Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antibacterial and Antibiofilm Activities of a Novel Synthetic Cyclic Lipopeptide against Cariogenic Streptococcus mutans UA159

doi: 10.1128/AAC.00776-17

Figure Lengend Snippet: In vitro susceptibilities of planktonic S. mutans UA159

Article Snippet: These results showed that CLP-4 is a promising agent that can effectively inhibit planktonic growth of S. mutans . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Antimicrobial agent MIC and MBC (μg/ml) by inoculum density of: 6 × 10 5 CFU/ml 2 × 10 7 CFU/ml MIC MBC MIC MBC CLP-4 2.8 6 5 20 Erythromycin 0.016 0.6 0.062 1 Chlorhexidine dihydrochloride 1.25 3.5 1.25 5 Open in a separate window In vitro susceptibilities of planktonic S. mutans UA159 table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Streptococcus mutans strain Serotype MIC (μg/ml) MBC (μg/ml) Reference or source UA159 c 2.8 6 56 ATCC 25175 c 3.0 9 57 LM7 e 2.0 8 58 JF243 c 3.0 <5 59 568-2v-5 2.0 <5 60 764 2.0 5 C. M. Levesque 768 2.0 6 C. M. Levesque Open in a separate window S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Techniques: In Vitro

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antibacterial and Antibiofilm Activities of a Novel Synthetic Cyclic Lipopeptide against Cariogenic Streptococcus mutans UA159

doi: 10.1128/AAC.00776-17

Figure Lengend Snippet: S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Article Snippet: These results showed that CLP-4 is a promising agent that can effectively inhibit planktonic growth of S. mutans . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Antimicrobial agent MIC and MBC (μg/ml) by inoculum density of: 6 × 10 5 CFU/ml 2 × 10 7 CFU/ml MIC MBC MIC MBC CLP-4 2.8 6 5 20 Erythromycin 0.016 0.6 0.062 1 Chlorhexidine dihydrochloride 1.25 3.5 1.25 5 Open in a separate window In vitro susceptibilities of planktonic S. mutans UA159 table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Streptococcus mutans strain Serotype MIC (μg/ml) MBC (μg/ml) Reference or source UA159 c 2.8 6 56 ATCC 25175 c 3.0 9 57 LM7 e 2.0 8 58 JF243 c 3.0 <5 59 568-2v-5 2.0 <5 60 764 2.0 5 C. M. Levesque 768 2.0 6 C. M. Levesque Open in a separate window S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Techniques: In Vitro

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antibacterial and Antibiofilm Activities of a Novel Synthetic Cyclic Lipopeptide against Cariogenic Streptococcus mutans UA159

doi: 10.1128/AAC.00776-17

Figure Lengend Snippet: Comparative killing kinetics of CLP-4. S. mutans UA159 cultures at a cell density of 6 × 105 CFU/ml were challenged with 5, 10, and 25 μg/ml CLP-4 under conditions of active growth in CDM supplemented with 0.5% (wt/vol) glucose (A) and against growth-arrested cells in CDM lacking any carbon source (B). Samples at time zero were enumerated prior to peptide treatment. Data shown are the means and standard deviations of three biological replicates from three independent experiments.

Article Snippet: These results showed that CLP-4 is a promising agent that can effectively inhibit planktonic growth of S. mutans . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Antimicrobial agent MIC and MBC (μg/ml) by inoculum density of: 6 × 10 5 CFU/ml 2 × 10 7 CFU/ml MIC MBC MIC MBC CLP-4 2.8 6 5 20 Erythromycin 0.016 0.6 0.062 1 Chlorhexidine dihydrochloride 1.25 3.5 1.25 5 Open in a separate window In vitro susceptibilities of planktonic S. mutans UA159 table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Streptococcus mutans strain Serotype MIC (μg/ml) MBC (μg/ml) Reference or source UA159 c 2.8 6 56 ATCC 25175 c 3.0 9 57 LM7 e 2.0 8 58 JF243 c 3.0 <5 59 568-2v-5 2.0 <5 60 764 2.0 5 C. M. Levesque 768 2.0 6 C. M. Levesque Open in a separate window S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Techniques:

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antibacterial and Antibiofilm Activities of a Novel Synthetic Cyclic Lipopeptide against Cariogenic Streptococcus mutans UA159

doi: 10.1128/AAC.00776-17

Figure Lengend Snippet: CLP-4 prevents S. mutans biofilm formation. (A) Biofilms inoculated with 2 × 107 CFU/ml were grown for 24 h in the presence of CLP-4, chlorhexidine, or erythromycin at concentrations ranging between 0.6× and 2× their respective MICs. Biofilm formation was quantified using crystal violet staining and expressed in percentage relative to untreated control. Shown are the means and standard deviations of three biological replicates from three independent experiments. *, P < 0.05; ***, P < 0.001 compared to untreated control. (B) Corresponding growth curve kinetics showing the MIC of CLP-4 on S. mutans UA159.

Article Snippet: These results showed that CLP-4 is a promising agent that can effectively inhibit planktonic growth of S. mutans . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Antimicrobial agent MIC and MBC (μg/ml) by inoculum density of: 6 × 10 5 CFU/ml 2 × 10 7 CFU/ml MIC MBC MIC MBC CLP-4 2.8 6 5 20 Erythromycin 0.016 0.6 0.062 1 Chlorhexidine dihydrochloride 1.25 3.5 1.25 5 Open in a separate window In vitro susceptibilities of planktonic S. mutans UA159 table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Streptococcus mutans strain Serotype MIC (μg/ml) MBC (μg/ml) Reference or source UA159 c 2.8 6 56 ATCC 25175 c 3.0 9 57 LM7 e 2.0 8 58 JF243 c 3.0 <5 59 568-2v-5 2.0 <5 60 764 2.0 5 C. M. Levesque 768 2.0 6 C. M. Levesque Open in a separate window S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Techniques: Staining, Control

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antibacterial and Antibiofilm Activities of a Novel Synthetic Cyclic Lipopeptide against Cariogenic Streptococcus mutans UA159

doi: 10.1128/AAC.00776-17

Figure Lengend Snippet: Effects of CLP-4 on preformed biofilms. S. mutans UA159 biofilms were established for 24 h and then treated with increasing concentrations (1× to 10× the MIC) of CLP-4, chlorhexidine, or erythromycin. (A) Antibiofilm activities were assessed by quantifying the cell viability of treated biofilms by colony enumeration on agar plates. The means and standard deviations of three biological replicates from three independent experiments are shown. **, P < 0.01; ***, P < 0.001 compared to untreated control. (B) Biofilms treated with 10× the MICs for each antimicrobial were fluorescently labeled using the LIVE/DEAD BacLight viability stain and visualized by confocal laser scanning microscopy. Shown are the top-down three-dimensional (3D) volume rendering of biofilms at a total magnification of ×400. Bottom images represent optical planes in the xz, and vertical thin images represent yz dimensions. Membrane-compromised bacteria are stained red with propidium iodide, while intact bacteria are stained green with SYTO 9. Areas highlighted by dashed lines indicate regions of interest (ROIs) viewed at a higher magnification. Dimensions shown are 387.5 μm by 387.5 μm by 16 μm. (C) ROIs are presented at ×2,300 magnification. Dimensions shown are 68.1 μm by 68.1 μm by 16 μm.

Article Snippet: These results showed that CLP-4 is a promising agent that can effectively inhibit planktonic growth of S. mutans . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Antimicrobial agent MIC and MBC (μg/ml) by inoculum density of: 6 × 10 5 CFU/ml 2 × 10 7 CFU/ml MIC MBC MIC MBC CLP-4 2.8 6 5 20 Erythromycin 0.016 0.6 0.062 1 Chlorhexidine dihydrochloride 1.25 3.5 1.25 5 Open in a separate window In vitro susceptibilities of planktonic S. mutans UA159 table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Streptococcus mutans strain Serotype MIC (μg/ml) MBC (μg/ml) Reference or source UA159 c 2.8 6 56 ATCC 25175 c 3.0 9 57 LM7 e 2.0 8 58 JF243 c 3.0 <5 59 568-2v-5 2.0 <5 60 764 2.0 5 C. M. Levesque 768 2.0 6 C. M. Levesque Open in a separate window S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Techniques: Control, Labeling, Staining, Confocal Laser Scanning Microscopy, Membrane, Bacteria

Journal:

Article Title: Arylamine N -acetyltransferases: a new inhibitor of apoptosis in HepG2 cells *

doi: 10.1631/jzus.B0820090

Figure Lengend Snippet: Effect of 0.4 μg/ml camptothecin (CAM) on the morphology of HepG2 cells observed using confocal microscopy with acridine orange (AO)/ethidium bromide (EB) double staining. (a) Control; (b) 0.4 µg/ml CAM group

Article Snippet: Cell line Human hepatocarcinoma cell line HepG2 was originally purchased from American Type Culture Collection (ATCC), USA and was maintained by the Institute of Cancer Studies of Heilongjiang Cancer Hospital, China.

