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Image Search Results
Journal: PLoS Pathogens
Article Title: High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and de-ubiquitination of FancD2
doi: 10.1371/journal.ppat.1007442
Figure Lengend Snippet: (A) Cells were treated with increasing concentrations of cisplatin for 72 hr and % survival was measured using the Crystal Violet assay. LXSN, E6, E7, E6E7 expressing cells showed respective IC50 of 2.5, 0.74, 1.27 and 0.73 uM; 72 hr. (B-D) Cells were transfected with siControl or siFancD2 for 48hrs and plated for western blotting and cisplatin survival assay. (B) Western blot showing FancD2 knockdown in the cells harvested at the time of reading survival assay. (C) Survival curves of LXSN, E6, E7 and E6E7 cells transfected with siRNA (siFancD2 or siControl) and treated with indicated doses of cisplatin for 48 hr. (D) Survival curve of siControl cells treated with indicated doses of cisplatin for 48 hrs.
Article Snippet: Primary antibodies against p53 (Cell Signaling Technology, 9282), pRb (BD Pharmingen, 554136),
Techniques: Crystal Violet Assay, Expressing, Transfection, Western Blot, Clonogenic Cell Survival Assay, Knockdown
Journal: PLoS Pathogens
Article Title: High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and de-ubiquitination of FancD2
doi: 10.1371/journal.ppat.1007442
Figure Lengend Snippet: (A) Immunoblot showing FancD2/ FancI expression and monoubiquitination status in transduced HFK cells which were either untreated or treated with 3 uM cisplatin for 24 hr. Ub refers to the monoubiquitinated forms of FancD2 and FancI, and non-Ub refers to the non-ubiquitinated forms. Ratios of monoubiquitinated to non-ubiquitinated FancD2 (D2 Ub: Non-Ub) and total FancD2 (Ub + Non-Ub) levels are indicated beneath the corresponding lanes. (B) Immunoblot of soluble and chromatin-bound fractions prepared from transduced HFK cells that were either untreated or treated with 3 uM cisplatin for 24 hr. Vinculin and Histone H3 act as loading controls respectively for soluble and chromatin-bound fractions. (C) Immunoblot of whole cell lysates showing levels of phosphorylated S556 FancI, total FancI, Ub-PCNA, non-Ub PCNA, and UHFR1. Vinculin acts as a loading control.
Article Snippet: Primary antibodies against p53 (Cell Signaling Technology, 9282), pRb (BD Pharmingen, 554136),
Techniques: Western Blot, Expressing, Control
Journal: PLoS Pathogens
Article Title: High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and de-ubiquitination of FancD2
doi: 10.1371/journal.ppat.1007442
Figure Lengend Snippet: (A) HFK cells were treated with cisplatin (3 uM) for 24 hr and immunostained with FancD2 (red), pH2AX (green) and DAPI (blue). Representative images are shown. (B) Cells with >5 foci were counted, and the percentage of positive cells is plotted (n = 3, mean ± SEM). (C) Quantification of percentage of FancD2 foci co-localization with pH2AX with or without cisplatin treatment. * (p-value ≤ 0.05) and ** (p-value ≤ 0.01) denote a statistically significant difference from the similarly treated LXSN control cells. Error bars represent standard error of the mean. Quantification was based on data observed from ≥ 15 nuclei from three independent experiments. (D) U2OS-DR cells (transduced with LXSN or E6/E7) were transfected with I-SceI expression plasmid for 24 hr before fixation. Cells were immunostained with pH2AX, FancD2, and DAPI (blue). Cells with a single large pH2AX focus (red) were examined for the colocalization with FancD2 (green). Representative images are shown. (E) Quantification of the frequency of colocalization of FancD2 with pH2AX foci in U2OS-DR cells. Data represent mean ± SEM and was based on observations from ≥ 50 cells from at least three independent experiments. (F-H) U2OS-DR cells transduced with the indicated constructs were transfected with the siControl or siFancD2. They were transfected with I-SceI expression plasmid and then fixed after 24 hr of transfection and stained with Rad51 and pH2AX antibodies. (F) Cell lysates were subjected to western blotting to confirm depletion of FancD2. (G) Cells with a single large pH2AX focus (red) were inspected for the colocalization with Rad51 (green). Representative images are shown. (H) Quantification of the frequency of colocalization of Rad51 with pH2AX foci. Data represent mean ± SEM and was based on observations from ≥ 25 cells from at least three independent experiments. * and ** indicate significance respectively at p<0.05 and p<0.01 (compared to LXSN) whereas n.s. indicates non-significant.
