Journal: bioRxiv

Article Title: SARS-CoV-2 susceptibility of cell lines and substrates commonly used in diagnosis and isolation of influenza and other viruses

doi: 10.1101/2021.01.04.425336

Figure Lengend Snippet: Whole cell lysate from uninoculated Vero E6, CV-1, A549, Mv1Lu, CRFK, MDCK-NBL-2 and MDCK-SIAT1 cell lines were immunoblotted for endogenous ACE2 expression. Recombinant hACE2 (Sino Biological) was used as a positive control for detection of hACE2. 20 μg of cell lysates or 0.2 ng of recombinant hACE2 protein were loaded. β-actin was also immunoblotted from samples as a loading control.

Article Snippet: Cell lysates and recombinant ACE2 protein control (Sino Biological) were immunoblotted for ACE2 and β-actin using primary antibodies (1:500 polyclonal goat anti-human ACE2, R&D Systems, AF933; 1:1000 monoclonal mouse anti-β-Actin, Abcam, AB8226) followed by secondary antibodies (1:4000 donkey anti-goat, Abcam; 1:4000 goat anti-mouse, Biorad).

Techniques: Expressing, Recombinant, Positive Control

Journal: bioRxiv

Article Title: SARS-CoV-2 susceptibility of cell lines and substrates commonly used in diagnosis and isolation of influenza and other viruses

doi: 10.1101/2021.01.04.425336

Figure Lengend Snippet: ACE2 protein sequences from human, rhesus macaque, African green monkey, cat, dog, American mink, mouse, and chicken were aligned using MUSCLE. Residues involved in interaction with SARS-CoV-2 spike protein (based on ref ( – )) are shown using hACE2 numbering, and residues varying from hACE2 are highlighted in yellow. A gap in alignment is indicated with a dash. Percent identity to hACE2 across the entire protein is shown.

Article Snippet: Cell lysates and recombinant ACE2 protein control (Sino Biological) were immunoblotted for ACE2 and β-actin using primary antibodies (1:500 polyclonal goat anti-human ACE2, R&D Systems, AF933; 1:1000 monoclonal mouse anti-β-Actin, Abcam, AB8226) followed by secondary antibodies (1:4000 donkey anti-goat, Abcam; 1:4000 goat anti-mouse, Biorad).

Techniques:

Journal: Scientific Reports

Article Title: Development of Potent Forchlorfenuron Analogs and Their Cytotoxic Effect in Cancer Cell Lines

doi: 10.1038/s41598-020-59824-4

Figure Lengend Snippet: The effect of FCF analogs on HER2 expression. ( A ) ECC-1 or HCH-1 cells were incubated with UR214-7 (33 µM), UR214-9 (33 µM) or FCF (300 µM) for 24 h. The levels of each protein were determined by Western blot analysis. ( B ) HCH-1 cells were incubated with UR14-9 (10 µM) or FCF (100 µM) for 48 h (top) or transfected with septin-2 targeting siRNA or non-targeting control siRNA for 24 or 48 h (bottom). Cell lysates were collected. The cellular levels of HER2 were determined by human HER2 enzyme-linked immunosorbent assay. ( C ) The cells were transfected with septin-2 targeting siRNA or non-targeting control siRNA. At 48 h post transfection, cell population and relative expression of septin-2 were determined by sulforhodamine B assay (top) and Western blot analysis (bottom), respectively.

Article Snippet: Following the treatment, cell lysates and supernatants were collected and subjected to HE4 assay (Human HE4 Quantikine ELISA Kit, R&D systems).

Techniques: Expressing, Incubation, Western Blot, Transfection, Control, Enzyme-linked Immunosorbent Assay, Sulforhodamine B Assay

Journal: Scientific Reports

Article Title: Development of Potent Forchlorfenuron Analogs and Their Cytotoxic Effect in Cancer Cell Lines

doi: 10.1038/s41598-020-59824-4

Figure Lengend Snippet: The effect of FCF analogs on the expression of HE4. ( A ) HE4 overexpression clones (OVCAR8-C5) were treated with or without FCF (300 µM) for 7 h, after which the cell lysates and matching culture media were collected and analyzed for the levels of HE4 using enzyme immunoassays. The amount of secreted HE4 was normalized to the protein concentration of respective cell lysate. ( B ) ECC-1 and HCH-1 cells were treated with the indicated concentrations of FCF for 5 h and the secreted levels of HE4 were measured. ( C ) ECC-1 cells were treated with either UR214-1, UR214-7 or FCF at the concentrations indicated for 5 h at which point the secreted levels of HE4 were determined. ( D ) ECC-1 cells were washed and incubated with FCF (300 µM) for 5 h in basal media. Relative HE4 expression was determined by real time PCR (normalized to B2M). ( E ) ECC-1 cells were transfected with septin-2 targeting siRNA or non-targeting control siRNA for 48 h (bottom) or for 72 h (top). Relative gene expression of HE4 was determined by real time PCR (normalized to TBP; bottom). The intracellular levels of HE4 were measured using enzyme immunoassays (top).

Article Snippet: Following the treatment, cell lysates and supernatants were collected and subjected to HE4 assay (Human HE4 Quantikine ELISA Kit, R&D systems).

