Journal: Life Science Alliance

Article Title: CHRAC/ACF contribute to the repressive ground state of chromatin

doi: 10.26508/lsa.201800024

Figure Lengend Snippet: (A) Schematic illustration of the transgenes used for testing the effects of ACF1 recruitment to multiple reporters in Kc167 cells. ACF1 is fused to the GAL4 DNA-binding domain (GAL4 DBD) at the N-terminus. A transgene containing the GAL4 DBD alone is used as a control. All constructs contain a V5 tag. (B) Western blot of whole cell extracts from transiently transfected Kc167 cells. ACF1 antibody was used for the detection of ACF1 transgenes and Lamin (LAM) was used for the loading control. The first three lanes represent ACF1 targeting construct and controls; all of them possess a V5 tag. “ACF1 (no tag)” is a construct that expresses ACF1 without the V5 tag and “CTRL” lane refers to cells transfected with water. Asterisks indicate expressed transgenic products. (C) Immunofluorescence microscopy of the cells transiently transfected as in (A). The cells were stained with DAPI and V5 antibody for the detection of the constructs. mCherry antibody was used as a control. Green horizontal bar indicates the scale (10 μm). (D) Comparison of the correlation between repression and reporter expression upon ACF1 (GAL4-ACF1, black) or HP1 tethering (GAL4-HP1, red). Lines represent linear regression. Equations describe the fitted lines. P -value refers to the significance of the difference in the slope between the two regression lines. (E) Jitter plots represent the distribution of log2 fold-changes of reporters integrated in GREEN (N = 12), GRAY (N = 17), and RED (N = 70) chromatin domains for the case of tethered ACF1 (GAL4-ACF1). Black horizontal bars represent median. (F) Same as (E) but for ACF1 overexpression (ACF1). (G) Same as (E) but for HP1 tethering (GAL4-HP1).

Article Snippet: The cells were washed twice with PBS and blocked with PBS/0.1% Triton X-100/5% normal donkey serum (Jackson Immuno Research)/5% nonfat milk for 2 h. After a brief wash with PBS, the cells were incubated overnight at RT with primary antibodies αV5 (1:1,000, GenScript, Cat. No. A00623) and αmCherry ( ) (1:20).

Techniques: Binding Assay, Control, Construct, Western Blot, Transfection, Transgenic Assay, Immunofluorescence, Microscopy, Staining, Comparison, Expressing, Over Expression

Journal: bioRxiv

Article Title: Inhibitory TIGIT signalling is dependent on T cell receptor activation

doi: 10.1101/2025.05.08.652881

Figure Lengend Snippet: a. Schematic depicting the expression of TIGIT and APEX2 in Jurkat T cells. TIGIT is expressed fused to APEX2 with either a non-cleavable GSW linker or a cleavable T2A linker, as shown in the corresponding gene and protein structures. Ext = Extracellular, Int = Intracellular. b. Flow cytometric analysis showing the expression of TIGIT in TIGIT-GSW-APEX2 (GSW, dark grey), TIGIT-T2A-APEX2 (T2A, light grey), and parental Jurkat T cells (black) together with an isotype-matched control (dashed black). c. Western blot showing TIGIT and biotinylated proteins in GSW and T2A Jurkat cells preincubated with biotin-phenol (BP, 1mM, 30 min) and/or H 2 O 2 (0.25 mM, 2 min). Controls without BP or H 2 O 2 are included for GSW cells. A loading control blot for α-Tubulin (DM1A) is also shown below from the same lysates. Molecular weight markers are depicted on the left. d. Representative Total Internal Reflection Fluorescence (TIRF) microscopy images of TIGIT (magenta) at the immune synapse of GSW and T2A Jurkat cells that have interacted with Planar Lipid Bilayers (PLB) loaded with ICAM-1 (100 molecules/μm 2 ) and CD155 (400 molecules/μm 2 ) for 20 mins. Cells were preincubated with BP and H 2 O 2 was added to the cells after 20 mins of interaction with PLBs, where indicated. NeutrAvidin (green) is used to mark biotinylated proteins. Controls without BP or H 2 O 2 are included for GSW cells. A merged fluorescence-BF image is also provided. Scale bars = 5 μm. e, f. Mean pixel intensity values of TIGIT ( e ) and NeutrAvidin ( f ) in Jurkat cells from the TIRF imaging shown in d (±S.D.; n=3 independent experiments; significant adjusted P values from a one-way ANOVA with Tukey’s multiple comparisons are shown; violin plots display individual cell variability). g. Confocal microscopy images showing TIGIT (magenta) in GSW and T2A Jurkat cells that were conjugated with different Raji cell populations (expressing either CD111 or CD155) for 5 mins, as indicated on the left of the panel. Jurkat cells were preincubated with BP, and H 2 O 2 was added after conjugation with Raji cells. NeutrAvidin (green) is used to mark biotinylated proteins and a V5 stain labels expressed ligands on Raji cells (blue). A merged fluorescence-BF image is provided. Scale bars = 5 μm. h,i. Mean log 2 fold change in synaptic TIGIT ( h ) and NeutrAvidin ( i ) enrichment in Jurkat cells, from the conjugates shown in g (±S.D.; n=3 independent experiments; adjusted P values from a one-way ANOVA with Tukey’s multiple comparisons are given; ns = not significant; violin plots display individual cell variability). j. ELISA data showing the amount of IL-2 released from either parental or different forms of TIGIT-APEX2 expressing Jurkat cells after co-incubation with SEE-pulsed Raji cells for 6 h. GSW cells were also pre-incubated with an antagonistic TIGIT antibody (αT) or an isotype-matched control (iso), as indicated (±S.D.; n=4-5 independent experiments; adjusted P values from a one-way ANOVA with Tukey’s multiple comparisons are given; ns = not significant; nd = not detected).

