Journal: medRxiv

Article Title: ARF1 prevents aberrant type I IFN induction by regulating STING activation and recycling

doi: 10.1101/2023.04.28.23289152

Figure Lengend Snippet: a , Induction of the ISRE promoter in THP-1-Dual WT (grey) and THP-1-Dual KO-cGAS (pink) cells transduced with lentiviral vectors expressing ARF1 WT or R99C as indicated, quantified by Lucia luciferase (LLuc) activity 72 h post transduction. IFN-β (1000 U/mL, 16 h) and cGAMP (10 µg/ml, 16 h) served as positive controls. Bars represent mean of n=3 ± SEM (biological replicates). Lower panel: Corresponding immunoblots of WCLs stained by anti-FLAG, anti-STING, anti-cGAS and anti-GAPDH. b , Exemplary electron microscopy analysis of HEK293T cells transiently transfected with ARF1 WT or ARF1 R99C as indicated. Mitochondria (m) are highlighted in insets in bottom panels. Electron-dense granules and inflated cristae are highlighted by black and white arrows, respectively. Annotations: cp, cytoplasm; er, endoplasmic reticulum; g , Golgi apparatus; lv, large vesicle; ly, lysosome; m, mitochondria; nc, nucleus. c, Exemplary immunoblots showing fractionation of ARF1 WT, R99C, Q71L and vector transfected HEK293T cells as indicated. WCLs and fraction blots stained by anti-FLAG, anti-TFAM (mitochondria), anti-LAMIN B1 (nucleus) and anti-GAPDH (cytosol). d, qPCR of mtDNA (MT-D-Loop) in the cytosolic fraction of ( c ) relative to total normalized cellular mtDNA (mtDNA/nuclear DNA) using the ΔΔCT method. n = 3 ± SEM. e , qPCR of mtDNA (MT-D-Loop) in the cytosolic fraction of primary fibroblasts from healthy donors (n2, f1, I7) or a patient (Patient 1) relative to total normalized cellular mtDNA (mtDNA/nuclear DNA) using the ΔΔCT method. n = 5 ± SEM. f, qPCR of representative ISG OAS1 in U2OS cells stably expressing STING and depleted of mtDNA by ddC, or untreated (NT), upon transfection with empty vector, ARF1 WT or R99C n=3 ± SEM. g , Exemplary immunoblot of WCLs of HEK293T cells transiently expressing ARF1 WT, R99C or vector. Blots were stained with anti-MFN1, anti-RHOT1, anti-FLAG and anti-GAPDH. Quantification of the band intensities for MFN1 normalized to the band intensities of GAPDH. Bars represent mean of n = 6 ± SEM (biological replicates). h , Exemplary immunoblots showing fractionation of HEK293T cells expressing ARF1 WT, R99C or vector control as well as VCP. WCLs and fraction blots stained by anti-FLAG, anti-HA, anti-TFAM (mitochondria), anti LAMIN B1 (nucleus) and anti-GAPDH (cytosol). i , qPCR of mtDNA (MT-D-Loop) in the cytosolic fraction of (h) relative to total normalized cellular mtDNA (mtDNA/nuclear DNA) using 725 the ΔΔCT method. n = 5 ± SEM.

Article Snippet: U2OS cells (ATCC) were maintained in McCoy medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum.

Techniques: Transduction, Expressing, Luciferase, Activity Assay, Western Blot, Staining, Electron Microscopy, Transfection, Fractionation, Plasmid Preparation, Stable Transfection, Control

Journal: International Journal of Nanomedicine

Article Title: Anti-FGFR1 aptamer-tagged superparamagnetic conjugates for anticancer hyperthermia therapy

doi: 10.2147/IJN.S125231

Figure Lengend Snippet: Fluorescence microscopy images of aptamer A11-G18T binding to FGFR1-expressing cells. Notes: U2OS (upper row), U2OS-R1 (middle row) and NIH-3T3 (lower row) cells were incubated with fluorescein-labeled A11-G18T anti-FGFR1 aptamer, as examined by fluorescence microscopy. Columns from the left: blue DAPI staining of nuclei, red phalloidin staining of actin, green fluorescein signal (staining of surface FGFR1) and a merged picture. Scale bars correspond to 50 μm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; Phalloidin, fluorescent-labeled phalloidin; AF, FITC-labeled A11-G18T anti-FGFR1 aptamer; FGFR1, fibroblast growth factor receptor type-1; FITC, fluorescein isothiocyanate.

Article Snippet: Human osteosarcoma cell line (U2OS) was obtained from American Type Culture Collection (Manassas, VA, USA) and cultured as recommended by the supplier.