Techniques: Expressing, Over Expression, Clone Assay, Enzyme Immunoassay, Protein Concentration, Incubation, Real-time Polymerase Chain Reaction, Transfection, Control, Gene Expression

Journal: Cells

Article Title: WIP1 Promotes Homologous Recombination and Modulates Sensitivity to PARP Inhibitors

doi: 10.3390/cells8101258

Figure Lengend Snippet: WIP1 plays role in DNA double-strand break repair in S-phase cells. ( A ) Quantification of 53BP1 foci in replicating (EdU+) cells after irradiation. U2OS parental cell lines with or without combined treatment with WIP1i and two independent WIP1 knockout cell lines were pulse-labeled with EdU for 30 min before irradiation. Cells were fixed after pre-extraction at indicated time-points and stained with 53BP1 antibody. Click chemistry was used to visualize EdU. Mean of median foci number +/- SD is plotted (n ≥ 3). Statistical significance evaluated by two tailed t -test. ( B ) Quantification of 53BP1 foci in non-replicating (EdU-) cells after irradiation. As in A. ( C ) Quantification of 53BP1 foci in replicating (EdU+) cells after irradiation. U2OS parental, WIP1 knockout and cell lines complemented with wild-type or phosphatase-dead (D314A) mutant of WIP1 were irradiated and analyzed as in A. ( D ) Quantification of 53BP1 foci in non-replicating (EdU-) cells after irradiation. U2OS parental, WIP1 knockout and cell lines complemented with wild-type or phosphatase-dead (D314A) mutant of WIP1 were irradiated and analyzed as in A. ( E ) Traffic light reporter assay in U2OS cells after transfection with indicated siRNA. Cells were transfected with ISceI together with BFP-donor vector with or without pretreatment with 1 μM WIP1i 2 days after siRNA transfection. Efficiency of repair was analyzed by FACS 3 days after ISceI and BFP-donor transfection. Plotted is mean +/− SD. Statistical significance evaluated by two-tailed t -test. ( F ) Efficiency of repair by HR and NHEJ in Traffic light reporter assay as in E. ( G ) Representative plots from Traffic light reporter assay in E.

Article Snippet: Following antibodies were used: WIP1 antibody (clone F-10, sc-376257), p21 (sc-397), p53 (clone D01, sc-126), BRCA1 (sc-6954), rabbit-53BP1 (sc-22760), RAD51 (sc-6862) and TFIIH (sc-293, used as loading control) from Santa Cruz Biotechnology (Dallas, TX, USA); phoshpo-Thr543-53BP1 (#3428), phospho-S15-p53 (#9284) from Cell Signaling Technology (Danvers, MA, USA); RPA2 (clone 9H8, ab2175), and phospho-Ser1524-BRCA1 (ab2401) from Abcam (Cambridge, UK), γH2AX (05-636), and mouse monoclonal 53BP1 (MAB3802) from Merck Millipore (Burlington, MA, USA); phospho-S824-KAP1 (GTX63711), KAP1 (GTX62973) and PP4C (GTX114659) from Genetex (Irvine, CA, USA); secondary Alexa Fluor conjugated antibodies from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: Irradiation, Knock-Out, Labeling, Extraction, Staining, Two Tailed Test, Mutagenesis, Reporter Assay, Transfection, Plasmid Preparation

Journal: Cells

Article Title: WIP1 Promotes Homologous Recombination and Modulates Sensitivity to PARP Inhibitors

doi: 10.3390/cells8101258

Figure Lengend Snippet: WIP1 delays recruitment of BRCA1 and dephosphorylation of 53BP1 at T543. ( A ) Co-immunoprecipitation of WIP1 and 53BP1. HEK293 cells were transfected with either empty GFP or GFP-WIP1, subjected to immunoprecipitation using GFP-Trap 24 h after transfection and by Western blotting with 53BP1 antibody. Ponceau staining with indicated positions of GFP (empty arrowhead) and GFP-WIP1 (full arrowhead) are shown. ( B ) HEK293 cells transfected with EGFP or EGFP-WIP1 were exposed to 3 Gy of IR, collected at indicated times and proteins were immunoprecipitated by GFP Trap. ( C ) Quantification of 53BP1 pT543 signal intensity in replicating (EdU+) cells after irradiation. U2OS parental and WIP1 knockout cell lines were pulse-labeled with EdU for 30 min before irradiation. Cells were fixed after pre-extraction at indicated time-points after IR and stained with p53BP1 T543 antibody. Click chemistry was used to visualize EdU. Mean of median total intensity +/− SD is plotted. ( D ) Western blot analysis of whole cell lysates of U2OS cells transfected with GAPDH or PP4C siRNA in response to irradiation and/or WIP1 inhibitor. ( E ) Quantification of 53BP1 pT543 signal intensity in replicating (EdU+) cells after irradiation. U2OS parental and WIP1 knockout cell lines were transfected with control or PP4C siRNA 2 days before irradiation. Cells were processed and analyzed as in C. ( F ) Quantification of RPA2 foci in replicating (EdU+) cells after irradiation. U2OS parental cell lines with or without combined treatment with WIP1i were pulse-labeled with EdU for 30 minutes before irradiation. Cells were fixed after pre-extraction at indicated time-points and stained with RPA2 and RAD51 antibodies. Click chemistry was used to visualize EdU. Mean of median foci number +/− SD is plotted. ( G ) Quantification of RAD51 foci in replicating (EdU+) cells after irradiation as in F.

