oci ly19  (ATCC)

Journal: Cell Reports Medicine

Article Title: LTA4H improves the tumor microenvironment and prevents HCC progression via targeting the HNRNPA1/LTBP1/TGF-β axis

doi: 10.1016/j.xcrm.2025.102000

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Article Snippet: CD54 (ICAM-1); Mouse; 163Dy , Fluidigm , 3163020B.

Techniques: Phospho-proteomics, Microarray, Recombinant, Lysis, Protease Inhibitor, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Reverse Transcription, Activity Assay, Immunoprecipitation, Extraction, Purification, In Vitro, Transfection, Sequencing, Mass Cytometry, ChIP-sequencing, Transgenic Assay, Software

Journal: Cell Reports Medicine

Article Title: LTA4H improves the tumor microenvironment and prevents HCC progression via targeting the HNRNPA1/LTBP1/TGF-β axis

doi: 10.1016/j.xcrm.2025.102000

Figure Lengend Snippet:

Article Snippet: CD54 (ICAM-1); Mouse; 163Dy , Fluidigm , 3163020B.

Techniques: Phospho-proteomics, Microarray, Recombinant, Lysis, Protease Inhibitor, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Reverse Transcription, Activity Assay, Immunoprecipitation, Extraction, Purification, In Vitro, Transfection, Sequencing, Mass Cytometry, ChIP-sequencing, Transgenic Assay, Software

Journal: Cell reports

Article Title: Distinct bidirectional regulation of LFA1 and α4β7 by Rap1 and integrin adaptors in T cells under shear flow

doi: 10.1016/j.celrep.2023.112580

Figure Lengend Snippet: (A) In vivo short homing assay with fluorescently labeled T cells from kindlin-3-deficient (Knd3-KO), talin1-deficient (Tln-KO), or Rap1a/b double-deficient (Rap1-KO) mice. Labeled T cells harvested from spleen (SPL), surface LN (SLN), mesenteric LN (MLN), and Peyer’s patches (PPs) were analyzed. The ratio against control T cells in tissues was calculated as homing efficiency. All experiments were performed in quadruplicate and statistically analyzed by Student’s t test. Data are mean ± SD. Total analyzed cell numbers are 96,883 cells (SPL), 24,600 cells (SLN), 25,771 cells (MLN), and 10,949 cells (PP). (B) Relative distributions of transferred Knd3-KO, Tln-KO, or Rap1-KO T cells in LNs. HEVs III, >30 μm (diameter); HEVs IV, 20–30 μm with branches; HEVs V, < 20 μm; and parenchyma (outside of vessels). For the control experiment, dyes were swapped. All experiments were performed in duplicate at minimum and statistically analyzed by chi-squared test. Total counted cell numbers were 2,667 for WT, 1,384 for Knd3-KO (left), 4,071 for WT, 397 for Tln-KO (center), 1,984 for WT, and 163 for Rap1-KO T cells (right). (C) Representative images of transferred T cell distribution in LN slices. Transferred WT T cells (blue) were distributed in HEV orders III, IV, V (green), and parenchyma. Knd3-KO T cells (red) were sparsely distributed, especially in narrow HEVs (order IV and V) and parenchyma. Bar, 30 μm. (D) Residual Knd3-KO T cells in HEVs were reduced by treatment with integrin-blocking Ab against integrin αL. Experiments were performed in duplicate and statistically analyzed by chi-squared test. Total cell numbers were 617 for Knd3-KO and 105 for Knd3-KO T cells with antibody blocking. (E) Detachment assay of WT, Knd3-KO, Tln-KO, or Rap1-KO T cells on 180 sites/μm 2 of ICAM1 at 2 and 5 dyn/cm 2 . Total numbers of input cells were 1,138 for the Knd3-KO control, 994 for Knd3-KO, 902 for the Tln-KO control, 721 for Tln-KO, 676 for the Rap1-KO control, and 805 for Rap1-KO T cells. All experiments were performed in triplicate at minimum and statistically analyzed by Student’s t test. (F) Detachment assay of WT, Knd3-KO, Tln-KO, or Rap1-KO T cells on 1,000 sites/μm 2 of ICAM1 at 2 and 5 dyn/cm 2 . The total numbers of input cells were 2,282 for the control, 1,754 for Knd3-KO, 2,236 for Tln-KO, and 2,060 for Rap1-KO T cells. All experiments were performed in quadruplicate and statistically analyzed by Student’s t test. Data are mean ± SD.