Techniques: Fluorescence, Microscopy, Binding Assay, Expressing, Incubation, Labeling, Staining

Journal: International Journal of Nanomedicine

Article Title: Anti-FGFR1 aptamer-tagged superparamagnetic conjugates for anticancer hyperthermia therapy

doi: 10.2147/IJN.S125231

Figure Lengend Snippet: Aptamer-targeted hyperthermia compared to unselected oligonucleotide library. Notes: Aptamer-targeted hyperthermia study on U2OS-R1 cells. All samples were washed prior to the magnetic field treatment as described in the Methods section. Nontreated cells (first column) were used as 100% viability control. Samples: columns 2–4 represent different negative controls: column 3 (- nanoparticles, + A11-G18T, + magnetic field) validates the effect of free A11-G18T aptamer without the nanoparticles, while column 4 (+ nanoparticles, + Lib, + magnetic field) employs conjugates prepared with unselected randomized oligonucleotide library (Lib) instead of the selected aptamer. The last column shows aptamer-targeted nanoparticles-induced hyperthermia effect. Statistics: **statistically significant difference between the sample and the nontreated control (first column) at multiplicity adjusted P -value <0.01. The experiments were performed in duplicate.

Article Snippet: Human osteosarcoma cell line (U2OS) was obtained from American Type Culture Collection (Manassas, VA, USA) and cultured as recommended by the supplier.

Techniques: Control

Journal: Oncogene

Article Title: RSK2 regulates endocytosis of FGF receptor 1 by phosphorylation on serine 789.

doi: 10.1038/onc.2013.425

Figure Lengend Snippet: Figure 1. RSK2 interacts with FGFR1. (a) Schematic presentation of FGFR1 and RSK2. Pink lines indicate the intracellular parts of FGFR1, which were used as baits in yeast two-hybrid screens. The K514R mutation in bait 2 is indicated with a star (*). The blue line indicates the selected interaction domain identified in the yeast two-hybrid screen. TM, transmembrane region; TK, tyrosine kinase domains; D, Immunoglobulin-like domain; NTKD, N-terminal kinase domain; CTKD, C-terminal kinase domain. The N-terminal (N) and the C-terminal end (C) of FGFR1 and RSK2 are indicated. (b) Co-immunoprecipitations of FGFR1 and RSK2 using rabbit a-FGFR1 (left panel) or goat a-RSK2 (right panel) antibodies. U2OS-R1 cells were serum-starved for 6 h before treatment (as indicated) with FGF1 for 15 min. Cell lysates were then subjected to immunoprecipitation reactions followed by SDS-PAGE and western blotting with indicated antibodies. Immunoprecipitation reactions with rabbit IgG (left panel) or goat IgG (right panel) were used as controls. (c) Pull-downs of RSK2 with recombinant GST-tagged C-terminal part of FGFR1. Cell lysates from untreated BJ cells or BJ cells treated with FGF1 for 15 min (as indicated) were incubated with recombinant GST-tagged C-terminal tail of wild-type FGFR1 (Ct-FGFR1), FGFR1 S777A mutant (Ct-FGFR1 S777A), FGFR1 S777D mutant (Ct-FGFR1 S777D) or GST alone. Protein complexes were pulled down using glutathione-conjugated sepharose resin and analyzed by western blotting with indicated antibodies.

Article Snippet: Cells The human osteosarcoma cell line, U2OS, and the human normal foreskin fibroblast cell line, BJ, were from ATCC (Manassas, VA, USA).

Techniques: Mutagenesis, Two Hybrid Screening, Immunoprecipitation, SDS Page, Western Blot, Recombinant, Incubation

Journal: Oncogene

Article Title: RSK2 regulates endocytosis of FGF receptor 1 by phosphorylation on serine 789.

doi: 10.1038/onc.2013.425

Figure Lengend Snippet: Figure 2. The association between RSK2 and FGFR1 depends on RSK2 phosphorylation. (a) Serum starved U2OS-R1 cells were pretreated, as indicated, for 30 min with 100 nM PD173074, 20 mM U0126 or 10 mM BI-D1870 and then treated for 15 min with 100 ng/ml FGF1 or EGF or 10% serum. Cell lysates were then subjected to immunoprecipitation reactions using anti-FGFR1 antibodies followed by SDS-PAGE and western blotting with indicated antibodies. (b) Serum-starved U2OS-R1 cells were stimulated with 100 ng/ml FGF1 and 10 U/ml heparin for indicated periods of time. The cells were then lysed, and the lysates were analyzed by SDS-PAGE and western blotting with indicated antibodies. Western blots were quantified and the bands corresponding to the phosphorylated proteins were normalized to the bands corresponding to total ERK1/2 and presented in the graphs as a fraction of their maximal response. In the case of p-RSK1/2 the background band at time point zero was subtracted before normalizing to the total ERK1/2 levels. The graph represents the mean±standard deviation of three independent experiments.

Article Snippet: Cells The human osteosarcoma cell line, U2OS, and the human normal foreskin fibroblast cell line, BJ, were from ATCC (Manassas, VA, USA).