Article Snippet: Following antibodies were used: WIP1 antibody (clone F-10, sc-376257), p21 (sc-397), p53 (clone D01, sc-126), BRCA1 (sc-6954), rabbit-53BP1 (sc-22760), RAD51 (sc-6862) and TFIIH (sc-293, used as loading control) from Santa Cruz Biotechnology (Dallas, TX, USA); phoshpo-Thr543-53BP1 (#3428), phospho-S15-p53 (#9284) from Cell Signaling Technology (Danvers, MA, USA); RPA2 (clone 9H8, ab2175), and phospho-Ser1524-BRCA1 (ab2401) from Abcam (Cambridge, UK), γH2AX (05-636), and mouse monoclonal 53BP1 (MAB3802) from Merck Millipore (Burlington, MA, USA); phospho-S824-KAP1 (GTX63711), KAP1 (GTX62973) and PP4C (GTX114659) from Genetex (Irvine, CA, USA); secondary Alexa Fluor conjugated antibodies from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: De-Phosphorylation Assay, Immunoprecipitation, Transfection, Western Blot, Staining, Irradiation, Knock-Out, Labeling, Extraction, Control

Journal: Cells

Article Title: WIP1 Promotes Homologous Recombination and Modulates Sensitivity to PARP Inhibitors

doi: 10.3390/cells8101258

Figure Lengend Snippet: WIP1 deficient cells are more sensitive to PARP inhibition. ( A ) Cell survival of parental U2OS, two independent U2OS-WIP1-KO cell lines with or without combined treatment with WIP1i was evaluated 7 days after treatment with indicated doses of olaparib using resazurin viability assay. Plotted is mean +/− SD, n ≥ 3. Statistical significance evaluated by two-way ANOVA. ( B ) Cell survival of parental U2OS, U2OS-WIP1-KO cells and cell lines complemented with wild-type or phosphatase-dead (D314A) mutant of WIP1 in response to 5 μM olaparib as in A. Statistical significance evaluated by two-tailed t -test (n ≥ 3). ( C ) Percentage of dead cells was evaluated by Hoechst 33258 staining and FACS analysis 7 days after treatment with 5 μM olaparib in U2OS cell line with or without combined treatment with WIP1i. Plotted is mean +/− SD. ( D ) Cell survival of RPE and MCF7 cell lines with or without combined treatment with WIP1i was evaluated 7 days after treatment with indicated doses of olaparib using resazurin viability assay. Plotted is mean +/− SD. N ≥ 3. Statistical significance evaluated by two-way ANOVA. ( E ) Cells were transfected with control siRNA (siNC) or siRNA to PP4C (siPP4C). Cell survival was evaluated after 7 days of treatment with olaparib and DMSO or WIP inhibitor. Statistical significance evaluated by two-tailed t -test (n = 3). ( F ) Quantification of 53BP1 foci number 3 days after treatment with olaparib. U2OS cells were treated with indicated doses of olaparib together with or without WIP1i for 3 days, fixed, stained with 53BP1 antibody and percentage of cells having 0–3, 3–10 and >10 foci were quantified. Mean +/− SD is plotted, n ≥ 3. ( G ) Quantification of 53BP1 foci after treatment with olaparib. U2OS-WIP1-KO cells and cell lines complemented with wild-type or phosphatase-dead (D314A) mutant of WIP1 were treated with WIP1i and olaparib for 3 days, fixed after pre-extraction and stained with 53BP1 antibody. Number of 53BP1 foci in S/G2 cells was evaluated using DAPI content of >2 n to gate S-G2 cells. Mean of median foci number +/− SD is plotted, n ≥ 3. Statistical significance evaluated by two-tailed t -test. ( H ) Response of U2OS and U2OS-WIP1-KO cell lines to treatment with 5 μM olaparib for 24–72h was analyzed by Western blotting using indicated antibodies. I ) Quantification of 53BP1 foci 3 days after treatment with olaparib. MCF7 cells were transfected with indicated siRNAs and treated after 2 days with WIP1i and olaparib alone or combined for further 3 days. Cells were fixed after pre-extraction and stained with 53BP1 antibody. Number of 53BP1 foci in S/G2 cells was evaluated using DAPI content of >2 n to gate S-G2 cells. Mean of median foci number +/− SD is plotted. Statistical significance evaluated by two-tailed t -test.