Techniques: Confocal Microscopy, Double Staining, Control

Journal:

Article Title: Arylamine N -acetyltransferases: a new inhibitor of apoptosis in HepG2 cells *

doi: 10.1631/jzus.B0820090

Figure Lengend Snippet: Effect of 0.4 μg/ml camptothecin (CAM) on the apoptosis rate of HepG2 cells observed using flow cytometry with PI single staining. (a) Control; (b) 0.4 µg/ml CAM group %G1: Percent of G1 phase; %S: Percent of S phase; %G2: Percent of G2 phase; %Tot: Apoptosis rate, i.e., percent of sub-G0 phase; Cell No.: Cell numbers determined

Article Snippet: Cell line Human hepatocarcinoma cell line HepG2 was originally purchased from American Type Culture Collection (ATCC), USA and was maintained by the Institute of Cancer Studies of Heilongjiang Cancer Hospital, China.

Techniques: Flow Cytometry, Staining, Control

Journal:

Article Title: Arylamine N -acetyltransferases: a new inhibitor of apoptosis in HepG2 cells *

doi: 10.1631/jzus.B0820090

Figure Lengend Snippet: The effect of CAM on NAT activity in HepG2 cells observed using HPLC. (a) 2-AAF standard; (b) Control; (c) 0.4 µg/ml CAM group

Article Snippet: Cell line Human hepatocarcinoma cell line HepG2 was originally purchased from American Type Culture Collection (ATCC), USA and was maintained by the Institute of Cancer Studies of Heilongjiang Cancer Hospital, China.

Techniques: Activity Assay, Control

Journal:

Article Title: Arylamine N -acetyltransferases: a new inhibitor of apoptosis in HepG2 cells *

doi: 10.1631/jzus.B0820090

Figure Lengend Snippet: Changes of NAT activity in apoptotic HepG2 cells induced by CAM

Article Snippet: Cell line Human hepatocarcinoma cell line HepG2 was originally purchased from American Type Culture Collection (ATCC), USA and was maintained by the Institute of Cancer Studies of Heilongjiang Cancer Hospital, China.

Techniques: Activity Assay

Journal:

Article Title: Arylamine N -acetyltransferases: a new inhibitor of apoptosis in HepG2 cells *

doi: 10.1631/jzus.B0820090

Figure Lengend Snippet: The effect of iodoacetamide on the induction of apoptosis in HepG2 cells by CAM observed using flow cytometry. (a) Control; (b) 0.08 μg/ml CAM, apoptosis rate: 10.4%; (c) 0.08 μg/ml CAM+100 μmol/L idoacetamide, apoptosis rate: 17.4%; (d) 0.08 μg/ml CAM+500 μmol/L iodoacetamide, apoptosis rate: 30.6%; (e) 500 μmol/L iodoacetamide %G1: Percent of G1 phase; %S: Percent of S phase; %G2: Percent of G2 phase; %Tot: Apoptosis rate, i.e., percent of sub-G0 phase; Cell No., Cell numbers determined

Article Snippet: Cell line Human hepatocarcinoma cell line HepG2 was originally purchased from American Type Culture Collection (ATCC), USA and was maintained by the Institute of Cancer Studies of Heilongjiang Cancer Hospital, China.

Techniques: Flow Cytometry, Control

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Down-regulation of human endogenous retrovirus type K (HERV-K) viral env RNA in pancreatic cancer cells decreases cell proliferation and tumor growth

doi: 10.1158/1078-0432.CCR-17-0001

Figure Lengend Snippet: Detection of HERV-K Env protein expression in pancreatic cancer cells. A, expression of HERV-K Env protein was detected by immunofluorescence staining in seven PC cell lines as well as HPDE-E6E7 cells using 6H5 mAb (left panel). The expression of HERV-K Env SU protein on the cell membrane of Panc-1 or Panc-2 cells was demonstrated by confocal microscopy after immunofluorescence staining with 6H5 mAb; the control mIgG showed no staining (right panel). DAPI (blue color) was used for nuclear counterstain. B, expression of HERV-K Env protein was detected by immunoblot in seven PC cell lines but not in HPDE-E6E7 cells, using 6H5 mAb. ACTB was used as the control. The expression of HERV-K Env protein from high to low is AsPC-1 (3.58 fold), BxPC-3 (2.70 fold), MAI-PaCa-2 (2.11 fold), SU8686 (2.02 fold), Colo-357 (1.67 fold), Panc-1 (1.10 fold), and Panc-2 (1 fold). No expression of HERV-K was detected in HPDE-E6 E7 cells using Image J. C, expression of HERV-K Env protein on the various pancreatic cell lines was determined by cell ELISA using 6H5 mAb (left panel); mIgG was used as the control (right panel). The expression of HERV-K Env protein was detected to a greater extent in all seven PC cell lines tested than in HPDE-E6E7. D, strong expression of HERV-K was detected by immunohistochemistry in most PC tissues containing poorly differentiated adenocarcinoma. HERV-K was not expressed in normal or matched uninvolved, non-neoplastic pancreatic tissues. The expression of HERV- K was compared in a patient with moderate differentiated adenocarcinoma including tumor biopsy and matched non-neoplastic pancreatic tissues.

Article Snippet: The human PC cell lines Panc-1, Panc-2, Colo-357, SU8686, AsPC-1, MIA PaCa-2 and BxPC-3 were obtained from the American Type Culture Collection (ATCC) (Manassas, VA).

Techniques: Expressing, Immunofluorescence, Staining, Membrane, Confocal Microscopy, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Immunohistochemistry

Journal: ACS applied materials & interfaces

Article Title: Adaptation of Operational Parameters of Cold Atmospheric Plasma for in Vitro Treatment of Cancer Cells

doi: 10.1021/acsami.7b18653

Figure Lengend Snippet: CAP treatment induces a significant reduction in cell viability over time as a function of the exposure time. MDA-MB-231 breast cancer cells (a) and U87 glioblastoma (b) cells were treated with a CAP output voltage of 3.16 kV for 30, 60, 90, and 180 s. Immediately following treatment, RealTime-Glo MT Cell Viability Assay (Promega) was performed with a continuous read method to evaluate the response of each cell line in real time. The data were normalized to the untreated control recorded for each time point. The % cell viability of the untreated control assumed as 100% is indicated by the dotted line.

Article Snippet: Cell Culture The human cancer cell lines, U87 (glioblastoma) and MDA-MB-231 (breast adenocarcinoma), were obtained from the American Type Culture Collection (ATCC) (Manassas, VA).

Techniques: Viability Assay, Control

Journal: ACS applied materials & interfaces

Article Title: Adaptation of Operational Parameters of Cold Atmospheric Plasma for in Vitro Treatment of Cancer Cells

doi: 10.1021/acsami.7b18653

Figure Lengend Snippet: CAP exposure and apoptotic cell death. Annexin V assay of MD-MB-231 breast cancer cells (a) and U87 glioblastoma cells (b) treated with 60 s CAP at 3.16 kV. Apoptosis is presented as the percent stained fraction after treatment with respect to the untreated control.

Article Snippet: Cell Culture The human cancer cell lines, U87 (glioblastoma) and MDA-MB-231 (breast adenocarcinoma), were obtained from the American Type Culture Collection (ATCC) (Manassas, VA).

Techniques: Annexin V Assay, Staining, Control

Journal: ACS applied materials & interfaces

Article Title: Adaptation of Operational Parameters of Cold Atmospheric Plasma for in Vitro Treatment of Cancer Cells

doi: 10.1021/acsami.7b18653

Figure Lengend Snippet: CAP effect on cell viability varies as a function of discharge voltage. Breast cancer cells (MDA-MB-231) (a) and glioblastoma cells (U87) (b) were treated with a CAP output voltage of 3.16 and 3.71 kV for 60 s. A side-by-side comparison (c) revealed a difference in viability between the two cell lines as a function of the discharge voltage. RealTime-Glo MT Cell Viability Assay (Promega) was performed with a continuous read method for evaluating the cell response. The data were normalized to the untreated control condition (assumed at 100% viability) indicated by the dotted line.

Article Snippet: Cell Culture The human cancer cell lines, U87 (glioblastoma) and MDA-MB-231 (breast adenocarcinoma), were obtained from the American Type Culture Collection (ATCC) (Manassas, VA).

Techniques: Comparison, Viability Assay, Control

Journal: ACS applied materials & interfaces

Article Title: Adaptation of Operational Parameters of Cold Atmospheric Plasma for in Vitro Treatment of Cancer Cells

doi: 10.1021/acsami.7b18653

Figure Lengend Snippet: Accumulation of NO2− in cancer cells. MDA-MB-231 breast cancer cells (a,b) and U87 glioblastoma cells (c,d) were treated with a CAP output voltage of 3.16 or 3.71 kV for 60 s and were monitored over the course of 48 h. Accumulation of NO2− in cancer cells after CAP exposure was estimated by Griess reagent assay. NO2− levels in CAP-treated cells were measured and compared with those of untreated cells (control 1) and plasma-treated media (control 2).