Article Snippet: Primary antibodies against p53 (Cell Signaling Technology, 9282), pRb (BD Pharmingen, 554136),
Techniques: Control, Transduction, Transfection, Expressing, Plasmid Preparation, Construct, Staining, Western Blot
Journal: PLoS Pathogens
Article Title: High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and de-ubiquitination of FancD2
doi: 10.1371/journal.ppat.1007442
Figure Lengend Snippet: (A) Outline of an experiment to evaluate FancD2 monoubiquitination/de-ubiquitination pattern. Transduced HFK cells were untreated or treated with cisplatin (1.5 uM for 24 hr) or exposed to 10 mJ/cm 2 UVB and allowed to repair. Whole-cell lysates were prepared for immunoblot at various time points during the experiments (represented by Δ). (B-C) Immunoblots of HFKs subjected to the experiment outlined in , following cisplatin withdrawal (B) or recovery after UVB exposure (C). Ratios of monoubiquitinated to non-ubiquitinated FancD2 (D2 Ub: non-Ub) are indicated beneath the corresponding lanes. FancD2 Ub:non-Ub ratio in cells following cisplatin withdrawal and UVB exposure was plotted alongside Figure 5B and 5C. ** (p< 0.01) denotes a statistically significant difference from 0 hr cisplatin withdrawal. ‘ns’ denotes non-significant differences. (D) USP1 immunoblot in cells untreated and treated with cisplatin. (E) Immunoblot of soluble and chromatin-bound fractions prepared from transduced HFK cells subjected to the experiment outlined in . Vinculin and Histone H3 act as loading controls respectively for soluble and chromatin-bound fractions. (F) Immunoblot showing levels of p-FancI-S565 and total FancI in cisplatin-treated or untreated cells. (G) Immunoblot for p-ATR, pCHK1, FancD2 and p-FancI-S565 following cisplatin withdrawal for 18 and 24 hrs. Actin or vinculin act as a loading control for immunoblots (B-G). (H) Proposed mechanisms for delayed de-ubiquitination of FancD2 in E6 cells.
Article Snippet: Primary antibodies against p53 (Cell Signaling Technology, 9282), pRb (BD Pharmingen, 554136),
Techniques: Ubiquitin Proteomics, Western Blot, Control
Journal: PLoS Pathogens
Article Title: High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and de-ubiquitination of FancD2
doi: 10.1371/journal.ppat.1007442
Figure Lengend Snippet: (A) Outline of synchronization assay in HFK cells using double-thymidine block. Cells were synchronized by double-thymine block and released at various time points. Immunoblot for FancD2, FancI, cyclin A and vinculin of LXSN (B) and E6 cells (C). Cell-cycle phases at each time point were determined by flow cytometry of DNA content (D). Asynchronous (Asyn.) cells, which have not undergone thymidine block, were included for comparison.
Article Snippet: Primary antibodies against p53 (Cell Signaling Technology, 9282), pRb (BD Pharmingen, 554136),
Techniques: Blocking Assay, Western Blot, Flow Cytometry, Comparison
Journal: PLoS Pathogens
Article Title: High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and de-ubiquitination of FancD2
doi: 10.1371/journal.ppat.1007442
Figure Lengend Snippet: (A) Immunoblot for p53 (upper panel) and RT-PCR analysis of 16E6 and GAPDH expression (lower panel) in HFK cells transduced with LXSN, E6 and E6 mutant (8S/9A/10T). (B-C) Immunoblot showing FancD2 expression and monoubiquitination status in cells which were either untreated or treated with cisplatin (B) or nutlin (C) at 1.5 uM for 24 hr. (D-E) Cells were untreated or treated with cisplatin (E) or exposed to 10 mJ/cm 2 UVB (D) and processed similarly as in .