Article Snippet: For immunofluorescence and flow cytometry experiments the following antibodies were used: αTIGIT (MBSA43; 2.5 μg/mL), αV5 (Rabbit polyclonal; NB600-381; Novus Biologicals; 1 μg/mL), αCD19 (HIB19; 740287; BD Biosciences; 1 μg/mL), αCD3ε (SK7, 344822; Biolegend; 1 μg/mL), αIST1 (51002-1-AP, Proteintech, 2.5 μg/mL), αSdcBP (68096-1-Ig; Proteintech, 2.5 μg/mL), NeutrAvidin (22832; Thermo Fisher Scientific; 2.5 μg/mL), and myc (9E10; 626810/ab206486; BioLegend/Abcam, 2.5 μg/mL).

Techniques: Expressing, Control, Western Blot, Molecular Weight, Fluorescence, Microscopy, Imaging, Confocal Microscopy, Conjugation Assay, Staining, Enzyme-linked Immunosorbent Assay, Incubation

Journal: bioRxiv

Article Title: Inhibitory TIGIT signalling is dependent on T cell receptor activation

doi: 10.1101/2025.05.08.652881

Figure Lengend Snippet: a. Schematic depicting the cell conjugates used to assess the TIGIT interactome upon ligation with its ligand CD155. TIGIT-GSW-APEX2 (GSW) cells are conjugated with either Raji-CD111 or Raji-CD155 cells in cells stimulated with superantigen (SEE). b. Western blot showing TIGIT and biotinylated proteins in GSW Jurkat cells interacting with either Raji-CD111 or Raji-CD155 for 5 mins. We show the banding patterns for both the total cell lysates (TCL) and eluate from streptavidin enrichments, following proximity labelling. Total protein loading controls are shown on the right. Molecular weight markers are indicated on the left. c. Principal component analysis depicting the relative positions of each replicate of the conditions analysed according to the variation in the data, with principal components 1 (PC1) and 2 (PC2) plotted. The conditions are colour coded, and replicates are shape coded, as indicated. d. Heatmap depicting the overall Pearson correlations between each of the conditions and individual replicates shown in c . e. Volcano plot showing the differentially proximal proteins to TIGIT in Raji-CD155 conjugates vs Raji-CD111 conjugates. Proteins that were significantly more abundant in Raji-CD155 conjugates are labelled in red and those significantly more abundant in Raji-CD111 conjugates are in blue. Detected proteins not significantly different between the two proteomes are in grey (ns; Cut-off = log 2 fold change of 1.5, P value = 0.1, as indicated by dashed lines). Both GSW proteomes were pre-filtered against the TIGIT-T2A-APEX2 (T2A) proteome to remove non-specific interactors (Cut-off = log 2 fold change of 2; P value = 0.05). f. Heatmap depicting the measured abundances of the regulated proteins depicted in e , abundances are scaled across individual proteins. g. GO analysis depicting significantly enriched Biological Process GO terms with the indicated associated proteins within proteins significantly proximal to TIGIT in Raji-CD155 vs Raji-CD111 conjugates. h. Confocal microscopy images showing TIGIT (magenta) and either IST1 or SdcBP (green) in GSW expressing Jurkat cells that were conjugated with different Raji cells (expressing either CD111 or CD155) for 5 mins, as indicated on the left of the panel. A V5 stain labels expressed ligands on Raji cells (blue). A merged fluorescence-BF image is provided. Scale bars = 5 μm. i,j. Mean log 2 fold-change in synaptic IST1 ( i ) and SdcBP ( j ) enrichment in Jurkat cells, from the conjugates shown in h (±S.D.; n=3 independent experiments; adjusted P values from a one-way ANOVA with Tukey’s multiple comparisons are given; violin plots display individual cell variability).