Techniques: Phospho-proteomics, Immunoprecipitation, SDS Page, Western Blot, Standard Deviation

Journal: Oncogene

Article Title: RSK2 regulates endocytosis of FGF receptor 1 by phosphorylation on serine 789.

doi: 10.1038/onc.2013.425

Figure Lengend Snippet: Figure 3. Mapping of interaction site of RSK2 in FGFR1. (a) Amino acid sequence of the C-terminal tail of FGFR1. A putative RSK2 phosphorylation motif is underlined. (b and c) U2OS cells were transiently transfected with wild-type FGFR1 (WT) or FGFR1 double (b) or single mutants (c) in which serines (S) and threonines (T) were substituted for alanines (A) or aspartic acids (D). Cell lysates from untreated cells or cells treated with 100 ng/ml FGF1 for 15 min (as indicated) were subjected to immunoprecipitation reactions using anti-FGFR1 antibodies followed by SDS-PAGE and western blotting with indicated antibodies. Western blots were quantified and the bands corresponding to RSK2 were normalized to total FGFR1. The amount of RSK2 is presented relative to the amount of RSK2 in untreated, wild- type FGFR1 transfected cells. The histogram represents the mean±standard deviation of two independent experiments.

Article Snippet: Cells The human osteosarcoma cell line, U2OS, and the human normal foreskin fibroblast cell line, BJ, were from ATCC (Manassas, VA, USA).

Techniques: Sequencing, Phospho-proteomics, Transfection, Immunoprecipitation, SDS Page, Western Blot, Standard Deviation

Journal: Oncogene

Article Title: RSK2 regulates endocytosis of FGF receptor 1 by phosphorylation on serine 789.

doi: 10.1038/onc.2013.425

Figure Lengend Snippet: Figure 4. FGFR1 is phosphorylated by RSK2 (a) In vitro phosphorylation of recombinant GST-tagged C-terminal part of FGFR1 (Ct-FGFR1) by recombinant RSK2. 1 mg of recombinant Ct-FGFR1 wild-type (WT), FGFR1 S777A, FGFR1 S777D or GST alone were incubated with recombinant active RSK2 and 200 mCi/ml [g-33P]ATP. The proteins were resolved by SDS-PAGE, electroblotted onto PVDF membranes and radioactive bands were visualized by autoradiography. The input of GST proteins is shown by Coomassie blue staining of the membrane. (b) In vitro phosphorylation of Ct-FGFR1 by recombinant RSK2 in the presence of increasing concentrations of RSK kinase inhibitor, BI-D1870. Reactions were performed as described in (a). Increasing concentrations of BI-D1870 was added as indicated. Recombinant inactive RSK2 kinase (x) was used as a control. (c) MS analysis of phosphorylation site for RSK2 in the C-terminal part of FGFR1. Selected peptides of the C-terminal part of FGFR1 were subjected to in vitro phosphorylation experiments. Sequence of the C-terminal part of FGFR1 with denoted peptides is shown. Serine residue 789 is indicated with *. The graphs represent examples of MS analysis of peptide 2 subjected to in vitro phosphorylation assay in the absence (upper panel) or presence (lower panel) of RSK2. An example of the MS/MS analysis of peptide 2 is shown in the table to the right. [Dhy] denotes dehydration (Dm ¼ 18), [2O ] denotes oxidation (Dm ¼ 36), [Pho] denotes phosphorylation (Dm ¼ 79.96), y denotes y-ion series, b denotes b-ion series. Ions observed in experiment are shown in grey. (d) FGFR1 is phosphorylated at serine 789 in vivo. Serum-starved U2OS (control), U2OS-R1, U2OS-R1 S789A or U2OS-R1 S789D cells were left untreated or treated with FGF1 or EGF for 15 min (as indicated). The cells were then lysed and the lysates were subjected to immunoprecipitation reactions using anti-FGFR1 antibodies followed by SDS-PAGE and western blotting with indicated antibodies. (e) FGFR1 is phosphorylated at serine 789 by RSK2 in vivo. Serum-starved U2OS-R1 cells were treated with 10 mM BI-D1870 or 20mM U0126 MEK1/2 inhibitor for 30min prior to FGF1 or EGF treatment (as indicated). The cells were then lysed and the lysates were subjected to immunoprecipitation reactions using anti-FGFR1 antibodies followed by SDS-PAGE and western blotting with indicated antibodies.

Article Snippet: Cells The human osteosarcoma cell line, U2OS, and the human normal foreskin fibroblast cell line, BJ, were from ATCC (Manassas, VA, USA).