Article Snippet: Cell Culture The human cancer cell lines, U87 (glioblastoma) and MDA-MB-231 (breast adenocarcinoma), were obtained from the American Type Culture Collection (ATCC) (Manassas, VA).

Techniques: Control, Clinical Proteomics

Journal: ACS applied materials & interfaces

Article Title: Adaptation of Operational Parameters of Cold Atmospheric Plasma for in Vitro Treatment of Cancer Cells

doi: 10.1021/acsami.7b18653

Figure Lengend Snippet: CAP-generated H2O2 becomes metabolically degraded by cancer cells. Cells were treated with a CAP output voltage of 3.16 or 3.71 kV for 60 s, and the effect of H2O2 was observed over the course of 48 h. CAP-treated MDA-MB-231 breast cancer cells (a,b) and U87 glioblastoma cells (c,d) metabolically degraded H2O2 starting immediately after CAP exposure. Levels measured in the CAP-treated cells were compared with those of control 1 (untreated cells) and 2 (CAP treated media).

Article Snippet: Cell Culture The human cancer cell lines, U87 (glioblastoma) and MDA-MB-231 (breast adenocarcinoma), were obtained from the American Type Culture Collection (ATCC) (Manassas, VA).

Techniques: Generated, Metabolic Labelling, Control

Journal: ACS applied materials & interfaces

Article Title: Adaptation of Operational Parameters of Cold Atmospheric Plasma for in Vitro Treatment of Cancer Cells

doi: 10.1021/acsami.7b18653

Figure Lengend Snippet: CAP exposure reduces the mitochondrial membrane potential in cancer cells. Immunofluorescence staining of actin (phalloidin) green, nucleus (DAPI) blue, and MitoTracker Red of breast cancer MDA-MB-231 cells (a,b) and glioblastoma U87 cells (e,f) at 24 h. Subcellular distribution of MitoTracker Red was revealed by confocal microscopy. The white arrows in (b,f) point to cells exhibiting a decrease in the red pixel intensity, which is indicative of a loss in mitochondrial membrane potential. Pixel intensity values of 50 cells (two frames per experiment) reveal the distribution of mitochondrial membrane potentials at 6, 12, and 24 h after CAP exposure (c,d); scale bar (bottom image): 10 μm.

Article Snippet: Cell Culture The human cancer cell lines, U87 (glioblastoma) and MDA-MB-231 (breast adenocarcinoma), were obtained from the American Type Culture Collection (ATCC) (Manassas, VA).

Techniques: Membrane, Immunofluorescence, Staining, Confocal Microscopy

Journal: ACS applied materials & interfaces

Article Title: Adaptation of Operational Parameters of Cold Atmospheric Plasma for in Vitro Treatment of Cancer Cells

doi: 10.1021/acsami.7b18653

Figure Lengend Snippet: CAP exposure reduces protein synthesis in cancer cells. Cells were treated with 3.16 kV CAP for 60 s and compared to the untreated (no CAP) control condition. The Click-iT Plus OPP Alexa Fluor 647 Protein Synthesis Assay Kit was used to measure protein synthesis. Fluorescence intensities of 50 MDA-MB-231 breast cancer cells and 50 U87 glioblastoma cells from two experiments were quantified. Subcellular distribution of Click-iT Plus OPP staining (green) was revealed by confocal microscopy (a,b,d,e). Expression of OPP decreased between 12 and 24 h in MDA-MB-231 cells (c) and between 6 and 24 h in U87 cells (f). A decrease in pixel intensity correlates with a decrease in the level of protein synthesis. White arrows in (b,e) indicate the loss of OPP (green) in cells. The cell nucleus is visualized with DAPI in blue. Scale bar (bottom image): 10 μm.

Article Snippet: Cell Culture The human cancer cell lines, U87 (glioblastoma) and MDA-MB-231 (breast adenocarcinoma), were obtained from the American Type Culture Collection (ATCC) (Manassas, VA).

Techniques: Control, Fluorescence, Staining, Confocal Microscopy, Expressing

Journal: mBio

Article Title: The Cellular NMD Pathway Restricts Zika Virus Infection and Is Targeted by the Viral Capsid Protein

doi: 10.1128/mBio.02126-18

Figure Lengend Snippet: The capsid protein of ZIKV interacts with the NMD pathway. (a) Ugandan ZIKV capsid (Ug Cap) (MR 766) and French Polynesian ZIKV capsid (Fp Cap) (H/PF/2013) PPI maps that show significant enrichment for host NMD-associated factors (purple), as identified by AP-MS (SAINTq probability score of > 0.9 and false-discovery rate [FDR] of <0.05). Ten interactions between Fp Cap and host NMD factors (hypergeometrical test, P value = 7.16 × 10 −10 ) and eight interactions between Ug Cap and host NMD factors ( P value = 3.45 × 10 −7 ) were identified. (b) Coimmunoprecipitation (co-IP) and Western blot analysis of HEK293T cells transfected with vector or Flag-tagged ZIKV capsid (H/PF/2013, Asian lineage) and harvested at 48 hpt to immunoprecipitate endogenous UPF3B. The upper band detected in the IP Capsid blot represents a nonspecific artifact. α-Flag, anti-Flag antibody. (c) Co-IP and Western blot analysis of HEK293T cells transfected with vector or Flag-tagged ZIKV capsid and harvested at 48 hpt to immunoprecipitate endogenous UPF1. (d) Myc tag co-IP and Western blot analysis of HEK293T cells transfected with Strep-tagged ZIKV capsid and Myc-UPF1 (wild type), Myc-UPF1-C126S (RNA-binding mutant) or Myc-UPF1-G495R/G497E (ATPase/helicase mutant) and harvested at 48 hpt to immunoprecipitate ZIKV capsid. (e) Western blot analysis of UPF1 levels in mock-infected and ZIKV-infected (PRVABC59, MOI of 1) Huh7 cells or mock-infected and ZIKV-infected (P6-740, MOI of 1) NPCs harvested at 48 hpi, with β-actin and ZIKV envelope (ZIKV E) or ZIKV capsid (ZIKV C) protein serving as loading and infection controls, respectively. Densitometric analyses were performed using ImageJ to quantify relative band intensities. Data are represented as means plus SEM. The P values were calculated by unpaired Student’s t test. * * , P ≤ 0.01; ** * , P ≤ 0.001. n = 3 independent experiments.

Article Snippet: HEK293T cells (ATCC) were maintained in DMEM/H21 medium supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 mM sodium pyruvate or DMEM with 10% FBS, 2 mM l- glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Protein-Protein interactions, Co-Immunoprecipitation Assay, Western Blot, Transfection, Plasmid Preparation, RNA Binding Assay, Mutagenesis, Infection

Journal: mBio

Article Title: The Cellular NMD Pathway Restricts Zika Virus Infection and Is Targeted by the Viral Capsid Protein

doi: 10.1128/mBio.02126-18

Figure Lengend Snippet: ZIKV capsid degrades UPF1, the master regulator of NMD, via a proteasome-dependent mechanism. (a) Western blot analysis of UPF1 levels in subcellular fractionated HEK293T cells transfected with vector or Flag-tagged ZIKV capsid (H/PF/2013, Asian lineage) for 48 h. GAPDH was used as a cytoplasmic marker, and SP1 was used as a nuclear marker to ensure optimal fractionation. Densitometric analyses were performed using ImageJ to quantify relative band intensities. Data are represented as means plus SEM. P values were calculated by unpaired Student’s t test. * * , P ≤ 0.01; ns, not significant. n = 3 independent experiments. (b) Western blot analysis of nuclear UPF1 levels in fractionated HEK293T cells transfected with vector or Flag-tagged ZIKV capsid for 48 h. Cells were treated with DMSO or increasing concentrations of the proteasome inhibitor bortezomib (Borte) for 24 h before harvest. Densitometric analyses were performed using ImageJ to quantify relative band intensities. Data are represented as means plus SEM. P values were calculated by one-way ANOVA with multiple comparisons. * , P ≤ 0.05; ns, not significant. n = 3 independent experiments. (c) Representative 3D confocal microscopy images of the nuclei of Huh7-Lunet cells transfected with Strep-tagged ZIKV capsid. Cells were treated at 24 hpt with DMSO or 10 nM bortezomib and processed for immunostaining at 48 hpt with antibodies against Strep tag (turquoise) and endogenous UPF1 (purple). DAPI (blue) was used to stain and define the nuclei. Each channel was reconstructed digitally for visualization of the 3D colocalization. The thresholded Mander’s correlation coefficients were determined, and P values were calculated by unpaired Student’s t test. * * , P ≤ 0.01. n = 8 cells per condition. Scale bars represent 3 μm.

