk2−/− cells stably expressing EYFP and ECFP were mixed in equal numbers and plated onto slides and then either left untreated, treated with hypoxia overnight, or treated and then allowed to grow for 1 or 2 days under normal culture conditions. Blue and yellow cells were counted at each time point. Cell numbers relative to the starting population are plotted above.

Article Snippet: Total Chk2 (1:1,000) (Upstate Biotechnology), phospho-Thr 68 Chk2 (1:800) (Cell Signaling), Cdc25A (1:500) (Santa Cruz), total Cdc25C (1:1,000) (Santa Cruz), phospho-S216 Cdc25C (1:100) (Santa Cruz), α-tubulin (1:5,000) (Research Diagnostics Inc.), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:5,000) (Research Diagnostics Inc.) were all detected following separation on an 8% acrylamide gel.

Techniques: Stable Transfection, Expressing

Journal:

Article Title: DNA Damage during Reoxygenation Elicits a Chk2-Dependent Checkpoint Response

doi: 10.1128/MCB.26.5.1598-1609.2006

Figure Lengend Snippet: Reoxygenation-dependent damage response is attenuated by ROS scavenger NAC. (A) The comet assay detects increasing damage during reoxygenation in HCT116 Chk2+/+ and Chk2−/− cells. Hypoxic samples were trypsinized and lysed under hypoxia conditions. Reoxygenation samples were harvested after indicated times. (B) HCT116 cells were treated with 10 mM NAC prior to hypoxia (H) and reoxygenation (R). After fixation, cell cycle distributions were assessed by FACS analysis of PI-stained DNA.

Article Snippet: Total Chk2 (1:1,000) (Upstate Biotechnology), phospho-Thr 68 Chk2 (1:800) (Cell Signaling), Cdc25A (1:500) (Santa Cruz), total Cdc25C (1:1,000) (Santa Cruz), phospho-S216 Cdc25C (1:100) (Santa Cruz), α-tubulin (1:5,000) (Research Diagnostics Inc.), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:5,000) (Research Diagnostics Inc.) were all detected following separation on an 8% acrylamide gel.

Techniques: Single Cell Gel Electrophoresis, Staining

Journal: Molecular Cancer

Article Title: MutS homologue hMSH4: interaction with eIF3f and a role in NHEJ-mediated DSB repair

doi: 10.1186/1476-4598-12-51

Figure Lengend Snippet: Immunoblotting analysis of IR-induced AKT (Ser473) activation. ( A ) The levels of AKT activation were measured by AKT Ser473 phosphorylation in 293T, 293T/eIF3f and 293T/eIF3f-hMSH4 cells treated with 10 Gy IR in comparison to untreated controls. Levels of p53 Ser15 phosphorylation and the total protein levels of AKT, hMRE11 and p53 were also analyzed. α-tubulin was used as a loading control. ( B ) The levels of Chk2 activation (Chk2 Thr68 phosphorylation) in 293T, 293T/eIF3f and 293T/eIF3f-hMSH4 cells in response to 10 Gy IR. Cell lysates were prepared at 1 hr post IR treatment. Untreated cells were analyzed as controls, while α-tubulin was used as a loading control.

Article Snippet: Antibodies used in this study include: α-Flag M2 (Sigma), α-α-tubulin (Sigma), α-GST (GE Healthcare Life Sciences, Piscataway, NJ), α-eIF3f (Rockland Immunochemical Inc.), α-Myc (Clontech), and α-hMSH4 [ ], α-p-AKT (Ser473) (Cell Signaling Technology, Danvers, MA), α-pS15-p53 (Cell Signaling Technology), α-total p53 (Cell Signaling Technology), α-phospho-Chk2 Thr68 (Cell Signaling Technology). α-hMRE11 (Novus Biologicals, Littleton, CO), α-HDAC3 (Abcam), α-prefolding 3 (VBP1) (K-13, Santa Cruz), α-Rad51 (ab-1) (Novus).