Article Snippet: Monoclonal Abs against αL (KBA), ICAM1 (YN1/1.7.4), MAdCAM1 (MECA-89), and L-selectin (MEL-14) were purified from supernatants of hybridomas (ATCC) for blocking experiments or flow cytometric analysis.

Techniques: In Vivo, Labeling, Control, Blocking Assay

Journal: Cell reports

Article Title: Distinct bidirectional regulation of LFA1 and α4β7 by Rap1 and integrin adaptors in T cells under shear flow

doi: 10.1016/j.celrep.2023.112580

Figure Lengend Snippet: (A) A schematic cartoon of the flow experiment. The upstream part of a flow chamber was masked by a silicon rubber for measurement of cell influx in the flow. T cells were loaded in the glass syringe and infused into the flow chamber at the shear stress indicated. After shear flow was stable, bright-field images (640 × 480 pixels) were acquired at 30 frames/s (fps). Captured images were background subtracted and split into the influx area (non-coated area) and the capture area (ligand-coated area). Cell influx was determined by cell numbers in each frame and normalized for time and area (cell numbers/s/mm 2 ). (B)Capture (rolling) events on PNAd + CD34 (700 sites/μm 2 , 240 sites/μm 2 , 80 sites/μm 2 , and 25 sites/μm 2 ) of WT T cells (267 cells, 242 cells, 177 cells, and 28 cells, respectively) were normalized by area (capture area) and influx at a flow rate of 3 dyn/cm 2 . All experiments were performed in duplicate and statistically analyzed by Student’s t test. Data are mean ± SD. (C)Rolling velocities from individual cell trajectories were plotted at 25–700 sites/μm 2 of PNAd + CD34 in (B) at a flow rate of 3 dyn/cm 2 . Bars (red) indicate average. All experiments were performed in duplicate and statistically analyzed by Student’s t test. (D) Relative capture frequencies on PNAd + CD34 (240 sites/μm 2 ) and ICAM1 (100 sites/μm 2 ) at a flow rate of 3 dyn/cm 2 with CCL21. Capture events/area of Knd3-KO (124 cells), Tln-KO (116 cells), or Rap1-KO T cells (145 cells) were normalized to that of WT (160 cells) and influx. All experiments were performed in triplicate and statistically analyzed by chi-squared test. Data are mean ± SD. (E) Capture (rolling) events of WT T cells on PNAd + CD34 (240 sites/μm 2 ) with or without ICAM1 (100 sites/μm 2 ) at a flow rate of 3 dyn/cm 2 without CCL21; 187 cells (on PNAd + CD34) and 227 cells (on PNAd + CD34 and ICAM1) were analyzed by Student’s t test in triplicate. Data are mean ± SD. (F) Relative capture (rolling) frequencies of αL-KO cells on PNAd + CD34 (120 sites/μm 2 ) and ICAM1 (100 sites/μm 2 ) at a flow rate of 3 dyn/cm 2 without CCL21. WT (283 cells) and αL-KO T cells (202 cells) were analyzed by Student’s t test in quadruplicate. Data are mean ± SD. (G) Relative capture (rolling) frequencies of WT (535 cells), Knd3-KO (431 cells), Tln-KO (326 cells), or Rap1-KO T cells (323 cells) on PNAd + CD34 (240 sites/μm 2 ) and ICAM1 (100 sites/μm 2 ) at a flow rate of 3 dyn/cm 2 without CCL21. All experiments were performed in quadruplicate and statistically analyzed by Student’s t test. Data are mean ± SD. (H) Relative capture frequencies of WT (274 cells for the Knd3KO control, 320 cells each for the Tln-KO control and Rap1-KO), Knd3-KO (220 cells), Tln-KO (255 cells), or Rap1-KO (279 cells) naive T cells on MAdCAM1 (270 sites/μm 2 ) at a flow rate of 3 dyn/cm 2 . All experiments were performed in quadruplicate and statistically analyzed by chi-squared test. Data are mean ± SD.

Article Snippet: Monoclonal Abs against αL (KBA), ICAM1 (YN1/1.7.4), MAdCAM1 (MECA-89), and L-selectin (MEL-14) were purified from supernatants of hybridomas (ATCC) for blocking experiments or flow cytometric analysis.