Techniques: In Vitro, Phospho-proteomics, Recombinant, Incubation, SDS Page, Autoradiography, Staining, Membrane, Control, Sequencing, Residue, Tandem Mass Spectroscopy, In Vivo, Immunoprecipitation, Western Blot

Journal: Oncogene

Article Title: RSK2 regulates endocytosis of FGF receptor 1 by phosphorylation on serine 789.

doi: 10.1038/onc.2013.425

Figure Lengend Snippet: Figure 5. Disruption of RSK2 leads to prolonged FGFR1 tyrosine phosphorylation. (a and b) Serum-starved U2OS-R1 cells were pretreated with 10 mM BI-D1870 (a) or 100 mM SL 0101-1 (b) in the presence of 2 mg/ml Brefeldin A. Next, the cells were treated with 100 ng/ml FGF1 for indicated periods of time. The cells were then lysed and lysates were analyzed by SDS-PAGE and western blotting with indicated antibodies. Western blots were quantified and the bands corresponding to tyrosine phosphorylated FGFR1 were normalized to GAPDH. The amount of tyrosine phosphorylated FGFR1 is presented as percentages of that in cells stimulated with FGF1 for 15 min in the absence of inhibitor. The graph represents the mean±standard deviation of three independent experiments. (c) Non-targeting siRNA (scr) or RSK2-specific siRNA were transfected into U2OS-R1 cells. 48–72 h after transfection cells were serum-starved and pretreated for 30 min with Brefeldin A prior to stimulation with FGF1 for indicated periods of time. The cells were then lysed and lysates were analyzed by SDS-PAGE and western blotting with indicated antibodies. Western blots were quantified and the bands corresponding to tyrosine-phosphorylated FGFR1 were normalized to GAPDH. The amount of tyrosine-phosphorylated FGFR1 is presented as percentages of that in scr-transfected cells stimulated with FGF1 for 15 min. The histogram represents the mean±standard deviation of three independent experiments.

Article Snippet: Cells The human osteosarcoma cell line, U2OS, and the human normal foreskin fibroblast cell line, BJ, were from ATCC (Manassas, VA, USA).

Techniques: Disruption, Phospho-proteomics, SDS Page, Western Blot, Standard Deviation, Transfection

Journal: Oncogene

Article Title: RSK2 regulates endocytosis of FGF receptor 1 by phosphorylation on serine 789.

doi: 10.1038/onc.2013.425

Figure Lengend Snippet: Figure 6. RSK2 activity influences the level of FGFR1 ubiquitination. (a) Serum-starved U2OS-R1 cells were pretreated with 10 mM BI-D1870 RSK2 kinase inhibitor for 30 min prior to FGF1 (100 ng/ ml) treatment for 15 min. Cells were lysed at 95 1C for 5 min in 1% SDS in PBS. Equal amounts of lysate and 2 IP-buffer were mixed and subjected to immunoprecipitation reactions using anti-FGFR1 antibodies followed by SDS-PAGE and western blotting with indicated antibodies. Western blots were quantified and the bands corresponding to ubiquitinated FGFR1 (Ub) were normalized to total FGFR1. The histogram represents the mean±standard deviation of three independent experiments. *Po0.05, **Po0.01. (b) Serum- starved U2OS-R1 and U2OS-R1 S789A cells were stimulated with 100 ng/ml FGF1 for 15 min. Cells were lysed at 95 1C for 5 min in 1% SDS in PBS. Equal amounts of lysate and 2 IP-buffer were mixed and subjected to immunoprecipitation reactions using anti-FGFR1 antibodies followed by SDS-PAGE and western blotting with indicated antibodies. Western blots were quantified and the bands corresponding to ubiquitinated FGFR1 (Ub) were normalized to total FGFR1. The amount of ubiquitinated receptor is presented relative to untreated cells. The histogram represents the mean±standard deviation of three independent experiments. a.u., arbitrary units. *Po0.05, **Po0.01, ***Po0.001.

Article Snippet: Cells The human osteosarcoma cell line, U2OS, and the human normal foreskin fibroblast cell line, BJ, were from ATCC (Manassas, VA, USA).

Techniques: Activity Assay, Ubiquitin Proteomics, Immunoprecipitation, SDS Page, Western Blot, Standard Deviation

Journal: Oncogene

Article Title: RSK2 regulates endocytosis of FGF receptor 1 by phosphorylation on serine 789.