Article Snippet: Primary antibodies against p53 (Cell Signaling Technology, 9282), pRb (BD Pharmingen, 554136),
Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Transduction, Mutagenesis
Journal: Nature Communications
Article Title: The HMGB1 protein induces a metabolic type of tumour cell death by blocking aerobic respiration
doi: 10.1038/ncomms10764
Figure Lengend Snippet: ( a ) HMGB1 was purified from NK-92 Cl cells by chromatography ( n =2). Arrow=HMGB1 containing fraction (eluate #38). ( b ) Immunoblot showing the membrane containing eluates 37–42. A specific HMGB1 band at 30 kD was detected only in eluate #38 . ( c ) Cytotoxicity assay after 72 h incubation with HMGB1 ( n =3). A 1:40 dilution of the purified HMGB1 (#38) was used (corresponding to ∼16 nM). Recombinant human HMGB1 was used at 80 nM. ( d ) Supernatants from activated human peripheral blood NK cells (cross-linked with anti-Nkp30 antibody) from two donors were tested for their cytotoxic capacity in a crystal violet assay (72 h, n =3). Glycyrrhizin (200 μM) was used as an inhibitor of HMGB1. ( e ) Immunoblot of the supernatants used in d . HMGB1 was specifically secreted on Nkp30 crosslinking. *, non-specific band. ** P <0.002 ( t -test). Error bars represent s.d.
Article Snippet: HMGB1 was detected by immunoblot analysis using
Techniques: Purification, Chromatography, Western Blot, Cytotoxicity Assay, Incubation, Recombinant, Crystal Violet Assay
Journal: Nature Communications
Article Title: The HMGB1 protein induces a metabolic type of tumour cell death by blocking aerobic respiration
doi: 10.1038/ncomms10764
Figure Lengend Snippet: ( a ) HPLC-purified HMGB1 (80 nM, 24 h, n =3) from the supernatant of Nkp30-stimulated blood donor NK cells was diluted in the IgG1 control supernatant and shows substantial cytotoxicity. ( b ) Silver gel showing purity of HMGB1 in eluate #38 (0.5 μg protein loaded). * P <0.05 ( t -test). Error bars represent the s.d.
Article Snippet: HMGB1 was detected by immunoblot analysis using
Techniques: Purification
Journal: Nature Communications
Article Title: The HMGB1 protein induces a metabolic type of tumour cell death by blocking aerobic respiration
doi: 10.1038/ncomms10764
Figure Lengend Snippet: ( a ) Electron microscopy showing the ultrastructure of mitochondria in CRC cells (SW480) treated with HMGB1 (24 h, 160 nM). Upper bars, 5 μm; lower bars, 1 μm; black arrow, normal mitochondria; asterisk, giant mitochondria. ( b ) ATP Luciferase-assay after incubation with 80 nM HMGB1 for the indicated times ( n =6 for SW480 and HCT116, n =3 for HT29). Error bars represent s.d.
Article Snippet: HMGB1 was detected by immunoblot analysis using
Techniques: Electron Microscopy, Luciferase, Incubation
Journal: Nature Communications
Article Title: The HMGB1 protein induces a metabolic type of tumour cell death by blocking aerobic respiration
doi: 10.1038/ncomms10764
Figure Lengend Snippet: ( a ) Activities of respiratory chain complexes measured in mitochondrial fractions of CRC cell lines after treatment with HMGB1 (80 nM, 24 h, n =5). ( b ) Tissue slices were generated from a fresh surgical human colon carcinoma specimen and treated with HMGB1 (160 nM, 72 h). After homogenization of tissue slices, COX activity was measured in the mitochondrial fractions ( n =8). ( c – f ) CRC cells and tissue slices were treated with HMGB1 as described in a and b , respectively. Then, cyanide sensitive respiration was measured. * P <0.05, ** P <0.001 ( t -test). Error bars represent s.d.