Article Snippet: HEK293T cells (ATCC) were maintained in DMEM/H21 medium supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 mM sodium pyruvate or DMEM with 10% FBS, 2 mM l- glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Western Blot, Transfection, Plasmid Preparation, Marker, Fractionation, Confocal Microscopy, Immunostaining, Strep-tag, Staining

Journal: Advanced Science

Article Title: TRAF3IP3 Induces ER Stress‐Mediated Apoptosis with Protective Autophagy to Inhibit Lung Adenocarcinoma Proliferation

doi: 10.1002/advs.202411020

Figure Lengend Snippet: TRAF3IP3 interacts with STRN3 and promotes STRN3 localization of endoplasmic reticulum. A) STRN3 was identified as the binding partner of TRAF3IP3 by two replicate co‐IP mass spectrometry experiments. B) Molecular docking of TRAF3IP3 and STRN3. The purple molecule stands for TRAF3IP3, and the sky‐blue molecule stands for STRN3. C) Exogenous reciprocal coimmunoprecipitation (co‐IP) of TRAF3IP3 and STRN3 in 293T cells overexpressing Flag‐TRAF3IP3 and/or HA‐STRN3. D) Immunofluorescence staining of DAPI (blue), TRAF3IP3 (red) and STRN3 (green) in LUAD cell lines. E) The colocalization curves showed the colocalization level of STRN3 and TRAF3IP3 in the representative image. F) Schematic representation of wild‐type TRAF3IP3 and TRAF3IP3 deletion truncations. G Lysates from HEK293T cells transfected with HA‐tagged STRN3 and Flag‐tagged wild‐type TRAF3IP3 or TRAF3IP3 deletion truncations (1‐235, 236–450, 451–520, 521–551) were collected for coimmunoprecipitation and immunoblotting. H) A549 cell was transfected with indicated expression vectors, and STRN3, GRP78, ATF4, and Bax levels were detected by western blotting. I) Wild‐type TRAF3IP3 or deletion truncations were transfected into A549 cells, and confocal microscopy analysis of GRP78, STRN3 and DAPI was conducted. J) The colocalization curves showed the colocalization level of GRP78 and STRN3 in the representative image. K,L) A549 cell was transfected with control vector, wild‐type TRAF3IP3, specific containing transmembrane domain, and transmembrane domain‐deleted (rTM) plasmids; cell lysates were subjected to immunoblot with indicated antibodies.

Article Snippet: LUAD cell lines (PC9, A549 and H1299), bronchial epithelioid cells (BEAS‐2B), and Human embryonic kidney (HEK) 293T cells were all purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Mass Spectrometry, Immunofluorescence, Staining, Transfection, Western Blot, Expressing, Confocal Microscopy, Control, Plasmid Preparation

Journal: Communications Biology

Article Title: Anti-tumor effects of P-LPK-CPT, a peptide-camptothecin conjugate, in colorectal cancer

doi: 10.1038/s42003-022-04191-1

Figure Lengend Snippet: a The FITC-P-LPK conjugate emitted stronger green fluorescence in CRC cells than normal NCM460 cells. Bar,10 μm. b The FITC-P-LPK conjugate selectively binds to CRC tissues (HE staining) Bar, 50 μm. c , d The fluorescence intensity of the P-LPK peptide in CRC cells ( c ) and tissues ( d ) was significantly higher than that of normal cells and tissues ( n = 3, means ± SD, NCM460: P-CON vs P-LPK, p = 0.9385; Colo320HSR: P-CON vs P-LPK, p < 0.0001; HCT116: P-CON vs P-LPK, p < 0.0001; LoVo: P-CON vs P-LPK, p = 0.0219; Adjacent normal tissues: P-CON vs P-LPK, p = 0.9934;Colon cancer tissues: P-CON vs P-LPK, p < 0.0001) (* p < 0.05, **** p < 0.0001). e The binding site of the P-LPK peptide in HCT116 cells was investigated after labeling the peptide with Rhodamine. Bar, 25 μm.

Article Snippet: The human CRC cell lines Colo320HSR, HCT116, LoVo, HT29, and SW480 were all obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Fluorescence, Staining, Binding Assay, Labeling

Journal: Communications Biology

Article Title: Anti-tumor effects of P-LPK-CPT, a peptide-camptothecin conjugate, in colorectal cancer

doi: 10.1038/s42003-022-04191-1

Figure Lengend Snippet: a – c Illustration of how the P-LPK-CPT conjugate was synthesized (Details reference to materials and methods). d The colonal forming capability after different treatments with the P-LPK-CPT conjugate of HCT116, LoVo and NCM460 cells. e Cell proliferation was measured by CCK-8 ( n = 5, means ± SD, HCT116: CPT vs P-LPK-CPT, p = 0.0005; P-CON-CPT vs P-LPK-CPT, p = 0.0002; LoVo: CPT vs P-LPK-CPT, p < 0.0001; P-CON-CPT vs P-LPK-CPT, p = 0.0004) (** p < 0.01, *** p < 0.001, **** p < 0.0001). f The proportion of cells in the DNA synthesis state was qualified and one-way analysis of variance is used to test the difference between groups ( n = 3, means ± SD, HCT116: CPT vs P-LPK-CPT, p = 0.0017; P-CON-CPT vs P-LPK-CPT, p = 0.0015; LoVo: CPT vs P-LPK-CPT, p = 0.0016; P-CON-CPT vs P-LPK-CPT, p = 0.0018) (** p < 0.01).

Article Snippet: The human CRC cell lines Colo320HSR, HCT116, LoVo, HT29, and SW480 were all obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Synthesized, CCK-8 Assay, DNA Synthesis

Journal: Communications Biology

Article Title: Anti-tumor effects of P-LPK-CPT, a peptide-camptothecin conjugate, in colorectal cancer

doi: 10.1038/s42003-022-04191-1

Figure Lengend Snippet: a , b Western blotting analysis of SLC1A5 in NCM460, HCT116, LoVo, SW480, HT29 and Colo320HSR cells. After knocking down SLC1A5 in HCT116 and HT29, the binding intensity of FITC-P-LPK to cells was observed by confocal microscopy. Bar, 10 μm. c , d Effects of different endocytosis inhibitors on the internalization of FITC-P-LPK. HCT116 cells were pre-treated with 10 µM chlorpromazine, 50 μM methyl-β-cyclodextrin, 20 µM Amiloride hydrochloride for 30 min at 37 °C. Subsequently, the cells were incubated with FITC-P-LPK for 2 h. Then the fluorescence intensity was observed by confocal microscopy and detected using a multi-well plate reader ( n = 5, means ± SD, NC vs CPZ, p = 0.0001; NC vs MβCD, p = 0.9069; NC vs EIPA, p = 0.9812) (**** p < 0.0001). Bar, 10 μm. e The competition experiment with SLC1A5 substrates glutamine. FITC-P-LPK was incubated with increasing concentrations (0–32 mM) of glutamine in HCT116 cells at 37 °C for 2 h. The fluorescence intensity was measured in a multi-well plate reader ( n = 5, means ± SD).

Article Snippet: The human CRC cell lines Colo320HSR, HCT116, LoVo, HT29, and SW480 were all obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Western Blot, Binding Assay, Confocal Microscopy, Incubation, Fluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: Antagonism of estrogen-related receptor-α inhibits mitochondrial oxidative phosphorylation and reduces M2 macrophage infiltration in endometrial cancer

doi: 10.1136/jitc-2025-012521

Figure Lengend Snippet: The ERRα-PTPMT1 axis promotes M2 macrophage recruitment via ROS-dependent mitochondrial OXPHOS activation. ( A–B ) Bioinformatic analysis of pathways associated with both ERRα and PTPMT1. Among the top 15 pathways, oxidative phosphorylation (OXPHOS) had the highest comprehensive score (excluding disease pathways) for both genes. ( C–F ) The associations of the oxygen consumption rate (OCR), basal capacity, maximal respiration capacity, spare respiratory capacity, and ATP production regulated by the ERRα-PTPMT1 axis are shown by the Seahorse XF Cell Mito Stress Test Kit. KLE -NC and HEC-1A -NC cells were used as the control groups. The data are shown as the means±SDs. Representative data from three independent experiments are shown, each performed with three technical replicates. P values were calculated using one-way ANOVA. ( G–L ) The ROS levels in the different ERRα/PTPMT1 expression groups were analyzed using flow cytometry. KLE -NC and HEC-1A -NC cells were used as control groups. The data are shown as the means±SDs. Representative data from three independent experiments are shown, each performed with three technical replicates. P values were calculated using one-way ANOVA. ( M–P ) ROS levels in the different ERRα/PTPMT1 expression groups were analyzed using laser scanning confocal microscopy. KLE -NC and HEC-1A -NC cells were used as the control group. The data are shown as the means±SDs. For each experimental group, five fields of view were randomly selected and analyzed across three independent experiments. P values were calculated using one-way ANOVA. ( Q–R ) Effects of ROS produced in endometrial cancer cells on M2 macrophage chemotaxis in a co-culture model (scale bar 200 µm). The data are shown as the means±SDs. Representative data from three independent experiments are shown; for each experiment, five fields of view were randomly selected for microscopic counting. P values were calculated by Student’s t-test. *p<0.05 and ***p<0.001. ANOVA, analysis of variance; ECC, endometrial cancer cell; ERRα, estrogen-related receptor α; CL, cardiolipin; NAC, N-acetylcysteine; NC, normal control; PG, phosphatidylglycerol; PTPMT1, protein tyrosine phosphatase mitochondrial 1; ROS, reactive oxygen species.