Techniques: Western Blot, Activation Assay, Phospho-proteomics, Comparison, Control

Journal: Experimental dermatology

Article Title: Curcuminoids Activate p38 Map Kinases and Promote UVB-Dependent Signaling in Keratinocytes

doi: 10.1111/j.1600-0625.2010.01081.x

Figure Lengend Snippet: A) PHKs were treated for two hours with 20μM curcumin or tetrahydrocurcumin (THC) at 50, 100, 200 μM. Control 1 was lysed at the beginning of the time course, 2-at the end. Cells subjected to western blot analysis to detect phosphorylated p53 (serine 15) and total p53. N=3 B) PHKs were treated with DMSO or 200μM tetrahydrocurcumin (THC) for the indicated times (hours). Cells were analyzed as in A. N=2. C) PHKs were not stimulated or stimulated with 20μM curcumin, 200μM tetrahydrocurcumin (THC), or DMSO. Where indicated, cells were irradiated with UVB (18 mJ/cm2). Two hours post-irradiation, cells were lysed and subjected to analysis as in A. N=2 D) Densitometric analysis of p53 activation in experiment C. Curcuminoids promote UVB-induced phosphorylation of p53. Error bars indicate standard deviation. Asterisk indicates a p=0.09 compared to UV alone. E) PHKs were treated with 200μM tetrahydrocurcumin (THC) or DMSO for the indicated times (hours). Cell lysates were analyzed by western blotting to detect levels of p21 and β-actin. N=2

Article Snippet: Antibodies - α-p44/42 MAP kinase antibody and α-phospho-p44/42 MAP kinase antibody (9101, 9102, Cell Signaling Technology) 1:1000 to detect total and phosphorylated p44/42 MAP kinase proteins. α-p38 antibody, α-phospho-p38 antibody (9212, 9211, Cell Signaling Technology), 1:1000 to detect total and phosphorylated p38 MAP kinase proteins. α-p21 mouse mAb(F-5), sc-6246, at 1:500 (Santa Cruz Biotechnology). α-p53 mouse mAb (1C12), 2524, Cell Signaling at 1:1000. α-phospho-p53 antibody (serine 15) 9284, Cell Signaling, at 1:500. α-β-actin (Abcam) antibody was used at 1:5000.

Techniques: Control, Western Blot, Irradiation, Activation Assay, Phospho-proteomics, Standard Deviation

Journal: Experimental dermatology

Article Title: Curcuminoids Activate p38 Map Kinases and Promote UVB-Dependent Signaling in Keratinocytes

doi: 10.1111/j.1600-0625.2010.01081.x

Figure Lengend Snippet: PHKs were not stimulated or stimulated with DMSO or 200μM tetrahydrocurcumin (THC). Where indicated, cells were irradiated subsequently with UVB (18 mJ/cm2). Duplicate cell sets were exposed to the p38 MAP kinase inhibitor SB202190 or its control compound SB202474 for 30 minutes prior to lysis. Two hours post-irradiation, cells were lysed and subjected to SDS-PAGE followed by western blotting to detect phosphorylated p38, total p38, phosphorylated p53 (serine 15), and total p53. N=3.

Article Snippet: Antibodies - α-p44/42 MAP kinase antibody and α-phospho-p44/42 MAP kinase antibody (9101, 9102, Cell Signaling Technology) 1:1000 to detect total and phosphorylated p44/42 MAP kinase proteins. α-p38 antibody, α-phospho-p38 antibody (9212, 9211, Cell Signaling Technology), 1:1000 to detect total and phosphorylated p38 MAP kinase proteins. α-p21 mouse mAb(F-5), sc-6246, at 1:500 (Santa Cruz Biotechnology). α-p53 mouse mAb (1C12), 2524, Cell Signaling at 1:1000. α-phospho-p53 antibody (serine 15) 9284, Cell Signaling, at 1:500. α-β-actin (Abcam) antibody was used at 1:5000.

Techniques: Irradiation, Control, Lysis, SDS Page, Western Blot

Journal: Experimental dermatology

Article Title: Curcuminoids Activate p38 Map Kinases and Promote UVB-Dependent Signaling in Keratinocytes

doi: 10.1111/j.1600-0625.2010.01081.x

Figure Lengend Snippet: Curcuminoids differentially regulate p44/42 and p38 MAP kinases. Activation of p38 leads to p53 phosphorylation, and increased p21 levels. The result of these cellular events is a block in G2/M and decreased S-phase. Curcuminoids enhance p53 function in PHKs.