doi: 10.1038/onc.2013.425

Figure Lengend Snippet: Figure 7. RSK2 activity influences endocytosis of FGFR1. (a–d) Serum-starved, U2OS-R1 cells, grown on gelatinized plates were incubated with 10–20 ng/ml 125I-FGF1 or 125I-FGF2 (as indicated) for indicated periods of time. When inhibitors were used, the cells were pretreated for 1 h prior to addition of 125I-FGF, and the inhibitor was kept throughout the experiment. Internalized and surface-bound 125I-FGF were separated as described in Materials and methods and the ratio was plotted as function of time. Graphs represent means±standard deviation of 2 (a), 5 (b) or 3 (d) independent experiments, each performed in triplicate. (e) Serum-starved U2OS-R1 and U2OS-R1 S789A cells grown on gelatinized plates were incubated with 20 ng/ml 125I-FGF2 for indicated periods of time. Internalized and surface-bound 125I-FGF were separated as described in Materials and methods, and the ratio was plotted as function of time. Graphs represent means±standard deviation of 3 independent experiments, each performed in triplicate. (f) Serum-starved U2OS-R1 cells, grown on coverslips were incubated with DyLight 549 labeled FGF1 (DL-FGF1) at 37 1C for 20 min. The cells were then washed with a high salt, low pH buffer to remove surface bound DL-FGF1 before fixation followed by staining with Hoechst and examination by confocal microscopy. Inhibitors were added as indicated. When inhibitors were used, the cells were pretreated for 1 h prior to addition of DL-FGF1, and the inhibitor was kept throughout the experiment. Scale bar, 5 mm. (g) The uptake of DL-FGF1 was measured as DL549 intensity in U2OS-R1 cell treated as described in (f). The mean intensity of DL549 in inhibitor-treated cells is presented in the histogram as the percentage of DL549 intensity in untreated cells. The histogram represents the means±s.e.m. of 3 independent experiments. In total, 223 cells were analyzed for control, 249 for BI-D1870 treatment and 174 for U0126 treatment. Confocal scanning was performed with identical settings. ***Po0.001 (h) Serum-starved U2OS-R1 and U2OS-R1 S789A cells, grown on coverslips were incubated with (DL-FGF1) at 37 1C for 20 min. The cells were then washed with a high salt, low pH buffer to remove surface bound DL-FGF1 before fixation followed by staining with Hoechst and examination by confocal microscopy. Scale bar, 5 mm. (i) The uptake of DL-FGF1 was measured as DL549 intensity in U2OS-R1 and U2OS-R1 S789A cells. The mean intensity of DL549 in U2OS-R1 S789A cells are presented in the histogram as the percentage of DL549 intensity in U2OS-R1 cells. The histogram represents the means±s.e.m. of 3 independent experiments. In total, 182 cells were analyzed for U2OS-R1 and 165 for U2OS-R1 S789A. Confocal scanning was performed with identical settings. ***Po0.001.

Article Snippet: Cells The human osteosarcoma cell line, U2OS, and the human normal foreskin fibroblast cell line, BJ, were from ATCC (Manassas, VA, USA).

Techniques: Activity Assay, Incubation, Standard Deviation, Labeling, Staining, Confocal Microscopy, Control

Journal: Oncogene

Article Title: RSK2 regulates endocytosis of FGF receptor 1 by phosphorylation on serine 789.

doi: 10.1038/onc.2013.425

Figure Lengend Snippet: Figure 8. RSK2 activity influences transport of DL-FGF1 to early endosomes. (a) Serum-starved U2OS-R1 cells, grown on coverslips were incubated with DyLight 549 labeled FGF1 (DL-FGF1) at 4 1C for 1 h. Excess DL-FGF1 was then washed off before immediate transfer of the cells to 37 1C for indicated periods of time. The cells were then fixed, stained with anti-EEA1 antibody (red) and Hoechst (blue) and examined by confocal microscopy. When BI-D1870 was used, the cells were pretreated for 30 min prior to addition of DL-FGF1, and the inhibitor was kept throughout the experiment. Scale bar, 5 mm. Enlarged areas are highlighted with a yellow box. Manders’ overlap coefficient was calculated and the histogram represents the means±s.e.m. of 3 independent experiments. 60–120 cells were analyzed for each time point for each condition in each experiment. *Po0.05, **Po0.01. (b) Serum-starved U2OS-R1 and U2OS-R1 S789A cells, grown on coverslips were incubated with DyLight 549 labeled FGF1 (DL-FGF1) at 4 1C for 1 h. Excess DL-FGF1 was then washed off before immediate transfer of the cells to 37 1C for indicated periods of time. The cells were then fixed, stained with anti-EEA1 antibody (red) and Hoechst (blue) and examined by confocal microscopy. Scale bar, 5 mm. Enlarged areas are highlighted with a yellow box. Manders’ overlap coefficient was calculated, and the histogram represents the means±s.e.m. of 3 independent experiments. 60–120 cells were analyzed for each time point for each condition in each experiment. **Po0.01, ***Pp0.001.

Article Snippet: Cells The human osteosarcoma cell line, U2OS, and the human normal foreskin fibroblast cell line, BJ, were from ATCC (Manassas, VA, USA).

Techniques: Activity Assay, Incubation, Labeling, Staining, Confocal Microscopy

Journal: Molecular and Cellular Biology

Article Title: LDB1 Enforces Stability on Direct and Indirect Oncoprotein Partners in Leukemia