Article Snippet: Human KLE (American Type Culture Collection, Maryland, USA), HEC-1A (National Collection of Authenticated Cell Cultures, Shanghai, China) and THP-1 (National Collection of Authenticated Cell Cultures, Shanghai, China) cells were cultured in DMEM/F12 or McCoy’s 5A medium, both of which contained 1% penicillin-streptomycin (#15140122; Gibco) and 10% fetal bovine serum (FBS) (#10091148; Gibco, New Zealand), at 37°C in a moist atmosphere (5% CO 2 , 95% air).

Techniques: Activation Assay, Phospho-proteomics, Control, Expressing, Flow Cytometry, Confocal Microscopy, Produced, Chemotaxis Assay, Co-Culture Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Antagonism of estrogen-related receptor-α inhibits mitochondrial oxidative phosphorylation and reduces M2 macrophage infiltration in endometrial cancer

doi: 10.1136/jitc-2025-012521

Figure Lengend Snippet: The ERRα-PTPMT1 axis affects the CL levels and mitochondrial structure. ( A ) 36 classes of lipids in KLE and HEC-1A cells were detected using lipidomics. ( B ) The cardiolipin content in different ERRα/PTPMT1 expression groups was analyzed using lipidomics. KLE -NC and HEC-1A -NC cells were used as control groups. Each group contains six biological replicates. P values were calculated using one-way ANOVA. ( C–F ) The significant differences in cardiolipin subclass contents in the different ERRα/PTPMT1 expression groups were analyzed using lipidomics (fold change >1.2, p<0.05 and VIP>1). KLE -NC and HEC-1A -NC cells were used as control groups. ( G ) A Venn diagram shows that two CL species—CL (14:0/16:0/18:0/18:2) and CL (14:0/16:0/16:0/18:1)—were significantly elevated in all ERRα-overexpressing or PTPMT1-overexpressing groups compared with untreated NC groups. ( H ) The phosphatidylglycerol content in the different ERRα/PTPMT1 expression groups was analyzed using lipidomics. KLE -NC and HEC-1A -NC cells were used as control groups. Each group contains six technical replicates. P values were calculated using one-way ANOVA. ( I ) Schematic diagram of the relationship between the contents of CLs and PG in the test samples. Each group contains six technical replicates. ( J–L ) Mitochondrial cardiolipin was stained with 10-N-nonyl acridine orange. Images (scale bar 20 µm) were captured using a laser scanning confocal microscope, and the fluorescence intensity was analyzed by ImageJ. The data are shown as the means±SDs. Representative data from three independent experiments are shown, five fields of view were randomly selected for microscopic counting. P values were calculated using one-way ANOVA. ( M ) Observation of the mitochondrial structure in different ERRα/PTPMT1 expression groups using transmission electron microscopy (scale bar 2 µm). For each experimental group, five fields of view were randomly selected and analyzed across three independent experiments. *p<0.05. ANOVA, analysis of variance; ERRα, estrogen-related receptor α; CL, cardiolipin; NC, normal control; PG, phosphatidylglycerol; PTPMT1, protein tyrosine phosphatase mitochondrial 1.

Article Snippet: Human KLE (American Type Culture Collection, Maryland, USA), HEC-1A (National Collection of Authenticated Cell Cultures, Shanghai, China) and THP-1 (National Collection of Authenticated Cell Cultures, Shanghai, China) cells were cultured in DMEM/F12 or McCoy’s 5A medium, both of which contained 1% penicillin-streptomycin (#15140122; Gibco) and 10% fetal bovine serum (FBS) (#10091148; Gibco, New Zealand), at 37°C in a moist atmosphere (5% CO 2 , 95% air).

Techniques: Expressing, Control, Staining, Microscopy, Fluorescence, Transmission Assay, Electron Microscopy

Journal: Oncotarget

Article Title: PICT-1 is a key nucleolar sensor in DNA damage response signaling that regulates apoptosis through the RPL11-MDM2-p53 pathway

doi: 10.18632/oncotarget.13082

Figure Lengend Snippet: A-B. HEK293 cells were exposed to UVB radiation (10 J/m 2 ) (A) or MMC (10 μg/mL) (B), then PICT-1 was immunostained and detected using confocal microscopy. PICT-1: red; DAPI: blue; Scale bar = 10 μm. C-D. HEK293 cells were exposed to UVB radiation (10 J/m 2 ) (C) or MMC (10 μg/mL) (D) and PICT-1 was detected by western blotting at the indicated time points. E. HEK293 cells were exposed to UVB radiation (10 J/m 2 ) and PICT-1 mRNA was quantified using qRT-PCR at the indicated time points. Results are presented as mean ± SD from three independent experiments. F. Naive or pre-treated (10 μM MG132 or 20 μM CA-074Me, 10 min) cells were exposed to UVB light. 6 h after UVB exposure, cell lysates were analyzed by western blotting using indicated antibodies.

Article Snippet: HEK293 and U251 cells were purchased from the American Type Culture Collection (ATCC) and maintained at 37°C with 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum.

Techniques: Confocal Microscopy, Western Blot, Quantitative RT-PCR

Journal: Oncotarget

Article Title: PICT-1 is a key nucleolar sensor in DNA damage response signaling that regulates apoptosis through the RPL11-MDM2-p53 pathway

doi: 10.18632/oncotarget.13082

Figure Lengend Snippet: A. HEK293 cells were immunostained with anti-ATM, DNA-PKcs or Ku70 antibodies. Cells were imaged using confocal microscopy. Scale bar = 10 μm. B. HEK293 cells were fractionated into nuclear and nucleolar fractions. C. Lysates were analyzed by western blotting using the indicated antibodies. Lamin B was used as a nucleoplasmic marker. D. HEK293 cells were transfected with DsRedC1-PICT-1, then immunostained with anti-ATM, DNA-PKcs or Ku70 antibodies. Cells were imaged using confocal microscopy. Scale bar = 10 μm. E-F. HEK293 cells were transfected with pEGFPC1 or pEGFPC1-PICT-1 plasmids for 24 h. Cell lysates were then immunoprecipitated with an anti-GFP antibody and subjected to western blot analysis with anti-ATM or anti-Ku70 antibodies, respectively. G-H. HEK293 cells were transfected with pFLAG-CMV2-PICT-1 for 24 h. Cell lysates were then immunoprecipitated with anti-ATM or anti-Ku70 antibodies and subjected to western blot analysis with FLAG antibody.

Article Snippet: HEK293 and U251 cells were purchased from the American Type Culture Collection (ATCC) and maintained at 37°C with 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum.

Techniques: Confocal Microscopy, Western Blot, Marker, Transfection, Immunoprecipitation

Journal: Oncotarget

Article Title: PICT-1 is a key nucleolar sensor in DNA damage response signaling that regulates apoptosis through the RPL11-MDM2-p53 pathway

doi: 10.18632/oncotarget.13082

Figure Lengend Snippet: A. HEK293 cells were transfected with pFLAG-CMV2-PICT-1 plasmid for 24 h, then UVB-irradiated (10 J/m 2 ). FLAG-PICT-1 was immunoprecipitated with an anti-FLAG antibody at the indicated time points. Phosphorylated and total FLAG-PICT-1 was detected using anti-phospho-(Ser/Thr/Tyr) antibody or FLAG antibody. Relative band intensities were quantified by densitometry and the ratios of phosphorylated to total PICT-1 are shown. B. Cells were transfected with pFLAG-CMV2-PICT-1 or pFLAG-CMV2-PICT-1 (S233A, T289A) plasmids and exposed to UVB light for 1 h. FLAG-PICT-1 was immunoprecipitated and phosphorylated FLAG-PICT-1 was detected using anti-phospho-(Ser/Thr/Tyr) antibody. Wt: FLAG-PICT-1; A: FLAG-PICT-1 (S233A, T289A).

Article Snippet: HEK293 and U251 cells were purchased from the American Type Culture Collection (ATCC) and maintained at 37°C with 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum.