Article Snippet: Antibodies - α-p44/42 MAP kinase antibody and α-phospho-p44/42 MAP kinase antibody (9101, 9102, Cell Signaling Technology) 1:1000 to detect total and phosphorylated p44/42 MAP kinase proteins. α-p38 antibody, α-phospho-p38 antibody (9212, 9211, Cell Signaling Technology), 1:1000 to detect total and phosphorylated p38 MAP kinase proteins. α-p21 mouse mAb(F-5), sc-6246, at 1:500 (Santa Cruz Biotechnology). α-p53 mouse mAb (1C12), 2524, Cell Signaling at 1:1000. α-phospho-p53 antibody (serine 15) 9284, Cell Signaling, at 1:500. α-β-actin (Abcam) antibody was used at 1:5000.

Techniques: Activation Assay, Phospho-proteomics, Blocking Assay

Journal: Cell Death & Disease

Article Title: SMAR1 coordinates HDAC6-induced deacetylation of Ku70 and dictates cell fate upon irradiation

doi: 10.1038/cddis.2014.397

Figure Lengend Snippet: SMAR1 modulates the deacetylation of Ku70 via HDAC6. ( a ) Acetylation-specific IP assay in control cells, cells overexpressed for SMAR1 (Ad-SM), NS and sh3 lentivirus-transduced cells. Cell lysates were immunoprecipitated with anti-acetyllysine (AcK) antibody and immunoblotted with Ku70 antibody. Graph (lower panel) represents the densitometry quantification of mean Ku70 acetylation±S.D. of three independent experiments using QuantityOne software (VersaDoc imaging system, BioRad). ( b ) Sequential IP assay to check the in vivo association of SMAR1, Ku70 and HDAC6 in one complex. HCT116 cell lysate (1 mg) was first immunoprecipitated with SMAR1 antibody and eluate was subsequently probed with indicated antibodies, followed by second IP with HDAC6 antibody and immunoblotting with mentioned antibodies. ( c ) IP assay in NS and sh3 lentivirus-transduced HCT116 cells to investigate the association between Ku70 and HDAC6. Whole-cell lysates were immunoprecipitated with either Ku70 or HDAC6, and then immunoblotted with indicated antibodies. ( d ) In silico analysis of HDAC6–SMAR1–Ku70 complex. C-terminal of SMAR1 (green spheres) is sandwiched between Ku70 (magenta) and HDAC6 (cyan) (upper panel). Anterior and posterior view of the triple complex (lower panel). ( e ) Protein lysates extracted from control and tubacin-treated (10 μ M, 2 h) HCT116 cells, which were previously transduced with either Ad-SM or sh3 lentivirus were analyzed for Ku70 acetylation by IP with anti-AcK antibody and immunoblotting for Ku70. ( f ) Western blot analysis to check IR-induced recruitment of SMAR1 and Ku70 to the chromatin in HCT116 cells that were previously transduced with either NS or sh3 lentivirus

Article Snippet: The following primary antibodies were used: SMAR1/BANP (Bethyl Laboratories, Montgomery, AL, USA), Ku70, Ku80, active caspase-3, ATM, phospho-ATM Ser1981, cleaved PARP, F1 α , active Bax (6A7), cytochrome c , α -tubulin, DNA-PKcs (Santa Cruz, Santa Cruz, CA, USA), Flag, actin, HDAC6 (Sigma), acetyllysine, H3, H4, phospho-Chk2 Thr68, total Chk2, phospho-Cdc25C Ser216, phospho-Cdc12 Tyr15, phospho-H3 Ser10, Bax (Cell Signaling, Frankfurt, Germany) and γ H2AX-FITC (upstate).

Techniques: Immunoprecipitation, Software, Imaging, In Vivo, Western Blot, In Silico, Transduction

Journal: Cell Death & Disease

Article Title: SMAR1 coordinates HDAC6-induced deacetylation of Ku70 and dictates cell fate upon irradiation

doi: 10.1038/cddis.2014.397

Figure Lengend Snippet: Proposed model for the role of SMAR1 in IR-induced DNA damage repair. ( a ) SMAR1 forms a triple complex with Ku70 and HDAC6, thus maintains Ku70 in a deacetylated state. Upon IR, SMAR1 gets phosphorylated by ATM at Ser370 and favors the recruitment of deacetylated Ku70 to the DSB sites. Moreover, SMAR1 also favors G2/M arrest, thus providing damaged cells ample time for efficient repair. On the other hand, deacetylated Ku70 interacts with Bax and regulates Bax-mediated apoptosis. ( b ) SMAR1 knockdown results in increased acetylation of Ku70 due to perturbation of triple complex between SMAR1, Ku70 and HDAC6. Acetylated Ku70 does not bind to DSB sites, leading to inefficient DNA repair and does not interact with Bax as well, resulting in apoptotic translocation of Bax to mitochondria