Article Snippet: HMGB1 was detected by immunoblot analysis using
Techniques: Generated, Homogenization, Activity Assay
Journal: Nature Communications
Article Title: The HMGB1 protein induces a metabolic type of tumour cell death by blocking aerobic respiration
doi: 10.1038/ncomms10764
Figure Lengend Snippet: ( a ) Activities of glycolytic enzymes measured in cytosolic fractions after treatment with HMGB1 (80 nM, 24 h, n =5). ENO, enolase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HK, hexokinase; LDH, lactate dehydrogenase; PGM, phosphoglycerate mutase; PFK, phosphofructokinase; TPI, triose-phosphate isomerase. ( b ) Colon cancer tissue slices from a fresh surgical specimen were treated with HMGB1 (160 nM, 72 h). Tetrameric PK M2 activity was measured in eight homogenates. ( c ) PK M2 activity in CRC cells was measured after 24 h incubation with the supernatant derived from stimulated NK cells from blood donor #2 . Glycyrrhizin (200 μM) was used as a HMGB1 inhibitor ( n =3). ( d ) 5- 3 H-glucose turn-over was assessed after treatment with HMGB1 (80 nM, 24 h, n =3). ( e ) The experiment using SW480 cells was performed as outlined in d . Glycyrrhizin (200 μM) was used as an inhibitor of HMGB1 ( n =3). ( f ) Enrichment of 14 C in the messenger RNA of crude extracts after treatment with HMGB1 (80 nM, 24 h, n =3). ( g ) Isolation of the PK M2-HMGB1 complex: ultrafiltration of a solution containing 2 μM human PK M2 and 2 μM human HMGB1. The filtrated PK M2-HMGB1 complex was exposed to western blotting. ( h ) Cells were treated with 125 I-HMGB1 (80 nM, 24 h, n =3) with or without the PKM2 tetramer inhibitor P-M2tide (10 μM, 24 h) or PKM2 activator ML-265 (100 μM, 24 h). * P <0.05, ** P <0.002, *** P <0.00008 ( t -test). Error bars represent s.d.
Article Snippet: HMGB1 was detected by immunoblot analysis using
Techniques: Activity Assay, Incubation, Derivative Assay, Isolation, Western Blot
Journal: Nature Communications
Article Title: The HMGB1 protein induces a metabolic type of tumour cell death by blocking aerobic respiration
doi: 10.1038/ncomms10764
Figure Lengend Snippet: ( a ) The calculations were performed for the following HMGB1 constructs: leftmost: A box (magenta), tyrosines unphosphorylated; centre left: A box, tyrosines phosphorylated; centre right: B box (green), tyrosines unphosphorylated; rightmost: B box, tyrosines phosphorylated. PK M2 monomer, yellow; three additional PK M2 domains in the PK M2 tetramer, cyan. ( b ) Electrostatic isopotential contours (+1 kT/e: blue; −1 kT/e: red) of the HMGB1 A and B boxes with: leftmost) A box, unphosphorylated tyrosines; centre left) A box, phosphorylated tyrosines; centre right) B box, unphosphorylated tyrosines; rightmost) B box, phosphorylated tyrosines. ( c ) Left: distances from HMGB1 Box B residues pTyr 116 and pTyr 162 (numbering according to PDB file: 2YRQ, corresponding to residues 109 and 155, respectively, in the human sequence) to PK M2 K433 for the best ranked docked pose and of PK M2 Y105 to the nearest charged residue from the HMGB1 box B. Right: a rotated view with electrostatic isopotential contours (+1 kT/e: blue; −1 kT/e: red) of PK M2. ( d ) The calculations was performed as outlined in a , here in the presence of FBP (left) or with Tyr 105 phosphorylated (centre) or in the absence of FBP and with unphosphorylated Tyr 105 (right).
Article Snippet: HMGB1 was detected by immunoblot analysis using
Techniques: Construct, Sequencing
Journal: Nature Communications
Article Title: The HMGB1 protein induces a metabolic type of tumour cell death by blocking aerobic respiration
doi: 10.1038/ncomms10764
Figure Lengend Snippet: ( a ) Viability assay performed with respiratory chain deficient cells ( ρ 0 ) and control (wild type) cells treated with HMGB1 (160 nM, 72 h, n =3). ** P <0.0001 ( t -test). ( b ) The amount of ATP production was calculated from O 2 consumption and from 13 C-lactate efflux derived from 13 C-labelled glucose . ( c ) The amount of ATP production was calculated from 13 C-lactate efflux derived from 13 C-labelled glutamine (Methods section). Cells were treated with HMGB1 (80 nM, 24 h, n =3, * P <0.02, t -test). ( d ) Survival of cells after treatment with oligomycin (10 ng ml −1 ) and HMGB1 (80 nM; both 72 h, n =3, ** P <0.0001, t -test). ( e ) Crystal violet survival assay in glucose-free medium after treatment with HMGB1 (80 nM, SW480 and HCT116; 160 nM, HT29; 24 h). L-DON (1 μM) was added as indicated ( n =3, * P <0.02, t -test). ( f ) After siRNA-mediated knockdown of ME1 the HT29 cells were treated with HMGB1 (80 nM, 72 h, n =3, * P <0.0003, ** P <0.000002, t -test). ( g ) Immunoblot with anti-ME1 antibody to confirm the knockdown. Error bars represent s.d.