Techniques: Transfection, Plasmid Preparation, Irradiation, Immunoprecipitation

Journal: Oncotarget

Article Title: PICT-1 is a key nucleolar sensor in DNA damage response signaling that regulates apoptosis through the RPL11-MDM2-p53 pathway

doi: 10.18632/oncotarget.13082

Figure Lengend Snippet: A. Naive or pre-treated (wortmannin or LY294002, 30 min) cells were incubated with MMC (10 μg/mL) for 6 h. Endogenous PICT-1 was detected using confocal microscopy. Scale bar = 10 μm. B. Naive or pre-treated (KU55933 or NU7026, 30 min) cells were incubated with MMC (10 μg/mL) for 6 h. Endogenous PICT-1 was detected using confocal microscopy. Scale bar = 10 μm. C. HEK293 cells were infected with ATM shRNA lentiviruses, and western blot was performed to detect the ATM expression. D. HEK293 cells infected with ATM shRNA#3 or control shRNA were transfected with pFLAG-CMV2-PICT-1 for 24h and subjected to UVB radiation, co-IP was performed using anti-FLAG antibody and phosphorylated PICT-1 was detected. E. HEK293 cells infected with ATM shRNA#3 or control shRNA were subjected to UVB radiation and endogenous PICT-1 was observed by confocal microscopy at 0 h and 6 h post-radiation. Scale bar = 10 μm.

Article Snippet: HEK293 and U251 cells were purchased from the American Type Culture Collection (ATCC) and maintained at 37°C with 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum.

Techniques: Incubation, Confocal Microscopy, Infection, shRNA, Western Blot, Expressing, Control, Transfection, Co-Immunoprecipitation Assay

Journal: Oncotarget

Article Title: PICT-1 is a key nucleolar sensor in DNA damage response signaling that regulates apoptosis through the RPL11-MDM2-p53 pathway

doi: 10.18632/oncotarget.13082

Figure Lengend Snippet: A-D. HEK293 cells were transfected with pFLAG-CMV2-PICT-1, pFLAG-CMV2-PICT-1 (S233A, T289A) or pFLAG-CMV2-PICT-1 (S233D, T289D) for 24 h. Cells were then treated with MMC (10 μg/mL). A-C. FLAG-fused proteins were stained with anti-FLAG antibodies and detected using confocal microscopy. (D) FLAG-fused proteins were detected by western blotting 3 h post-MMC treatment. Wt: FLAG-PICT-1; A: FLAG-PICT-1 (S233A, T289A); D: FLAG-PICT-1 (S233D, T289D). E. HEK293 cells were transfected with pFLAG-CMV2-PICT-1, pFLAG-CMV2-PICT-1 (S233A), (S233D), (T289A) or (T289D) for 24 h. Cells were then treated with UVB, FLAG-fused proteins were stained with anti-FLAG antibodies and detected using confocal microscopy.

Article Snippet: HEK293 and U251 cells were purchased from the American Type Culture Collection (ATCC) and maintained at 37°C with 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum.

Techniques: Transfection, Staining, Confocal Microscopy, Western Blot

Journal: Oncotarget

Article Title: PICT-1 is a key nucleolar sensor in DNA damage response signaling that regulates apoptosis through the RPL11-MDM2-p53 pathway

doi: 10.18632/oncotarget.13082

Figure Lengend Snippet: A. HEK293 cells were transfected with pFLAG-CMV2-RPL11 for 24 h and treated with MMC (10 μg/mL). FLAG-RPL11 and MDM2 were detected with antibodies against FLAG (green) or MDM2 (red) by confocal microscopy at the indicated time points. Scale bar = 20 μm. B. U251 cells were treated with MMC (10 μg/mL). Cell lysates were immunoprecipitated using anti-MDM2 antibody and RPL11 was detected by western blot at the indicated time points. C. HEK293 cells were treated with MMC (10 μg/mL) and p53 were detected by western blot at the indicated time points. D. HEK293 cells were treated with MMC (10 μg/mL) in the presence or absence of wortmannin or LY294002 for 6 h, and p53 was then detected by western blot.

Article Snippet: HEK293 and U251 cells were purchased from the American Type Culture Collection (ATCC) and maintained at 37°C with 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum.

Techniques: Transfection, Confocal Microscopy, Immunoprecipitation, Western Blot

Journal: Oncotarget

Article Title: PICT-1 is a key nucleolar sensor in DNA damage response signaling that regulates apoptosis through the RPL11-MDM2-p53 pathway

doi: 10.18632/oncotarget.13082

Figure Lengend Snippet: A. HEK293 cells were transfected with pFLAG-CMV2-PICT-1 for 24 h. Cells were then either left untreated or were treated with MMC (10 μg/mL). The same amounts of FLAG-PICT-1 protein were immunoprecipitated in both groups, and co-precipitated RPL11 protein was detected by western blotting. B. Cells were treated as in (A), but MMC treatment occurred in the presence or absence of wortmannin or LY294002 for 6 h. C. HEK293 cells were transfected with pFLAG-CMV2-PICT-1, pFLAG-CMV2-PICT-1 (S233A, T289A) or pFLAG-CMV2-PICT-1 (S233D, T289D) for 24 h. Cells were left untreated or were incubated with MMC (10 μg/mL). The same amounts of FLAG-PICT-1 protein were immnoprecipitated in each group, and co-precipitated RPL11 proteins were detected by western blotting. D. HEK293 cells were treated as in (C), and p53 was detected by western blotting. Relative p53 band intensities were quantified by densitometry and the ratios of the p53 to ß-actin are shown (* p <0.05). Wt: FLAG-PICT-1; A: FLAG-PICT-1 (S233A, T289A); D: FLAG-PICT-1 (S233D, T289D).

Article Snippet: HEK293 and U251 cells were purchased from the American Type Culture Collection (ATCC) and maintained at 37°C with 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum.

Techniques: Transfection, Immunoprecipitation, Western Blot, Incubation

Journal: Oncotarget

Article Title: PICT-1 is a key nucleolar sensor in DNA damage response signaling that regulates apoptosis through the RPL11-MDM2-p53 pathway

doi: 10.18632/oncotarget.13082

Figure Lengend Snippet: HEK293 cells were transfected with pFLAG-CMV2-PICT-1, pFLAG-CMV2-PICT-1 (S233A, T289A) or pFLAG-CMV2-PICT-1 (S233D, T289D) for 24 h, and then treated with MMC (10 μg/mL) for another 12 h. Cell apoptosis was detected with the Annexin V/PI staining by flow cytometry. A. Representative plots. B. Experiments were performed in triplicate, and the data are presented as mean ± SD (*p < 0.05). C. HEK293 cells were transfected with pFLAG-CMV2 or pFLAG-CMV2-PICT-1 for 24 h, and then 47S precursor rRNA (pre-rRNA) was detected by qRT-PCR. The experiment was repeated three times and the expression level in control cells was set to 1 (* p <0.05). D. pFLAG-CMV2 or pFLAG-CMV2-PICT-1 together with pHrD-IRES-Luc were co-transfected into HEK293 cells for 48 h. Protein concentration was normalized across lysates, and luciferase activity was measured. Data (arbitrary units) from three independent experiments are presented as mean ± SD (* p <0.05).

Article Snippet: HEK293 and U251 cells were purchased from the American Type Culture Collection (ATCC) and maintained at 37°C with 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum.

Techniques: Transfection, Staining, Flow Cytometry, Quantitative RT-PCR, Expressing, Control, Protein Concentration, Luciferase, Activity Assay

Journal: The Journal of Cell Biology

Article Title: β-Arrestin–Dependent Endocytosis of Proteinase-Activated Receptor 2 Is Required for Intracellular Targeting of Activated Erk1/2

doi:

Figure Lengend Snippet: PAR2-mediated activation of ERK1/2. (a) KNRK-PAR2+ARR-GFP cells (•) and KNRK-PAR2+ARR 319-418 -GFP cells (○) and hBRIE cells (□) were incubated with 50 nM trypsin for 0–60 min at 37°C, and ERK activity was measured using the MBP assay. (b–d) Western blots using antibodies to pERK1/2. (b) KNRK-PAR2+ARR-GFP cells (•), KNRK-PAR2 cells (⋄), and KNRK-PAR2+ARR 319-418 -GFP cells (○) were incubated with 50 nM trypsin. (c) hBRIE cells (□), hBRIE+ARR-GFP (⋄), and hBRIE+ARR 319-418 -GFP (♦) were incubated with 50 nM trypsin for 0–30 min at 37°C. (d) KNRK-PAR2+ARR-GFP cells (•) and KNRK-PAR2+ARR 319-418 -GFP cells (○) were incubated 50 μM AP for 0–30 min at 37°C. * P < 0.05 compared with cells expressing PAR2 alone or PAR2 plus ARR-GFP cells, n = 4.

Article Snippet: Kirsten murine sarcoma virus–transformed rat kidney epithelial cells (KNRK) were from the American Type Tissue Culture Collection (ATCC CRL 1569).

