Journal: Developmental cell

Article Title: T cell cytoskeletal forces shape synapse topography for targeted lysis via membrane curvature bias of perforin.

doi: 10.1016/j.devcel.2022.08.012

Figure Lengend Snippet: Figure 5. Lytic granules are delivered to negatively curved degranulation pockets and perforin preferentially perforates positively curved membrane (A) Airyscan confocal image of a fixed conjugate formed between OT-I CTLs expressing a membrane-localized tdTomato and a cognate EL4 tumor cell labeled with CellBrite Fix 488 and embedded in a 3D collagen matrix. Scale bar, 5 mm. (B) En face mean curvatures of the CTL and tumor cell synapse* shown in (A). Projections of cytolytic granules nearest synapse are shown as white circles. Scale bar, 3 mm. (C) Mean synaptic curvatures at the projected point of granule arrival for CTLs interacting with (top) cognate tumor cells and (bottom) cognate tumor cytoplasts in the DPA assay. Each data point represents the mean curvature for an individual time point within the perforation window. Boxplots show median, IQR, and range for n = 9 (tumor cells) and n = 3 (tumor cytoplasts) interactions. p values from Mann-Whitney test. (D) Confocal image of a fixed conjugate formed between a human CTL labeled with CellBrite Fix 555 and a cognate melanoma cell embedded in 3D collagen matrix. Scale bar, 10 mm. (E) En face mean curvatures of the human CTL and melanoma cell synapse shown in (D). Projections of cytolytic granules nearest synapse are shown as white circles. Scale bar, 3 mm. (F) Mean synaptic curvatures at the projected point of granule arrival for fixed conjugates of human CTLs and cognate melanoma cells embedded in 3D collagen matrix and imaged at super-resolution. p values from Mann-Whitney test for n = 11 conjugates. (G) Schematic illustrating the outside-out high-speed pressure patch clamp assay with perforin solution delivered by a second pipette. (H) Tumor membrane patch potentials following exposure to perforin under positive, negative, or no pressure as indicated. Each trace represents a single membrane patch; n = 5, n = 5, and n = 6 for positive, negative, and no pressure, respectively.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Alexa Fluor 647 anti-human/mouse Granzyme B Mouse mAb, clone: GB11 Biolegend Cat. #: 372219; RRID: AB_2728386 Anti-Tubulin antibody Rat mAb, clone: YL1/2 Abcam Cat. #: ab6160; RRID: AB_305328 Alexa Fluor 647 Goat anti-rat IgG Antibody, clone: Poly4054 Biolegend Cat. #: 405416; RRID: AB_2562967 CD69-monoclonal antibody (H1.2F3), PE Thermofisher Cat. #: 12-0691-82; RRID: AB_465732 Chemicals, peptides, and recombinant proteins Blebbistatin Sigma-Aldrich Cat. #: B0560 para-amino-Blebbistatin Sapphire Bioscience Cat. # 22699 ML-7 Sigma-Aldrich Cat. #: I2764 Y-27632 Sigma-Aldrich Cat. #: SCM075 Nocodazole Sigma-Aldrich Cat. #: M1404 Dynarrestin Sigma-Aldrich Cat. #: SML2332 OVA257-264 peptide, SIINFEKL AusPep Cat. #: 2711 Corning Collagen I Rat Tail Natural In Vitro Technologies Cat. #: FAL354236 Recombinant mouse IL-2 BioLegend Cat. #: 575408 CellTracker Orange 5-(and-6)-(((4chloromethyl) benzoyl) amino) tetramethylrhodamine (CMTMR) Invitrogen Cat. #: C2927 CellTracker Deep Red Invitrogen Cat. #: C34565 CellTracker Green 5-chloromethylfluorescein diacetate (CMFDA) Invitrogen Cat. #: C7025 CellTrace Violet Invitrogen Cat. #: C34571 CellBrite Fix 488 Membrane Dye Biotium Cat. #: 30090 CellBrite Fix 555 Membrane Dye Biotium Cat. #: 30088 LysotrackerTM Deep Red Invitrogen Cat. #: L12492 Propidium Iodide (PI) Invitrogen Cat. #: P3566 SiR-Tubulin Cytoskeleton, Inc. Cat. #: CY-SC006 Recombinant perforin Lopez et al. (2013b); gift from Ruby Law (Monash University) N/A Ficoll 400 Fisher Scientific Cat. #: BP525-25 Hoechst 33342, Trihydochloride, Trihydrate (Hoechst) Invitrogen Cat. #: H1399 Concanavalin A Sigma-Aldrich Cat. #: C5275 Triton X-114 Sigma-Aldrich Cat. #: X114 Latrunculin A Abcam Cat. #: ab144290 AlexaFluorTM 488 Phalloidin Invitrogen Cat. #: A12379 Fibronectin Sigma-Aldrich Cat. #: F0895 Critical commercial assays Nucleobond Xtra midi kit Scientifix Cat. #: 740410.50 Cytofix/Cytoperm kit BD Biosciences Cat. #: 554715 LegendPlex Mouse Cytokine Release Syndrome Panel BioLegend Cat. #: 741024 (Continued on next page) e1 Developmental Cell 57, 2237–2247.e1–e8, September 26, 2022

Techniques: Membrane, Expressing, Labeling, MANN-WHITNEY, Patch Clamp, Transferring

Journal: Acta Naturae

Article Title: A Highly Productive CHO Cell Line Secreting Human Blood Clotting Factor IX

doi:

Figure Lengend Snippet: The integrity of the intracellular and extracellular FIX polypeptide chain for the p1.1-F9-T2/S cell line, FIX secretion level and the change in clotting activity for various producer cell lines; the phylogenetic tree for the Chinese hamster VKORC1 protein. Panel A – Western blotting of the secreted and intracellular FIX. SDS-PAGE under reducing conditions, detection by polyclonal anti-FIX antibodies, the molecular weight of the marker bands is shown in kDa. Denotation: “K + ” – recombinant FIX standard; “control-” – untransfected CHO DG44 cells. The mature FIX position is depicted by an arrow. Panel B – FIX secretion level and the degree of propeptide processing for cell populations and clonal lines. ELISA-determined specific productivity is shown as bars (left axis). Specific productivity as the clotting activity is shown as a broken line (right axis). The percentage of FIX molecules without the propeptide was determined by ELISA and is shown as numbers above the bars. Specific productivity for both methods is shown as the mean value; error bars represent the standard deviation, n=2. The scheme of production of cell populations and clonal lines is shown with arrows. Panel C – The taxonomic tree for a mammalian VKORC1 protein visualized using the Tree Viewer software (NCBI, USA). The scale bar represents the evolutionary distance measured as the number of substitutions per amino acid residue. Panel D – multiple alignment of the amino acid sequences of VKORC1 variants for the selected mammalian species. Conservative amino acid residues are shown against a black background

Article Snippet: ELISA measurement of FIX concentration The FIX concentration was measured using rabbit anti-FIX polyclonal antibodies (LifeSpan BioSciences, USA) (50 ng/well) as an immobilized antibody according to the procedure described in [ ].

Techniques: Coagulation, Activity Assay, Western Blot, SDS Page, Molecular Weight, Marker, Recombinant, Control, Enzyme-linked Immunosorbent Assay, Standard Deviation, Software, Residue