Article Snippet: HMGB1 was detected by immunoblot analysis using
Techniques: Viability Assay, Derivative Assay, Clonogenic Cell Survival Assay, Western Blot
Journal: Nature Communications
Article Title: The HMGB1 protein induces a metabolic type of tumour cell death by blocking aerobic respiration
doi: 10.1038/ncomms10764
Figure Lengend Snippet: ( a ) Systemic (intraperitoneal) HMGB1 treatment of CD1 nude mice bearing subcutaneous SW480 ( n =20, P <0.05 ( t -test) HMGB1 versus control) or HT29 ( n =20; P <0.05 ( t -test) for HMGB1+L-DON versus control or L-DON or HMGB1) xenograft tumours was started when tumours were palpable (time point 0). Control mice were injected with saline. The tumour volumes were determined at regular intervals. Scale bars, 1 cm. ( b ) ME1 expression levels and local invasion depth of cancer tissue (pT stage) in patients with rectal carcinoma;+low, ++ moderate, +++ strong expression of ME1. ( c ) Association of ME1 expression levels and lymph node metastasis (pN stage) in patients with rectal carcinoma. Error bars represent s.d.
Article Snippet: HMGB1 was detected by immunoblot analysis using
Techniques: Injection, Expressing
Journal: bioRxiv
Article Title: High-Risk Human Papillomavirus Oncogenes Disrupt the Fanconi Anemia DNA repair Pathway by Impairing Localization and Deubiquitination of FancD2
doi: 10.1101/457176
Figure Lengend Snippet: (A) Cells were treated with increasing concentrations of cisplatin for 72 hr and % survival was measured using the Crystal Violet assay. LXSN, E6, E7, E6E7 expressing cells showed respective IC50 of 2.5, 0.74, 1.27 and 0.73 uM; 72 hr. (B-C) Cells were transfected with siControl or siFancD2 for 48hrs and plated for western blotting and cisplatin survival assay. (B) Western blot showing FancD2 knockdown in the cells harvested at the time of reading survival assay. (C) Survival curves of LXSN, E6, E7 and E6E7 cells transfected with siRNA and treated with indicated doses of cisplatin for 48 hr. (D)
Article Snippet: Cells were treated with
Techniques: Crystal Violet Assay, Expressing, Transfection, Western Blot, Clonogenic Cell Survival Assay
Journal: bioRxiv
Article Title: High-Risk Human Papillomavirus Oncogenes Disrupt the Fanconi Anemia DNA repair Pathway by Impairing Localization and Deubiquitination of FancD2
doi: 10.1101/457176
Figure Lengend Snippet: (A) Outline of an experiment to evaluate repair of cisplatin-induced ICLs using the modified alkaline comet assay. Cells were treated with 1.5 μM cisplatin for 2 hr and then incubated in cisplatin-free medium. Triangles (Δ) represent the time points (0, 24, 48 and 72 hr after cisplatin release) when cells were harvested and frozen. Immediately before comet analysis, cells were thawed and irradiated with 12 Gy to deliver a fixed level of random DSBs. (B) Representative comet images of untreated and cisplatin treated+ irradiated cells at 0hr and 72 hr. The insets are the magnified comets. A mean tail moment from a representative assay is shown. (C) Quantification of % ICL present in LXSN and E6/E7 expressing HFK cells at different post-treatment incubation time. The percentage of ICLs remaining was determined as described in Methods. Data represent mean ± SEM of at least 3 independent biological replicates.