Techniques: Shear, Control

Journal: Cell reports

Article Title: Distinct bidirectional regulation of LFA1 and α4β7 by Rap1 and integrin adaptors in T cells under shear flow

doi: 10.1016/j.celrep.2023.112580

Figure Lengend Snippet: (A) Rap1 activity was measured in a cultured T cell expressing GFP-RalGDS on PNAd + CD34 (120 sites/μm 2 ) and ICAM1 (100 sites/μm 2 ) at a flow rate of 2 dyn/cm 2 . Bar, 100 μm. The zero time point was set at a turning point where rolling velocity started to decelerate (capture). (B) Magnified images of the pre-capture (blue dotted rectangle) and post-capture (orange dotted rectangle) events shown in (A). Bar, 10 μm. (C) Time course of Rap1 activity (blue) and displacement (orange) in (A). Plot of intensities of the Rap1 affinity probe with the displacement curve of the rolling T cell indicated that increased intensities occurred concurrently with capture. Rap1 activity was normalized at time zero. (D) Rap1 activity from individual cultured T cells (n = 14) and the average (thick red) on PNAd + CD34 (120 sites/μm 2 ) and ICAM1 (100 sites/μm 2 ) at a flow rate of 2 dyn/cm 2 . (E) Statistical analysis of Rap1 activities of pre- and post-capture events in (D) by Student’s t test. Pre-capture and post-capture events were determined from average intensities in 10 frames (0.2 s) before the capture and 10 frames after the capture, respectively. (F) Rap1 activity from individually cultured T cells (n = 14) and the average (thick blue) on PNAd + CD34 (120 sites/μm 2 ) at a flow rate of 2 dyn/cm 2 . (G) Statistical analysis of Rap1 activities of pre- and post-capture events in (F) by Student’s t test. (H) Rap1 activity from individually cultured C3G/RasGRP2-KO T cells (n = 14) and the average (thick red) on PNAd + CD34 (120 sites/μm 2 ) and ICAM1 (100 sites/μm 2 ) at a flow rate of 2 dyn/cm 2 . (I) Statistical analysis of Rap1 activities of pre- and post-capture events in (H) by Student’s t test. (J) Rap1 activity from individual cultured Tln-KO T cells (n = 14) and the average (thick red) on PNAd + CD34 (120 sites/μm 2 ) and ICAM1 (100 sites/μm 2 ) at a flow rate of 2 dyn/cm 2 . (K) Statistical analysis of Rap1 activities of pre- and post-capture events in (J) by Student’s t test. (L) Accumulation of talin1 from individual T cells (n = 12) and the average on PNAd + CD34 (120 sites/μm 2 ) and ICAM1 (100 sites/μm 2 ) at a flow rate of 2 dyn/cm 2 . (M) Statistical analysis of talin1 accumulation of pre- and post-capture events in (L) was performed by Student’s t test, as in (E). (N) Accumulation of talin1 from individual T cells (n = 12) and the average on PNAd + CD34 (120 sites/μm 2 ). (O) Statistical analysis of talin1 accumulation of pre- and post-capture events in (N) by Student’s t test.

Article Snippet: Monoclonal Abs against αL (KBA), ICAM1 (YN1/1.7.4), MAdCAM1 (MECA-89), and L-selectin (MEL-14) were purified from supernatants of hybridomas (ATCC) for blocking experiments or flow cytometric analysis.

Techniques: Activity Assay, Cell Culture, Expressing

Journal: Cell reports

Article Title: Distinct bidirectional regulation of LFA1 and α4β7 by Rap1 and integrin adaptors in T cells under shear flow