Article Snippet: Following antibodies were used: WIP1 antibody (clone F-10, sc-376257), p21 (sc-397), p53 (clone D01, sc-126), BRCA1 (sc-6954), rabbit-53BP1 (sc-22760), RAD51 (sc-6862) and TFIIH (sc-293, used as loading control) from Santa Cruz Biotechnology (Dallas, TX, USA); phoshpo-Thr543-53BP1 (#3428), phospho-S15-p53 (#9284) from Cell Signaling Technology (Danvers, MA, USA); RPA2 (clone 9H8, ab2175), and phospho-Ser1524-BRCA1 (ab2401) from Abcam (Cambridge, UK), γH2AX (05-636), and mouse monoclonal 53BP1 (MAB3802) from Merck Millipore (Burlington, MA, USA); phospho-S824-KAP1 (GTX63711), KAP1 (GTX62973) and PP4C (GTX114659) from Genetex (Irvine, CA, USA); secondary Alexa Fluor conjugated antibodies from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: Inhibition, Viability Assay, Mutagenesis, Two Tailed Test, Staining, Transfection, Control, Extraction, Western Blot

Journal: Journal of Cell Science

Article Title: Nuclear entry and export of FIH are mediated by HIF1α and exportin1, respectively

doi: 10.1242/jcs.219782

Figure Lengend Snippet: Evidence that hypoxia induces nuclear entry of FIH. (A) Protein levels of HIF1α, HIF2α, FIH and PHD2 in U2OS cells during hypoxia (1% O 2 ) treatment at the indicated time points. GAPDH was used as a loading control. (B) Immunofluorescence staining of FIH (green) in U2OS cells under hypoxic conditions (1% O 2 ) at the indicated time points. TO-PRO-3 (blue) was used to stain nuclei. (C) Protein levels of FIH and HIF1α from cytoplasmic or nuclear fractions in U2OS cells in normoxia or hypoxia (1% O 2 , 3 h). β-tubulin and PARP were used as loading controls for the cytoplasmic and nuclear fractions, respectively. Figures beneath lanes 2 and 4 indicate relative intensities of nuclear FIH in normoxia and hypoxia. Note that different quantities of cytoplasmic and nuclear extracts were loaded. Scale bars: 20 µm.

Article Snippet: Cell extracts were then precleared with protein G beads and incubated with antibodies against importin β1 (2 μl/mg protein lysate; Cell Signaling Technology, 8673, rabbit polyclonal), exportin1 (2 μg/mg protein lysate; Sigma-Aldrich, E7784, rabbit polyclonal), HIF1α (2 μg/mg protein lysate; Novus Biologicals, NB100-479, rabbit polyclonal) or p300 (2 μg/mg protein lysate, Millipore, 05-257, mouse monoclonal RW128) for 16 h at 4°C.

Techniques: Control, Immunofluorescence, Staining

Journal: Journal of Cell Science

Article Title: Nuclear entry and export of FIH are mediated by HIF1α and exportin1, respectively

doi: 10.1242/jcs.219782

Figure Lengend Snippet: Nuclear entry of FIH is mainly HIF1α-dependent, and requires inhibition of FIH enzymatic activity. (A) U2OS cells were transfected with the indicated siRNAs for 3 days, followed by culture in normoxia (20% O 2 ) or hypoxia (1% O 2 , 3h). Images show immunofluorescence staining of FIH (green) in U2OS cells after the indicated treatments. TO-PRO-3 (blue) was used to stain nuclei. (B) Immunofluorescence staining of FIH (green), HIF1α N803OH (green) and HIF1α (red) in U2OS cells treated with DMSO, DM-NOFD (1 mM), IOX2 (0.25 mM) or DM-NOFD (1 mM) plus IOX2 (0.25 mM) for 3 h. TO-PRO-3 (blue) was used to stain nuclei. (C) Protein levels of FIH, HIF1α and HIF1α N803OH in U2OS cells after the indicated treatments. β-tubulin was used as a loading control. Total cell lysates from the treated U2OS cells were immunoprecipitated with an anti-HIF1α antibody. Scale bars: 20 µm.

Article Snippet: Cell extracts were then precleared with protein G beads and incubated with antibodies against importin β1 (2 μl/mg protein lysate; Cell Signaling Technology, 8673, rabbit polyclonal), exportin1 (2 μg/mg protein lysate; Sigma-Aldrich, E7784, rabbit polyclonal), HIF1α (2 μg/mg protein lysate; Novus Biologicals, NB100-479, rabbit polyclonal) or p300 (2 μg/mg protein lysate, Millipore, 05-257, mouse monoclonal RW128) for 16 h at 4°C.

Techniques: Inhibition, Activity Assay, Transfection, Immunofluorescence, Staining, Control, Immunoprecipitation

Journal: Journal of Cell Science

Article Title: Nuclear entry and export of FIH are mediated by HIF1α and exportin1, respectively

doi: 10.1242/jcs.219782

Figure Lengend Snippet: FIH complexes with importin β1 via HIF1α for nuclear import. (A) U2OS cells were transfected with control siRNA or importin β1 siRNA for 3 days, followed by treatment with DMSO or DMOG (1 mM) for 3 h. Images show immunofluorescence staining of FIH (red) or HIF1α (red) in U2OS cells after the indicated treatments. DAPI (blue) was used to stain nuclei. (B) U2OS cells were transfected with control siRNA or HIF1α siRNA for 3 days, followed by treatment with DMSO or DMOG (1 mM) for 3 h. Total cell lysates from the U2OS cells were immunoprecipitated with an anti-importin β antibody or control IgG. FIH, HIF1α and importin β levels are indicated. Scale bars: 10 µm.