Article Snippet: Cells were treated with
Techniques: Modification, Alkaline Single Cell Gel Electrophoresis, Incubation, Irradiation, Expressing
Journal: bioRxiv
Article Title: High-Risk Human Papillomavirus Oncogenes Disrupt the Fanconi Anemia DNA repair Pathway by Impairing Localization and Deubiquitination of FancD2
doi: 10.1101/457176
Figure Lengend Snippet: (A) Immunoblot showing FancD2/FancI expression and monoubiquitination status in transduced HFK cells which were either untreated or treated with 3 uM cisplatin for 24 hr. Ub refers to the monoubiquitinated forms of FancD2 and FancI, and non-Ub refers to the non-ubiquitinated forms. Ratios of monoubiquitinated to non-ubiquitinated FancD2 (D2 Ub: Non-Ub) and total FancD2 (Ub + Non-Ub) levels are indicated beneath the corresponding lanes. (B) Immunoblot of soluble and chromatin-bound fractions prepared from transduced HFK cells that were either untreated or treated with 3 uM cisplatin for 24 hr. Vinculin and Histone H3 act as loading controls respectively for soluble and chromatin-bound fractions. (C) Immunoblot of whole cell lysates showing levels of phosphorylated S556 FancI, Ub-PCNA, non-Ub PCNA, and UHFR1. Vinculin acts as a loading control.
Article Snippet: Cells were treated with
Techniques: Western Blot, Expressing
Journal: bioRxiv
Article Title: High-Risk Human Papillomavirus Oncogenes Disrupt the Fanconi Anemia DNA repair Pathway by Impairing Localization and Deubiquitination of FancD2
doi: 10.1101/457176
Figure Lengend Snippet: (A)HFK cells were treated with cisplatin (3 uM) for 24 hr and immunostained with FancD2 (red), pH2AX (green) and DAPI (blue). Representative images are shown. White arrows denote mislocalization of FancD2 away from pH2AX. (B) Cells with >5 foci were counted, and the percentage of positive cells is plotted (n = 3, mean ± SEM). (C) Schematic of I-SceI colocalization assay. The DR-GFP reporter cassette (integrated into U2OS genome) consists of two copies of nonfunctional GFP gene. The first copy is inactive due to the presence of a stop codon within the I-SceI cleavage site, while the second copy (iGFP) is truncated at both ends. Exogenous expression of I-SceI in U2OS cells with one integrated copy of the ISceI recognition site produces a single persistent DSB. Recruitment of repair protein (green) to this enlarged pH2AX focus (red) can be visualized by IF. (D) U2OS-DR cells (transduced with LXSN or E6/E7) were transfected with I-SceI expression plasmid for 24 hr before fixation. Cells were immunostained with pH2AX, FancD2, and DAPI (blue). Cells with a single large pH2AX focus (red) were examined for the colocalization with FancD2 (green). Representative images are shown. (E) Quantification of the frequency of colocalization of FancD2 with pH2AX foci in U2OS-DR cells. Data represent mean ± SEM and was based on observations from ≥ 50 cells from at least three independent experiments. (F-H) U2OS-DR cells transduced with the indicated constructs were transfected with the siControl or siFancD2. They were transfected with I-SceI expression plasmid and then fixed after 24 hr of transfection and stained with Rad51 and pH2AX antibodies. (F) Cell lysates were subjected to western blotting to confirm depletion of FancD2. (G) Cells with a single large pH2AX focus (red) were inspected for the colocalization with Rad51 (green). Representative images are shown. (H) Quantification of the frequency of colocalization of Rad51 with pH2AX foci. Data represent mean ± SEM and was based on observations from ≥ 25 cells from at least three independent experiments. * and ** indicate significance respectively at p<0.05 and p<0.01 (compared to LXSN) whereas n.s. indicates non-significant.