Article Snippet: The following primary antibodies were used: SMAR1/BANP (Bethyl Laboratories, Montgomery, AL, USA), Ku70, Ku80, active caspase-3, ATM, phospho-ATM Ser1981, cleaved PARP, F1 α , active Bax (6A7), cytochrome c , α -tubulin, DNA-PKcs (Santa Cruz, Santa Cruz, CA, USA), Flag, actin, HDAC6 (Sigma), acetyllysine, H3, H4, phospho-Chk2 Thr68, total Chk2, phospho-Cdc25C Ser216, phospho-Cdc12 Tyr15, phospho-H3 Ser10, Bax (Cell Signaling, Frankfurt, Germany) and γ H2AX-FITC (upstate).

Techniques: Translocation Assay

Journal: Molecular Cancer

Article Title: MutS homologue hMSH4: interaction with eIF3f and a role in NHEJ-mediated DSB repair

doi: 10.1186/1476-4598-12-51

Figure Lengend Snippet: The interaction between hMSH4 and eIF3f. ( A ) Yeast-two hybrid analysis of the hMSH4-eIF3f interaction. A series of hMSH4 truncation mutants were utilized to determine the eIF3f-interacting domain on hMSH4. Positive interactions were ascertained by the transcription activation of ADE2 and HIS3 reporter genes. ( B ) Co-IP analysis of hMSH4-eIF3f interaction in human cells. Immunoblotting with α-tubulin was used as a loading control. ( C ) Co-IP analysis of the interaction between eIF3f and hMSH4sv. Full-length hMSH4 and hMSH4sv were expressed separately with eIF3f and the respective interactions were analyzed by co-IP. ( D ) GST pull-down analysis of hMSH4-eIF3f interaction and determination of the hMSH4-interacting domain on eIF3f. The full-length eIF3f and four truncated fragments were expressed as GST-fusion proteins. GST pull-down experiments were performed with Glutathione-Sepharose 4B beads.

Article Snippet: Antibodies used in this study include: α-Flag M2 (Sigma), α-α-tubulin (Sigma), α-GST (GE Healthcare Life Sciences, Piscataway, NJ), α-eIF3f (Rockland Immunochemical Inc.), α-Myc (Clontech), and α-hMSH4 [ ], α-p-AKT (Ser473) (Cell Signaling Technology, Danvers, MA), α-pS15-p53 (Cell Signaling Technology), α-total p53 (Cell Signaling Technology), α-phospho-Chk2 Thr68 (Cell Signaling Technology). α-hMRE11 (Novus Biologicals, Littleton, CO), α-HDAC3 (Abcam), α-prefolding 3 (VBP1) (K-13, Santa Cruz), α-Rad51 (ab-1) (Novus).

Techniques: Activation Assay, Co-Immunoprecipitation Assay, Western Blot, Control

Journal: Molecular Cancer

Article Title: MutS homologue hMSH4: interaction with eIF3f and a role in NHEJ-mediated DSB repair

doi: 10.1186/1476-4598-12-51

Figure Lengend Snippet: eIF3f facilitates hMSH4 stabilization. ( A ) The effect of RNAi-mediated down-regulation of eIF3f on the levels of endogenous hMSH4 was analyzed in A549 cells. A mixture of eIF3f sh-1 and sh-2 RNAi constructs was used for transient transfection, and cells were collected and analyzed by immunoblotting at 48 hrs post-transfection. Immunoblotting with α-tubulin was used as a loading control. ( B ) Stable expression of eIF3f and hMSH4 in 293T/eIF3f and 293T/eIF3f-hMSH4 cell lines (from selected single clones). ( C ) Western blotting analysis of the levels of HDAC3, hRad51, and VBP1 expression in 293T, 293T/eIF3f, and 293T/eIF3f-hMSH4 cells. ( D ) Effects of reduced eIF3f expression on the levels of hMSH4 in the stable cell line 293T/eIF3f-hMSH4. Reduction of eIF3f expression was achieved by transient transfection of eIF3f RNAi constructs. ( E ) Nuclear and cytoplasmic distribution of hMSH4 and eIF3f proteins in response to IR. 293T/eIF3f-hMSH4 cells treated with 1 or 10 Gy IR were fractionated at 6 hrs post-treatment and the levels of hMSH4 and eIF3f in the nuclear and cytoplasmic fractions were determined by immunoblotting. α-tubulin was used as a marker for the cytoplasmic fraction.