Article Snippet: Cell extracts were then precleared with protein G beads and incubated with antibodies against importin β1 (2 μl/mg protein lysate; Cell Signaling Technology, 8673, rabbit polyclonal), exportin1 (2 μg/mg protein lysate; Sigma-Aldrich, E7784, rabbit polyclonal), HIF1α (2 μg/mg protein lysate; Novus Biologicals, NB100-479, rabbit polyclonal) or p300 (2 μg/mg protein lysate, Millipore, 05-257, mouse monoclonal RW128) for 16 h at 4°C.

Techniques: Transfection, Control, Immunofluorescence, Staining, Immunoprecipitation

Journal: Journal of Cell Science

Article Title: Nuclear entry and export of FIH are mediated by HIF1α and exportin1, respectively

doi: 10.1242/jcs.219782

Figure Lengend Snippet: FIH exits the nucleus via a Leptomycin B-sensitive exportin1-dependent pathway. (A) Immunofluorescence staining of FIH (green) and HIF1α (red) in MCF7 cells after the indicated hypoxia (0.5% O 2 ) and re-oxygenation treatments. TO-PRO-3 (blue) was used to stain nuclei. (B) Total cell lysates from U2OS cells were immunoprecipitated with an anti-exportin1 antibody or control IgG. FIH, exportin1 and β-tubulin levels are indicated. (C) Immunofluorescence staining of FIH (green) in FIH-null mouse embryonic fibroblasts (MEFs) transfected with HA-FIH 1–349 or HA-FIH ΔNES followed by normoxia, hypoxia (1% O 2 , 3 h) or 3 h of hypoxia followed by re-oxygenation for 1 h. TO-PRO-3 (blue) was used to stain nuclei. Arrows indicate nuclear localization of signal. (D) Total cell lysates from U2OS cells transfected with control vector, HA-FIH 1–349 or HA-FIH ΔNES were immunoprecipitated with an anti-exportin 1 antibody. HA-FIH, exportin1 and β-tubulin levels are indicated. FL, full length; IgG L , IgG light chain. Scale bars: 20 µm.

Article Snippet: Cell extracts were then precleared with protein G beads and incubated with antibodies against importin β1 (2 μl/mg protein lysate; Cell Signaling Technology, 8673, rabbit polyclonal), exportin1 (2 μg/mg protein lysate; Sigma-Aldrich, E7784, rabbit polyclonal), HIF1α (2 μg/mg protein lysate; Novus Biologicals, NB100-479, rabbit polyclonal) or p300 (2 μg/mg protein lysate, Millipore, 05-257, mouse monoclonal RW128) for 16 h at 4°C.

Techniques: Immunofluorescence, Staining, Immunoprecipitation, Control, Transfection, Plasmid Preparation

Journal: Heliyon

Article Title: Over-expressed, N-terminally truncated BRAF is detected in the nucleus of cells with nuclear phosphorylated MEK and ERK

doi: 10.1016/j.heliyon.2018.e01065

Figure Lengend Snippet: Localisation of GFP-RAF fusion proteins (A) GFP fluorescence imaging. NIH3T3 cells were transfected with vectors expressing GFP alone or the various RAF-GFP fusion proteins shown on the right. Cells were subjected to fluorescence imaging, generating images for GFP and DAPI, which were then merged. Scale bars, 50 μm. (B) Quantitation of GFP compartmentalisation. NIH3T3 cells were transfected with the vectors indicated and treated ± LMB. Following fluorescence microscopy, GFP fluorescence was categorized by the user as mostly nuclear (N > C), equally distributed (N=C) or mostly cytoplasmic (N < C). Over 200 cells were visualized for each transfection. Bar chart indicates mean (n = 3) ± SEM. Representative images of cells in each category are shown on the left. Scale bars, 50 μm. (C) Nuclear/cytoplasmic fractionation of GFP, GFP-FL- WT BRAF and GFP-ΔBRAF. NIH3T3 cells were transfected with the vectors indicated and subjected to subcellular fractionation 48 hours later. Fractions were analysed by western blot for GFP or with α-Tubulin or PARP to confirm purity of cytoplasmic and nuclear fractions. (D) Nuclear/cytoplasmic fractionation of GFP-ΔCRAF. NIH3T3 cells were transfected with the vectors indicated, fractions prepared and analysed by Western blot with antibodies for GFP, α-Tubulin or PARP. (E) Nuclear/cytoplasmic fractionation of endogenous BRAF. Whole cell Triton X-100 soluble lysates (WCL) of NIH3T3 cells were prepared as well as nuclear/cytoplasmic fractions and analysed with antibodies for BRAF, α-Tubulin or PARP. (F) Quantitation of western blot data. The Nuclear:Cytoplasmic (N:C) proportions were determined using Image J analysis of western blot signals. Density signals were adjusted for loading using signals for α-Tubulin (cytoplasmic loading) or PARP (nuclear loading). Values for the nuclear fractions were then divided by the total values for each sample. Data represent mean ± SEM (GFP, n = 8; GFP-FL-BRAF, n = 9; GFP-ΔBRAF, n = 10; GFP-ΔCRAF, n = 4; endogenous, n = 5).