Article Snippet: Cells were treated with
Techniques: Expressing, Transduction, Transfection, Plasmid Preparation, Construct, Staining, Western Blot
Journal: bioRxiv
Article Title: High-Risk Human Papillomavirus Oncogenes Disrupt the Fanconi Anemia DNA repair Pathway by Impairing Localization and Deubiquitination of FancD2
doi: 10.1101/457176
Figure Lengend Snippet: (A) Outline of an experiment to evaluate FancD2 monoubiquitination/de-ubiquitination pattern. Transduced HFK cells were untreated or treated with cisplatin (1.5 uM for 24 hr) or exposed to 10 mJ/cm 2 UVB and allowed to repair. Whole-cell lysates were prepared for immunoblot at various time points during the experiments (represented by Δ). (B-C) Immunoblots of HFKs subjected to the experiment outlined in , following cisplatin withdrawal (B) or recovery after UVB exposure (C). Ratios of monoubiquitinated to non-ubiquitinated FancD2 (D2 Ub: non-Ub) are indicated beneath the corresponding lanes. (D) USP1 immunoblot in cells untreated and treated with cisplatin. (E) Immunoblot of soluble and chromatin-bound fractions prepared from transduced HFK cells subjected to the experiment outlined in . Vinculin and Histone H3 act as loading controls respectively for soluble and chromatin-bound fractions. (F) Immunoblot showing levels of p-FancI-S565 in cisplatin-treated or untreated cells. (G) Immunoblot for p-ATR, pCHK1, FancD2 and p-FancI-S565 following cisplatin withdrawal for 18 and 24 hrs. Actin or vinculin act as a loading control for immunoblots (B-G). (H) Proposed mechanisms for delayed de-ubiquitination of FancD2 in E6 cells.
Article Snippet: Cells were treated with
Techniques: Western Blot
Journal: bioRxiv
Article Title: High-Risk Human Papillomavirus Oncogenes Disrupt the Fanconi Anemia DNA repair Pathway by Impairing Localization and Deubiquitination of FancD2
doi: 10.1101/457176
Figure Lengend Snippet: (A) Immunoblot for p53 in HFK cells transduced with LXSN, E6 and E6 mutant (8S/9A/10T). (B-C) Immunoblot showing FancD2 expression and monoubiquitination status in cells which were either untreated or treated with cisplatin (B) or nutlin (C) at 1.5 uM for 24 hr. (D-E) Cells were untreated or treated with cisplatin (E) or exposed to 10 mJ/cm 2 UVB (D) and processed similarly as in . See also Fig S6A.
Article Snippet: Cells were treated with
Techniques: Western Blot, Transduction, Mutagenesis, Expressing
Journal: bioRxiv
Article Title: High-Risk Human Papillomavirus Oncogenes Disrupt the Fanconi Anemia DNA repair Pathway by Impairing Localization and Deubiquitination of FancD2
doi: 10.1101/457176
Figure Lengend Snippet: (A-C) HFKs were transduced with LXSN, E6 or E6/E7 (A) Immunoblot showing Palb2 expression. Actin serves as a loading control. (B) Cells were untreated or treated with cisplatin (3 uM) for 24 hr and immunostained with Palb2, pH2AX, and DAPI. Cells with >5 Palb2 foci were counted and the percentage of positive cells is plotted (n = 3, mean ± SD). * and ** indicate significance respectively at p<0.05 and p<0.01 (compared to LXSN) whereas ns indicates non-significant. (C) HFKs were transfected with siControl or siPalb2. Immunoblot confirming depletion of Palb2 (upper panel). Cisplatin survival assay for siRNA-transfected cells (lower panel).
Article Snippet: Cells were treated with
Techniques: Transduction, Western Blot, Expressing, Transfection, Clonogenic Cell Survival Assay
Journal: Frontiers in Microbiology
Article Title: Natural Anti-biofilm Agents: Strategies to Control Biofilm-Forming Pathogens
doi: 10.3389/fmicb.2020.566325
Figure Lengend Snippet: Anti-biofilm activity of phytocompounds with their mechanism of action.
Article Snippet: Curcumin , Curcuma longa L. , In vitro (crystal violet biofilm assay, pellicle formation assay, surface motility assay, mixed culture biofilm assay) In vivo ( Caenorhabditis elegans model organism, C. elegans killing assay) , Acinetobacter baumannii , C. albicans , Inhibits pellicle formation, Pilli motility, and ring biofilm formation Molecular docking analysis proved that curcumin interacts with the biofilm response regulator BfmR , MIC > 500 μg/ml for
Techniques: Activity Assay, In Vitro, In Vivo, Infection, Knock-Out, Expressing, Inhibition, Biofilm Production Assay, Microdilution Assay, Kinase Assay, Membrane, Staining, Microscopy, Cell Surface Hydrophobicity, Clonogenic Cell Survival Assay, Reporter Assay, Motility Assay, Gene Expression, Tube Formation Assay, Growth Assay, Control, Binding Assay, Bacteria, Mutagenesis, Crystal Violet Assay, Clinical Proteomics