doi: 10.1016/j.celrep.2023.112580

Figure Lengend Snippet: (A) Detachment assays with WT, Rasa3/Sipa1 double-deficient (Rasa3/Sipa1-KO), Rasa3/Sipa1/Talin1 triple-deficient (Rasa3/Sipa1/Tln-KO), or Rasa3/Sipa1/Rap1a/Rap1b quadruple-deficient (Rasa3/Sipa1/Rap1a/Rap1b-KO) T cells on ICAM1 (180 sites/μm 2 ) without CCL21 at a flow rate of 2–5 dyn/cm 2 . Total numbers of input cells were 1,163 for the control, 762 for Rasa3/Sipa1-KO, 946 for Rasa3/Sipa1/Tln-KO, and 955 for Rasa3/Sipa1/Rap1a/Rap1b-KO T cells. All experiments were performed in triplicate and statistically analyzed by Student’s t test (Rasa3/Sipa1-KO and WT at 5 dyn/cm 2 ). Data are mean ± SD. (B) Relative capture frequencies of WT (239 cells) or Rasa3/Sipa1-KO T cells (300 cells) on PNAd + CD34 (120 sites/μm 2 ) and ICAM1 (100 sites/μm 2 ) without CCL21 (left) under 2 dyn/cm 2 flow. Relative capture frequencies of WT (88 cells) or Rasa3/Sipa1-KO T cells (115 cells) on PNAd + CD34 and ICAM1 with CCL21 (right) at a flow rate of 2 dyn/cm 2 . All experiments were performed in duplicate at minimum and statistically analyzed by chi-squared test. Data are mean ± SD. (C) Relative capture frequency of WT (20 cells), Rasa3/Sipa1-KO (79 cells), or Rasa3/Sipa1/Tln-KO T cells (9 cells) on ICAM1 (100 sites/μm 2 ) without CCL21 at a flow rate of 2 dyn/cm 2 . All experiments were performed in triplicate and statistically analyzed by chi-squared t test. Data are mean ± SD. (D) Binding assays to soluble ICAM1 of WT and mutant T cells were performed in the presence of EDTA (5 mM), Ca 2+ (0.5 mM) and Mg 2+ (0.5 mM), and Mn 2+ (1 mM). Data are mean ± SD for the three independent experiments. (E) Single-molecule analysis of LFA1-ICAM1 interaction of WT or Rasa3/Sipa1-KO T cells in the presence of Ca 2+ and Mg 2+ (0.5 mM each). The site density of ICAM1 was 300 molecules/μm 2 . Data are mean ± SD. (Left) Frequencies of LFA1-ICAM1 interaction events normalized by adhesion area. Each circle indicates events per area from a single cell. n = 38 (WT) and n = 29 (Rasa3/Sipa1-KO). Statistical significance was calculated by Student’s t test. (Middle) Histogram of frequencies of LFA1-ICAM1 binding events with lifetimes from 1 to 10 s (yellow) or longer than 10 s (pink) of WT (n = 138) and Rasa3/Sipa1-KO (n = 309) T cells. Statistical significance was calculated by chi-squared t test. (Right) The dissociation rate constants ( k off , s −1 ) and average lifetime (τ, s) of LFA1-ICAM1 interaction on WT and Rasa3/Sipa1-KO T cells.

Article Snippet: Monoclonal Abs against αL (KBA), ICAM1 (YN1/1.7.4), MAdCAM1 (MECA-89), and L-selectin (MEL-14) were purified from supernatants of hybridomas (ATCC) for blocking experiments or flow cytometric analysis.

Techniques: Control, Binding Assay, Mutagenesis

Journal: Cell reports

Article Title: Distinct bidirectional regulation of LFA1 and α4β7 by Rap1 and integrin adaptors in T cells under shear flow

doi: 10.1016/j.celrep.2023.112580

Figure Lengend Snippet: (A) A multistep adhesion cascade by L-selectin and LFA1. L-selectin interacting with PNAd + CD34 mediates a capture and initiates tether/fast rolling with a minimal level of activated Rap1 and talin1 recruitment. Simultaneous occupancy of LFA1 with ICAM1 and talin1 results in conformational changes of LFA1 from bent/low affinity to extended/intermediate affinity and transmits the outside-in signaling through subsecond activation of Rap1, which recruits talin1, increasing the capture and decreasing rolling velocities (slow rolling). A further increase of Rap1 activation by CCL21 results in more recruitment of talin1, leading to talin1/kindlin-3-dependent arrest by high-affinity bindings of LFA1 to ICAM1. Rap1 activation and inactivation occurs via RasGRP2/C3G and Rasa3/Sipa1, respectively. (B) Rolling and arrest by α4β7. Interactions with α4β7 with MAdCAM1 support tether/rolling in a manner independent of Rap1, talin1, and kindlin-3, all of which require subsequent chemokine-induced arrest. Ligand-induced arrest without chemokines also occurs in a lesser degree, which requires Rap1 and talin1 with a marginal contribution of kindlin-3. High basal levels of activated Rap1 due to the loss of Rasa3/Sipa1 generates high-affinity α4β7, which results in inhibition of adhesive interactions under flow (see text). Sequential activation processes by outside-in and inside-out signaling induce efficient rolling and arrest by α4β7.

Article Snippet: Monoclonal Abs against αL (KBA), ICAM1 (YN1/1.7.4), MAdCAM1 (MECA-89), and L-selectin (MEL-14) were purified from supernatants of hybridomas (ATCC) for blocking experiments or flow cytometric analysis.

Techniques: Activation Assay, Inhibition, Adhesive