Article Snippet: For immunofluorescence and flow cytometry experiments the following antibodies were used: αTIGIT (MBSA43; 2.5 μg/mL), αV5 (Rabbit polyclonal; NB600-381; Novus Biologicals; 1 μg/mL), αCD19 (HIB19; 740287; BD Biosciences; 1 μg/mL), αCD3ε (SK7, 344822; Biolegend; 1 μg/mL), αIST1 (51002-1-AP, Proteintech, 2.5 μg/mL), αSdcBP (68096-1-Ig; Proteintech, 2.5 μg/mL), NeutrAvidin (22832; Thermo Fisher Scientific; 2.5 μg/mL), and myc (9E10; 626810/ab206486; BioLegend/Abcam, 2.5 μg/mL).

Techniques: Ligation, Western Blot, Molecular Weight, Confocal Microscopy, Expressing, Staining, Fluorescence

Journal: Developmental cell

Article Title: PUF partner interactions at a conserved interface shape the RNA binding landscape and cell fate in Caenorhabditis elegans

doi: 10.1016/j.devcel.2024.01.005

Figure Lengend Snippet: (A) Tethering assay. FBF-2 is tethered by λN22 binding to boxB hairpins in the reporter RNA. Modified from Aoki et al. 201891. (B) Above, location of gonadal arm (black box) within animal. Below, location of distal region (dotted box) within gonadal arm. Asterisk marks distal end. Germline stem cells (GSCs) reside at the most distal end of germline and their daughters begin differentiation as they move proximally. (C) Diagram of distal gonad, showing distance from the distal end in microns below and extents of abundant FBF-2 (black line) and lower FBF-2 (dotted line) above. (D-E) Representative z-projections of extruded germlines. 20μm scale bar in (D) applies to all images. Dotted line marks gonad boundary; asterisk marks distal end. Top, GFP reporter expression (green); bottom, FBF-2FLAG staining (magenta). (D) Untethered wild-type FBF-2. (E) Tethered wild-type FBF-2. (F) ImageJ quantitation of reporter signal from tethered vs untethered FBF-2. GFP abundance plotted against distance from distal end. Solid line shows mean abundance and shading shows 95% confidence interval. Each plot represents three biological replicates with at least 10 gonad arms per replicate. P-values are given for pooled data in 0-35, 35-70 and 70-100 μm regions (black bars). P-values: *** p < 0.001, ** p < 0.01, * p < 0.05, ns (not significant) p > 0.05. Exact p-values in Table S4. (G-H) Representative z-projections of extruded germlines, as in (3D-E). (G) Untethered Y479A. (H) Tethered Y479A. (I) Quantitation of tethered vs untethered Y479A. P-values as in (F). (J) Western blots after FBF-2 and NTL-1 co-immunoprecipitation. Left, input lysates (1%); right, FLAG IP (10%). NTL-1:V5 co-immunoprecipitates with both wild-type and Y479A.

Article Snippet: mouse monoclonal αV5 , Bio-Rad , Cat# MCA1360, RRID:AB_322378.

Techniques: Binding Assay, Modification, Expressing, Staining, Quantitation Assay, Western Blot, Immunoprecipitation

Journal: Developmental cell

Article Title: PUF partner interactions at a conserved interface shape the RNA binding landscape and cell fate in Caenorhabditis elegans

doi: 10.1016/j.devcel.2024.01.005

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: mouse monoclonal αV5 , Bio-Rad , Cat# MCA1360, RRID:AB_322378.

Techniques: Virus, Recombinant, Protease Inhibitor, Software, Sequencing