Article Snippet: For assessing the RAF kinase activity of GFP-ΔBRAF fractions or control GFP, nuclear/cytoplasmic fractions (GFP-ΔBRAF) or whole cell lysates (GFP) were subjected to immunoprecipitation using GFP–TRAP beads (Chromotek, ABIN509407) under their recommended conditions and the RAF kinase cascade assay was undertaken as previously described .

Techniques: Fluorescence, Imaging, Transfection, Expressing, Quantitation Assay, Microscopy, Fractionation, Western Blot

Journal: Heliyon

Article Title: Over-expressed, N-terminally truncated BRAF is detected in the nucleus of cells with nuclear phosphorylated MEK and ERK

doi: 10.1016/j.heliyon.2018.e01065

Figure Lengend Snippet: Localisation of oncogenic BRAF. (A) GFP fluorescence imaging of V600E BRAF. NIH3T3 cells were transfected with vectors expressing either GFP-FL- WT BRAF as in Fig. 1 B or GFP- V600E BRAF (schematic on the left). Cells were subjected to fluorescence imaging, generating images for GFP and DAPI, which were then merged. Representative images of GFP- V600E BRAF-expressing cells are shown on the left. Scale bars, 50 μm. GFP fluorescence was categorized as N > C, N = C or N < C. Over 200 cells were visualized for each transfection. The bar chart indicates mean (n = 3) ± SEM. The data for GFP-FL- WT BRAF are the same as that shown in Fig. 1 B. (B) Fractionation of GFP- V600E BRAF. NIH3T3 cells were transfected with the vectors indicated and nuclear/cytoplasmic fractions were analysed with the antibodies indicated. Western blot data were quantified using Image J analysis to generate the N:C proportion as indicated in the bar chart. Data represent mean ± SEM (GFP-FL- WT BRAF, n = 9; GFP-ΔBRAF, n = 10; GFP- V600E BRAF, n = 4). The quantitative data for GFP-FL- WT BRAF and GFP-ΔBRAF are the same as that shown in Fig. 1 F. (C) Fractionation of BRAF oncogenic fusion protein. NIH3T3 cells transiently expressing KIAA1549-BRAF (SK:B, shown above) or full-length, wild-type BRAF in pcDNA3.1 were subjected to fractionation and fractions were analysed with an antibody detecting the C-terminus of BRAF or with the α-Tubulin/PARP antibodies. Western blot data were quantified using Image J analysis to generate the N:C proportions presented in the bar chart. Data represent mean ± SEM (n = 3 for both FL- WT BRAF and SK:B).

Article Snippet: For assessing the RAF kinase activity of GFP-ΔBRAF fractions or control GFP, nuclear/cytoplasmic fractions (GFP-ΔBRAF) or whole cell lysates (GFP) were subjected to immunoprecipitation using GFP–TRAP beads (Chromotek, ABIN509407) under their recommended conditions and the RAF kinase cascade assay was undertaken as previously described .

Techniques: Fluorescence, Imaging, Transfection, Expressing, Fractionation, Western Blot

Journal: Heliyon

Article Title: Over-expressed, N-terminally truncated BRAF is detected in the nucleus of cells with nuclear phosphorylated MEK and ERK

doi: 10.1016/j.heliyon.2018.e01065

Figure Lengend Snippet: GFP-ΔBRAF nuclear import is independent of a classical NLS. (A) Comparison of the amino acid sequences of a putative bipartite NLS within BRAF and RAF homologues from the indicated species. Sequences were taken from Ensembl ( www.ensembl.org/index.html ). (B) Ribbon diagram of the putative bipartite NLS within human BRAF. Expanded views of the exposed motifs at basic residues 690-691 (top expanded view) and 698-704 (bottom expanded view) are shown. (C) Mutation of the putative bipartite NLS in GFP-ΔBRAF. Four different mutants were generated within the putative NLS sequence at residues 726-741 of GFP-ΔBRAF. These vectors, along with GFP-ΔBRAF and GFP-ΔCRAF were transfected into NIH3T3 cells and nuclear and cytoplasmic fractions prepared. Western blots were analysed with the antibodies indicated. Western blot data were quantified using Image J analysis to generate the N:C proportion for the fusion proteins indicated in the bar chart on the right. Data represent mean ± SEM (GFP-ΔBRAF, n = 10; GFP-ΔCRAF, n = 4; all mutants, n = 3). The quantitative data for GFP-ΔBRAF and GFP-ΔCRAF are the same as that shown in Fig. 1 F. (D) Representative fluorescence microscopy images of GFP-ΔBRAF and GFP-ΔBRAF carrying the AA-AAA mutations. Scale bars, 50 μm. (E) FRAP analysis of GFP-ΔBRAF. NIH3T3 cells transfected with vectors expressing either GFP-ΔBRAF or monomeric GFP were subjected to FRAP. Mean mobile fractions and recovery half times are shown. Data for individual cells are shown in Fig. S3.

Article Snippet: For assessing the RAF kinase activity of GFP-ΔBRAF fractions or control GFP, nuclear/cytoplasmic fractions (GFP-ΔBRAF) or whole cell lysates (GFP) were subjected to immunoprecipitation using GFP–TRAP beads (Chromotek, ABIN509407) under their recommended conditions and the RAF kinase cascade assay was undertaken as previously described .