doi: 10.1128/MCB.00652-19

Figure Lengend Snippet: Halo-tagged proteins expressed from recombinant lentiviruses have physiological abundance and localization and can be precisely quantified in lysates or live cells. (A) Cell lysates were prepared from untransduced Jurkat cells; Jurkat cells transduced with Halo-LMO2; untransduced K562 cells; K562 cells transduced with Halo-LMO2; and KOPT-K1, LOUCY, and U937 cells. Lysates were subjected to 4% to 15% SDS-PAGE, transferred, and blotted with anti-LDB1, anti-LMO2 monoclonal antibody, anti-Halo, anti-TAL1, and anti-VCP (gel loading control). Molecular weight standards were run on the same gel and are shown at the right. (B) The top panel shows an immunoblot of lysates prepared from U2OS with a Halo knock-in at CTCF (lane 1) or lysates from Jurkat cells transduced with the constructs shown in the grid above (lanes 2 to 14). Empty vector control is shown as EBFPII-Hygro (lane 2). Lanes 2 to 9 show lysates from Jurkat cells lentivirally expressing Halo-CTCF at increasing MOIs. Lanes 10 to 14 show lysates from Jurkat cells lentivirally expressing Halo-SOX2 at increasing MOIs. The top panel shows gradient SDS-PAGE, transfer, and blotting with anti-CTCF antibody. The middle panel shows direct in-gel Halo fluorescence. Live cells prepared as described for panel A were labeled with cell-permeative fluorescent ligand R110, lysed, and subjected to gradient SDS-PAGE. The gel was visualized for green fluorescence as described in Materials and Methods. (C) Live Jurkat cells expressing Halo-tagged proteins were labeled with R110 Halo ligand, washed, and subjected to flow cytometry. Histograms show FITC fluorescence of the various cells in comparison to the labeled U2OS cells, which have a Halo tag knocked into the CTCF gene. (D) Table showing calculated copy numbers of Halo-tagged proteins based on the absolute values derived from the Halo knock-in cell line. Lentiviral Halo CTCF and Halo SOX2 were quantified from cell populations infected at intermediate and low MOIs, respectively. (E) Confocal microscopy of Jurkat cells lentivirally expressing Halo-LMO2 and labeled with Halo ligand, R110, and nuclear stain (Syto 17 Red). The right panel shows a merged image. Voxel quantification showed that 95% of Halo-LMO2 was nuclear.

Article Snippet: Catalog no. S7579 pBluescript SK Stratagene Iscove's modified Dulbecco's medium (IMDM) Gibco Catalog no. 12200-036 RPMI 1640 Gibco Catalog no. 31800-022 Penicillin-streptomycin solution 10× Corning Catalog no. 30-022-CI Geneticin Gibco Catalog no. 10131-027 0.05% trypsin, 0.53 mM EDTA 1× [−]sodium bicarbonate Corning Catalog no. 20116004 Puromycin dihydrochloride Fisher Bioreagents Catalog no. BP2956-100 Pierce protease inhibitor tablets Thermo Scientific Catalog no. A32965 Hygromycin B-PBS (50 mg/ml) Invitrogen Catalog no. 10687010 Anti-FLAG M2 resin Sigma Catalog no. A2220 Protein A/G resin Santa Cruz Polyvinylidene difluoride (PVDF) membrane GE Catalog no. 10600022 SuperSignal PicoWest Plus Thermo/Pierce Catalog no. 1863099 Experimental models: cell lines Human: HEK 293 ATCC Human: Jurkat ATCC Human: K562 ATCC Human: KOPTK1 ATCC Human: LOUCY ATCC Human: U937 ATCC Human: U2OS C32 Halo-CTCF Hansen et al. ( 27 ) U2OS endogenous knock-in cell line where all endogenous copies of CTCF have been N-terminally tagged with FLAG-HaloTag; clone 32 Software and algorithms Flowjo 10.3 analysis software FlowJo, LLC Contact corresponding author for URL Ideas software Amnis Corporation Contact corresponding author for URL ImageLab 5.2.1 BioRad Contact corresponding author for URL Imaris Bitplane Inc. Other CytoFLEX benchtop cytometer Beckman Contact corresponding author for URL Leica TCS SP8 confocal imaging system Leica Contact corresponding author for URL ImageStream Mk II Amnis Contact corresponding author for URL Open in a separate window Reagents and resources Experimental model and subject details. (i) Cell lines.

Techniques: Recombinant, Transduction, SDS Page, Control, Molecular Weight, Western Blot, Knock-In, Construct, Plasmid Preparation, Expressing, Fluorescence, Labeling, Flow Cytometry, Comparison, Derivative Assay, Infection, Confocal Microscopy, Staining

Journal: Molecular and Cellular Biology

Article Title: LDB1 Enforces Stability on Direct and Indirect Oncoprotein Partners in Leukemia