Techniques: Activation Assay, Incubation, Activity Assay, Western Blot, Expressing

Journal: The Journal of Cell Biology

Article Title: β-Arrestin–Dependent Endocytosis of Proteinase-Activated Receptor 2 Is Required for Intracellular Targeting of Activated Erk1/2

doi:

Figure Lengend Snippet: PAR2-mediated Ca 2+ mobilization in KNRK-PAR2 cells and KNRK-PAR2δ(ST363/6A) cells. (a) Concentration-response analysis for KNRK-PAR2 (•) and KNRK-PAR2 (δST363/6A) (▴) cells. (b–d) Each line shows [Ca 2+ ] i for individual KNRK-PAR2 cells (left) and KNRK-PAR2(δST363/6A) cells (right). (b) Note that the response to trypsin is prolonged in KNRK-PAR2(δST363/6A) cells. (c) Homologous desensitization in cells pretreated with 10 μM AP for 5 min before addition of 10 nM trypsin. (d) Heterologous desensitization in cells pretreated with 1 μM PDB for 20 min before addition of 10 nM trypsin. Note that KNRK-PAR2(δST363/6A) cells are resistant to desensitization.

Article Snippet: Kirsten murine sarcoma virus–transformed rat kidney epithelial cells (KNRK) were from the American Type Tissue Culture Collection (ATCC CRL 1569).

Techniques: Concentration Assay

Journal: The Journal of Cell Biology

Article Title: β-Arrestin–Dependent Endocytosis of Proteinase-Activated Receptor 2 Is Required for Intracellular Targeting of Activated Erk1/2

doi:

Figure Lengend Snippet: Agonist-induced PAR2 internalization. (a) Kinetics of PAR2 endocytosis in KNRK-PAR2+ARR-GFP cells (•), KNRK-PAR2 cells (⋄) KNRK-PAR2+ ARR 319-418 -GFP cells (○), KNRK-PAR2(δST363/6A) cells (▴), and hBRIE cells (□). Cells were incubated with 50 μM AP for 0–30 min at 37°C and endocytosis was determined by measuring surface Flag immunoreactivity by flow cytometry. * P < 0.05 compared with cells expressing PAR2 alone or PAR2 plus ARR-GFP cells, n = 3. (b–d) Localization of PAR2 and β-arrestin by immunofluorescence and confocal microscopy. KNRK-PAR2+ARR-GFP cells (b), KNRK-PAR2(δST363/6A)+ ARR-GFP cells (c), or KNRK-PAR2(δST363/6A) (d) were incubated with 50 nM trypsin for 0–30 min at 37°C. PAR2 was localized by immunofluorescence and β-arrestin was detected using GFP (b and c) or by immunofluorescence (d). The same cells are shown in each row and the images in the right panel are formed by superimposition of the images from the other two panels in the same row. Representative of two experiments. In KNRK-PAR2+ARR-GFP cells, note redistribution of β-arrestin to the plasma membrane at 5 min (arrowheads) and to endosomes at 30 min (arrows), where it colocalizes with PAR2. In KNRK-PAR2(δST363/6A)+ARR-GFP cells and in KNRK-PAR2(δST363/6A), note that PAR2 remains at the plasma membrane (arrowheads) and β-arrestin remains in the cytosol (arrows) with no colocalization. Bar, 10 μm.

Article Snippet: Kirsten murine sarcoma virus–transformed rat kidney epithelial cells (KNRK) were from the American Type Tissue Culture Collection (ATCC CRL 1569).

Techniques: Incubation, Flow Cytometry, Expressing, Immunofluorescence, Confocal Microscopy, Clinical Proteomics, Membrane

Journal: The Journal of Cell Biology

Article Title: β-Arrestin–Dependent Endocytosis of Proteinase-Activated Receptor 2 Is Required for Intracellular Targeting of Activated Erk1/2

doi:

Figure Lengend Snippet: (a) PAR2-mediated ERK1/2 activity. KNRK-PAR2 (⋄) and KNRK-PAR2(δST363/6A) (▴) cells were treated with 50 nM trypsin for 0–30 min, and ERK activity was measured using the MBP assay. (b) Activation of ERK1/2. KNRK-PAR2(δST363/6A) (▴), KNRK-PAR2 (δST363/6A)+ARR-GFP (▵), and KNRK-PAR2 (δST363/6A)+ARR 319-418 -GFP (♦) cells were treated with 50 nM trypsin for 0–30 min and phosphorylation was assessed using antibodies to pERK1/2. * P < 0.05 compared with KNRK-PAR2 cells, n = 3.

Article Snippet: Kirsten murine sarcoma virus–transformed rat kidney epithelial cells (KNRK) were from the American Type Tissue Culture Collection (ATCC CRL 1569).

Techniques: Activity Assay, Activation Assay, Phospho-proteomics

Journal: The Journal of Cell Biology

Article Title: β-Arrestin–Dependent Endocytosis of Proteinase-Activated Receptor 2 Is Required for Intracellular Targeting of Activated Erk1/2

doi:

Figure Lengend Snippet: Mechanism of PAR2-mediated activation of ERK1/2. (a) KNRK-PAR2, hBRIE, KNRK-PAR2+ARR 319-418 , and KNRK-PAR2(δST363/6A) cells were untreated (control, con), or incubated with 100 nM GF109203X (GFX), 20 nM LY379196 (LY), 100 ng/ml PTX, 10 μM genistein (GEN), 20 μM tyrphostin 25 (TP), or were cotransfected with N17ras. Cells were incubated with 50 nM trypsin for 5 min. * P < 0.05 compared with untreated cells, n = 4. (b–f) Analysis of KNRK-PAR2 and KNRK-PAR2(δST363/6A) cells by immunoprecipitation (IP) and Western blotting (WB). Cells were incubated with 50 nM trypsin for 5 min. Extracts were immunoprecipitated with antibodies to PYK2 (b), Shc (c and d), and src (e–g). Blots were probed for phosphotyrosine (b–e), PYK2 (f), and src (g).

Article Snippet: Kirsten murine sarcoma virus–transformed rat kidney epithelial cells (KNRK) were from the American Type Tissue Culture Collection (ATCC CRL 1569).

Techniques: Activation Assay, Control, Incubation, Immunoprecipitation, Western Blot

Journal: The Journal of Cell Biology

Article Title: β-Arrestin–Dependent Endocytosis of Proteinase-Activated Receptor 2 Is Required for Intracellular Targeting of Activated Erk1/2

doi:

Figure Lengend Snippet: Nuclear translocation of ERK1/2. (a–d) Subcellular fractionation of activated ERK1/2. KNRK-PAR2+ARR-GFP cells (•), KNRK-PAR2+ ARR 319-418 -GFP cells (○), or PAR2(δST363/6A) cells (s) were incubated with 50 nM trypsin for 0–30 min at 37°C, and pERK was determined in the cytosolic (a) and nuclear (b) fractions ( n = 3). hBRIE cells were incubated with 50 nM trypsin (□) or 10% serum (♦), and pERK was determined in the cytosolic (c) and nuclear (d) fractions. (e) KNRK-PAR2 and KNRK-PAR2(δST363/6A) cells, transiently transfected with ERK2-GFP, were incubated with 50 nM trypsin at 37°C, and translocation of ERK2-GFP was observed by confocal imaging. Representative of eight experiments. In KNRK-PAR2 cells, note that ERK2-GFP remains cytosolic but, in KNRK-PAR2(δST363/6A) cells, it redistributes to the nucleus. (f and g) Proliferative responses to AP and serum. KNRK-PAR2 (f, □) and KNRK-PAR2(δST363/6A) cells (f, ▪) or hBRIE cells (g) were incubated with 50 μM AP or 20% FCS for 24 h, and incorporation of [ 3 H]thymidine and cell number were measured. * P < 0.05 compared with untreated cells or KNRK-PAR2 cells, n = 3.

Article Snippet: Kirsten murine sarcoma virus–transformed rat kidney epithelial cells (KNRK) were from the American Type Tissue Culture Collection (ATCC CRL 1569).

Techniques: Translocation Assay, Fractionation, Incubation, Transfection, Imaging

Journal: The Journal of Cell Biology

Article Title: β-Arrestin–Dependent Endocytosis of Proteinase-Activated Receptor 2 Is Required for Intracellular Targeting of Activated Erk1/2

doi:

Figure Lengend Snippet: Trypsin induced association of β-arrestin and raf-1. (a and b) Localization of β-arrestin and raf-1 by immunofluorescence and confocal microscopy. KNRK-PAR2+ARR-GFP cells (a) or KNRK-PAR2(δST363/6A)+ ARR-GFP cells (b) were incubated with 50 nM trypsin for 0 or 5 min at 37°C. β-Arrestin was localized using GFP and raf-1 was localized by immunofluorescence. The same cells are shown in each row and the images in the right panel are formed by superimposition of images from the other two panels in the same row. Representative of two experiments. In KNRK-PAR2+ARR-GFP cells, note the redistribution of β-arrestin and raf-1 from the cytosol at 0 min (arrows) to the plasma membrane at 5 min (arrowheads), where they colocalize. In KNRK-PAR2 (δST363/6A)+ARR-GFP cells, note that β-arrestins remain in the cytosol (arrows) and raf-1 redistributes to the plasma membrane at 5 min (arrowheads). (c–f) Coimmunoprecipitation of raf-1 and β-arrestin. Cells were incubated with 50 nM trypsin for 0–30 min at 37°C, lysed, immunoprecipitated (IP) using antibodies to GFP or β-arrestin-1/2, and analyzed by Western blotting (WB) with a raf-1 antibody. (c) In KNRK-PAR2 cells, but not in KNRK-PAR2(δST363/6A) cells, β-arrestin and raf-1 coprecipitated. (d) Similarly, in KNRK-PAR2+ARR-GFP cells but not KNRK-PAR2(δST363/6A)+ARR-GFP cells ARR-GFP and raf-1 coprecipitated with antibodies to GFP. (e) In KNRK-PAR2+ARR 319-418 -GFP cells, endogenous β-arrestin and raf-1 coprecipitated, but ARR 319-418 and raf-1 did not coprecipitate. (f) In hBRIE+ARR-GFP cells, ARR-GFP and raf-1 coprecipitated. Bars, 10 μm.