Techniques: Comparison, Mutagenesis, Generated, Sequencing, Transfection, Western Blot, Fluorescence, Microscopy, Expressing

Journal: Heliyon

Article Title: Over-expressed, N-terminally truncated BRAF is detected in the nucleus of cells with nuclear phosphorylated MEK and ERK

doi: 10.1016/j.heliyon.2018.e01065

Figure Lengend Snippet: Effect of CRAF, ARAF, G12D KRAS and 14-3-3 on GFP-BRAF localisation. (A) GFP-FL- WT BRAF or GFP-ΔBRAF expression in Craf KO or wild-type MEFs followed by fluorescence microscopy quantitation. (B) GFP-FL- WT BRAF or GFP-ΔBRAF in Araf KO or wild-type MEFs followed by fluorescence microscopy quantitation. (C) GFP-FL- WT BRAF or GFP-ΔBRAF expression in wild-type or KRAS G12D MEFs followed by fluorescence microscopy quantitation. (D) GFP-FL-BRAF with or without the R188L, S729A or S356A mutations or GFP-ΔBRAF expressed in NIH3T3 cells followed by fluorescence microscopy quantitation. For (A–D), cells were categorized as in Fig. 1 B and bar charts show mean (n = 3) ± SEM. In (A–C), three different MEFs of each genotype were examined. (E) Cells from (D) were subjected to nuclear/cytoplasmic fractionation and western blots analysed with antibodies against GFP or with GAPDH or Histone H1 to confirm purity of fractions. Western blot data were quantified using Image J analysis to generate the N:C proportion indicated in the bar chart on the right. Data represent mean ± SEM (n = 3 for each vector).

Article Snippet: For assessing the RAF kinase activity of GFP-ΔBRAF fractions or control GFP, nuclear/cytoplasmic fractions (GFP-ΔBRAF) or whole cell lysates (GFP) were subjected to immunoprecipitation using GFP–TRAP beads (Chromotek, ABIN509407) under their recommended conditions and the RAF kinase cascade assay was undertaken as previously described .

Techniques: Expressing, Fluorescence, Microscopy, Quantitation Assay, Fractionation, Western Blot, Plasmid Preparation

Journal: Heliyon

Article Title: Over-expressed, N-terminally truncated BRAF is detected in the nucleus of cells with nuclear phosphorylated MEK and ERK

doi: 10.1016/j.heliyon.2018.e01065

Figure Lengend Snippet: GFP-ΔBRAF nuclear compartmentalisation is suppressed by the CR1 domain. (A) Diagrams indicate the full length and truncated versions of human BRAF-GFP generated. (B) The vectors from (A) were transfected into NIH3T3 cells and cells were categorised as in Fig. 1 B. Bar chart indicates mean (n = 3) ± SEM. (C) Cells from (B) were also subjected to nuclear/cytoplasmic fractionation, which were analysed by western blot with the antibodies indicated. Western blot data were quantified using Image J analysis to generate the N:C proportion indicated in the bar chart on the right. Data represent mean ± SEM (n = 3 for each vector).

Article Snippet: For assessing the RAF kinase activity of GFP-ΔBRAF fractions or control GFP, nuclear/cytoplasmic fractions (GFP-ΔBRAF) or whole cell lysates (GFP) were subjected to immunoprecipitation using GFP–TRAP beads (Chromotek, ABIN509407) under their recommended conditions and the RAF kinase cascade assay was undertaken as previously described .

Techniques: Generated, Transfection, Fractionation, Western Blot, Plasmid Preparation

Journal: Heliyon

Article Title: Over-expressed, N-terminally truncated BRAF is detected in the nucleus of cells with nuclear phosphorylated MEK and ERK

doi: 10.1016/j.heliyon.2018.e01065

Figure Lengend Snippet: Localisation of phosphorylated MEK and ERK. (A) Nuclear GFP-ΔBRAF has kinase activity towards the MEK-ERK pathway. NIH3T3 cells were transfected with vectors expressing GFP or GFP-ΔBRAF. Whole cell lysates were prepared from the GFP-transfected cells while nuclear/cytoplasmic fractions were prepared from the GFP-ΔBRAF transfected cells. GFP expression levels and purity of fractions are indicated by the western blots on the bottom. Samples were subjected to immunoprecipitation for GFP and kinase cascade assays were performed. Data shows mean ± SD of three independent experiments. (B) Nuclear GFP-ΔBRAF binds to phosphorylated and non-phosphorylated MEK and ERK. NIH3T3 cells were transfected with vectors expressing GFP or GFP-ΔBRAF, nuclear/cytoplasmic fractions were prepared and analysed for GFP, α-Tubulin or PARP as well as components of the MAPK pathway. A portion of the fractions was subjected to GFP-TRAP immunoprecipitation and immunoprecipitates were subjected to western blot analysis with the antibodies indicated. (C) Phosphorylated MEK and ERK accumulate in the nucleus of cells expressing GFP-ΔBRAF. NIH3T3 cells were transfected with the vectors indicated. Nuclear and cytoplasmic fractions were prepared and analysed by western blot with the indicated antibodies. (D) Quantitation of western blot data from (C). Image J analysis was used to determine density signals of each band on western blots which were adjusted for loading using signals for α-Tubulin (cytoplasmic loading) or PARP (nuclear loading). In the upper bar chart, PP-MEK value were adjusted for total MEK values while in the lower bar chart PP-ERK values were adjusted for ERK2 values. The data are presented as Arbitrary Units (AU) and represent mean ± SEM (n = 3 for each condition).