doi: 10.1128/MCB.00652-19

Figure Lengend Snippet: Reagents and resources

Article Snippet: Catalog no. S7579 pBluescript SK Stratagene Iscove's modified Dulbecco's medium (IMDM) Gibco Catalog no. 12200-036 RPMI 1640 Gibco Catalog no. 31800-022 Penicillin-streptomycin solution 10× Corning Catalog no. 30-022-CI Geneticin Gibco Catalog no. 10131-027 0.05% trypsin, 0.53 mM EDTA 1× [−]sodium bicarbonate Corning Catalog no. 20116004 Puromycin dihydrochloride Fisher Bioreagents Catalog no. BP2956-100 Pierce protease inhibitor tablets Thermo Scientific Catalog no. A32965 Hygromycin B-PBS (50 mg/ml) Invitrogen Catalog no. 10687010 Anti-FLAG M2 resin Sigma Catalog no. A2220 Protein A/G resin Santa Cruz Polyvinylidene difluoride (PVDF) membrane GE Catalog no. 10600022 SuperSignal PicoWest Plus Thermo/Pierce Catalog no. 1863099 Experimental models: cell lines Human: HEK 293 ATCC Human: Jurkat ATCC Human: K562 ATCC Human: KOPTK1 ATCC Human: LOUCY ATCC Human: U937 ATCC Human: U2OS C32 Halo-CTCF Hansen et al. ( 27 ) U2OS endogenous knock-in cell line where all endogenous copies of CTCF have been N-terminally tagged with FLAG-HaloTag; clone 32 Software and algorithms Flowjo 10.3 analysis software FlowJo, LLC Contact corresponding author for URL Ideas software Amnis Corporation Contact corresponding author for URL ImageLab 5.2.1 BioRad Contact corresponding author for URL Imaris Bitplane Inc. Other CytoFLEX benchtop cytometer Beckman Contact corresponding author for URL Leica TCS SP8 confocal imaging system Leica Contact corresponding author for URL ImageStream Mk II Amnis Contact corresponding author for URL Open in a separate window Reagents and resources Experimental model and subject details. (i) Cell lines.

Techniques: Recombinant, Staining, Modification, Protease Inhibitor, Membrane, Knock-In, Software, Cytometry, Imaging

Journal: The Journal of Cell Biology

Article Title: TLNRD1 is a CCM complex component and regulates endothelial barrier integrity

doi: 10.1083/jcb.202310030

Figure Lengend Snippet: TLNRD1, CCM2, PDCD10, and ITG1BP1 localize at the tip of MYO10 filopodia. U2OS cells expressing mScarlet-MYO10 with TLNRD1-GFP, CCM2-GFP, PDCD10-GFP, or ITG1BP1-GFP were plated on fibronectin for 2 h, fixed and stained to visualize F-actin. Samples were imaged using structured illumination microscopy. Representative maximum intensity projections are displayed; scale bars: (main) 5 µm; (inset) 1 µm. The yellow squares highlight magnified ROIs. The yellow arrows indicate the filopodia tips.

Article Snippet: U2OS cells were purchased from DSMZ (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig DE, ACC 785).

Techniques: Expressing, Staining, Microscopy

Journal: The Journal of Cell Biology

Article Title: TLNRD1 is a CCM complex component and regulates endothelial barrier integrity

doi: 10.1083/jcb.202310030

Figure Lengend Snippet: The TLNRD1–CCM2 interaction involves the TLNRD1 4-helix bundle and a C-terminal helix in CCM2. (A and B) U2OS cells expressing TLNRD1-GFP and mito-mScarlet (CTRL), mito-PDCD10-mScarlet, or mito-CCM2-mScarlet were imaged using a spinning disk confocal microscope. (A) Representative single Z-planes are displayed. Dashed yellow lines highlight the cell outlines. The yellow squares highlight magnified ROIs. Scale bars: (main) 25 µm and (inset) 5 µm. (B) 3D colocalization analysis was performed using the JACoP Fiji plugin (three biological repeats, n > 31 image stacks per condition). The results are shown as Tukey boxplots. The whiskers (shown here as vertical lines) extend to data points no further from the box than 1.5× the interquartile range. The P values were determined using a randomization test. NS indicates no statistical difference between the mean values of the highlighted condition and the control. (C) HUVECs expressing TLNRD1-GFP and CCM2-mCherry were stained for F-actin and DAPI and imaged using an Airyscan confocal microscope. A single Z-plane is displayed. The yellow squares highlight a magnified ROI. Scale bars: (main) 25 µm and (inset) 5 µm. (D) GFP-pulldown in HEK293T cells expressing GFP-TLNRD1, GFP-TLNRD1 4H . GFP-TLNRD1 5H or GFP alone. CCM2 recruitment to the bait proteins was assessed by western blotting (representative of three biological repeats). (E) CCM2 schematic showing the boundaries of the phosphotyrosine binding (PTB) domain, the harmonin homology domain (HHD), and the C-terminal helix (CTH). (F) A GST-pulldown assay was used where Glutathione agarose-bound GST-CCM2 fragments (beads: B) were incubated with recombinant TLNRD (input: I). After multiple washes, proteins bound to the beads (pellet: P) were eluted. Red boxes highlight areas of interest in the gel. (G) A fluorescence polarization assay was used to determine the K d of the interaction between TLNRD1, TLNRD1 4H , or TLN1 R7R8 with SUMO-CCM2 CTH . K d values (nM) are shown in parentheses. ND, not determined. Source data are available for this figure: .

Article Snippet: U2OS cells were purchased from DSMZ (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig DE, ACC 785).