Article Snippet: Kirsten murine sarcoma virus–transformed rat kidney epithelial cells (KNRK) were from the American Type Tissue Culture Collection (ATCC CRL 1569).

Techniques: Immunofluorescence, Confocal Microscopy, Incubation, Clinical Proteomics, Membrane, Immunoprecipitation, Western Blot

Journal: The Journal of Cell Biology

Article Title: β-Arrestin–Dependent Endocytosis of Proteinase-Activated Receptor 2 Is Required for Intracellular Targeting of Activated Erk1/2

doi:

Figure Lengend Snippet: Gel filtration analysis of an ERK signaling complex. KNRK-PAR2 (a and b), KNRK-PAR2(δST363/6A) cells (c and d), or hBRIE cells (e and f), or KNRK-PAR2+ARR 319-418 -GFP cells (g) were untreated (a, c, and e) or incubated with 50 μM AP (b, d, f, and g) for 5 min. Cell lysates were fractionated on a S300 Sephacryl column. The presence of pERK, raf-1, β-arrestin-1, and PAR2 in each fraction was determined by Western blotting (inset). PAR2 was detected using HA.11 antibody in KNRK cells and 2N antibody in hBRIE cells. Results are expressed as a percentage of the total protein for each partition coefficient (σ) ( n = 3). The bracketed columns represent regions where proteins coeluted. Representative Western blots are shown of fractions containing the complex in KNRK-PAR2 cells (★, σ = 0.34), hBRIE cells (⋆, σ = 0.31–34), and in KNRK-PAR2+ARR 319-418 -GFP (*, σ = 0.45). (h) The Stoke's radii of the four molecular mass standards and the complex in KNRK-PAR2 cells (★, ∼6.2), hBRIE cells (⋆, ∼6.2–6.6), and KNRK-PAR2+ARR 319-418 -GFP (*, ∼5 nm) are graphed as a function of the error function complement of σ.

Article Snippet: Kirsten murine sarcoma virus–transformed rat kidney epithelial cells (KNRK) were from the American Type Tissue Culture Collection (ATCC CRL 1569).

Techniques: Filtration, Incubation, Western Blot

Journal: The Journal of Cell Biology

Article Title: β-Arrestin–Dependent Endocytosis of Proteinase-Activated Receptor 2 Is Required for Intracellular Targeting of Activated Erk1/2

doi:

Figure Lengend Snippet: Coprecipitation of components of the β-arrestin–containing signaling complex. (a) Fractions from the gel filtration columns of AP-stimulated KNRK-PAR2 cells at partition coefficients 0.31–0.34 were pooled, concentrated, and immunoprecipitated with pERK or β-arrestin-1/2 antibodies. Western blots were probed for PAR2 using the HA.11 antibody, β-arrestin-1, raf-1, and pERK. (b) Fractions from the gel filtration columns of AP-stimulated hBRIE cells at partition coefficients 0.31–0.34 were similarly processed, immunoprecipitated with a β-arrestin-1/2 antibody, and blotted with antibodies to pERK and raf-1.

Article Snippet: Kirsten murine sarcoma virus–transformed rat kidney epithelial cells (KNRK) were from the American Type Tissue Culture Collection (ATCC CRL 1569).

Techniques: Filtration, Immunoprecipitation, Western Blot

Journal: Scientific Reports

Article Title: OPN promotes the aggressiveness of non-small-cell lung cancer cells through the activation of the RON tyrosine kinase

doi: 10.1038/s41598-019-54843-2

Figure Lengend Snippet: Effect of OPN expression level on the aggressive property of NSCLC cells. ( a ) A significant increase of in vitro Matrigel invasion in OPN-overexpressing SK-MES-1 cells. ( b ) Knockdown of OPN in A549 cells significantly reduced cellular Matrigel invasion. ( c ) OPN overexpression in SK-MES-1 cells increased cellular migration when assessed using ECIS after electric wounding (red dotted line), as indicated by resistance. ( d ) Knockdown of OPN markedly inhibited the post-wound migration capacity of A549 cells in the ECIS system which showed decreased resistance. ( e ) OPN overexpression in SK-MES-1 cells increased migration capacity after cultivation for 24 hours. ( f ) Knockdown of OPN significantly reduced cellular migration capacity of A549 cells. The results represent the mean values ± SD of three independent experiments. * p < 0.05, ** p < 0.01.

Article Snippet: Human lung adenocarcinoma A549 cells and squamous SK-MES-1 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, In Vitro, Knockdown, Over Expression, Migration

Journal: Scientific Reports

Article Title: OPN promotes the aggressiveness of non-small-cell lung cancer cells through the activation of the RON tyrosine kinase

doi: 10.1038/s41598-019-54843-2

Figure Lengend Snippet: Alteration of the EMT-related protein levels by OPN was dependent on the phosphorylated activation of RON. ( a , b ) The levels of total RON and p-RON (Y1238 + Y1239) proteins were significantly increased in OPN-overexpressed A549 cells but decreased in A549 cells which were treated with OPN shRNA, as indicated by western blotting. ( c , d ) The expression of OPN, RON and EMT markers (E-cadherin, N-cadherin, β-catenin, Slug, Twist) in A549 cells. OPN downregulated E-cadherin expression and upregulated the expression of N-cadherin, β-catenin, Slug, Twist. The OPN-induced differential expression of the EMT markers was reversed following the knockdown of RON by siRNA1 and siRNA2. Western blotting developed using different antibodies or gels were sepreated by white space.

Article Snippet: Human lung adenocarcinoma A549 cells and squamous SK-MES-1 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Activation Assay, shRNA, Western Blot, Expressing, Quantitative Proteomics, Knockdown

Journal: Scientific Reports

Article Title: OPN promotes the aggressiveness of non-small-cell lung cancer cells through the activation of the RON tyrosine kinase

doi: 10.1038/s41598-019-54843-2

Figure Lengend Snippet: Co-localization and interaction of OPN and RON proteins. ( a ) OPN (shown in green) and RON (shown in red) were probed in lung carcinoma and matched adjacent normal tissue. Nuclei were stained with Hoechst dye 33258. Co-localization was investigated by confocal microscopy. ( b ) Immunoprecipitation (IP) of OPN from whole cell lysate of A549 cells. An irrelevant IgG was used as a control of the anti-OPN antibody during IP. Immunoprecipitated proteins and the whole cell lysate were then analyzed by western blotting. Western blotting developed using different antibodies or gels were sepreated by white space.

Article Snippet: Human lung adenocarcinoma A549 cells and squamous SK-MES-1 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Staining, Confocal Microscopy, Immunoprecipitation, Control, Western Blot

Journal: Scientific Reports

Article Title: OPN promotes the aggressiveness of non-small-cell lung cancer cells through the activation of the RON tyrosine kinase

doi: 10.1038/s41598-019-54843-2

Figure Lengend Snippet: Secreted OPN promoted the malignant phenotypes of lung cancer cells mediated by the RON signaling pathway. ( a , b ) Functions of the OPN-overexpressing A549 cells including invasion and migration, respectively, were dramatically inhibited in the presence of OPN-neutralizing antibody (OPN-Ab) respectively compared to IgG-neutralizing antibody (IgG-Ab) control groups ( p < 0.01). OPN-overexpressing A549 cells were treated with media containing 20 μg/ml of OPN-Ab and IgG-Ab, respectively, to block the secreted OPN protein. ( c , d ) The elevated levels of invasion and migration A549 cells by OPN overexpression were abolished after cells were exposed to 10 μM of the RON inhibitor MK8033 (RONi) for 24 hours compared to the control groups (p < 0.001). ( e , f ) knockdown of RON by siRNA (kd1 and kd2, respectively) in OPN overexpressing A549 cells significantly reduced the invasion and migration capacities of the cells, respectively. The results represent the mean values ± SD. **p < 0.01, ***p < 0.001.

Article Snippet: Human lung adenocarcinoma A549 cells and squamous SK-MES-1 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Migration, Control, Blocking Assay, Over Expression, Knockdown