Article Snippet: For assessing the RAF kinase activity of GFP-ΔBRAF fractions or control GFP, nuclear/cytoplasmic fractions (GFP-ΔBRAF) or whole cell lysates (GFP) were subjected to immunoprecipitation using GFP–TRAP beads (Chromotek, ABIN509407) under their recommended conditions and the RAF kinase cascade assay was undertaken as previously described .

Techniques: Activity Assay, Transfection, Expressing, Western Blot, Immunoprecipitation, Quantitation Assay

Journal: Heliyon

Article Title: Over-expressed, N-terminally truncated BRAF is detected in the nucleus of cells with nuclear phosphorylated MEK and ERK

doi: 10.1016/j.heliyon.2018.e01065

Figure Lengend Snippet: High Content Microscopy (HCM). (A) Localisation of BRAF-GFP using HCM. NIH3T3 cells expressing either GFP-ΔBRAF or GFP-FL- WT BRAF were treated with LMB for 3 hours and then subjected to HCM analysis. GFP staining was categorised into one of three categories as indicated where N > C represents >1.5 class nuclear staining, N=C represents 1–1.5 class nuclear staining and N < C represents <1 class nuclear staining. Mean values ± SEM are shown. (B) Localisation of PP-ERK. NIH3T3 cells expressing GFP-FL- WT BRAF or GFP-ΔBRAF were treated with LMB for 3 hours, immunostained for PP-ERK1/2 and subjected to HCM. Representative digital images are shown. Cells were sorted into bins as described in Methods. The data are population averages (in AFU) from 3 repeat experiments, each with duplicate adenovirus infections and 500–600 cells per infection. Correlation coefficients (r) were determined for each dataset, converted to Z scores and compared to generate the p value indicated. (C) Localisation of PP-MEK. NIH3T3 cells expressing GFP-FL- WT BRAF or GFP-ΔBRAF were treated with LMB for 3 hours, immunostained for PP-MEK and analysed by HCM. Representative digital images are shown. Cells were sorted into bins as described in Methods. The data in the graph are population averages (in AFU) from 3 repeat experiments, each with duplicate adenovirus infections and 500–600 cells per infection. Correlation coefficients (r) were determined for each dataset, converted to Z scores and compared to generate the p value indicated.

Article Snippet: For assessing the RAF kinase activity of GFP-ΔBRAF fractions or control GFP, nuclear/cytoplasmic fractions (GFP-ΔBRAF) or whole cell lysates (GFP) were subjected to immunoprecipitation using GFP–TRAP beads (Chromotek, ABIN509407) under their recommended conditions and the RAF kinase cascade assay was undertaken as previously described .

Techniques: Microscopy, Expressing, Staining, Infection

Journal: Cell calcium

Article Title: Ca 2+ -dependent binding of S100A6 to cofilin-1 regulates actin filament polymerization-depolymerization dynamics.

doi: 10.1016/j.ceca.2021.102457

Figure Lengend Snippet: Fig. 1. Interaction of S100A6 with cofilin-1 in NIH3T3 fibroblasts. (A) Pull-down assay with the use of protein lysate from NIH3T3 cells and S100A6 affinity resin (upper panel) or empty resin (lower panel). Lanes: 1-input, 2-unbound fraction, 3-last wash, 4-first wash with 0.5 M NaCl, 5-last wash with 0.5 M NaCl, 6-first wash with 1 M NaCl, 7- last wash with 1 M NaCl, 8- elution in buffer containing EGTA. Fractions were analyzed by SDS-PAGE (15% gel) fol lowed by immunoblotting developed with anti- cofilin-1 antibody. (B) Co-immunoprecipitation of S100A6 with cofilin-1 from NIH3T3 cell lysate. 30 μg of protein lysate was used directly for immunoblotting (input; lane 1 in both upper and lower panel) and 2.5 mg of protein lysate was incubated with (upper panel) or without (control, lower panel) anti-S100A6 monoclonal antibody and then with protein A/G agarose. In both panels, lane 2 shows unbound fraction, lane 3-last wash and lane 4-elution. Proteins were identified by immunoblotting using anti- cofilin-1 antibody. (C) Presence of S100A6- cofilin-1 complexes in NIH3T3 cells studied by PLA. Complexes of examined proteins are visualized in red; cell nuclei, stained with DAPI, are in blue. Scale bar is 20 μm.

Article Snippet: For co-immunoprecipitation assays 2.5 mg of protein lysate from NIH3T3 cells, obtained using the Plasma Membrane Protein Extraction Kit (Abcam) according to the manufacturer’s instruction was incubated with protein A/G-Agarose (Santa Cruz Biotechnology) for 1 h at 4◦C, as described by Jurewicz et al. [31].

Techniques: Pull Down Assay, SDS Page, Western Blot, Immunoprecipitation, Incubation, Control, Staining