Article Snippet: Antibodies used in this study include: α-Flag M2 (Sigma), α-α-tubulin (Sigma), α-GST (GE Healthcare Life Sciences, Piscataway, NJ), α-eIF3f (Rockland Immunochemical Inc.), α-Myc (Clontech), and α-hMSH4 [ ], α-p-AKT (Ser473) (Cell Signaling Technology, Danvers, MA), α-pS15-p53 (Cell Signaling Technology), α-total p53 (Cell Signaling Technology), α-phospho-Chk2 Thr68 (Cell Signaling Technology). α-hMRE11 (Novus Biologicals, Littleton, CO), α-HDAC3 (Abcam), α-prefolding 3 (VBP1) (K-13, Santa Cruz), α-Rad51 (ab-1) (Novus).

Techniques: Construct, Transfection, Western Blot, Control, Expressing, Clone Assay, Stable Transfection, Marker

Journal: Molecular Cancer

Article Title: MutS homologue hMSH4: interaction with eIF3f and a role in NHEJ-mediated DSB repair

doi: 10.1186/1476-4598-12-51

Figure Lengend Snippet: Effects of eIF3f - hMSH4 on cellular response to IR. ( A ) Clonogenic survival analysis of 293T, 293T/eIF3f and 293T/eIF3f-hMSH4 cells treated with 1 or 2 Gy IR. Colonies that contained at least 50 cells were counted and the percentage of cell survival was determined in reference to untreated control cells. The means of three individual experiments and the corresponding standard deviations (error bars) are presented. ( B ) Examination of γ-H2AX foci formation at 6 or 24 hrs post-exposure to 10 Gy IR. Percentages of cells possessing 15 or more foci/nucleus are graphically presented, and statistically significant differences are indicated with asterisks (* p < 0.05 and ** p < 0.01; Student’s t -test).

Article Snippet: Antibodies used in this study include: α-Flag M2 (Sigma), α-α-tubulin (Sigma), α-GST (GE Healthcare Life Sciences, Piscataway, NJ), α-eIF3f (Rockland Immunochemical Inc.), α-Myc (Clontech), and α-hMSH4 [ ], α-p-AKT (Ser473) (Cell Signaling Technology, Danvers, MA), α-pS15-p53 (Cell Signaling Technology), α-total p53 (Cell Signaling Technology), α-phospho-Chk2 Thr68 (Cell Signaling Technology). α-hMRE11 (Novus Biologicals, Littleton, CO), α-HDAC3 (Abcam), α-prefolding 3 (VBP1) (K-13, Santa Cruz), α-Rad51 (ab-1) (Novus).

Techniques: Control

Journal: Molecular Cancer

Article Title: MutS homologue hMSH4: interaction with eIF3f and a role in NHEJ-mediated DSB repair

doi: 10.1186/1476-4598-12-51

Figure Lengend Snippet: In vitro NHEJ assay. ( A ) Determination of NHEJ activities in extracts prepared from 293T, 293T/eIF3f and 293T/eIF3f-hMSH4 cells. DNA end joining reactions were performed by incubation of cell extracts with Sal I-digested plasmid DNA, and these reactions were terminated at the indicated time points. End joining products were separated by agarose gel electrophoresis. ‘S’ signifies linear DNA substrate, ‘D’ for joint dimer, and ‘M’ indicates all other higher order joint products. ( B ) Analysis of NHEJ activities in 293T extracts complemented with either 5 μg of BSA or 293T/eIF3f-hMSH4 extracts under identical buffer conditions. A representative gel image was shown on the left, and the relative NHEJ activities were quantified and graphed as a function of time (on the right).