Techniques: Expressing, Microscopy, Control, Staining, Western Blot, Binding Assay, GST Pulldown Assay, Incubation, Recombinant, Fluorescence

Journal: The Journal of Cell Biology

Article Title: TLNRD1 is a CCM complex component and regulates endothelial barrier integrity

doi: 10.1083/jcb.202310030

Figure Lengend Snippet: TLNRD1 interacts with CCM2. (A) U2OS cells expressing mito-PDCD10-mScarlet, mito-CCM2-mScarlet, or mito-TLNRD1-mScarlet were imaged using a spinning disk confocal microscope. (B) U2OS cells treated with siRNA targeting KRIT1 or siRNA control. KRIT1 levels were then analyzed using Western blots. A representative western blot is displayed. (C) U2OS cells treated with siRNA targeting KRIT1 or siRNA control and expressing TLNRD1-GFP and mito-CCM2-mScarlet were imaged using a spinning disk confocal microscope. 3D colocalization analyses were performed using the JACoP Fiji plugin, and results are displayed as Tukey boxplots (three biological repeats, n > 21 image stacks per condition). (D) Glutathione agarose-bound GST-CCM2 (beads: B) was incubated with recombinant TLNRD1, TLNRD1 4H , or TLN1 R7R8 (input: I). After multiple washes, proteins bound to the beads (pellet: P) were visualized. The various fractions were then analyzed using SDS-PAGE followed by Coomassie staining. A representative gel of three independent repeats is displayed. Red boxes highlight areas of interest in the gel. (E) U2OS cells expressing various GFP-tagged CCM2 constructs and mito-TLNRD1-mScarlet or mito-mScarlet (CTRL) were imaged using a spinning disk confocal microscope. Representative single Z-planes are displayed. The yellow squares highlight magnified ROIs. Scale bars: (main) 25 µm and (inset) 5 µm. (F) 3D colocalization analyses were performed using the JACoP Fiji plugin, and results are displayed as Tukey boxplots (three biological repeats, n > 38 image stacks per condition). The whiskers (shown here as vertical lines) extend to data points no further from the box than 1.5× the interquartile range. For all panels, the P values were determined using a randomization test. NS indicates no statistical difference between the mean values of the highlighted condition and the control. Source data are available for this figure: .

Article Snippet: U2OS cells were purchased from DSMZ (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig DE, ACC 785).

Techniques: Expressing, Microscopy, Control, Western Blot, Incubation, Recombinant, SDS Page, Staining, Construct

Journal: The Journal of Cell Biology

Article Title: TLNRD1 is a CCM complex component and regulates endothelial barrier integrity

doi: 10.1083/jcb.202310030

Figure Lengend Snippet: The TLNRD1–CCM2 binding interface involves a hydrophobic groove on TLNRD1 and hydrophobic residues of CCM2. (A and B) Modeling of the TLNRD1–CCM2 complex using ColabFold using the TLNRD1 crystal structure (PDB accession no. 6XZ4 ) as a template. (A) Overall view of the predicted complex. The TLNRD1 monomers are colored blue, and the CCM2 CTH helices are colored pink. The TLNRD1–CCM2 binding area is magnified, and the residues contributing to the interface are shown as sticks. (B) TLNRD1 has a hydrophobic channel (green) on the surface, which could facilitate CCM2 (pink) binding. The TLNRD1 four-helix module was colored by hydrophobicity using the AA index database (entry FASG890101 ) in PyMOL, where green denotes hydrophobic residues and white polar residues. CCM2 CTH is shown as sticks and predominantly contacts the hydrophobic region on TLNRD1 4H . (C) Comparison of the hydrophobic channel on the surface of TLNRD1 4H and the equivalent region on TLN1 R8 . The TLNRD1 2T mutant was designed to mimic the surface of TLN1 R8 . The green color denotes hydrophobic residues. On the TLNRD1 2E , the mutated basic residues are highlighted in blue. (D) Fluorescence polarization was used to determine the K d of the interaction between TLNRD1 and various SUMO-CCM2 CTH constructs (WT, I428S, I432D, and W412A/D422A). K d values (nM) are shown in parentheses. ND, not determined. (E) Fluorescence polarization was used to determine the K d of the interaction between CCM2 CTH and various TLNRD1 4H constructs (WT, 2T, and 2E). K d values (nM) are shown in parentheses. ND, not determined. (F) U2OS cells expressing various GFP-tagged CCM2 constructs and mito-TLNRD1-mScarlet or mito-mScarlet (CTRL) were imaged using a spinning disk confocal microscope. Representative single Z-planes are displayed. See also . Scale bars: (main) 25 µm and (inset) 5 µm. (G) U2OS cells expressing various GFP-tagged TLNRD1 constructs and mito-CCM2-mScarlet or mito-mScarlet (CTRL) were imaged using a spinning disk confocal microscope. Representative maximum intensity projections are displayed. Scale bars: (main) 25 µm and (inset) 5 µm.

Article Snippet: U2OS cells were purchased from DSMZ (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig DE, ACC 785).

Techniques: Binding Assay, Comparison, Mutagenesis, Fluorescence, Construct, Expressing, Microscopy