Article Snippet: Antibodies used in this study include: α-Flag M2 (Sigma), α-α-tubulin (Sigma), α-GST (GE Healthcare Life Sciences, Piscataway, NJ), α-eIF3f (Rockland Immunochemical Inc.), α-Myc (Clontech), and α-hMSH4 [ ], α-p-AKT (Ser473) (Cell Signaling Technology, Danvers, MA), α-pS15-p53 (Cell Signaling Technology), α-total p53 (Cell Signaling Technology), α-phospho-Chk2 Thr68 (Cell Signaling Technology). α-hMRE11 (Novus Biologicals, Littleton, CO), α-HDAC3 (Abcam), α-prefolding 3 (VBP1) (K-13, Santa Cruz), α-Rad51 (ab-1) (Novus).

Techniques: In Vitro, NHEJ Assay, Incubation, Plasmid Preparation, Agarose Gel Electrophoresis

Journal: Molecular Cancer

Article Title: MutS homologue hMSH4: interaction with eIF3f and a role in NHEJ-mediated DSB repair

doi: 10.1186/1476-4598-12-51

Figure Lengend Snippet: Effects of eIF3f-hMSH4 on IR-induced cell cycle arrest. ( A ) Cell cycle analysis of 293T, 293T/eIF3f, and 293T/eIF3f-hMSH4 cells treated with different doses of IR. Cell cycle analysis was conducted either at 12 hrs or 24 hrs post-IR treatment, and percentages of cells in the G2/M phase are indicated. ( B ) Effect of IR treatment on eIF3f-hMSH4 interaction. 293T/eIF3f cells were transfected to express Myc-hMSH4, and cells were then irradiated with 10 Gy IR at 48 hrs post-transfection. Cell lysates were prepared, 2 hrs post-IR treatment, for subsequent co-IP analysis.

Article Snippet: Antibodies used in this study include: α-Flag M2 (Sigma), α-α-tubulin (Sigma), α-GST (GE Healthcare Life Sciences, Piscataway, NJ), α-eIF3f (Rockland Immunochemical Inc.), α-Myc (Clontech), and α-hMSH4 [ ], α-p-AKT (Ser473) (Cell Signaling Technology, Danvers, MA), α-pS15-p53 (Cell Signaling Technology), α-total p53 (Cell Signaling Technology), α-phospho-Chk2 Thr68 (Cell Signaling Technology). α-hMRE11 (Novus Biologicals, Littleton, CO), α-HDAC3 (Abcam), α-prefolding 3 (VBP1) (K-13, Santa Cruz), α-Rad51 (ab-1) (Novus).

Techniques: Cell Cycle Assay, Transfection, Irradiation, Co-Immunoprecipitation Assay

Journal: Molecular Cancer

Article Title: MutS homologue hMSH4: interaction with eIF3f and a role in NHEJ-mediated DSB repair

doi: 10.1186/1476-4598-12-51

Figure Lengend Snippet: Immunoblotting analysis of IR-induced AKT (Ser473) activation. ( A ) The levels of AKT activation were measured by AKT Ser473 phosphorylation in 293T, 293T/eIF3f and 293T/eIF3f-hMSH4 cells treated with 10 Gy IR in comparison to untreated controls. Levels of p53 Ser15 phosphorylation and the total protein levels of AKT, hMRE11 and p53 were also analyzed. α-tubulin was used as a loading control. ( B ) The levels of Chk2 activation (Chk2 Thr68 phosphorylation) in 293T, 293T/eIF3f and 293T/eIF3f-hMSH4 cells in response to 10 Gy IR. Cell lysates were prepared at 1 hr post IR treatment. Untreated cells were analyzed as controls, while α-tubulin was used as a loading control.

Article Snippet: Antibodies used in this study include: α-Flag M2 (Sigma), α-α-tubulin (Sigma), α-GST (GE Healthcare Life Sciences, Piscataway, NJ), α-eIF3f (Rockland Immunochemical Inc.), α-Myc (Clontech), and α-hMSH4 [ ], α-p-AKT (Ser473) (Cell Signaling Technology, Danvers, MA), α-pS15-p53 (Cell Signaling Technology), α-total p53 (Cell Signaling Technology), α-phospho-Chk2 Thr68 (Cell Signaling Technology). α-hMRE11 (Novus Biologicals, Littleton, CO), α-HDAC3 (Abcam), α-prefolding 3 (VBP1) (K-13, Santa Cruz), α-Rad51 (ab-1) (Novus).

Techniques: Western Blot, Activation Assay, Phospho-proteomics, Comparison, Control